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Maximizer Manual - Biovest Hollow Fiber

1. dormallya appears aiid Decoines the active mode automatically after Fill Flush and Inoculation oo are completed However if you need to directly activate 6 iHo mode ahi START then PROCESS Ra The pH temperature EC Cycling and other process control functions become active Override Controls are available during any of the four modes of operation described above If an override applies to a parameter displayed on the Monitor screen its status of being manually overriden is indicated by an asterisk to the left of the monitored parameter An example of this is shown in figure 5 2 The F3 pump is overriden to a speed of 0 ml hr Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 5 5 User Interface N SETUP Key Press SET UP to access two screens S 4sbem Tests andLogeine Inberual figures 5 3 and 5 4 Whichever screen was last viewed is the first one to be displayed Press ENTER to cycle between the two screens Press MONITOR to exit from either screen These screens may be accessed at any time during an active culture run System Tests Six instrument self tests should be performed on the MAXIMIZER before each culture run is started These tests ensure the functioning of the LCD audible alarm LEDs keypad response and printer connection The sixth test allows you to examine the revision level of the microprocessor PROMs Figure 5 3 System Tests Menu Screen Instrument Setup
2. 24 Press ENTER to display the Monitor screen The following actions have automatically occurred m Elapsed time has reset to H6 HE E SETUP has ended and FUMHIMG is the active mode of operation m Active Process Control is active Active Process Control LED is on so logging alarms setpoint control and other activities are now active m See Note on next page Figure 6 51 Instructions Screen 3 for Pre inoculation Phase 25 Press ALARM to silence the audible alarm and deactivate the external alarm relay Alarms normally occur such as temperature pH and DO immediately when mode FUMM IHG mode begins Review the alarms stored in the queue to ensure unusual alarms are not present 26 Press MONITOR and watch temperature pH and DO readings After approximately 20 minutes they will be equilibrated and likely within the 27 Press ALARM Individually restore each stored alarm by using arrow keys to locate each parameter that is in its alarm range and pressing ENTER to restore its alarm Press MONITOR P N 700037 000 Rev H Biovest International Inc Page 6 60 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures ee ey 28 If pH does not reach the entered setpoint and equilibrate within its alarm range in order for the pH alarm to be restored it is likely that pH must be calibrated which should be done regardless to ensure pH is properly controlled After FLIMMIM amp mode s elapsed time has reached approximately equ
3. As Needed Outgas biohazardous applications only slight vacuum P N 700037 000 Rev H Biovest International Inc Page A 2 Operations Manual for the ACUSYST MAXIMIZER Appendix A Specifications ee ens Cultureware and Accessories Disposable pre sterilized Flowpath with Pharmed pump tubing bioreactor gas exchanger silicone tubing Tygon tubing ultrasonics Reusable non sterile Electrode pH DO temperature probe cases bottles cap assemblies vent filters Tygon gassing lines Approximate Flowpath Volumes 600058 215 IC EC Circuit Volume 570 ml EC Circuit Volume 230 ml at low vol EC Circuit Volume 300 ml at high vol 600058 221 IC EC Circuit Volume 615 ml EC Circuit Volume 275 ml EC low vol EC Circuit Volume 345 ml EC high vol 600068 215 IC EC Circuit Volume 680 ml EC Circuit Volume 340 ml at low vol EC Circuit Volume 480 ml at high vol 600068 221 IC EC Circuit Volume 770 ml EC Circuit Volume 430 ml EC low vol EC Circuit Volume 570 ml EC high vol 70 ml transfer volume 140 ml transfer volume Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page A 3 Appendix A Specifications aaa Peristaltic Pump Rate Ranges Range Increment Process Control Pump Rates 7016 Pump Heads Outflow Pump 0 4320 ml hr 1 ml hr Media Pump O 800 ml hr 1 ml hr 7014 Pump Heads Factor 1 Pump 0 200 ml hr 1
4. Outflow line In port on bottle cap assembly Harvest line In port on bottle cap assembly Media line Out port on bottle cap assembly Factor line Out port on bottle cap assembly 5 Turn off the appropriate pump by setting the rate to zero using Override Controls 6 On the appropriate line close the clamp near the bottle or sterile protector 7 After the connections are sterile disconnect the tubing line from the cap assembly or protection Keep the tubing from the flowpath covered with the sterile gauze at all times 8 Remove the Luer plug from the new bottle and attach the line Place the Luer plug in the old bottle s opening P N 700037 000 Rev H Biovest International Inc Page 6 68 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures er ee ey 9 Open the clamp 10 Clear the override or return the pump rate to its previous override value Note Air may be removed from the Factor 3 tubing before it reaches the BRX and collects in the ECS To do this you will need to put a temporary clamp on the Factor 3 tubing downstream from the FACTOR PRIME port and attach a syringe to the FACTOR PRIME port After the fresh Factor 3 bottle has been attached steps 1 9 above open the FACTOR PRIME clamp and set the Override Controls to run the Factor 3 pump at high speed until the air is in the syringe Then override the Factor 3 pump rate to zero close the FACTOR PRIME clamp remove the temporary clamp and clear the
5. ACUSYST MAXIMIZER Operations Manual S T www biovest com e B O V E Telephone 763 786 0302 After Hours 763 786 0302 voice prompt 8500 Evergreen Boulevard Fax 763 786 0915 Minneapolis MN 55433 6000 USA Email accountservices biovest com Copyright Notice 2013 Biovest International Inc All rights reserved This document may not be reproduced in any form without the prior written consent of Biovest International Inc Biovest International makes no warranties with respect to this documentation and disclaims any implied warranties of merchantability and fitness for a particular purpose Information in this document is subject to change without notice Biovest International assumes no responsibility for any errors that may appear in this document Trademark Acknowledgements AcuSysT MAximizeEr REsCu ACULINK DATA LOG and ACUDATA are trademarks of Biovest International Inc Luer and Luer Lok are trademarks of Becton Dickinson and Co Acrodisc 1s a trademark of Gelman Sciences Masterflex is a trademark of Cole Parmer Inc Tygon is a registered trademark of Norton Performance Plastics PharMed 1s a registered trademark of Saint Gobain Performance Plastics Corp Revision History Document 700037 000 This manual describes the Biovest International ACUSYST MAXIMIZER p n 500011 XXX rev M instruments The revision history of this manual is shown below Rev A 3 91 Initial Release Rev B 3 93 Changed p
6. Page 6 64 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures ee Probe Calibration For pH probe calibrations use samples from the pre BRX sample line for DO probe calibrations use samples from the post BRX sample line Calibrate probes according to the following schedule pH probe 2 3 times per week DO probe 1 2 times per week The instructions below are specific to calibrating either the pH or DO probe These instructions do not cover details of aseptic connection removing samples from the flowpath opening and closing clamps etc that were covered in the preceding section Sampling CAUTION Calibrate in advance the offline pH meter or blood gas analyzer or equivalent as recommended by the manufacturer If this is not done the use of this equipment to calibrate the MAXIMIZER pH or DO probe can result in faulty pH control or DO readings 1 Check PRE BRX and POST BRX clamps to verify that the desired clamp is open pre for pH post for DO Only one of these clamps should be open at one time If the desired IC sample can be taken with the clamp that happens to be open the pre sample can be taken immediately Any time the open clamp changes wait approximately 2 minutes before proceeding to step 2 to allow the medium in the common tubing line to change This can be monitored by changes to the DO reading on the Monitor screen 2 Press CALIBRATE to display the following screen This screen appears only for t
7. Use 4 f to move the cursor between the four programming options Allowed EC Cycling parameters see chapter 6 Optimization Procedures m Delay 0 00 00 999 23 59 in increments of 1 minute m Rise Time 5 999 minutes in increments of 1 minute m Fall Time 10 999 minutes in increments of 1 minute m Transfer Volume 1 140 ml in increments of 1 ml Figure 5 11 EC Cycling Parameters Screen P N 700037 000 Rev H Biovest International Inc Page 5 12 Operations Manual for the ACUSYST MAXIMIZER User Interface CCC O Ewe ay Factor 3 F3 Serum Pump Control This pump delivers factor 3 solution growth supplemented medium to the EC in a continuous or discontinuous pulsatile manner Aliet the a Helge period elapses the pump runs at the desired flow Rabe The Or and Ceg i gt fields are used to define a continuous or pulsatile pumping process To run the F3 Pump continuously the amp r and Eg 1e times must be set to the same non zero time This is the most common mode of operation for this pump To run the F3 pump in pulses at your desired EAL set the fri field to the length of time the pump should turn and set the fei field to tiie combined and Off time Pulses are the only means of ae Daon rates and they allow or infrequent and large or small doses of factor to be pumped Cir piege With fabs ae the F3 3 Pump w will l begin tu iae two days a TN iid abi minutes after H mode began and at a rate of
8. bit2 EC gas solenoid bit3 EC vent solenoid where 0 closed 1 open pH Set This indicates the pH setpoint and its corresponding A D reading A pH value of 7 00 at 37 C should have an A D reading of 128 Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 7 21 Troubleshooting Figure 7 31 Diagnostics Screen 3 This screen is called Screen 3 The fields represent the following information EC DN Time This is the time in minutes that the cycling control routine will attempt to move EC fluid from the high setpoint to the low setpoint Default value 15 EC UP Time This is the time in minutes that the cycling control routine will attempt to move EC fluid from the low setpoint to the high setpoint Default value 15 EC LOW and EC HIGH Setpoint These are the A D readings where EC cycling toggles from a Rise phase to a Fall phase The default values are 68 for LOW Setpoint and 96 for HIGH Setpoint They correspond to Expansion Chamber volumes of 108 ml and 178 ml respectively P N 700037 000 Rev H Biovest International Inc Page 7 22 Operations Manual for the ACUSYST MAXIMIZER Troubleshooting eee PROBLEMS WITH BIOLOGICAL PERFORMANCE pH Control Because pH is controlled by several factors you may have to consider many aspects of the culture to resolve problems with pH control The following 1s a list of major areas to examine Contact Account Services for more deta
9. check the Glossary for definitions Notes designated as NOTES are used to call attention to unique or important applications to which you should pay particular attention Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 1 1 Introduction SSS SERVICE AND SUPPORT Contact Biovest International s Account Services department for m Technical Service m Field Service Customer Service m Sales Information We are always willing to discuss your questions suggestions or problems with the ACUSYST MAXIMIZER cell culture system Account Services 1s available weekdays from 8 00 5 00 Central Time GMT 6 Our contact information is on the manual s cover page After hours telephone callers hear recorded instructions for emergency technical assistance Keep a copy of our telephone number posted near the MAXIMIZER for quick reference Technical Service Contact us with any question you have when operating the MAXIMIZER instrument or cultureware Please be ready to provide as much detailed information as possible including instrument serial number cultureware manufacturing lot number printed or electronic run log process alarms chronology leading to the situation relevant previous run experiences etc Customer Service Appendix B has ordering information for common items Field Service Appendix F has important information about Calibration and Preventive Maintenance services
10. m If there was no leak the test passes see top screen of Figure 6 23 Go to step 16 m If there was a leak the test fails see bottom screen of Figure 6 23 Go to step 12 Figure 6 23 Pressurization Test Passed Screen and Pressure Test Failed Screen Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 33 Operating Procedures ey 12 If the probe case assembly Pressurization Test fails press ALARM to silence the audible alarm and deactivate the external alarm relay 13 A screen similar to Figure 6 24 appears ca te ae Figure 6 24 Pressuirzation Test Failure Alarm Screen 14 The leak must be located and fixed before proceeding with the run Otherwise later on the cultureware will likely leak cell culture medium and become contaminated which could jeopardize the cultureware s warranty Potential sources of a leak are a Tightness of the lower and upper sections of the probe case don t over tighten them b Proper installation of the o rings c Damage to the o rings d Tightness of the Luer connections e The instrument itself if it did not successfully pass the instrument Pressurization Test described earlier in this chapter Perform this test if necessary 15 After each attempt to fix the leak repeat the Pressurization Test to confirm the leak has been fixed a If necessary press ALARM b Use or to locate the Pressure dros detected alarm c Press
11. m When the Delay time of EC Cycling is greater than the Delay time of the Continuous Batch or Timed Batch harvest strategy these two harvesting methods will not begin until after EC Cycling has started m Using the Override Controls described below to set a Low or Hold condition on EC Cycling may prevent these harvest strategies from being used This potential pause to harvesting depends on the phase of EC Cycling that is in progress when either of these two EC Cycling overrides is set Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 5 15 User Interface _ E E EEEeeay Continuous Harvesting When this selection 1s made the screen in figure 5 15 will be displayed by pressing ENTER Yes even though this harvest method is called Continuous it nevertheless is possible to program a pulsatile harvest pumping method in addition to the fully continuous method The basic difference between Pulsatile Continuous Harvesting and Continuous Batch or Timed Batch is Pulsatile Continuous Harvesting occurs irrespective of the EC Cycling process In other words the Harvest Pump operates regardless of the fluctuating volume in the Expansion Chamber Continuous Batch and Timed Batch operate the Harvest Pump relative to the Expansion Chamber volume See the following page for further details about Continuous Batch and Timed Batch Fully continuous harvesting is the
12. peracetic acid etc is necessary for making sterile connections Sterile gauze or cotton pads may be used with the disinfectant to protect the culture during these procedures A laminar flow hood autoclave and some kind of pH meter benchtop monitor or blood gas analyzer are absolutely required for certain procedures Assay equipment and reagents for the measurement of secreted product glucose and lactate are used to adjust process control parameters Calibration of the optional oxygen electrode can be performed offline with an instrument such as a blood gas analyzer Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 4 3 Instrument Setup e e Se PRINTER SETUP OPTIONAL Biovest International optionally offers a printer to document the run log It is used with the MAXIMIZER to make hardcopy records of process control conditions and alarms during the run Assemble the printer and tractor feed according to the printer s pre packaged instructions and connect the printer to the MAXIMIZER by attaching the parallel interface cable see Figure 4 3 for location of parallel port Load the printer with paper and plug it into an appropriate electrical outlet or uninterruptible power supply as described on page 4 1 SERIAL INTERFACE PORT The serial interface port is a 25 pin serial cable connector on the rear panel of the instrument The serial port provides for the transfer of information
13. s pH 2 Ensure the buffer solutions have not expired 3 If either A D value is nearly out of range the subsequent autoclave sterilization may render the probe unacceptable for continued use Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 27 Operating Procedures 7 SS SS DO Probe Evaluation 1 The instrument must be plugged in and turned on 2 Remove the electrical cap and the protective cap covering the probe s tip 3 Unscrew the probe s sleeve remove the probe s membrane cartridge discard the electrolyte rinse the probe and membrane cartridge pour electrolyte into the membrane cartridge without forming bubbles until it is full carefully slide the membrane cartridge on to the DO probe wipe off electrolyte from the outside of the membrane cartridge and screw on the sleeve Ensure no air bubbles are inside the membrane cartridge 4 Connect the DO cable to the probe and the bulkhead pO2 jack 5 Do not allow the DO probe to contact any metal parts inside the incubator This would not damage the probe but it would electrically ground the probe to earth ground and prevent it from being polarized 6 Connect the temperature probe to the bulkhead TEMP jack 7 Close the incubator door Allow at least 2 hours for the probe to reach 37 C 8 In addition to warming to 37 C the probe receives an electrical charge from the instrument The electrical charge pola
14. 3 ml hr The F3 Pump v turn for 30n minutes stop for 1 hour 30 minutes to complete the two hour CL time turn again for another 30 minutes stop again for another hour 30 minutes etc The long term effective pumping rate of these pulses will be 0 75 ml hr 3 ml hr at 25 on time Allowed F3 Pump parameters m Delay 0 00 00 999 23 59 in increments of 1 minute m Rate 0 200 ml hr in increments of 1 ml hr see page A 4 mE On 0 00 00 999 23 59 in increments of 1 minute On time must be lt Cycle time m Cycle 0 00 00 999 23 59 in increments of 1 minute Figure 5 12 Factor 3 Serum Control Screen Note The use of Cg 1 time in this screen should not be confused with and is unrelated to EC Cycling parameters described on page 5 12 Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 5 13 User Interface _ E Eeay Factor 2 Pump Control F2 This pump delivers factor 2 solution usually a glucose or glutamine concentrate to the IC in a continuous or discontinuous pulsatile manner The usage for these settings is the same as for the F3 Pump Control described on the previous page Allowed F2 Pump parameters Delay 0 00 00 999 23 59 in increments of 1 minute Rate 0 200 ml hr in increments of 1 ml see page A 4 On 0 00 00 999 23 59 in increments of 1 minute On time must be lt Cycle time m Cycle 0 00 00
15. A known control will ensure that cell death is due to toxicity and not some other factor Flasks should be gassed aseptically with 5 10 CO to prevent bicarbonate buffer in the medium from losing CO until the flask is equilibrated with the incubator environment While some cells survive this shock loss of CO which results in high pH it usually induces a lag period of cell growth and some cells will die A high percentage of serum may bind toxins and mask a potentially bad lot of medium A suggested protocol follows Other cytotoxicity tests may be used P N 700037 000 Rev H Biovest International Inc Page 6 74 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures ee ee Cytotoxicity Testing Procedure Run cytotoxicity assays of medium taken from the system against a control flask of the same type medium All flasks should have the necessary nutrients at the same concentrations 1 Label six T 25 flasks two control flasks two for ICS samples and two for ECS samples 2 Add 10 ml of control medium to each of the control flasks 3 Remove two 20 ml samples one from the IC circuit and one from the EC circuit using the technique described in the sampling procedure 4 Put half of the IC sample 10 ml in one IC sample flask and the other half in the second IC sample flask Put half of the EC sample in one EC sample flask and the other half in the second EC sample flask 5 Add serum to all flasks s
16. Pump Rate This screen allows you to enable press 1 or disable press 2 the base addition strategy for pH control and to designate a volume that must be pumped into the cultureware before the next strategy Medium Dilution is activated Base solution is automatically pumped at 0 of the Medium Pump rate or 5 ml hr whichever is greater when Gas 1 typically by itself is unable to maintain or raise pH to the pH setpoint If Base Addition is enabled Dy you oe enter ia tgs ml mo increments of 1 ml as the desired Hass Lj He shari of Oiluticm This volume of base is pumped Kee the i instrument activates Medium Dilution if it is enabled and its use is necessary pH continues to be lower than the setpoint despite base addition being used Base addition continues once mean Dilution P If Base Addition 1 IS enabled and the Fass Wolume before start of Dilution is setto as shown in Figure 5 10 then Base Addition andl Medium Dilution if enabled begin simultaneously when Gas 1 typically by itself is unable to maintain or raise pH to the pH setpoint After pH returns to the nese AA S Base Rien n stops a Papps stops turning and the Hase Wig lume before i of Oi lution is reset to the user defined volume for example 50 al If DH g goes out tof range again and Base Addition is activated another 50 ml of base may be pumped if pH remains below the setpoint during this time before Medium Dilution can be activat
17. service If you report an alarm condition to Account Services please include all of the displayed information Alarms 34 35 and 36 monitor internal instrument functions Even when they are stored in the alarm queue each new occurrence of these alarms causes another activation of the audible alarm and external alarm relay and it is recorded recorded by ACULINK DATA LOG or the printer If these alarms occur more than once in a short period of time contact Biovest s Account Services The remaining alarms do not re activate the alarm responses if they are stored in the alarm queue P N 700037 000 Rev H Biovest International Inc Page 2 Operations Manual for the ACUSYST MAXIMIZER Troubleshooting ee Some information about each of the 33 alarms is presented below Some of alarm descriptions will be useful to you but they are intended for qualified service personnel Figure 7 2 Format for Alarms 1 Through 8 Alarms numbered 1 8 indicate eight types of failures of the main control program to communicate with secondary programs If this alarm occurs once it may be of little concern because it could be caused by electrical noise However repeated occurrences need immediate attention The primary causes are improper DIP switch settings on the pump or monitor boards loose or faulty wire assemblies or a malfunction in various circuit boards This problem likely requires repair service Figure 7 3 Alarm Screen 9 This alarm mea
18. the integration chamber and associated tubing The Integration Circuit is separated from the Expansion Circuit by the walls of the hollow fibers The volume inside of the fibers of the bioreactor Liquid crystal display at the top of the control panel Used to show all of the user interface screens The process of removing high levels of waste or raising the pH by increasing the medium flow rate Molecular weight cutoff measured in daltons Refers to the pore size of the hollow fibers Effluent from the IC media containing metabolic by products which are small enough to pass through the pores of the fibers Biovest International Inc Operations Manual for the ACUSYST MAXIMIZER Glossary Oxygenator Peristaltic Pump Pre sample Pump Head Sample Sample Port Setpoint Tubing Raceway Vent Filters tBiovest International Inc See Gas Exchange Cartridge The metal and plastic pumps which move fluid into and out of the flowpath for media factor harvest and outflow The portion of the media that is removed before the sample to be analyzed The pre sample contains media which may not be representative of that which is in the culture The plastic portion of the peristaltic pump which encloses the pump segment of the tubing The media removed from the flowpath for metabolic or chemical analysis A controlled opening in the flowpath which permits removal of media for analysis A sample port may end in a
19. 2 A D a g aa o5 ep Ww 5 2 O aa x lt D ome PHIN LO ti O gt a amp mi a Lo zZ x m IC SAMPLE OUTFLOW MEDIA Factor 2 lt N m rY Lies Factor 1 lt N Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page A 5 Appendix A Specifications Schematic for 6 00068 xxx Cultureware Models E OE e CELL REMOVAL EC RETURN EC CHECK VALVE BYPASS so lt CHAREST EC CHECK VALVE BYPASS j INOCULATE e EC SAMPLE a k amp _ FACTOR PRIME i EC FLUSH IN FACTOR 3 PH OUT HI Extracapillary Circuit POST BRX DO PH IN LO GEX OUT Intracapillary Circuit IC SAMPLE IC RETURN OUTFLOW MEDIA Factor 2 Factor 1 P N 700037 000 Rev H Biovest International Inc Page A 6 Operations Manual for the ACUSYST MAXIMIZER Appendix B Ordering Information er eS These orderirng part numbers represent common items needed to operate the ACUSYST MAXIMIZER Other miscellaneous items also are available so if you need something not on the list please contact Biovest s Account Services group Part Number Description Of Part Instrument and Accessories 600052 XXX ACUSYST MAXIMIZER Cell Culture System 000 120 VAC 001 100 VAC 002 230 VAC 4190 000 Printer Package 100 120 VAC 600072 00
20. 50 DO Low 100 High 200 mmHg Temp Low 36 5 High 37 5 C Circulation pump 500 1000 ml min Media pump Basal 25 75 ml hr Dilution 0 ml hr Base F1 Disabled EC Cycling Delay 0 00 00 DDD HH MM Up 15 minutes Dn 15 minutes Transfer Vol 70 ml Serum Control F3 Rate 0 ml hr Delay 0 00 00 DDD HH MM ON 0 00 00 DDD HH MM Cycle 0 00 00 DDD HH MM F2 Control F2 Rate 0 ml hr Delay 0 00 00 DDD HH MM ON 0 00 00 DDD HH MM Cycle 0 00 00 DDD HH MM Harvest None option 4 Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 81 Operating Procedures 7 SS INOCULATION PROCEDURE Note Note CAUTION The preceding part of this manual describes certain procedures that prepare and test the flowpath before cells are added to the BRX Read them carefully and be certain the necessary steps have been performed before continuing with this procedure The last part in this section Optimization Procedures provides you with advice related to the inoculation of flowpath modules The following instructions are written for cells that have been grown in static culture using serum supplemented media Consult the Optimization Procedures part of this manual or contact Account Services if you have questions about using serum free or similar media Cells should be prepared so they will be in mid log phase of growth on the day of flowpath inoculation This can be determined by constructing a growth curve fo
21. 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 4 7 User Interface _ _ _ _ _ EEeEe ay This section describes the control features and screens that are accessed using the keypad of the ACUSYST MAXIMIZER Some of the keys and features are not explained in detail in this chapter but a brief description of their general use 1s provided More extensive coverage is provided in the chapter 6 Operating Procedures This is necessary for some features because it is easier to learn them as you proceed through the steps of a culture run Guidance for using some of these features is provided in chapter 6 Optimization Procedure Note Refer to Section 7 Troubleshooting for a complete list and description of alarms OVERVIEW OF LCD AND KEYPAD Refer to Figure 5 1 for the location of the LCD and keypad items LCD This is the screen at the top of the keypad Information is displayed as discrete screens that may or may not contain a flashing movable cursor Because the LCD has space for a limited amount of text some information must be presented as a series of separate screens Scroll through them by repetitively pressing ENTER for most series or and for others Keypad Numeric Panel This is a collection of keys numbered 0 through 9 with a key for decimal CLEAR and ENTER A selection from a menu is chosen by pressing the desired number To input values such as pH or temperature
22. 999 23 59 in increments of 1 minute Figure 5 13 Factor 2 Control Screen Note The use of lt 1 time in this screen should not be confused with and is unrelated to EC Cycling parameters described on page 5 12 P N 700037 000 Rev H Biovest International Inc Page 5 14 Operations Manual for the ACUSYST MAXIMIZER User Interface _ _ _ _ _ _ EEEee ay Harvest Schedule This screen allows you to select the type of harvesting strategy to be employed by Process Control One of the choices option 4 is no harvesting The other choices are referenced as Continuous Continuous Batch and Timed Batch and are described in detail on the following pages When any of the 4 selections are made the top of this screen displays the name of the selection after Harvest Bohecdiile When option 4 is chosen the Harvest Pump does not turn and the next Process Control screen displayed by pressing ENTER is pH Setpoint When options 1 3 are chosen the next Process Control screen displayed by pressing ENTER varies based on the harvest schedule option chosen and it provides the appropriate settings needed for the chosen harvesting method Figure 5 14 Harvest Schedule Selection Screen Note Because the Continuous Batch and Timed Batch strategies rely partly on EC Cycling to start the harvest pump certain conditions related to EC Cycling may influence timing aspects of these two harvesting methods
23. Circulation Rate Screen Note Cultureware that contains one BRX should not exceed circulation rates of 600 ml min Media ump Rates The Me Males screen comics two pump rates eared to pee IC medam by the Media Pump for nutrient feed Oi but ior for optional supplemental control of pH en pH Control beginning on page 6 1 Acceptable rates for both are between 0 800 ml hr in increments of 1 ml hr The Basal rate should be changed according to metabolic information periodic off line assays of media samples to measure for example glucose concentration As the culture expands and requires faster nutrient delivery increase the Basal rate as necessary When the fi iut ion rate lt Ea221 rate the Media Pump remains at the Basal rate to maintain athe chosen nutrient delivery rate The result is the disabling of the Medium Dilution method of maintaining the pH setpoint Excess medium will not be used to dilute lactate even if pH drops below its setpoint When the fi lution rate gt Eae21 rate the result is the enabling of the Medium Dilution ened of maintaining the pH setpoint Excess medium may be used to dilute lactate if pH drops below its setpoint Figure 5 9 Medium Pump Rates Screen P N 700037 000 Rev H Biovest International Inc Page 5 10 Operations Manual for the ACUSYST MAXIMIZER User Interface _ _ _ EEoe ay Factor 1 F1 Base Addition
24. Loading Pump Heads P N 700037 000 Rev H Biovest International Inc vili Operations Manual for the ACUSYST MAXIMIZER Table of Contents E Appendix F Maintenance ProODE SIONO eeii a a a aE E E e ers eauese dees F 1 DA PrOD O eer ererrrr EREEREER ETT F 1 DO PODC e eea a A E EEEE EEE ee EE ee E F 1 POMP TEADS Saarinen iea EEEE EE E E RARA EEE F 2 GOINNE 46 se actacaaaseeenneueaeeene E T one oe ess F 2 ACUSYST INSTRUMENT SERVICE scecesdeaecae55ee464cu urund bbb eee ee ees F 3 Installation Qualification Operational Qualification 05 F 3 CallOraUlOl 44444440 pe enen E EE E Su renee oe een ee ee et F 3 Preventive Maintenance ccc eee ee ee ee nee e eens F 3 FU Service COM ACh cic ctomeeeesausedueseses ener asdae E es F 3 Glossary Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Ix Introduction FF a Zz eee Welcome to ACUSYST Automated CUlture SYSTem technology from Biovest International the most advanced technology available in hollow fiber bioreactors The ACUSYST MAXIMIZER MAXIMIZER belongs to a product line that is directed toward the pilot plant scale of producing secreted biologicals from mammalian cells Average antibody harvests from this instrument can reach up to 1000 mg day The perfused hollow fiber cultures in this instrument are maintained by microprocessor control ABOUT THIS MANUAL This manual is designed to assist you in understand
25. Package includes e Six liter bottles e Four 10 liter bottles e Two 10 liter delivery cap assemblies e Four 1 liter delivery cap assemblies Biovest International Inc Operations Manual for the ACUSYST MAXIMIZER Appendix B Ordering Information Cap Assembly for Plastic Bottles e Four receiving cap assemblies fit any size bottle e One box of vent filters see 1825 000 Cap is polypropylene size 53B with two liquid ports accept male or female luer lock connections and one vent port filter included It is fully assembled including all necessary external and internal tubing clamps and color coded labels to deliver or collect fluid It is ready for use User only needs to autoclave sterilize it 400836 000 Miscellaneous Spare Parts 1825 000 3033 000 3429 027 3675 019 4197 000 4198 000 100201 000 102064 000 400136 000 400137 240 400148 000 600156 000 Services CALIB SERV SERV CON 400828 000 Biovest International Inc Delivery and Receiving Cap Assembly use with 2L Nalge bottle Sterilizable Reusable Vent Filters 50 per box White Sterility Protector Vent Plug each minimum 10 4 Amp Fuse 100 120 VAC units 2 Amp Fuse 230 VAC units Size 14 Pump Head Size 16 Pump Head Urethane Pump Head Coupler Pump Head White Teflon Washers pack of 12 Operations Manual Circulation Temperature Probe Circulation Pump Hardware e Metal Screws two e Plastic Threaded Knob one Incubator Gassing Lines
26. Page 6 6 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures Se eee ie ieee sear Note Biovest International offers a software package ACUDATA for IBM compatible computers It is based on a non linear regression growth rate formula Use ACUDATA during a culture run to calculate media pump rates that will maintain metabolite concentrations in the future based on recent nutrient consumption rates which ACUDATA also calculates See Appendix B of this manual for information and contact Account Services for more details Special consideration must be placed on any labile components of the medium Because glutamine is known to degrade spontaneously to ammonia especially with increasing temperatures it makes for a prime example You may choose to formulate your basal medium without glutamine and make a concentrated glutamine factor solution If so Process Control or Override Controls may be used to pump this factor into the flowpath at a rate which effectively delivers glutamine as if it were incorporated in the basal medium EXAMPLE If your basal medium normally has 4mM glutamine you can prepare a 160mM glutamine factor and set Process Control or Override Controls to add it at 0 of the basal medium rate The advantage of this technique is to allow you to store such an unstable component in a small volume which can be chilled in an ice bath to prevent its breakdown Note Although Biovest International offers bottles up to
27. Procedures beginning on page 6 15 explain the details of the six System Tests which are activated by pressing the respective menu numbers Printer Log Interval The second Set Up screen is shown in the figure below Figure 5 4 Logging Interval Screen Logging Interval is the user defined frequency for sending current instrument status information to the printer or ACULINK DATA LoG When set to logging is disabled The interval may be changed at any time during an active culture run Logging is active only in EHHH IHG mode This feature applies to a printer or ACULINK DATA LOG connected to the serial or parallel port Note The function of Logging Interval is different from the Printer test in the System Tests screen P N 700037 000 Rev H Biovest International Inc Page 5 6 Operations Manual for the ACUSYST MAXIMIZER User Interface a START SETUP Functions Press START then SET UP to display the screen shown in figure 5 5 This also causes me Active Set Up LED to turn on and it changes the miniMAX s status from IDLE to ETLE Press MONITOR to exit from this screen Figure 5 5 START SETUP Selection Screen Pressing 1 or 2 results in Da screens Bee instructions that are specific to the oon ancien The FRESSURSTEATION TEST must be performed before al 1 After the Preso Test i is complete the display returns to fic screen in onde to then perform Fill Flush See chapter 6 for operational details Pressurization Te
28. System Description al BOTTLES AND CAP ASSEMBLIES OPTIONAL Bottles Biovest International offers glass bottles to be used with the MAXIMIZER These bottles are provided with screw caps and plastic pouring rings that will survive repeated autoclave cycles Figure 3 5 Cap Assemblies In addition to the bottles offered by Biovest International two types of patented vented RESCu CaP assemblies are available Figure 3 5 Each type can be fitted with a 0 2 micron Acrodisc vent filter and two Luer plugs to ensure protection of sterility after steam sterilization One type of assembly the receiving cap has a short segment of tubing attached to the inlet port Bottles used for collection of outflow or harvest should have this type of cap assembly The other type of assembly has a longer segment of tubing plus a glass tube on the outlet port Bottles used to deliver feed medium to the flowpath should have this type of cap assembly with the correct length of tubing Figure 3 5 Bottle components and patented cap assemblies P N 700037 000 Rev H Biovest International Inc Page 3 8 Operations Manual for the ACUSYST MAXIMIZER System Description E O a a PRINTER PACKAGE OPTIONAL A printer and communications cable may be purchased from Biovest International see Appendix B This device may be used with the MAXIMIZER to record process control events and alarms with a user defined logging schedule It is useful when hardcopies are ne
29. activities normally occur after Idle Further oao nae Start Set Up is described three pages below dh TMC appears automatically after completing the Start Set Up activities and the NES Process Control LED also turns on automatically Elapsed time resets to 8 8 a This stage of the run is called Pre Inoculation phase a time when the mua is adjusting the cell culture medium to make it ready for inoculating the cultureware with the scale up culture by controlling pH temperature EC Cycling etc Ai a and other functions become enabled when the MAXIMIZER is in mode MOE appears after pressing START then INOCU LATE en also turns on the nee Inoculate LED Elapsed time resets to H H amp amp Inoculation phase is a short duration phase used to inoculate the E sath the scale up culture 3 re appears automatically after completing the Start Inoculate activities The Active Process Control LED turns on automatically again The MAXIMIZER resumes controlling process control parameters programmed in the series of Process Control screens MAXIMIZER remains in HLIMM PME ede until it is time to terminate the run Press progressing through these mode of operation repeats for the neat culture run P N 700037 000 Rev H Biovest International Inc Page 5 4 Operations Manual for the ACUSYST MAXIMIZER User Interface EEEEE Eee Note POLE SETUP and IHOCLULATE must be manually activated
30. again in the future the pH alarm will not be recognized which prevents what happens when an alarm occurs described on the previous page The Alarm LED remains lit when at least one alarm message is stored in the alarm queue Storing the alarm message continues to silence the alarm and allows you to perform normal activities while also evaluating what may have caused the alarm When an alarm is stored in the alarm queue and the Alarm LED is on and the audible alarm is silenced when a new alarm occurs that is not stored in the alarm queue the audible alarm and external alarm relay will activate Press alarm to silence the audible alarm and deactivate the external alarm relay Respond to this new alarm according to the same two choices described above There are a few critical alarms that monitor the instrument s internal circuitry If one of these alarms occurs and is then stored in the alarm queue subsequent occurrences of the same alarm will nevertheless cause the audible alarm and external alarm relay to be re activated Inform Biovest s Account Services of this situation When the last message in the queue has been restored the screen in figure 5 23 appears Press ENTER The Alarm LED turns off and the Monitor screen appears Note Chapter 7 Troubleshooting lists and describes all available alarms Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 5 27 User Interface N Di
31. ane HiH gas numbers We strongly suggest using the default values Ltd i HIGH i4 A typical run needs the full range of gas numbers i changed the i 5 value must be lt the HGH value When if value the HIGH value High the result is the same as overriding Gas to a single gas number Note Changing the default 2 and HISH values reduces the range of pH control Alarms Use this screen to set the desired i Hii and i alarm limits for pH DO and Temperature and setpoint for Temperature These alarms are active only in muiMM TH mode To enter a temperature setpoint outside of the default alarm range first change the i or HIGH temperature limit to a value outside of the setpoint Acceptable ranges and increments m pH alarm 6 00 8 00 pH units 0 01 units m DO alarm 0 200 mmHg 1 mmHg m Temperature alarm 24 1 45 8 C 0 1 C m Temperature setpoint 24 1 45 8 C 0 1 C Figure 5 7 Alarm and Temperature Setpoint Screen Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 5 9 User Interface EN Circulation Rate The Circulaiiar Rate screen allows you to set the speed of the Circulation Pump in ania Ke dcebeds in chapter 6 Oxygen Delivery this value should be raised during the active culture run to maintain a minimum desired post BRX DO level suggest 100 mmHg Acceptable Circulation Rate values are between 0 1000 ml min in increments of 1 ml min Figure 5 8
32. connected in its bulkhead jack Outflow and Harvest bottles full of media or are their vent filters wet which could prevent removal of liquid from the flowpath P N 700037 000 Rev H Biovest International Inc Page 7 8 Operations Manual for the ACUSYST MAXIMIZER Troubleshooting IIE IE ES EES ESD Figure 7 10 Alarm Screen 16 This alarm means the Expansion Chamber is below its minimum volume Take immediate action 1 Ifthe Expansion Chamber is empty and or the bioreactor is filled with air a Disconnect the EC gassing line from EC bulkhead port b On Monitor screen if i e is shown go to step d c On Monitor screen if 11 is shown disconnect IC gassing from IC bulkhead port and connect it to EC bulkhead port d Watch Integration Chamber volume and increase Media pump speed if necessary to maintain 1 5 inches of medium e When Expansion chamber has 108 ml clear the overrides and connect IC and EC gassing lines in their normal places f Check the suggestions below and if EC chamber volumes drains again contact Biovest s Account Services Verify the following items to try to correct the problem m Any clamps incorrectly open or closed particularly GEX Out Does the flowpath have any leaks cracks or loose tubing connections Gassing lines fully connected to IC and EC chambers and bulkhead ports Gassing lines attached to the correct bulkheads IC to IC and EC to EC Medium pump segment properly loaded
33. damaged A reusable pH probe has a finite lifespan however and will eventually fail Such failures may not be detected until after the pH probe has been aseptically installed into a cultureware and it contains circulating cell culture medium Replacement in this circumstance is possible but is time consuming can lead to contamination or may negatively impact the cell culture The advantage of reusable pH probes is their purchase price is low compared to disposable pH probes To proceed with a reusable pH probe go to the next section pH Probe Evaluation The disposable pH probe comes as an assembly including the probe probe case and electronics cable Disposable pH probes are provided with a certificate of compliance and are sterile and ready for use Cultureware also are provided in the same manner Disposable pH probes make run startup and turn around very fast The technician obtains one cultureware and one disposable pH probe aseptically connects them and starts the run At the end of the run the entire disposable pH probe assembly is discarded with the cultureware The disposable pH probe has many advantages during run startup and termination It also eliminates maintenance and storage issues as well as the risk that a reusable probe will not function during the next production run To proceed with a disposable pH probe skip ahead to Cultureware Sterile Assembly P N 700037 000 Rev H Biovest International Inc Page 6 26 Op
34. discarded so leave them in the cultureware as they are b remove the reusable plastic pH holder Save it for the next run 7 Remove the two metal screws and plastic knob from the circulation pump bracket and crank pin Save them for the next run 8 Slide the cultureware s circulation pump out of the metal bracket and off of the crank pin The cultureware s circulation pump has a cylindrical bearing that should slide off of the crank pin If it stays on the crank pin remove it too because it is discarded with the cultureware 9 Remove the six tubing lines from the pump heads 10 Set the six pump heads and their screws aside 11 Lift the cultureware up and over the metal circulation pump bracket and pull it out of the incubator 12 Open a biohazard bag and place it on a benchtop 13 Place the cultureware on the opened biohazard bag Do not close it yet P N 700037 000 Rev H Biovest International Inc Page 6 92 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures SS e 14 Remove the reusable pH probe case assembly and the DO probe case assembly if in use from the cultureware a put temporary clamps on the two tubing lines that connect to the pH probe case b put temporary clamps on the two tubing lines that connect to the DO probe case c disconnect the tubing from the probe cases and d reconnect the cultureware s open pH and DO tubing lines by installing the fittings saved from Culture
35. female Luer Lok fitting or a septum The value of a given metabolic parameter pH temperature which is to be maintained by the software A grooved plastic panel used for routing tubing into the incubator and refrigerator Either the filters used for introducing gas into the flowpath to the Integration and Expansion Chambers or the hydrophobic 0 2 micron pore filter Acrodisc on the cap assemblies which prevent pressure buildup P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER G 3
36. fiber in a bioreactor As the circulation pumps moves medium into the hollow fiber a high relative pressure is created within the ICS This high pressure forces medium through the pores of the fiber and into the ECS Figure 2 1 As medium flows into the ECS the pressure in the ICS drops along the length of the fiber Near the distal end of the BRX high relative pressure in the ECS forces medium to flow back into the ICS carrying with it the metabolic waste from the ECS This process is known as the Starling Effect and can be detrimental to the culture as it ends up pushing growth factors and cells to the distal end of the bioreactor Due to the ECS flow along the length of the BRX cells at the entrance of the BRX are exposed to greater levels of oxygen and nutrients than those at the outlet end P N 700037 000 Rev H Biovest International Inc Page 2 2 Operations Manual for the ACUSYST MAXIMIZER System Theory SSeS Acusyst TECHNOLOGY DIFFERENCE The MAXIMIZER compensates for Starling Effect and keeps the ECS flow channels open by a mixing process called EC Cycling This mixing creates a more homogeneous cellular environment allowing better cell growth and production Think of the MAXIMIZER as having two separate fluid circuits which intersect only at the fiber membrane of the BRX These two fluid circuits including their fluid reservoirs constitute the flowpath of the system Figure 2 2 Fi
37. fill a syringe with the desired volume of serum 2 Aseptically fill a 3ml syringe with unsupplemented medium 3 Disinfect the FACTOR PRIME port 4 Attach the serum syringe to the FACTOR PRIME port 5 When using the single BRX flowpath 600058 xxx a Open the FACTOR PRIME clamp b Inject the serum c Close the FACTOR PRIME clamp 6 When using the double BRX flowpath 600068 xxx a Put a temporary clamp on the tubing connected to the top EC port of one BRX a Open the FACTOR PRIME clamp b Inject half of the serum c Close the FACTOR PRIME clamp d Move the temporary clamp to the tubing connected to the top EC port of the other BRX e Open the FACTOR PRIME clamp f Inject the remaining serum g Close the FACTOR PRIME clamp h Remove the temporary clamp 7 Aseptically replace the empty serum syringe with the syringe containing unsupplemented medium 8 Open the FACTOR PRIME clamp 9 Inject the unsupplemented medium to flush residual serum in the tubing into the bioreactor s 10 Close the FACTOR PRIME clamp P N 700037 000 Rev H Biovest International Inc Page 6 78 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures 11 Leave the flush syringe on this port unless you are using sampling septa This syringe can later be used to purge air out of the line when F3 bottles are replaced 12 Be certain that the system 1s cycling properly This will ensure that the serum 1s distributed throughout
38. for the ACUSYST MAXIMIZER Troubleshooting Figure 7 6 Alarm Screen 12 This alarm means the pressure in the Integration Chamber is gt 280 mmHg Verify that the chamber is not flooded and its gassing line is attached properly This problem likely requires repair service Figure 7 7 Alarm Screen 13 This alarm means the current pH value 1s outside of the alarm limits set in Process Control Verify the following items to try to correct this problem Bulkhead IN port connected to GEX IN vent filter port Wet GEX Vent filter GEX Out clamp open Gas lines to and from GEX pinched or plugged Off gas port at the instrument s back panel not obstructed or plugged CO gas line to the instrument and its gas supply pinched disconnected closed CO gas supply properly set to deliver 15 1 ps1 m pH cable properly connected to the probe and bulkhead jack pH probe A D reading within acceptable range See Section 6 pH Probe Evaluation Calibration of the pH probe fixes problem Calibration procedure being properly performed Circulation pump turning smoothly Bubbles in pH probe case Bubbles in the bioreactor s or in the IC Medium Dilution set high enough to sufficiently dilute lactate Base Addition attempted Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 7 5 Troubleshooting Figure 7 8 Alarm Screen 14 This alarm means the current DO value is outside of the alarm
39. level Continuous Batch harvesting generally occurs more frequently than the Timed Batch method see next page although both harvesting methods remove fluid when the Expansion Chamber volume 1s near its low level Allowed parameters m Delay 0 00 00 999 23 59 in increments of 1 minute m Volume 0 35 ml in increments of 1 ml Figure 5 16 Continuous Batch Harvest Control Screen Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 5 17 User Interface _ SS EEEEEEaw Timed Batch Harvesting When this selection is made the screen in figure 5 17 will be displayed by pressing ENTER Delay has elapsed this harvesting method becomes active The Harvest Pump then will automatically run to remove the user defined iis inme each time the Harvest Pump turns on which generally does NOT occur each time the Expansion Chamber volume is near its lowest level during the EC Cycling s Rise and Fall process Unlike with Continuous Batch harvesting Timed Batch harvesting includes a Once fc 1 time reaches 2 2 28 the Harvest Pump waits until the next time the Expansion Chamber volume is near its low level Then the Harvest Pump turns on at the pre programmed high flow rate to remove the user defined Volume Several EC Cycling Low Levels may occur without the Harvest Pump turning on while the Harvest Pump is waiting for the Timed Batch Cycle time t
40. line 20 Press E C CYCLE then 5 to clear the EC Cycle override 21 Press MONITOR 22 Remove the temporary clamp applied in step 6 23 Reconnect the tubing line to the EC bulkhead port 24 Open the EC RETURN clamp 25 The cell removal procedure is finished 26 Reset the alarms that may have been activated once they are between their respective Low and High Alarm Limits 27 Optional Process the collected cell slurry because it contains product from the ECS Discard the cell pellet as a biohazard CAUTION Ifthe application has the potential to release a biohazardous agent contact Biovest s Account Services department Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 71 Operating Procedures ar a SS Alternate Cell Removal Procedure 1 Needed supplies a sterile container a bottle and cap assembly or a disposable bag that holds gt 250 ml and can connect to the Cell Removal line s female Luer fitting 2 Aseptically connect the sterile container from step 1 to the Cell Removal line 3 Place the sterile container on the floor 4 Close the EC RETURN clamp 5 Carefully shake the Expansion Chamber to loosen the cells and form a slurry 6 Open the CELL REMOVAL clamp 7 Gravity alone may cause the liquid and cell slurry in the Expansion Chamber to flow into the sterile container If not press E C CYCLE then 2 to cause EC pressure to momentarily increas
41. means that in the Running mode the temperature setpoint used by the instrument will be the one set in Process Control unless a Temperature override is active In all other modes Idle Setup and Inoculate the setpoint is 37 C unless a Temperature override is active When a temperature override is used an asterisk 1s displayed to the left of the word Set Point and the Temperature LED will be turned on An asterisk also appears on the Monitor screen next to TF To activate a Temperature override use f to move the cursor to the value under Set Point press the desired number keys the desired temperature and press ENTER The acceptable range of values is 24 1 45 8 C in increments of 0 1 C To remove the Temperature override use f to move the cursor to the asterisk and press CLEAR After the override has been cleared the value below Set Point returns to the current setpoint value NOTE If the Temperature override is set to a value outside of the alarm limits in Process Control an alarm will occur when the temperature exceeds these limits NOTE A temperature override may not be able to attain the desired value This limitation is bound by the temperature of ambient air and the temperature and flow rate of incoming media Contact Technical Services for further details Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 5 23 User Interface EN CALIBRATE K
42. ml hr Factor 2 Pump 0 200 ml hr 1 ml hr Factor 3 Pump O 200 ml hr 1 ml hr Harvest Pump 0 200 ml hr 1 ml hr Automated no direct user input Manual Override Pump Rates 7016 Pump Heads Outflow Pump O 4320 ml hr 1 ml hr Media Pump O 800 ml hr 1 ml hr 7014 Pump Heads Factor 1 Pump O 400 ml hr 1 ml hr Factor 2 Pump O 400 ml hr 1 ml hr Factor 3 Pump O 400 ml hr 1 ml hr Harvest Pump O 400 ml hr 1 ml hr Fill Flush Media Pump Rate 4320 ml hr automated Inoculation Outflow Pump Rate 1000 ml hr automated Circulation Pump Rate Range 0 1000 ml min 1 ml min Note Same range and increment for process control and manual override CAUTION Maximum user entered CP speed when using a single bioreactor cultureware must be lt 500 ml min to prevent excessive pressure tubing disconnection severe media leak and contamination Using CP speeds gt 500 ml min will void the cultureware s warranty Note All pumps rotate counterclockwise only P N 700037 000 Rev H Biovest International Inc Page A 4 Operations Manual for the ACUSYST MAXIMIZER Appendix A Specifications Schematic for 600058 xxx Cultureware Models A D D pe _ __ _ 1c ow dao CELL REMOVAL O Q N L op ve Of W nE 58 23 p 2 HARVEST 5 gt 00 IU o gt Ox INOCULATE DS EC SAMPLE FACTOR PRIME V EC FLUSH IN 6 amp 5 FACTOR3 w gt c amp vs PH OUT HI 5
43. monitors but also provides feedback control to the various parts of the system ensuring that environ mental conditions are maintained in their operational ranges Cell secreted product remains in a circuit separate from the waste products and may be harvested by activating a separate pump as required With these general concepts in mind the following detailed description will give you an understanding of how and why the MAXIMIZER work Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 2 1 System Theory HOLLOW FIBER TECHNOLOGY The core of the MAXIMIZER mammalian cell culture systems is the hollow fiber bioreactor BRX The BRX 1s a plastic cylinder containing several thousand hollow fibers attached at each end of the tube Cells are grown in the space outside of the fibers known as the extracapillary space or ECS Growth media are circulated through the inside of the fibers known as the intracapillary space or ICS Figure 2 1 The fibers provide a substrate upon which the cells can grow These fibers also permit the exchange of nutrients and wastes The fibers of the BRX are semi permeable and allow small molecular weight molecules such as glucose and ammonia to move freely across the fiber The cells growth factors and the large molecular weight products produced by the cells remain in the ECS In Out _ gt Intracapillary Space ICS Se Figure 2 1 Hollow
44. occurs the Alarm LED turns on the dam message is sent to the printer or ACULINK DATA LOG and the audible alarm and external alarm relay are activated Press ALARM to silence the audible alarm and deactivate the external alarm relay The display changes to show the most recent alarm message if more than one alarm is activated P N 700037 000 Rev H Biovest International Inc Page 5 26 Operations Manual for the ACUSYST MAXIMIZER User Interface _ _ _ EEEEEEEEEe Each time a new alarm occurs you have two choices to respond m Press ENTER to restore re enable a particular alarm For example a pH out of range alarm occurs Press or to display this particular alarm if necessary When this alarm is displayed pressing ENTER restores this alarm only If other alarm s are stored in the alarm queue they must be restored individually Press ENTER only when the monitored parameter pH in this example is no longer out of range Otherwise another pH out of range alarm will recur in a matter of several seconds m Leave the alarm message stored in alarm queue prevent its recurrence Do this when the monitored parameter pH in this example remains out of range In this case press MONITOR while the problem 1s examined Once pH is no longer out of range is within the alarm limits set in PROCESS CONTROL restore the pH alarm by following the instructions above If this is not done and pH goes out of range
45. on the computer and start the ACUSOFT Link program 5 To enable exporting the run log to ASCII format for use in various other computer programs from within the ACUSOFT Link program press SHIFT F2 and set the parallel port to 6 Further setup and operation details are provided in the operations manual for ACULINK DATA LOG If a printer is in use rather than ACULINK DATA LOG 1 Load paper 2 Plug in the parallel or serial cable to the printer and the MAXIMIZER 3 Turn on the printer s power 4 Configure the printer to be on line or selected depending on printer model Consult the printer s user manual for further information Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 15 Operating Procedures E System Checkout Before starting any culture run the following checkout list should be consulted and the appropriate procedures should be performed to ensure that everything is in working order If you have a certified service technician who can inspect the internal electronics and gassing system you may consult him her before performing the following checkout Pre Culture Checkout List 1 System Tests 2 Instrument Pressurization Test 3 Validation of pH Probe and DO Probe 4 Pressure Testing of pH and DO Probe Case Assemblies Note The pH and DO probes should be tested for satisfactory performance before installing them in the flowpath This testing is done pr
46. one set e GEX IN GEX OUT IC EC Calibration recommend annually Installation Qualification Calibration Operation Qual Service Contract renewable one year coverage Preventive Maintenance recommend every five years P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page B 3 Appendix C Relocation eS If you need to move the ACUSYST MAXIMIZER to another room or building you should consider the following recommendations before starting any cultures in the instrument Preparing the MAXIMIZER Turn off the power to the MAXIMIZER before disconnecting the power cord from the wall outlet m Remove any plastic cable ties from the CO gas line on the rear of the instrument before disconnecting the tubing Then remove the tubing carefully to avoid breaking the plastic fitting on the filter Allow ample work space to do this efficiently m Be certain you have the cables for temperature probe pH probe and DO probe as well as the four Tygon gassing lines m The gas filters on the rear of the MAXIMIZER do not have to be removed when the instrument is moved However use caution to avoid damaging the filters or their mounting brackets when you place the instrument on a cart and when you move through narrow doorways This also applies to the peristaltic pumps on the front of the instrument m Be certain you have the plastic screw cap and the 2 metal screws for the circulation pump bracket Biovest Internati
47. onscreen instructions For the instruction mommies LL L vE LaS disconnect the reusable Tygon tubing from Figure 6 59 Instructions Screen 3 for Inoculation 10 Tie Ouitlow pap starts again and the screen changes For the instruction Mm DMULL SHR LS open the clamp labeled INOC EC SAMPLE Figure 6 60 Instructions Screen 4 for Inoculation 11 Ifthe cultureware has more than one BRX apply a temporary clamp on the tubing that connects to the upper EC side port of the left hand BRX to direct the inoculum into only the top of the right hand BRX CAUTION Do NOT clamp the top IC end port of the BRX 12 Inject the cells Excess fluid passes into the lumen and is removed at the Integration Chamber by the Outflow pump As you inject cells maintain constant pressure on the syringe and rock the syringe gently to keep the cells suspended Avoid adding any bubbles to the BRX This step should require 1 2 minutes 13 Ifthe cultureware has two BRXs a Close the INOC EC SAMPLE clamp b Aseptically replace the empty syringe with one containing cells c Move the temporary clamp to the tubing that connects to the upper EC side port of the right hand BRX to direct the inoculum into only the top of the left hand BRX See CAUTION note above d Open the INOC EC SAMPLE clamp and repeat step 13 e Close the INOC EC SAMPLE clamp f Remove the temporary clamp P N 700037 000 Rev H Biovest International Inc Page 6 84 Operations Ma
48. or F1 Pump speed P N 700037 000 Rev H Biovest International Inc Page 6 2 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures ee ee Overview of pH Control Algorithm The following pH control algorithm functions when no overrides are active for Gas or the MP and F1 pumps These overrides take precedence over calculations made by the pH control algorithm and partially or completely disable pH control YES Enabled NO YES NO kN ps Reached YES Medium Dilution Enabled Medium Dilution Required Gas gt 1 YES Stop Medium Dilution and Base Addition Reset Base Volume Stop Base Addition Continue Present Method NO NO Figure 6 2 Flowchart of pH control algorithm Gassing The pH control algorithm uses a PID controller to automatically determine and adjust the required gas mixture variable CO air and air flow rate pH control preferentially uses this gas mixture and flow referred to as Gassing but supplements it with base addition and medium dilution which may be individually enabled Gassing is always in effect even when these supplementary methods of pH control are used and HIGH Default i bi is 1 Default HIGH is 14 As pH drops Gas automatically drops to compensate s continues to drop as pH continues to drop Once as reaches the Li value it cannot drop further If pH now drops below its setpoint the algorithm advances to Base Addition
49. override as described in step 10 11 Treat removed bottles appropriately Outflow bottles should be decontaminated All bottles should be washed as soon as possible Replacing LAVs and Septa LAVs should be replaced after 100 uses or 96 hours of continuous use 1 e when something has been connected to it long term which we do not recommend Septa should be replaced after 7 10 samples 1 Wear a mask hair restraint lintless lab coat and sterile gloves Aseptic technique is important in this procedure 2 Check the sample line clamp to make sure it is closed 3 Carefully wrap the old LAV or septum with gauze soaked in disinfectant Wipe the entire area tubing LAV septum and female Luer Lok connector 4 Holding the female Luer Lok connector in one hand carefully twist the old LAV septum counterclockwise until it is loose Do not remove at this time 5 Open the sterile package of the new LAV or septum and hold the removable protector between your fingers 6 Carefully remove and discard the old LAV or septum and replace it with the new item Twist it clockwise until secure 7 Sample site is once again ready for use Disinfect the surface of the new LAV and the inside of its cover Install the cover on the LAV 9 Suggest covering the septum with a disinfected cover such as a piece of aluminum foil Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 69 Op
50. oxygen uptake rate OUR for the culture see Appendix E Equally important is the post BRX DO value itself because it represents the lowest level of oxygen to which the cells may be exposed This may be used as a guideline for DO delivery to maintain a healthy culture DO delivery is regulated by the speed of the circulation pump Therefore as the post BRX DO value falls due to cellular activity the circulation pump rate can be increased to maintain a desired post culture minimum level of DO You may wish to consider maintaining different post BRX DO values for the growth and production phases of a MAXIMIZER culture Feed Strategy The most commonly used feeding strategy in perfusion is based on providing nutrients mainly glucose or diluting wastes mainly lactate In the case of nutrients a minimum concentration setpoint of glucose is established in advance of setting up the culture and the feed rate is manually adjusted to ensure that this level is attained by the next sampling period A similar maximum setpoint may be established for lactate so that an upper tolerable concentration 1s known Regardless of which metabolite is considered more important the MAXIMIZER medium rate must be changed manually to maintain the desired level of nutrients and or wastes A regular schedule of sample removal and analysis for metabolite concentrations provides important data for this determination P N 700037 000 Rev H Biovest International Inc
51. procedure is done 2 Press E C CYCLE then 2 to override EC Cycling to the Low Expansion Chamber volume Close the EC RETURN clamp Nn A W a gm OQ NM NM D C S D 77 o 5 r lt Q ar oO 5 O a Put a temporary clamp on the EC tubing that connects to the lower side port in port of the BRX s and close this clamp a Disconnect the tubing line from the EC bulkhead port 8 Aseptically attach an empty 60 ml syringe to the Cell Removal line 9 Carefully shake the Expansion Chamber to loosen the cells and form a slurry P N 700037 000 Rev H Biovest International Inc Page 6 70 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures SS 10 Open the CELL REMOVAL clamp and remove as much of this suspension as possible 11 Close the CELL REMOVAL clamp 12 Aseptically replace the first syringe with an empty 60 ml syringe 13 Repeat steps 10 and 11 Tilt the Expansion Chamber to the left to remove as much of the slurry as possible if necessary 14 Aseptically replace the second syringe with a 60 ml syringe filled with serum supplemented medium 15 Open the CELL REMOVAL clamp 16 Inject the 60 ml of medium up the Cell Removal line and into the Expansion Chamber 17 Close the CELL REMOVAL clamp 18 Replace this syringe with the second syringe filled with serum supplemented medium Repeat steps 15 and 16 19 Leave this syringe attached to the Cell Removal
52. pumps are loaded in the opposite orientation to pump these fluids in Note Wrap the tubing around the roller by following its existing curvature Tubing loaded against its existing curvature will have a tendency to twist resulting in greater inaccuracies in flow rates and premature wear of the pump tubing segment 5 With your left thumb securely hold the tubing in the right raceway Be sure that your hand is positioned as shown in the figures Correct hand position is essential to the successful completion of the procedure 6 With your right hand slide the pump key into the position as shown so that it presses down on the tubing Figure E 4 Figure E 4 Pump key position 7 Pushing the roller down with the key turn the pump key clockwise The key presses approximately half of the tubing s diameter in the pump casing Turn key until approximately the ten o clock position where there is a stop tab NOTE Hold the left pump segment loosely while you turn the pump Key As the pump key positions the tubing in place the left pump segment needs to be able to slide under your left thumb Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER E 3 Appendix E Loading Pump Heads E ey 8 Remove the pump key Hold the pump tubing firmly in both raceways with your left thumb as you also place the top half of the pump over the roller 9 Push the two halves of the pump head together The h
53. requirements and a thorough knowledge of your cells is important For further information on establishing process parameters contact Account Services P N 700037 000 Rev H Biovest International Inc Page 6 10 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures rr ee Sterilizing Bottles and Cap Assemblies All bottles and cap assemblies used in the MAXIMIZER system must be sterilized to minimize the potential for system contamination The time required for complete sterilization should be determined by autoclave validation All bottles except those being used for outflow and harvest should be sterilized with their original caps on to facilitate the sterile addition of media and or factor to the bottle The special delivery cap assemblies should be sterilized separately wrapped in aluminum foil or placed in an autoclave bag Note Because instruments and conditions may vary ensure sterility by validating your autoclave before carrying out this procedure Note Filters on the cap assemblies may be autoclaved up to five times 1 Validate your autoclave 2 Clean and wash bottles caps and cap assemblies using a tissue culture grade soap and water Use good laboratory procedures for cleaning tissue culture glassware Note Prior to washing cap assemblies remove the filter and two Luer plugs A syringe may be used to clean the IN and OUT ports on the cap assemblies Do not wash the filters Note The plastic pou
54. set points or various pump speeds first press the desired number keys then press ENTER If an error is made before ENTER is pressed press CLEAR to erase the unwanted entry Menu Keys The following keys are called menu keys because pressing them displays a list menu of items to be selected for further action m SET UP PROCESS CONTROL CALIBRATE HOLD Four Override Control keys Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 5 1 User Interface LEDs are present in the upper left hand corner of some keys They are red when turned on Their purposes are explained with their relevant keys in the sections throughout this chapter Arrow keys move the cursor to fields that need input or to the override indicator if it is present The cursor uses the wrap around function as a shortcut function to quickly move from one end of the screen to another end if desired for example pressing from a topmost field brings the cursor to a bottommost field The and keys are also used with ALARM screens to scroll through the stored alarm messages START has several functions START Figure 5 1 ACUSYST MAXIMIZER must be pressed immediately before keypad and LCD pressing SET UP PROCESS CONTROL or INOCULATE to activate these functions and cause the respective LED to turn on to confirm which of the three functions is active Only one of these functions may be active at a
55. that keep your MAXIMIZER operating optimally and consistently to ensure its highest production lowest operating cost and minimal unplanned downtime We offer a Full Service Contract to simplify maintenance of the instrument over its many years of usage If it is malfunctioning we provide full repair services MAXIMIZER is not user servicable Sales Support We re happy to help you purchase any of Biovest s products or services P N 700037 000 Rev H Biovest International Inc Page 1 2 Operations Manual for the ACUSYST MAXIMIZER System Theory ey The ACUSYST MAXIMIZER is a hollow fiber cell culture system Both suspension and anchorage dependent cells are grown in hollow fiber bioreactors which are maintained in the MAXIMIZER The MAXIMIZER is designed to maintain cells in an optimal environment one that mimics the mammalian body In fact the analogy of a body is used to describe how the system functions Cells are grown in the space outside of the fibers of a bioreactor Media the blood of the system carry fresh nutrients and oxygen to the cells while carrying away cell wastes such as ammonia and CO Media are circulated via the circulation pump acting as the heart of the MAXIMIZER The lungs of the system an oxygenator diffuses an air and CO gas mixture into the media oxygenating the media while maintaining the prescribed pH setpoint The microprocessor based control system is the brain of the MAXIMIZER It not only
56. the daily yield or concentration of product has reached a peak Then because dense cultures frequently need less serum the serum factor pump concentration may be lowered gradually to further optimize production The key to both of these strategies is to effect changes slowly to avoid sudden shifts in environmental conditions which might cause an adverse reaction by the culture OUTLINE OF A CULTURE RUN The following list shows the major events that occur in a typical culture run They are presented chronologically but due to the variability of each MAXIMIZER owner the chart cannot show the length of time required for each step to be completed Each step is described in detail P N 700037 000 Rev H Biovest International Inc Page 6 8 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures Milestones For A Culture Run Biological Setup Procedures m Cell line characterization m Sterilization of bottles and cap assemblies m Preparation of media Scale up for inoculation Instrument Setup Procedures m Instrument checkout Cultureware Setup Procedures Probe validation Sterilization of probe assemblies Completion of flowpath module Flowpath installation in MAXIMIZER Flowpath pressurization test Fill Flush procedure Maintenance Procedures Sampling Calibration Replacing bottles Replacing luer activated valve or septa Removal of cell debris Pre inoculation Procedures pH calibration DO ca
57. the manufacturer s recommendations or contact Biovest International s Account Services Scale up for Inoculation Before you proceed to Instrument Setup Procedures you should begin the scale up preparations for inoculating the MAXIMIZER As there is no single method suitable for all cell lines this manual will not recommend specific procedures Typical protocols for growing cells in T flasks spinner flasks or roller bottles are probably already in place at your facility Use them to prepare the inoculum so that the inoculation can proceed smoothly Account Services and the opening paragraphs of the Inoculation part of this section will be able to provide you with the necessary background information P N 700037 000 Rev H Biovest International Inc Page 6 14 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures SSS SSeS INSTRUMENT SETUP PROCEDURES Prepare Utilities 1 Regulate the two gas supplies to 15 1 psi 2 Open the CO required supply to the MAXIMIZER 3 Open the Backup Air optional supply to the MAXIMIZER Prepare Data Logging Equipment If ACULINK DATA LOG is in use 1 Connect the serial cable to the computer s serial port and the PC INTERFACE port of the ACULINK Instrument Interface Module 2 Connect the parallel cable to MAXIMIZER and any one of the four ACUSYST ports of the ACULINK Instrument Interface Module oS Plug the power supply into the ACULINK Instrument Interface Module 4 Turn
58. the pH and DO fields to validate probes Section 6 The fields represent the following information Incu Indicates the temperature of the air as it leaves the heater box Normal operating temperature is 39 55 C Range of A D reading second column is 176 250 Media Indicates the temperature of the media measured by the cultureware probe At 37 C the A D reading second column is 147 Heat This indicates the number of 50 msec increments that the incubator heating element is ON during a heater cycle time of 250 msec Completely OFF is shown as 0 while completely ON is shown as 5 pH The first column displays the pH while the second column displays the A D reading from the pH probe DO The first column displays the DO while the second column displays the A D reading from the DO probe P1 This is a collection of bits indicating various status values on the monitor board These are used for internal system communication only Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 7 19 Troubleshooting Figure 7 29 Diagnostics Screen 1 The screen shown in Figure 7 29 is called Screen 1 The fields represent the following information A D EC Read IC Read EC Err IC Err EC Valid IC Valid EC Set IC Set P N 700037 000 Rev H Page 7 20 This indicates the A D time out count and should always be zero These are the counts from the ultrasonics Normal volume in th
59. the plastic orange cap and sponge from the DO probe tip Be careful to avoid touching the membrane at the tip of the probe 6 Insert the DO probe down fully into the case as far as its design allows This includes gently but firmly forcing the probe tip through the probe case s o ring Install the locking ring but just hand tight 7 Insert the pH probe into its probe case These items have threads Only make them finger tight Do not use tools to tighten them together 8 Finish tightening the lower sections of both probe cases but just hand tight 9 Slide the DO probe case tubing lines onto the port fittings as far as possible Either tubing line can go on either port fitting See CAUTION note below P N 700037 000 Rev H Biovest International Inc Page 6 30 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures SSS 10 Connect the pH tubing lines to the pH probe case ports in the following orientation connect the pH IN LO tubing line to the lower port fitting of the probe case and connect the pH OUT HI tubing line to the higher port fitting of the probe case Follow the next step s instructions to actually connect the pH tubing lines to the probe case port fittings 11 Rotate the white locking rings to the unlocked position for both pH probe case tubing lines Press and hold the latch push the connector fully on to the port fitting release the latch and rotate the locking ring to the locked position Repeat t
60. ties should be used to secure all gas line connections Attach the supply of 100 carbon dioxide medical grade to the correct filter and set the incoming supply pressure to 15 psi 1 0 kg sq cm A port is available for attaching a barbed fitting to be used for backup air supply set to same pressure If there is a need to filter or incinerate exhaust gas an outgas port is available for a fitting and vacuum line Gas supply Medical grade carbon dioxide 100 1s recommended for the MAXIMIZER P N 700037 000 Rev H Biovest International Inc Page 4 2 Operations Manual for the ACUSYST MAXIMIZER Instrument Setup E Laboratory Supplies See Electrical and Gassing Requirements for making the proper pressure adjustments and connections to the instrument Cell culture equipment Various supplies will be needed to maintain the MAXIMIZER during a run Laboratory coats sterile gloves masks and other apparel are recommended to minimize the risk of contamination The importance of these types of precautions depend upon the cleanliness and sterility of the room where the MAXIMIZER is located as well as the presence or lack of antibiotics in the culture medium Inoculation will require sterile syringes 60 ml and sterile needles 15 gauge Two sizes of sterile syringes 10 ml and 3 ml are recommended for sampling if septa are used during sampling sterile needles 20 gauge will also be needed A disinfectant solution of alcohol beta iodine
61. with the door closed and 73 6 cm 29 0 inches with the door open To permit clearance of cables gas lines and the exhaust fan the rear of the instrument must have clearance of 10 cm 4 inches More space will be needed if the printer package is used See Figure 4 1 for benchtop area covered by the MAXIMIZER and printer Electrical and Gassing Requirements One electrical outlet is required for the MAXIMIZER The MAXIMIZER may be configured at the factory for the three types of power specifications shown in Figure 4 2 refer to Appendix A and B The in house electrical circuit must supply at least 500 watts of uninterrupted power If the printer 1s used an additional outlet will be needed It is recommended that an uninterruptible power supply on line 500 vA with sine wave form should be used with the MAXIMIZER Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 4 1 Instrument Setup 27 5 in 69 2 cm 16 5 in 41 9 cm z Soj CUSYST MAXIMIZER u Printer 6 O iN c Ct am Figure 4 1 Foot print dimensions for ACUSYST MAXIMIZER and printer SPECIFICATIONS VOLTAGE 120 VAC 100 VAC 230 VAC FREQUENCY 90 60 Hz 90 60 Hz Figure 4 2 Power requirements for the ACUSYST MAXIMIZER Attach the two gas filters to the rear of the instrument as shown by the label Figure 4 3 item 14 Remove the gas line plugs and connect the gas line to the proper inlet and outlet Cable
62. 0 AcuUDATA Software Package 600095 000 ACULINK DATA LoG Base Unit 600096 000 ACULINK DATA LOG Complete 100 120 VAC 600096 001 ACULINK DATA LoG Complete 230 VAC Cultureware Options 600058 215 Single 1 5 m Bioreactor Flowpath 600058 221 Single 2 1 m Bioreactor Flowpath 600068 215 Double 1 5 m Bioreactor Flowpath 600068 221 Double 2 1 m Bioreactor Flowpath Sterile pH Probe Items 600305 000 pH Probe Sterile Ready for Use Single Use 600306 000 pH Probe Holder Reusable use with 600305 000 only Reusable pH Probe Items 103037 000 pH Electrode Non sterile Reusable 103047 000 pH Electrode Storage Solution 250 mL 401090 000 pH Electrode Cable 600298 000 pH Electrode Holder 600300 000 pH probe Tubing Lines spare set of two 600301 000 pH Electrode Holder O rings spare set of three Dissolved Oxygen Probe Items 4192 001 Oxygen Electrode Re membrane Kit replaces 4192 000 102323 000 Oxygen Electrode Electrolyte 25 mL 3923 000 Oxygen Electrode Cable 5330 000 Oxygen Electrode Holder O ring spare qty 1 Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page B 1 Appendix B Ordering Information SS 40083 1 000 600057 000 600054 000 Oxygen Electrode amp Electrolyte Oxygen Electrode Holder DO Probe Tubing Lines spare set of two Miscellaneous Sterile Optional Items 600063 000 600063 001 600 100 072 600101 072 6003 18 000 In line Me
63. 10 liters in size you may feel that larger containers are necessary for weekends and or total feed rates greater than 400 ml hr 9 6 I day Consult Account Services for alternative sources of vessels and their appropriate sterilization conditions Harvesting Strategy The ACUSYST MAXIMIZER may be programmed for several types of harvesting protocols You must decide which method is best suited for harvesting the secreted product from your cell line Continuous harvest 1s the most common technique The two other options available through Process Control include harvesting at every low Expansion Chamber volume or each low Expansion Chamber volume that occurs after a user defined number of EC Cycles that are skipped Of course the PUMPS Override Controls allows you the flexibility to perform batch harvesting by overriding the HP manually to collect the desired volume regardless of the phase of EC Cycling see Section 5 User Interface Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 7 Operating Procedures a Be aware that removing fluid from the ECS is not a selective process All material in the ECS will be pumped out including serum secreted protein and loosely adherent cells For this reason the early stages of harvesting should be complemented with an equivalent replacement rate of serum factor As the culture matures the harvest rate or frequency may be increased gradually until
64. 6 Open the POST BRX DO clamp to prime the DO circuit during the pre inoculation period Be certain the EC FLUSH IN clamp is centered over its tubing close the clamp and ensure the tubing 1s completely pinched shut Wrap the EC FLUSH IN clamp with tape to keep it securely closed This clamp remains shut for the remainder of the run Figure 6 45 Instructions Screen 10 for Fill Flush 17 Re attach the reusable Tygon IC tubing to the IC bulkhead connector Open the IC Vent see note below and IC RETURN clamps Be certain the IC RETURN clamp does not pinch the circulation tubing after it has been opened Figure 6 46 Instructions Screen 11 for Fill Flush Note The IC Vent clamp s label reads IC not IC VENT Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 57 Operating Procedures a 18 The next screen tells you to load the remaining five pump heads with their respective tubing segments Remember that the Outflow and Harvest pumps are loaded differently from the other pumps because they remove media from the flowpath a Aseptically attach a waste bottle to the Outflow pump b Asepically attach a fresh medium bottle or bag to the Media pump if necessary c Aseptically attach Factor bottles to their respective tubing lines d Aseptically replace the Flush Collection container on the Harvest line with an empty sterile bottle e Open the OUTFLOW clamp f Discard the col
65. ACUSYST MAXIMIZER are explained with emphasis on pH control dissolved oxygen delivery media feed strategy and harvesting technique Then an outline is presented to show the basic sequence of activities of a typical culture run This is followed by detailed instructions on each item in that outline so you will be able to set up inoculate and operate a culture run in the MAXIMIZER pH Control MAXIMIZER actively controls pH to maintain the user defined setpoint when in MINH Ti mode pH is monitored on line using the pH probe in the cultureware and controlled in real time with a 10 second sampling and correction interval MAXIMIZER uses three techniques to control pH described below The primary method Gassing remains in use throughout the active culture run except for briefly stopping during the inoculation phase even when either or both of two optional supplemental methods of controlling pH are enabled and in use m Gassing pH is primarily controlled by delivering variable gas mixtures and flow rates through the gas exchanger referred to as gassing CO increases when pH 1s above its setpoint CO decreases or air flow rate increases when pH is below its setpoint Base Addition Optional Factor 1 Pump pumps an alkaline buffer solution to counter the effects of lactic acid The recommended base solution is 0 1 0 5 N NaOH or 8 5 NaHCO in distilled water Medium Dilution Optional Media Pump pumps additio
66. E EC SAMPLE PMOL SPIRE MEDIA PEE POST BRX DO PERS GR LH PRE BRX DO eek ERS OO EC not displayed onscreen DO IN LO not displayed onscreen GEX IN not displayed onscreen PH IN LO not displayed onscreen PH OUT HI not displayed onscreen CLOSE CLAMP BEFORE USE not displayed onscreen Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 43 Operating Procedures Cultureware Pressurization Test After the cultureware has been installed in the MAXIMIZER incubator it must be tested for leaks This procedure can be performed with the incubator door open or closed 1 Press START then SET UP Figure 6 27 Active Setup Menu Screen 2 Press 1 to select Pressurization Test Figure 6 28 Begin Pressurization Test Screen 3 Press ENTER to begin the test P N 700037 000 Rev H Biovest International Inc Page 6 44 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures ee 4 Open or close the clamps as instructed on the next five screens shown below to prepare the cultureware for the Pressurization Test Make certain that closed clamps are centered over the tubing and completely pinch it Press ENTER when instructed Figure 6 29 Instructions Screens 1 5 for Pressurization Test Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 45 Operating Procedures ey 5 The LCD updates to show the three automated stages of the
67. E EE Figure 7 22 Alarm Screen 31 This alarm means the calibration procedure measured too large a difference between the ideal pH probe value and the offline value Verify the following items to try to correct the problem Calibration of the pH probe fixes problem Calibration procedure being properly performed pH cable fully connected to the probe and its bulkhead jack pH cable faulty pH probe A D reading within acceptable range See Section 6 pH Probe Evaluation Note Because pH control is critical to the viability of a culture this alarm is extremely important If you are unable to resolve the problem control pH manually by using Gas overrides periodically checking pH offline and adjusting Gas as necessary Otherwise replace the pH cable or pH probe or contact Account Services Figure 7 23 Alarm Screen 32 This alarm means the calibration procedure measured too large a difference between the ideal DO probe value and the offline value Verify the following items to try to correct the problem m Calibration of the DO probe fixes problem m Calibration procedure being properly performed DO cable fully connected to the probe and its bulkhead jack m DO probe A D reading within acceptable range See Section 6 DO Probe Evaluation If not you must decide whether or not to replace the probe Contact Account Services for further details Biovest International Inc P N 700037 000 Rev H Operations Manual
68. ENTER to restore the alarm d Press START then SET UP e Press ENTER if necessary to return to the Active Setup Menu screen Figure 6 26 f Repeat the probe case assembly Pressurization Test using steps 6 11 16 When the probe case assembly Pressurization Test passes press ENTER to display the following screen Figure 6 25 Final Pressuirzation Test Screen P N 700037 000 Rev H Biovest International Inc Page 6 34 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures eee 17 Ignore the clamp instruction because it is irrelevant when pressure testing the probe case assembly 18 Press ENTER to return to the Active Setup Menu screen Figure 6 26 Figure 6 26 Active Setup Menu Screen 19 Disconnect the probe case assembly from the IC and EC gassing lines and connect the other probe case assembly 20 Repeat steps 6 11 to pressure test the other probe case assembly 21 When the other probe case assembly Pressurization Test passes disconnect it from the IC and EC gassing lines 22 Repeat steps 16 19 23 Reconnect the sterility protectors that were disconnected in step 2 Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 35 Operating Procedures EE Sterilization of Probes The sterilization instructions do not apply to the disposable pH probe After the probe case assemblies have passed pressure testing they can be sterilized Fill the probe cases
69. Front of peristaltic pump head 2 Remove the front half of the pump head 3 Remove the roller assembly and examine both sides Figure E 2 Assure that the Teflon washers one per axle are clean and in place Replace the roller Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER E 1 Appendix E Loading Pump Heads Tubing fo Raceways Teflon Washers one on each side Roller Assembly Figure E 2 Pump head roller assembly 4 With your right hand position the pump segment as shown in Figure E 3 At this point shift the pump segment counterclockwise from how you ultimately want it when loaded because the loading process shifts the pump segment clockwise Outflow from incubator Media container Factor 1 container Outflow container Fluids In Media to incubator Factor 1 to incubator Factor 2 to incubator Factor 3 to incubator Harvest container Factor 2 container Factor 3 container Harvest from incubator Figure E 3 Tubing placement P N 700037 000 Rev H Biovest International Inc E 2 Operations Manual for the ACUSYST MAXIMIZER Appendix E Loading Pump Heads a Se ee Note The orientation of each pump segment must be considered Fluids enter the pump head on the LEFT and exit on the RIGHT because all peristaltic pumps on all ACUSYST systems operate counterclockwise Outflow and harvest are loaded in one orientation to pump these fluids out while all other
70. H probe case Locate both black clips below the clip of the cultureware that is holding the pH probe case itself e when using the disposable pH probe holder press the two black clips on the two grooves of the reusable pH probe holder b Similar to the pH probe case the DO probe case ports are offset from one another one is higher than the other The difference is more subtle than for the pH probe case Orientation is important The liquid inlet port is lower than the liquid outlet port c Locate in the cultureware the two red Luer Lok fittings connected together by a polypropylene double female Luer fitting They are near the bottom of the GEX Two clear Tygon tubing lines connect to these red Luers DO IN tubing line and the unlabeled DO OUT tubing d The DO probe case inlet tubing line connects to the culturewares DO IN tubing line e The DO probe case outlet tubing line connects to the culturewares DO IN tubing line f Disinfect the white Luer Lok fittings of both DO probe case tubing lines g Rotate the DO probe case in the black ckips so the DO probe case inlet tubing line reaches the DO IN tubing line h Loosen white Luer Lok on DO inlet tubing line Loosen polypropylene fitting connected to red Luer Lok on the DO IN tubing line 1 Remove and discard the white Luer Lok short tubing and white plug J Disconnect DO IN red Luer Lok from polypropylene fitting and connect it to exposed female Luer of DO probe cas
71. If Base Addition is disabled the algorithm advances to Medium Dilution If Medium Dilution is also disabled Gi iuti or rate lt Basal rate the algorithm continues to use only Gassing to adjust pH pH may slowly drop which often is not detrimental to the culture unless it drops to a very low level which occasionally happens Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 3 Operating Procedures 1 a Base Addition Base Addition 1s optional and disabled by default Base Addition may be enabled or disabled as desired throughout the active culture run via its Process Control screen Enable or disable Base AGAON in its Process conn screen If enabled also enter the desired Ease Uolume before start of Oa lust that is to be pumped into the stem belie Medini Dilution i IS ae aed When gassing alone cannot keep pH at its setpoint current Gaz the LOW value and Base Addition is enabled the F1 Pump automatically starts pumping base at 40 of the Media Pump s rate or 5 ml hr meee IS I Fl peu continues to pump base until the desired amp Wo lune before skart of Hi lution has been pumped as long as DH re remains bowi its o At this point if ne is still below its setpoint Base Addition continues F1 keeps pumping base and Medium Dilution is also activated if it is enabled If pH returns to its ae a ne current i gt the Lid value before the desired amp a io Lune be or e
72. If you use luer activated valves remove the dust cover and wipe the surface of the LAV with an approved disinfectant see usage instructions that came with the LAVs If you use septa wipe only the surface that will be punctured by the needle 3 Attach the pre sample syringe to the sample port If you use caps remove the cap and protect it by covering it with sterile gauze If you use LAVs simply connect the syringe to the LAV which opens inside of the LAV If you use septa remove the needle guard then puncture the septum until the tip of the needle can be seen inside the sample port 4 Adjust the PRE BRX and POST BRX clamps to permit the desired IC sample Open the clamp to the sample port Remove the pre sample volume 3 ml for IC or EC samples 5 Close the sample clamp Replace the pre sample syringe with the sample syringe for offline analysis If you use LAVs wipe the LAV with an approved disinfectant after disconnecting the pre sample syringe and again before connecting the sample syringe It s unnecessary to reconnect the dust cover between the pre sample and sample syringes If you use septa you can detach the syringe from the needle and leave the needle in the septum while only the syringe is replaced 6 Open the sample clamp again Remove the analysis sample 7 Close the sample clamp Remove the sample syringe Replace the sample syringe with a sterile syringe This syringe may be used for the pre sample during your
73. Inc Page 2 4 Operations Manual for the ACUSYST MAXIMIZER System Theory y The Expansion Circuit In the MAXIMIZER there is a second fluid circuit called the Expansion Circuit EC This circuit is connected to the outside of the fibers where the cells are growing and product is being produced The EC consists of E the Expansion Chamber E two check valves which keep the flow within the circuit moving in one direction the ECS of the BRX the EC flowpath tubing EC Cycling In the first half of the cycle the IC is pressurized at the integration chamber Additional pressure is provided to the IC by the circulation pump As a result medium flows from the ICS inside of the fibers to the ECS outside of the fibers along the length of the BRX thereby moving media from the IC to the EC Thus cells are provided with fresh glucose glutamine dissolved oxygen and other small molecular weight nutrients As media move from the IC to the EC the media level in the integration chamber decreases and the media level in the Expansion Chamber increases This half of the cycle is therefore referred to as EC Rise When fluid in the Expansion Chamber reaches a defined upper limit an ultrasonic detector signals the microprocessor to lower the pressure in the integration chamber This reduces the pressure throughout the IC In addition pressure 1s applied to the Expansion Chamber to make the EC pressure higher than the IC pressure As a result m
74. Pressurization Test FRESSURTSINMG STAHETLIZE and TESTING Figure 6 30 Pressurization Duty Screens P N 700037 000 Rev H Biovest International Inc Page 6 46 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures 6 The Pressurization Test automatically finishes within 90 seconds m If there was no leak the test passes see top screen of Figure 6 31 Go to step 12 m If there was a leak the test fails see bottom screen of Figure 6 31 Go to step 7 Figure 6 31 Pressurization Test Passed Screen and Pressure Test Failed Screen If the cultureware Pressurization Test fails press ALARM to silence the audible alarm and deactivate the external alarm relay A screen similar to Figure 6 16 or 6 17 appears m If EC leaked the second number is gt 0 For example in Fig 6 32 m IfIC leaked the third number is gt 0 For example in Fig 6 33 Figure 6 33 Example Pressuirzation Test Alarm Screen IC Leak Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 47 Operating Procedures a 9 The leak must be located and fixed before proceeding with the run Otherwise later on the cultureware will likely leak cell culture medium and become contaminated which could jeopardize the cultureware s warranty Focus your efforts on a and wane the ae in either the IC circuit or EC as indicated in the F supe coe cle Lectecl alarm screen 10 Potentia
75. RR RES Sea eReS 3 2 ICU ATON EEEE E E E oe aan ea EE sone ee ee ee oe Gee eee 3 2 PUMP MOUI eare ireren nr Erene SEE ERRARTE EEEREN A 3 3 Keypad and LCD 4 airs oe en gear ennnen ad adadesae oop oes ane eee 3 3 Electrical and Gassing FeatureS ccc ccc eee eee nes 3 4 Cultureware Components 0 0 eee eee eee eee enes 3 5 Flowpath Modules irisccricrrerccassssssr osodir ariama Ee aia aa aa 3 5 Oxygen Probe and Case Optional 0 0 cc eee eee eee 3 7 PH Probe and Or aero ee aa 3 7 Bottles and Cap Assemblies Optional 0 0 0 0 ccc eee ee eee teens 3 8 BOUG sorana bau ceeded eee tate eee ee dade sopepe rate aes 3 8 Cap ASseMDICS nau caren auen sob oao4eeees a yea 28 aomae ee 3 8 Printer Package Optional o 66sssdadaideeeeueedwdadeaed ee ees soneanaaeaeaas 3 9 Serial Interface Port 2 0 ce eee eee e teen eee e eens 3 9 Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER ili Table of Contents CE Instrument Setup Facility Reg iremeEntS esscr canon at id idin EEIEIE E A 4 1 BONON S0dCC ceceeuenapeaaueee ceases oS nso ea eateeeeeesnacaaaaeaeee 4 1 Electrical and Gassing Requirements 00 cee eee eee eee ees 4 1 Laboratory Supplies 0 0 ce eee eee enes 4 3 Printer Setup Optional 0 0 ccc eee eee eee eee cece ene nnnnne 4 4 Serial Interface POM 64 040 cc tee ed wing dn EnEn 0 09 YH W Aire wie eden ee ee eee ee 4 4 ACULINK DA
76. T MAXIMIZER Hardware includes the instrument and its obvious features while cultureware includes the disposable flowpath and the reusable probes and holders Also included in this section are descriptions of the bottles cap assemblies and optional printer package HARDWARE COMPONENTS Refer to Figure 3 1 for front external view of MAXIMIZER Incubator Door Bulkhead Connections Keypad and LCD Circulation Pump Mounting Bracket Door Latch Pin Tubing Raceway Peristaltic Pumps Figure 3 1 Front view of ACUSYST MAXIMIZER door open Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 3 1 System Description E Instrument Cabinet Incubator Note The cabinet of the MAXIMIZER is made of molded plastic It may be cleaned with a mild detergent or alcohol cleaner For necessary counter space see Section 4 Instrument Setup for facility requirements The incubator module is a forced draft convection system designed to maintain the culture at a controlled temperature The exhaust fan for the incubator is located on the upper rear of the incubator Access to the interior of the incubator is gained by lifting the door from its lower right hand corner When the door is fully open the incubator fan and heater are shut off A viewing window on the door can be exposed to examine the incubator without disturbing its contents Inside the incubator are two rows of bulkhead connecti
77. TA LOG Setup Optional 0 ccc ete eens 4 7 User Interface Overview of LCD and Keypad ccc eee eee eens 5 1 LCD 6 one aae ad eee ee eas ba ee oa oe ea ae eee a oa be ee 5 1 KCVD EO boca bbe wha bbn5454000040 24 bboobd de eed EEE EEEER SAREE RO 5 1 Menu Key Operations 0 ccc ce ee ee eee eee eee een e eens 5 3 MONITOR Key a3 dc hectetete rea a rade ee eee ead eee oe aa 5 3 SETUR Key rererere eh nop ueer ere eed G CEOS EAA ES Oe ORES ee 5 6 START SETUP Functions 0 0 cece eee eee eee cece ee ee ee enn neee 5 7 PROCESS CONTROL Key 44 4 4 44000 409000000 0650404 00 oS GRRE REARS PRS 5 8 Override COMMU GIS 4 4404544425 44e4e0er eens esate seeesedueiidess 5 19 CALIBRATE KOV ser pP aF 5424 ndbocsesuss4ee sone sen eeeiusumeaneas anus 5 24 NOLD KG eer E pause aoa geeae sea ee eaeeeaneaaee es 5 25 ALARM KEY 4 6 582444 4 5940 00046 CER eee Re RIIET VENENAN EEA 5 26 DIAN OSUCS Rey 204 44544 4 4 4754444 eile eG shane bee de aoe eeu eae 5 28 P N 700037 000 Rev H Biovest International Inc IV Operations Manual for the ACUSYST MAXIMIZER Table of Contents T Operating Procedures Process C ntrol Strategy auc ean aa er idid oo o9 44o eE Aa 6 1 OEO e niio PETATE EREET EEEE TSTST ETETETT EEEE 6 1 Oxygen Delivery 0 ccc nanan 6 6 Feed Strategy sriririroiiiitnssnnnsrrrr E Aeir erian ARR 6 6 Harvesting Strategy ieee ee RR A ee ae eee ge be aS 6 7 Outline of a Culture RUN 2 eee eee eee eens 6 8 Bi
78. The following information applies to all metabolic calculations e e rt v where e 2 7183 r total inflow rate ml h t t2 tl rounded to the nearest integer tl time of previous sample t2 time of current sample v system volume Single bioreactor 1 5 m Flowpath 570 ml Double bioreactor 1 5 m Flowpath 615 ml medium rate medium volume medium volume t factor rate factor volume factor volume t glucose rate glucose volume glucose volume t serum rate serum volume serum volume t base rate base volume base volume t harvest rate harvest volume harvest volume t r medium rate factor rate glucose rate serum rate base rate Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page D 1 Appendix D Metabolic Calculations eee The remainder of this section describes individual calculations for rates of cellular metabolism and productivity GLUCOSE UPTAKE The calculation of glucose uptake is based on the following equation GUR where GUR Glucose Glucose Gm Gg Gs Gf glucose LACTATE PRODUCTION Glucose r Glucose e Glucose r Glucose l e Glucose Uptake Rate mg h current glucose reading mg ml previous glucose reading mg ml conc of glucose in medium mg ml conc of glucose in glucose supplement mg ml conc of glucose i
79. ZER Page 6 29 Operating Procedures 7 SS SS Probe Case Assemblies The pH and DO probe assemblies include the two probes and their respective probe cases The probes are reused as long as they pass the evaluation tests The probe cases are very durable and last many runs Only the o rings should be replaced for each run New o rings are included with each new cultureware so there 1s no reason to reuse them Each new cultureware also includes two sets of non sterile probe case tubing lines used to pressure test and autoclave sterilize the probe assemblies The probe assembly instructions do not apply to the disposable pH probe The instructions for assembling the DO probe and reusable pH probe are 1 Get the o rings and both sets of probe case tubing lines from the cultureware box Do NOT lift the cultureware by its sterile bag if it s necessary to move the cultureware to find these items Instead lift the cultureware by grasping it by its white plastic tray 2 Remove the o rings from their bags They do NOT need to remain sterile now Inspect the o rings for damage 3 Disassemble the probe cases and ensure they were cleaned at the end of the previous run and discard the old o rings if present Install the new o rings and oosely thread the lower section into the upper section see figures 6 19 and 6 20 4 Connect the threaded pH probe electrical cap finger tight and connect the DO probe twist lock electrical cap 5 Remove
80. ae ee ee ess 7 26 COIMUPOWALGs avr ssaaaeer eee E E E E E ween eenseeceeeene ses 7 26 Appendix A Specifications ACUSYST MAXIMIZER Specifications 0 ccc eee eee eens A 1 Instrument DiMeNnSIONS 0 ce ete e eee etn eenee A 1 mMstrument Wei Us aaa anteeeeteusedadess4edenaadudaaeeeeeeaeceaaess A 1 Operating Temperature 1 eee eee eee eens A 1 Electrical REGUITCMENTS 150446 c644eee0e0wer eee ee hitt ta ERRER A 1 Backup Power Requirements Instrument and Printer A 1 Wali RUSE 4 45 5 4 4 554 oo nyt dy ee eee eo Se bs 29444 SSH HHSHGSEEORDE SEDO O SS A 1 Heater BOX FUSE 1 eee ee eee ee ee ee eee eee ene A 2 Power SUDOIN FUSE 2x 4ccaes ee Gene ee ee h eo asaes 4c bee eee keane A 2 Pollution DOSTCC oi i4164255 csseg be beenaeannas eae eee epee a A 2 installation Category s44 4c22464 edee0ees rerent adadaaasaaegdaaan apd A 2 Battery VOC 4 24224 ose act GG eG aa eee hte aes eae be oe oe be eee EEEE RERA A 2 Gas Requirements 4666966444445 cn0e enn hd Ue eee he LEELA eee Cece ees A 2 Cultureware and Accessories 2 0 0 eee eee e teens A 3 Approximate Flowpath VolumeS 0c ccc cee ee eee eee A 3 Peristaltic Pump Rate Ranges ccc eee eee eens A 4 Schematic for 600058 xxx Cultureware Models 0 00 e eee A 5 Schematic for 600068 xxx Cultureware Models 00 cece ee eee A 6 Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER v
81. agnostics Key A special key exists to permit access to a series of four screens that display information that is automatically monitored by the instrument Most of this information is used by and meaningful to trained service personnel One of the diagnostic screens shown below is routinely used to test the pH and DO probes prior to autoclaving them for use in a culture run The pH probe is tested by assessing its A D value 126 as shown below The DO probe is tested by assessing its A D value 150 as shown below A D values represent the electrical signal from the probes and are reliable means of evaluating the probes functionality Operational details of this testing are provided in chapter 6 pH Probe Validation and DO Probe Validation Figure 5 25 Diagnostics Screen 0 for pH and DO Probe Validation Press START then HIDDEN KEY see figure 5 1 to display whichever of the four diagnostic screens was last viewed Press ENTER to scroll through the four diagnostics screens and find the one you want Press MONITOR at any time to exit from any of the Diagnostics screens Chapter 7 Troubleshooting provides details of the diagnostic screens P N 700037 000 Rev H Biovest International Inc Page 5 28 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures rr ee PROCESS CONTROL STRATEGY You may wish to review Section 1 System Theory before proceeding through this section Here the overall operating procedures of the
82. aintenance replaces all wearable components of the system and includes calibration Full Service Contract Biovest International offers a renewable annual service contract to ensure the instrument 1s maintained in normal working order by covering all parts labor and an amount of travel expense for the contract year Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page F 3 Glossary AcuSYST Bioreactor BRX Bulkhead Cap Assembly Circulation Pump Clamp Cultureware Expansion Chamber EC EC Cycling ECS Expansion Circuit EC Extracapillary Space ECS Factor tBiovest International Inc Automated Culture System Polycarbonate case containing thousands of hollow fibers where media is exchanged and a cell culture grows See Bioreactor Connection panel located near the top inside of the incubator Biovest International s patented vented screw cap for glass bottles May be used for feed or receiving bottles Bellows pump which circulates media throughout the IC flowpath Device which restricts flow through tubing The disposable and non disposable components of the flowpath and its associated sensors cables and tubing The fluid reservoir in the Expansion Circuit See Expansion Circuit Ultrafiltrative movement of media through the bioreactor fibers from the IC to the EC One cycle consists of an EC Rise and an EC Fall See Extracapilla
83. al Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 73 Operating Procedures E Osmolarity The effect of medium pump rate on osmolarity should be considered especially during long pre inoculation periods Slow medium rates lt 25ml hr have the potential of allowing the GEX to dehydrate the flowpath and consequently raise the osmolarity of the medium This problem can be resolved by raising the medium pump rate temporarily Contact Account Services if you have questions about this relationship Offgassing Optional If the gas exiting the oxygenator has the potential of carrying biohazardous material from the cells or media the off gassing tubing should be installed This is the black collared Tygon tubing supplied with the instrument It should be attached to the GEX OUT line and to the bulkhead fitting labeled OUT The barbed fitting on the rear of the instrument should be connected to slightly negative pressure and to a disposal system to ensure that gases are collected and properly treated Cytotoxicity Testing Optional A few days before inoculation samples of media should be removed from the ICS and ECS then tested for cytotoxic residue The cytotoxicity test 1s run to determine whether medium in the system has any toxic effect on cells All tests should be compared to medium which has previously been shown to be non toxic A certain percentage of cells will die even if the medium is not toxic
84. alves will snap together and the edges will be flush when they are properly connected Figure E 5 If you are concerned that the two halves did not go together as expected take the pump head apart remove the tubing and start again at step 4 Figure E 5 Completed pump head assembly CAUTION DO NOT pull stretch or otherwise manipulate the tubing inside the pump in an effort to get the two pump head halves together in step 9 The tubing will not return to its normal state after extended operation and the result will be decreased flow rate and variances between pumps P N 700037 000 Rev H Biovest International Inc E 4 Operations Manual for the ACUSYST MAXIMIZER Appendix F Maintenance PROBE STORAGE pH Probe DO Probe The reusable pH probe should be stored with its electrical cap attached and it s tip in pH storage solution see Ordering Information in Appendix B A storage bottle is provided with the probe to hold the storage soluttion Store new disposable pH probes in the same manner as for cultureware The DO probe should be stored with its electrical cap attached and the plastic cap and sponge covering its membrane tip Store the probe vertically with the tip downward Leave the electrolyte in the membrane cartridge if the probe will be used in the next 6 months If storage time will be longer store the probe without electrolyte in the membrane cartridge The DO probe requires replacement parts to work prope
85. anual for the ACUSYST MAXIMIZER Operating Procedures SS SSS 9 Press or to move between the pH and DO fields Press number keys to enter the offline pH or DO value in its corresponding field Press ENTER to input the offline value 10 After entering the value press the following key depending on what you want to do next m press MONITOR to leave the Calibrate mode m Press ENTER to perform another calibration 11 If you pressed ENTER in the previous step a screen similar to the following screen appears It shows which probe was most recently calibrated offline value was m er ABILE ly j i tter f pH was calibrated most recently E orii ugine rit ni DO v was calibrated most recently m The elapsed ti time en Le Lake is when START was pressed for the last sampling prior to oemei the offline value This also is logged by the printer or ACULINK DATA LOG if in use a Press 1 to begin a new calibration of the pH or DO probe Go to step 6 b Press 2 to re enter an offline value for a previous calibration Only do this if an error occurred during the first entry of the offline value In this case the log will show Sample Taken once followed by two Calibration entries Figure 6 56 Main Menu Calibration Screen after Data Entry Notes 1 Blood gas analyzers are compatible with Luer Lok syringes If you have only a pH meter you will need to modify the sampling method to accommodate the use of syringes during the offline meas
86. ard the foam protectors that are taped to the corners of the cultureware tray Remove and discard the green striped tape that stretches across most of the tubing f Carefully place the tubing from the tray onto the surface of the flow hood Inspect all clamps and sterility covers Check and finger tighten all fittings and connections Do not over tighten clear plastic components because using too much force can break them g Confirm the GEX clamp labeled CLOSE CLAMP BEFORE USE is open Now close this clamp It remains closed for the entire run h Check that all sterility caps and plugs are in place Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 37 Operating Procedures ea 1 Confirm there is no kinked tubing and all clamps are open J Close all clamps on tubing with open ends sample lines bottle connections etc k Remember to check the tightness of the 3 filters IC EC GEX IN five Luer plugs on the GEX and the Luer connection for the in line filter in the combined Media and F2 line If there is any question regarding cultureware sterility please contact Biovest International Account Services before proceeding 2 Working in a sterile hood attach either the autoclave sterilized reusable pH probe or the pre sterilized disposable pH probe to the cultureware a b c d e P N 700037 000 Rev H Page 6 38 Remove the black plastic holder
87. as well as to maintain a record of activities during each run Contact Account Services for ideas and examples of this type of documentation Daily m EC Cycling is inactive after inoculation Remember to activate 1t when necessary see page 6 95 Sample for metabolite and product assays and determine which pump speeds to change based on that information m Check the circulation pump to ensure that it is operating smoothly and does not have leaks Check the fluid in the Integration and Expansion Chambers to verify that the level is satisfactory and for signs of contamination m Check the IC and EC chamber vent filters to ensure proper gas line connections especially after inoculation and removal of cell debris Inspect the outflow and feed bottles for proper fluid levels Check CO tank and optional backup air supply Check ALARM screen and correct problems as needed Periodically Calibrate pH and DO probes m Replace the feed factor s outflow and harvest bottles m Replace sampling port septa m Remove unwanted cell debris from Expansion Chamber P N 700037 000 Rev H Biovest International Inc Page 6 88 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures ee End of Culture Procedures The following is a description of the procedures that are to be performed after a culture run is finished Instructions will be given for removing the cultureware discarding the disposable components and saving the reusable com
88. audible alarm e In ACULINK DATA LOG navigate to the correct Instrument Monitor screen that represents this MAXIMIZER f After 1 minute ACULINK DATA LOG should update and show the MAXIMIZER is running g Ifstep f happens the test passed h Press HOLD then 2 1 Press ALARM Press ENTER until all alarms are restored and the Monitor screen appears J ChangeLoeeing Trtervael tof mine P N 700037 000 Rev H Biovest International Inc Page 6 20 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures er ee rEOMS Test a Press 6 b LCD displays top screen of Figure 6 10 c Press ENTER d LCD displays middle screen of Figure 6 10 e Press ENTER f LCD displays bottom screen of Figure 6 10 g Press ENTER h If each of the three screens shows the correct software names part numbers and revision levels the test passed 1 Press ENTER Figure 6 10 PROMS Test Screens Showing Current Information Notes The PROMS test allows you to check the revision levels of the various Programmable Read Only Memory PROM chips This information will not change unless the instrument is serviced by Biovest personnel Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 21 Operating Procedures ar a SS Instrument Pressurization Test This procedure checks the integrity of the internal gassing system and should be performed before proceeding with the run to ensure the instrumen
89. ave cycles validation of hoods and autoclaves sterility tests medium and serum lots quality control procedures and any timetable that shows when sampling or bottle replacement is performed Be honest about the history of aseptic techniques used throughout the run Cultureware and instrument cleanup Remove the flowpath as described on page 6 90 Save the probe cables and gassing lines and thoroughly clean the pH and oxygen probe case assemblies Replace the oxygen electrode membrane and add fresh electrolyte The components of the probe holders can be soaked in sodium hydroxide solution 0 1N for 30 minutes to remove endotoxin residue then rinsed thoroughly in distilled water before they are washed with tissue culture detergent Properly dispose of the contents of all bottles that were attached to the flowpath Bottles and cap assemblies may be treated like the probe holders with sodium hydroxide solution Vent filters for cap assemblies should be discarded as a biohazard Disinfect the incubator the outer surfaces of the MAXIMIZER cabinet and the interior of the door Avoid spilling any solutions on the circulation pump rotor in the bulkhead connections and in the incubator exhaust vent Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 7 25 Troubleshooting EEN HARDWARE AND CULTUREWARE Pumps Note Cultureware To ensure optimum performance the peristaltic pumps should be
90. bottle contains an average of product material from the ECS throughout a specified period while the sample originates from a single point in time In addition product levels in the sample may vary with the phase of cycling Therefore to measure accurately from the flowpath samples should always be removed at the same time during the cycling process Cooling the harvest bottle is a good way to preserve product integrity by preventing thermal degradation It also has the advantage of stabilizing the changes in pH due to harvested cells as well as reducing the activity of proteases released by cell lysis If cell buildup in the harvest becomes a concern the bottles should be replaced as often as possible and the contents separated by centrifugation P N 700037 000 Rev H Biovest International Inc Page 6 100 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures EE Serum Requirements The pre treatment of BRX ECS with serum is an important step in conditioning the flowpath before inoculation Although the inoculum should contain the same amount of serum as in static culture lack of serum in the EC would dilute the inoculum to an undesirable level Therefore serum should be added to the EC prior to inoculation This procedure is described in Serum Pre treatment of the ECS Some components of serum are consumed while others notably enzymes are used as tools for biochemical reactions The serum pump should be started w
91. c FACTOR PRIME d CELL REMOVAL e Lower CHECK VALVE BYPASS 7 Open these clamps a FACTOR 3 b EC RETURN c Upper CHECK VALVE BYPASS 8 Override F3 Pump to 400 ml hr Collect supernatant from BRX s and proceed when the ECS is empty 10 Close the upper CHECK VALVE BYPASS clamp 11 Open the lower CHECK VALVE BYPASS clamp 12 Collect supernatant from Expansion Chamber Proceed when it s empty 13 Press HOLD then 2 14 Close all clamps and process the supernatant Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 91 Operating Procedures eE Removal of Cultureware Components The cultureware has disposable and reusable components Read the following procedures and be certain that you save the reusable components for use in future cultures CAUTION Ifthe application has the potential to release a biohazardous agent contact Biovest s Account Services department 1 Close all clamps particularly Outflow Media F1 F2 F3 and Harvest 2 Disconnect the In Out if used IC and EC gassing lines from their bulkhead and cultureware connections Save them for the next run 3 Disconnect all electronic cables from their bulkhead jacks 4 Remove the temperature cable and DO probe cable if it s in use Save them for the next run 5 For the reusable pH probe Remove the cable Save it for the next run 6 For the disposable pH probe a the cable case and the probe are
92. centrations are higher than suggested it may be necessary to increase the concentration of NaHCO to prevent problems with pH control Procedures for medium preparation will vary depending upon the medium being prepared Use the procedure most appropriate for your circumstances Sterilizing Medium As one of the key components of the system the growth medium used must be sterilized before it is pumped into the system Once sterilized medium can be transferred into sterile bags or bottles Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 13 Operating Procedures E Medium Quality Control The medium used in your system is one of the key ingredients to a successful experiment or production run If it is in any way contaminated the entire run may be at risk of contamination Thus you should always check your medium for contamination before pumping it into the system Because medium quality control requires one week to complete it is important to complete medium checks not only before beginning but also throughout your production run Several quality control methods can be used to check the quality of your medium If medium has been prepared and stored for over six weeks before being used it should be tested to be sure that it is still usable Certain nutrients in the medium may degrade during a long storage period If there are any questions regarding medium quality please refer to
93. cleaned after each culture run Media spills and small pieces of worn pump tubing may be trapped inside the plastic housing and should be removed Wipe the interior of each plastic shell with a cloth or paper towel soaked in disinfectant or alcohol Wipe the surface of the metal rollers with a cloth or paper towel soaked in alcohol Allow any moisture to evaporate before you re assemble the pump head Each pump head requires two white teflon washers Replacements must be installed before the next run See Appendix B Ordering Information Do not soak the pump head or rollers in alcohol because it will strip away the necessary lubricant Each cultureware assembly is assigned a lot number which may be used to trace defects specific to a certain build This number is located on the left corner of the tray near the Outflow tubing In addition the shipping box for the cultureware has a manufacturing number which identifies the sterilization lot These numbers should be recorded and used when any cultureware problem is brought to the attention of Account Services P N 700037 000 Rev H Biovest International Inc Page 7 26 Operations Manual for the ACUSYST MAXIMIZER Appendix A Specifications ee a AcCUSYST MAXIMIZER SPECIFICATIONS Note FOR INDOOR USE ONLY Instrument Dimensions Width 69 8 cm 27 5 inches Depth 66 0 cm 26 0 inches Height 52 0 cm 20 5 inches door closed 73 6 cm 29 0 inches door open Instrument W
94. d controlled conditions hi pH of pre BRX medium io dissolved oxygen mmHg F temperature C of circulating medium CYCLE phase of EC Cycling currently active RISE FALL LOW or HOLE ri pressure mmHg in Integration chamber pressure mmHg and volume ml in Expansion Chamber HE Harvest pump rate ml hr 3 Factor 1 pump rate ml hr CF Circulation pump rate ml min Factor 2 pump rate ml hr Me Media pump rate ml hr F3 Factor 3 pump rate ml hr OF Outflow pump rate ml hr Note The DO value displayed on the Monitor and Calibrate screen may change depending on whether the pre BRX or post BRX clamp is open see Figure 3 4 The DO value on the LCD does not reflect which of these clamps is open nor is this indicated in the run log Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 5 3 User Interface _ EEEsy Following is a synopsis of the four modes of operation and the sequence of when they normally occur during a culture run IOLE appears on the Monitor screen immediately after the Abort function is used when the previous run was terminated and until one of the active function begins either ACTIVE SET UP ACTIVE PROCESS CONTROL or ACTIVE INOCULATION TUF appears on the LCD after pressing START a SET UP vale also turns on he Active Set Up LED and resets elapsed time to Heii H4 Start Set Up
95. dderine ss 6 43 Cultureware Pressurization Test 0 ccc ccc eee eee nee 6 44 Fill Flush Procedure 0 ccc cee eee eee ee eee eee e naan 6 50 Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER V Table of Contents a Maintenance Procedures cc eee eee e eee e eens 6 62 SIMONE 6 20200 er eases cree RRR E anaes eeaeages cease ees 6 62 PROG CAIDA OIT 1 2 s ncer ee aenaeaseas co EESE EERENS ERE es 6 65 REDIACING BOUNCS soera on ud hae 66 eee eee een a ne ee eee 6 68 Replacing LAVs and Septa ccc eee eee ee eee ee eees 6 69 Removal of Cell Debris 0 eee eee eee e ee eees 6 70 Pre inoculation Procedures ccc eeren rannen 6 73 PH Courant sveeers tae ee e a end oder kane pees one Oe ee ea 6 73 DO CaliPrahlONencds444acas an bese see enag57468 esse eeeneseee du auderes 6 73 EC CUCM Oe ee aaa dod oo eo are E E aan one ae ee eG ee eee ee 6 73 OSIMOIIMIEY oc acecacaeeseea0nh ad aoae eee aene pea RAAE 6 74 Offgassing Optional ne e4064e60 su eo eur eetesess soe eeeedeedaannde ae 6 74 Cytotoxicity Testing Optional 0 0 ccc ccc eee eens 6 74 Sterility Testing Optional 0 0 eee eee teens 6 76 Serum Pre treatment of the ECS 0 eee eee ees 6 77 Process Control Summary during Pre Inoculation Phase 6 80 Inoculation PIOCCOUNG 5 ossecaceccteteccutetand Genes chbbbeeeaeeeeeeae er 6 82 Post inoculation Procedure
96. dia filters Small 4 Individually packaged In line Media filters Large 4 Individually packaged As Sterile Tubing Extension six feet long Sterile Tubing Extension six feet long Both extension lines are silicone and include e Male luer to connect to the cultureware e Male and female luers to connect to the container e Pinch clamp at the container connection end Luer Activated Valve LAV Kit 15 each of e Sterile and Individually Packaged LAVs e Polypropylene Luer Dust Covers Sterilizable Reusable Glass Bottles and Polypropylene Cap Assemblies Bottles are borosilicate with polypropylene pouring ring GL45 thread and volume markings Caps are polypropylene with two liquid ports accept male luer slip connection one vent port 1825 000 filter purchased separately and they include internal silicone tubing to deliver or collect fluid Each part number is a package of four items 6000 13 000 6000 14 000 600015 000 600016 000 600017 000 600018 000 600019 000 600020 000 60002 1 000 600022 000 600023 000 60006 1 000 P N 700037 000 Rev H Page B 2 Media Bottle 0 5 liter Media Bottle 1 0 liter Media Bottle 2 0 liters Media Bottle 5 0 liters Media Bottle 10 0 liters Delivery Cap Assembly 0 5 liter Delivery Cap Assembly 1 0 liter Delivery Cap Assembly 2 0 liters Delivery Cap Assembly 5 0 liters Delivery Cap Assembly 10 0 liters Receiving Cap Assembly use on any volume bottle Bottle amp Cap Startup
97. e 8 When the Expansion Chamber is reasonably empty close the CELL REMOVAL clamp 9 Press 5 to clear the EC Cycle override 10 Press MONITOR 10 Either leave the sterile container on the Cell Removal line or replace it with a sterile male Luer plug or syringe 11 Open the EC RETURN clamp 12 Allow EC Cycling to resume It will replenish the volume in the Expansion Chamber If the MP rate is quite slow compared to the EC Cycling flow rate using syringes to replenish the Expansion Chamber volume may provide better results 13 The cell removal procedure is finished 14 Reset the alarms that may have been activated once they are between their respective Low and High Alarm Limits 15 Optional Process the collected cell slurry because it contains product from the ECS Discard the cell pellet as a biohazard P N 700037 000 Rev H Biovest International Inc Page 6 72 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures PRE INOCULATION PROCEDURES After the flowpath has been flushed and filled with medium it may be run without cells for a period of time before it 1s inoculated This pre inoculation phase of the culture is begun automatically after the Fill amp Flush procedure and it uses Process Control parameters for pump rates cycling conditions and pH control You may use the default parameters for pH setpoint and pump rates or you may change them to suit your needs It is recommended that you change
98. e Expansion Chamber during cycling with default settings 1s 108 178 ml The corresponding counts are 64 96 Disregard This value indicates the number of faulty ultrasonic readings in a 30 second period and should always be less than 120 Disregard This value indicates the weighted valid reading from the ultrasonics If the ultrasonics are functioning properly the EC Read and EC Valid values will be within 2 A D readings of each other Disregard This value indicates the last pressure setting that was calculated for the Expansion Chamber during cycling operation At 100 mmHg the A D reading should be 33 Disregard Biovest International Inc Operations Manual for the ACUSYST MAXIMIZER Troubleshooting Figure 7 30 Diagnostics Screen 2 This screen is called Screen 2 The fields represent the following information Disable This is a collection of bits indicating whether gas temperature and EC control are enabled These are for internal system communication only IOA These are a collection of bits that control the gas solenoids bit0 CO bit high air bit2 medium air bit3 low air where 0 closed 1 open Media Set These indicate the media temperature setpoint and its corresponding A D reading A medium temperature of 37 0 should have an A D reading of 150 IOC These are a collection of bits that control the chamber pressurization and venting valves bit0 IC gas solenoid bit IC vent solenoid
99. e SESE UE de na it 7 WLior has been pumped m F Pump stops E Ease Uolume before start of OGilutior is reset to the entered value Gassing alone controls pH If pH again falls below its setpoint and Base Addition 1s activated the previous amount of base that was pumped 1s forgotten and the user defined volume now can be pumped again before Medium Dilution is re activated if it s enabled Base addition is usually unnecessary for most applications and may be left disabled However if pH is dropping more than seems safe you may wish to enable Base Addition to be prepared and prevent pH from dropping too far and jeopardizing culture viability Prepare a sterile bottle of base solution aseptically connect it to F1 tubing line and open the F1 clamp Then if Base Addition is needed it is ready to be automatically pumped P N 700037 000 Rev H Biovest International Inc Page 6 4 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures ee ee Medium Dilution Medium Dilution is optional and disabled by default Medium Dilution may be enabled or disabled as desired throughout the active culture run via its Process Control screen Medium Dilution is m Enabled when fii iution rate gt Basa rate m Disabled when fi ii hice rate lt asal rate When Medium Dilution is enabled it is activated for either situation E Gace Uo lunes b efore skart of Dilution has been pumped current Gas is the L LE Fae aad Ait remain
100. e cells Best results are often obtained by preparing the inoculum from cells that are in mid to late log phase of growth A standard growth curve is often useful in scheduling the progress of cells in static culture to coincide with the day of inoculating the flowpath Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 95 Operating Procedures a m An option when inoculating is to split the inoculum and inject half of the cells into the upper EC port which 1s the normal port and the other half of the cells into the lower EC port via the Factor Prime port This may lead to better distribution when the culture s expansion begins This effectiveness depends on several factors including how sticky the cells are and how much protein is in the EC medium among others Although the cultureware permits several options for adding cells this manual has described the simplest method Using a pressurized bottle to deliver cells to the BRXs may be dangerous if the bottle 1s weak or damaged It is possible to distribute cells along the entire length of the BRX by circulating a cell suspension with a vented bottle and peristaltic pump but this process requires more time than the standard technique caution must be observed when using the override functions to control pump rates and the longer inoculation time and pump action may cause cell damage m Forcing excess media from the ECS to the IC
101. e inlet tubing line k Rotate DO probe case in the black ckips so DO probe case outlet tubing line reaches the unlabeled DO OUT tubing line 1 Repeat steps h j to connect DO Out tubing line s female Luer to remaining red Luer Lok of unlabeled DO OUT tubing line m Save the removed double female Luer fitting for use at the end of the run P N 700037 000 Rev H Biovest International Inc Page 6 40 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures SSeS 5 Optional sterile items should be added to the cultureware while the cultureware is still in the sterile laminar cabinet Examples of these options are explained below Appendix B has ordering information for these items Use LAVs or tubing extension lines together or separately depending on your application and facility LAV Cover a Luer Activated Valves LAV allow you to connect and disconnect 4 syringes aseptically outside of a sterile laminar cabinet Usage e T instructions are provided with the package of LAVs male port b Use LAVs on cultureware s four syringe ports IC SAMPLE FACTOR PRIME INOCULATE EC SAMPLE CELL REMOVAL b Tubing extension lines provide longer tubing lengths to allow you to aseptically connect bags bottles and syringes in a nearby sterile laminar cabinet Use them to extend cultureware s ten tubing lines e MEDIA OUTFLOW CELL REMOVAL use 600101 072 F1 F2 F3 HARVEST IC SAMPLE FACTOR PRIME INOC EC SAMPLE u
102. ead the two metal screws until their heads touch the bracket 8 Thread the white plastic threaded cap 9 Press the tubing lines into channels at the edge of the incubator s opening 10 The cultureware is now ready for the Pressurization Test P N 700037 000 Rev H Biovest International Inc Page 6 42 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures ee Cultureware Labels Certain cultureware tubing lines and connections are labeled with unique names to differentiate them and make their usage clear These labels are referred to throughout the following procedures and in the LCD s onscreen instructions Because the LCD s space is limited some of its label names are shortened from what is printed on the labels that are on the cultureware The following table relates the actual names to the names shown on the LCD Use this information if there is any uncertainty about which label is being referred to by the onscreen instructions Not all cultureware labels are used in the onscreen instructions Label names in this manual primarily use the full cultureware label names Cultureware Label Name LCD Label Name CELL REMOVAL inkek PRE PEA E EC CHECK VALVE BYPASS mi Dek Ue iia bor oso EC FLUSH IN eae ais EC RETURN mil RETURN FACTOR 1 Fi FACTOR 2 Pa FACTOR 3 p FACTOR PRIME PHOTOR PRIME andi FACTOR PRIME GEX OUT Ges CHIT HARVEST MEPL SP IC PED hiert IC RETURN TED RE PLR IC SAMPLE TE SHAR LE INOCULAT
103. ed Figure 5 10 Factor 1 Base Addition Screen If you do not need to pump base to assist with pH control which is true for most applications and instead want to use the F1 Pump to deliver some other solution into the IC leave Base Addition disabled and control this pump s speed using a manual override Press PUMPS use the arrow keys to navigate to the F1 pump field enter the desired pump rate using the number keys and press ENTER Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 5 11 User Interface E EC Cycling This screen allows you to set the conditions used by the MAXIMIZER in regulating the fluid cycling process between IC and EC Default values are E Helsy y leiGe 7 days a ml E i gt 15 minute Rise time m 2 i5 15 minute Fall time During active process control UMM IMG mode the MAXIMIZER controls the volume in the Expansion Chamber at a low level setpoint 108 ml during the EC Cycling Delay period This is displayed as i on the Monitor screen When the elapsed time exceeds the delay period EC Cycling begins in a Rise phase using the transfer volume and Rise Fall times specified in this process control screen When the ultrasonic level detector senses that the desired transfer volume has been added to the Expansion Chamber the Rise phase is stopped and the Fall phase begins At the low level setpoint the Fall phase stops and the sequence repeats
104. ed Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 7 13 Troubleshooting eS Figure 7 20 Alarm Screen 26 This alarm means the printer is offline Ensure printer is selected or on line Check for paper out light on printer and jammed printer head Figure 7 21 Alarm Screen 27 This alarm means the software is unable to measure a high percentage of valid readings from the ultrasonics cable If this alarm occurs infrequently it may be of little concern because it could be caused by sudden vibration against the instrument moving the instrument slamming the door or by shaking the cultureware tray during cell removal or other tasks If this alarm occurs frequently verify the items below to try to correct the problem It is possible the chamber could drain or flood Watch the Expansion Chamber for a few Rise and Fall cycles to ensure its volume is properly controlled and remains between the Low 108 ml and the High volume 108 ml Transfer Volume Cultureware tray in a level position in the incubator Repositioning cultureware tray resolves problem Ensure nothing is under cultureware tray Cell debris or foam in the Expansion Chamber Performing Cell Removal Procedure resolves problem m Ultrasonics cable fully connected to its bulkhead jack P N 700037 000 Rev H Biovest International Inc Page 7 14 Operations Manual for the ACUSYST MAXIMIZER Troubleshooting EE E
105. ed above it will not withstand the dry heat conditions used in depyrogenating glassware If you employ this process you must remove the pouring ring before placing the bottle in the depyrogenating oven P N 700037 000 Rev H Biovest International Inc Page 6 12 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures ee Medium Preparation Be aware that it can take up to 6 8 weeks for sufficient medium for the run to be ready so plan the date to inoculate the MAXIMIZER accordingly The medium should meet any special requirements suggested by your cell line characterization and can be pre packaged or made in the laboratory The medium must contain all of the necessary nutrients adequate sodium bicarbonate buffer and any required pH indicators or antibiotics The choice of a medium is usually determined by cell requirements the user s experience and financial considerations Note A high glucose medium 3 0 4 5 g l is recommended for use in the MAXIMIZER to minimize the amount of medium required while supplying adequate glucose to the cell culture A glucose level lower than 3 0 g l may result in excessive medium usage Note pH control in the MAXIMIZER is based on a bicarbonate buffering system We suggest 3 5 g l of Sodium bicarbonate when using approximately 4 g L glucose At least 2 2 g l of Sodium bicarbonate should be present in the medium Other pH buffers such as HEPES or Tris should NOT be used If glucose con
106. ed oxygen to the IC The oxygenator also contains a connector for the temperature probe Oxygen Probe and Case Optional The MAXIMIZER can support a polarographic oxygen electrode When it is inserted into its plastic and stainless steel DO probe case it is considered a DO probe case assembly that is to be prepared for attachment to the flowpath The case exposes the tip of the electrode to a small volume of constantly flowing medium for optimal response time An electrical cable is provided to connect the probe to the appropriate bulkhead port in the incubator When the electrode is not in use it should be stored in a vertical position with the membrane tip at the bottom The protective cap should cover the membrane tip and the electrical cover should be in place Contact Account Services for more information pH Probe and Case A polarimetric pH electrode is furnished with a container of buffer When the electrode is inserted into its plastic and stainless steel pH probe case it is considered a pH probe case assembly very much like the oxygen probe case assembly The case is designed to expose the tip of the pH electrode to a small volume of constantly moving medium for rapid response time When the electrode is not in use it should be stored with the electrode tip in a buffer solution pH 4 and with the electrical cap in place Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 3
107. eded for compliance with regulatory agencies or for historical purposes run to run comparison troubleshooting daily flowpath variance in process control etc Included in this package is a box of printer paper compatible with the tractor feed mechanism SERIAL INTERFACE PORT Optionally connect to this port either of the following two devices e ACULINK DATA Loc Base Unit to electronically collect the run log and graph logged run data see Appendix B for ordering information or e A serial communications tractor fed printer Biovest does not supply this type of printer Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 3 9 Instrument Setup ey This section will explain what is needed to install the ACUSYST MAXIMIZER in the laboratory It will describe the specific electrical and gassing requirements as well as the procedures to test the instrument for damage that may have occurred during shipping Please remember to save the packing list that was enclosed with the box of accessories FACILITY REQUIREMENTS Bench space The MAXIMIZER should be placed on a level surface in a room with ambient temperature of 18 26 C 64 79 F If possible the location of the MAXIMIZER should provide easy access to an autoclave and a laminar flow hood The tabletop dimensions of the instrument are 69 8 cm 27 5 inches width and 66 cm 26 inches depth The height is 52 cm 20 5 inches
108. edium flows from the EC to the IC and removes small molecular weight wastes such as lactate and ammonia This portion of the cycle is called EC Fall When the level of media in the Expansion Chamber reaches its defined minimum level the process starts over Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 2 5 System Theory Note Figures 2 3 and 2 4 illustrate this process Arrows are shown to indicate net flow of media This alternating flow of media between IC and EC is called EC Cycling This patented process thoroughly mixes the media in the BRX ECS and greatly reduces the microenvironments and gradients Cire Pressure Only EC Fall nn ic EC IC Total IC Pressure gt EC Pressure Total IC Pressure lt EC Pressure IC amp Circ Pressure Figure 2 3 Figure 2 4 Process controls in the ACUSYST MAXIMIZER allow you to select time periods for the EC Rise and EC Fall phases of cycling as well as a desired transfer volume between IC and EC To maintain these conditions the instrument uses an ultrasonic level sensor to measure fluid level in the Expansion Chamber and automatically regulates the pressures applied to the Integration and Expansion Chambers P N 700037 000 Rev H Biovest International Inc Page 2 6 Operations Manual for the ACUSYST MAXIMIZER System Description ee ee This section will introduce you to the basic components of the AcUSYS
109. ee days of glucose concentration decreases or glucose uptake rate increases lactate concentration or lactate production rate increases etc Typically EC Cycling will begin some time during the first week after inoculation of the bioreactor s To activate EC Cycling 1 Open EC RETURN clamp 2 Press PROCESS CONTROL 3 Press ENTER until the following screen appears Figure 6 66 EC Cycling Parameters Screen 4 Use 4 f to move the cursor between the four programming options to the LiF EC Chamber volume increases duration and iiH EC Chamber volume decreases duration times in minutes Routine EC media have enough protein supplementation that the default Li and P N 700037 000 Rev H Biovest International Inc Page 6 98 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures SS well when using various EC media for the entire run and when using cultureware with one bioreactor two weeks of active EC Cycling time when using cultureware with two bioreactors After this time it may be beneficial to increase the transfer volume in steps of 15 35 ml every 2 3 days until the maximum transfer volume of 140 ml is in use unless metabolic activity worsens In such cases increase the transfer volume more slowly Reducing the initial transfer volume may be necessary especially if the EC medium is protein free or low in protein because the cell culture may require a longer period of time to become establi
110. efer to use the oxygen update or consumption rate OUR as a measure of a culture s metabolic status The calculation of OUR is based on the following equation OUR CP a pO2i where OUR Oxygen Uptake Rate mmol hr pO2out post BRX DO mmHg pO2 in pre BRX DO mmHg CP circulation pump rate ml min k oxygen solubility constant 13 333 rr famotmin PRODUCT FORMATION The calculation of product formation is based on the following equation PFR 24 x f x Product where PFR Product Formation Rate units day f Harvest pump flow rate ml h Product current product concentration units ml P N 700037 000 Rev H Biovest International Inc Page D 4 Operations Manual for the ACUSYST MAXIMIZER Appendix E Loading Pump Heads eS The following instructions show the proper technique to load tubing into the peristatic pump heads These techniques achieve the most consistent accuracy and precision in actual pumping flow rates relative to the desired pumps speeds entered in Process Control This consistency applies to all of the six pumps during a single production run as well as to these pumps for numerous production runs over time These techniques also result in the liquid flowing through the pump heads in the correct direction Be especially careful when reviewing figure E 3 1 Hold the pump in your left hand the side with the slot in the roller assembly facing up Figure E 1 Figure E l
111. eight 54 5 kg 120 1b Operating Temperature External Ambient 18 26 C 64 79 F Internal Incubator 24 1 45 8 C 75 114 F Electrical Requirements P N 600052 000 120 VAC 4 amp 60 Hz 500 watts P N 600052 001 100 VAC 4 amp 50 or 60 Hz 500 watts P N 600052 002 230 VAC 2 amp 50 or 60 Hz 500 watts CAUTION Supply voltage fluctuation not to exceed 10 of nominal voltage Refer to Appendix B Note Biovest International s printers require 120 VAC and 0 7 amps Backup Power Requirements Instrument and Printer Uninterruptible Power Supply UPS 500 watts sine wave form Main Fuse 120VAC 4 Amp 3AG Time Delay SLO BLO 4x 1 4 inch 100VAC 4 Amp 3AG Time Delay SLO BLO x 1 inch 230VAC 2 Amp Type T SLO BLO need 2 Metric 5 x 20 mm Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page A 1 Appendix A Specifications E Heater Box Fuse Not intended for replacement by Operator 1 5 Amp 3AG Time Delay SLO BLO 4 x 1 inch Power Supply Fuse Not intended for replacement by Operator 1 5 Amp 3AG Time Delay SLO BLO 4 x 1 inch 230VAC units only Pollution Degree II Installation Category II Battery Type Sanyo Lithium CR 2N 3 Volt M O Serviced by authorized personnel only Gas Requirements Necessary 100 Carbon Dioxide medical grade 15 psi 1 kg cm Backup optional 100 Air medical grade 15 psi 1 kg cm
112. ely on the Gas LED s status if it is lit a gas override is active 3 Gas numbers 0 and 15 may be set at any time even during an active culture run These numbers correspond to no gas flow and 100 carbon dioxide respectively see Figure 6 1 and should be used only at the direction of Biovest technical service personnel P N 700037 000 Rev H Biovest International Inc Page 5 20 Operations Manual for the ACUSYST MAXIMIZER User Interface _ _ _ _ _ EE Eoxoe ay E C Cycle Press E C CYCLE to display the following screen Figure 5 20 E C Cycle Override Control Screen These override functions are related to the fluid movement cycling between the IC and EC Press numbers 1 5 activates the relevant menu items immediately without the need to press ENTER Items 1 4 are the options to override EC Cycling Activating one of these four options turns on the E C Cycle LED Item 5 1s used to clear the EC Cycle override instead of needing to press CLEAR which also turns off the E C Cycle LED When items 1 4 are selected the Monitor screen shows an asterisk and message in the Cycle field to indicate which EC Cycle override is active In the override screen the word Off will be replaced to show the same override selection After an EC Cycle override has been cleared this word indicates whether process control is in Cycle or Low mode The Hold override
113. en choose menu item 6 Alarms The MAXIMIZER is capable of generating 33 alarms At the time an alarm condition occurs m Audible alarm makes a 1 second tone every 4 seconds m External alarm relay closes m Alarm message and details see next page are recorded by ACULINK DATA LOG or the printer if either is in use Therefore when an alarm occurs common practice should be to 1 Press ALARM to see what happened 2 Press MONITOR to silence the alarm and deactivate the external alarm relay 3 Resolve the alarm condition 4 Press ALARM and use the arrow keys to display the desired stored alarm 5 Press ENTER to restore the alarm so it can be activated in the future See chapter 5 section ALARM Key for further information about the information in alarm screens and responding to them Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 7 1 Troubleshooting E Each of the 33 alarm messages has the same format Each alarm has a E A name m f a unique number for each alarm name E delal Haie number for each alarm message and alarm condition E E iataz variable number for each alarm message and alarm condition elapsed time when alarm condition occurred Days Hours Muinutes format Figure 7 1 General Format for Alarm Screen datai and dataz the meaning of these values is not detailed in this manual They a are used by E Bova personnel to provide technical assistance or during field
114. equentially press f and START g LCD updates to show the name of each key h Press CLEAR and note the value in the lower right hand corner disappears 1 If the keys and LCD respond as described the tests passed j Press ENTER Figure 6 8 Keypad Test Screen Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 19 Operating Procedures SS naa Printer Test a Press 5 b LCD displays Frinter Testi c Printer advances paper by one page d Four lines of various letters numbers and characters are printed e If steps c and d occur the test passed f Press ENTER Figure 6 9 Printer Test Screen Notes 1 This test merely checks the communications between the MAXIMIZER and the printer It does not set the printer log interval 2 This test applies to a printer that is connected either to the serial or the parallel port 3 If the four printed lines are printed on a single line or there is an empty line between each printed line the printer is not configured properly which must be corrected 4 This test does not apply to ACULINK DATA LoG The MAXIMIZER does not have a built in method of testing ACULINK DATA LOG To test that it is working perform the following ACULINK DATA LOG Test a Follow instructions on page 6 16 to display Lcaeing Inierwal screen b Press 1 then ENTER c Press START and PROCESS CONTROL d Press ALARM to silence the
115. er likely must be replaced with a new sterile filter Do this aseptically 4 Ifthe filter is wet disconnect its corresponding gassing line from the bulkhead port to prevent additional medium from flowing into the instrument If this happens the instrument may require service Inspect the IC or EC port for the presence of cell culture medium 5 Drain IC chamber a If HOLD was pressed press 1 first b Override Outflow pump to maximum 4230 ml hr c Override Media pump to 0 ml hr d Clear overrides when Outflow pump pumps air from IC chamber 6 Drain EC chamber a Remove medium via the Cell Removal line using syringes or b If IC chamber volume is OK e override Outflow pump to 4230 ml hr e connect EC gassing line to EC chamber and instrument e override EC Cycling to Low e clear overrides when EC volume is 108 ml Contact Biovest s Account Services for technical assistance Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 7 7 Troubleshooting a Verify the following items to try to correct the cause of the System Volume Exceeded problem Any clamps incorrectly open or closed Any tubing pinched or plugged Vent lines fully connected to the IC and EC chambers and bulkhead ports Vent lines attached to the correct bulkheads 1 e IC to IC and EC to EC Any unintended pump overrides still active particularly Outflow Pump EC Chamber ultrasonics cable fully
116. erating Procedures E Removal of Cell Debris Note Note Cells that slough off the BRX s are removed to some extent by the harvest pump but most accumulate in the Expansion Chamber Here they can interfere with EC Cycling or disturb the environmental conditions within the ECS For these reasons the Cell Removal line is attached to the bottom of the Expansion Chamber If followed regularly the procedure described below will help you to minimize cellular buildup An alternate method is provided after the standard method This procedure instructs you to remove the entire contents of the Expansion Chamber cells plus ECS liquid then return fresh serum supplemented medium to the flowpath to avoid depleting the ECS of serum Contact Account Services if you have any questions about modifying this protocol for serum free cultures or for replenishing the ECS with conditioned medium Standard Cell Removal Procedure 1 Needed supplies at least two empty 60 ml syringes and possibly two 60 ml syringes filled with serum supplemented medium and the usual supplies for aseptic sampling During this procedure you will be working with the incubator door open long enough to activate the temperature alarm Also your handling of the cultureware in step 7 may activate other alarms related to EC Cycling In all of these cases press ALARM then MONITOR to silence the alarm until you have completed the entire procedure Reset the alarms after this
117. erations Manual for the ACUSYST MAXIMIZER Operating Procedures SaaS pH Probe Evaluation The probe evaluation instructions do not apply to the disposable pH probe 1 The instrument must be plugged in and turned on 2 Press START then HIDDEN KEY to display the diagnostics screens 3 Press ENTER until the following screen appears 1 column to the right of Standard reading from probe pH units m 2 column to the right of A D reading from probe Figure 6 17 Diagnostics Screen 0 Remove and save the storage solution bottle and electrical cap Connect the pH cable to the probe and the bulkhead pH jack Place the tip of the probe in a container with pH 7 0 buffer solution eo Place the container and probe so they remain upright without being touched or held Wait about 2 minutes then proceed for the 7 0 buffer test 8 A functional pH probe s A D reading in pH buffer 7 0 is 95 160 at room temperature Do not use the probe if the value is outside this range 9 Remove the pH probe from buffer 7 0 rinse it and place it in pH buffer 4 0 10 Repeat step 5 11 A functional pH probe s A D reading in pH buffer 4 0 1s lt 3 at room temperature Do not use the probe if the value is outside this range 12 Rinse the probe and put on the storage solution bottle and electrical cap Notes 1 Ignore the readings in pH units during the evaluation Acceptance is based only on A D values not whether pH agrees with buffer
118. es have been sterilized attach them to the cultureware inside a laminar flow hood Additionally in line media filters tubing extension lines and or sampling septa should be attached at this time if desired The following instructions describe the procedures to make these additions to the cultureware You need to supply sterile gloves disinfectant in addition to optional sterile in line filters tubing extension lines and any other sterile modifications necessary for a custom configuration See Appendix B for ordering information on in line filter assemblies and tubing extension lines CAUTION Do NOT lift the cultureware by its sterile bag Lift and carry the cultureware by holding it from the underside 1 To minimize system contamination a Use aseptic technique b Inspect the cultureware s sterile bag for holes or tears Do the same for the optional sampling septa and other sterile accessories that will be used c Disinfect the outside of the cultureware s bag and the packaging materials of the sterile accessories if in use and place them in the sterile hood CAUTION Fully open the bag and remove the cultureware from it while inside the sterile hood During this step cultureware tubing can be caught and pulled by the plastic bag Be very careful to not pull tubing off of their connectors d Confirm the green tape on the cultureware has brown stripes to indicate sterility conditions by Ethylene oxide e Remove and disc
119. ese calculations Pre BRX IC samples may be used to calibrate the pH probe Post BRX IC samples may be used to calibrate the DO probe Samples from the EC port are used for offline analysis of secreted product EC sample analysis is affected by EC chamber volume Remove EC samples at consistent EC chamber volumes to simplify data analysis Recommended schedules of sampling for metabolite and product analysis as well as for probe calibration are provided in Post inoculation Procedures Sampling involves the removal of stagnant medium in the sample port followed by removal of fresh flowpath medium for offline analysis The stagnant medium is called a pre sample and is to be discarded The pre sample volume from the IC and EC sample ports should be 3 ml Pre samples must be removed and discarded before the analysis sample is withdrawn This is true even if a post BRX sample immediately follows a pre BRX sample or vice versa P N 700037 000 Rev H Biovest International Inc Page 6 62 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures SS Use aseptic technique to perform all sampling operations according to the following protocol 1 Prepare sterile syringes disinfectant sterile gauze and sampling tubes If septa are attached to the sampling ports use 21 gauge needles on the syringes as described below 2 Disinfect the sampling port If you use caps wipe the junction of the cap and sample port fitting
120. establish baseline levels of metabolites Wait at least about 30 minutes after inoculation before removing this sample to allow their concentrations to stabilize Note Process Control sets the Gas at 7 after Inoculation Gas gradually increases during the first 15 20 minutes to establish pH control pH may go out of range while pH control is determing the correct Gas to use causing a pH out of range alarm Follow steps 25 27 to respond to the alarm Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 87 Operating Procedures SS POST INOCULATION PROCEDURES After inoculation the culture will require periodic changes to the feed rate factor rate harvest rate cycling conditions and circulation rate Although some of these parameters are automatically set into Process Control you must perform manual changes to others Information useful to these procedures was provided earlier in the descriptions on Oxygen Delivery Feed Strategy and Harvesting Strategy In addition the last part of this section Optimization Procedures contains more ideas on customizing various parameters to your cultures After a culture has been inoculated various system functions need to be examined to ensure that a healthy culture is maintained The following schedule is suggested for daily and periodic checkouts of the culture Note You may wish to prepare a printed form to remind you of these items
121. eware s tubing adapter between the tubing ends labeled PH IN LO and PH OUT HI The pH probe case ports are offset from one another one is higher than the other Orientation is important b Rotate the locking rings of the four quick coupler fittings to the unlocked position There are two in the pH probe assembly and two in the tubing adapter between the PH IN LO and PH OUT HI labels c Press and hold the latch of the pH probe s PH IN LO quick coupler fitting Remove the fitting from the probe case and discard it d Press and hold the latch of the cultureware s PH IN LO quick coupler fitting Disconnect this fitting from the cultureware s tubing adapter and connect it to the pH probe s exposed port from the previous step Ensure the latch faces upward e Release the latch and rotate the locking ring to the locked position f Repeat the previous two steps for the PH OUT HI connection g When using the reusable pH probe save the removed tubing adapter for use at the end of the run Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 39 Operating Procedures a 4 Attach the autoclave sterilized DO probe case assembly to the cultureware a Attach the two black plastic clips that were set aside as the first task of step 2 above to the correct locations depending on which type of pH probe is in use e when using the reusable pH probe press the two black clips on to the p
122. ey Press CALIBRATE to display the program and onscreen instructions to calibrate the pH and DO probes Calibration can be done at any time during an active culture run Calibrating either probe requires offline analysis of an IC sample of medium that is aseptically removed from the cultureware See chapter 6 Probe Calibration for further details P N 700037 000 Rev H Biovest International Inc Page 5 24 Operations Manual for the ACUSYST MAXIMIZER User Interface 1 rwva AZZ TE HOLD Key Pressing HOLD results in the following screen Figure 5 22 Hold Key Selection Screen Use the Hold feature to respond to an imminent or on going crisis or to terminate the instrument s current state during 71 for example or the active culture run An example of a crisis is a media leak from a tubing line Pressing HOLD stops these functions whether they re operating from process control or override settings where applicable All pumps stop m EC Cycling control stops pH control stops Alarm monitoring stops m Oxygenation stops Logging stops If HOLD was pressed to respond to a crisis after the cultureware has been inoculated it is important to resove the problem and resume normal operation as quickly as possibe to minimize the negative effects of no pumping no pH control etc to the cell culture After resolving the problem and to resume normal operation press 1 To terminate the instrument s current state or the act
123. f the cultureware are the oxygen and pH probe assemblies They are to be steam sterilized then attached to the flowpath under a laminar flow hood Each flowpath module is supplied with an accessories package This contains 60 ml syringes for serum pre treatment and inoculation sterile sampling port septa and tubing assemblies for the pH and DO probe cases Flowpath Modules The flowpath consists of two major circuits that meet at the hollow fiber BRX see Figure 3 4 The IC contains the Integration Chamber circulation pump pH probe assembly oxygenator medium tubing outflow tubing two factor lines and the lumen of the BRX fibers Within this major circuit lies a secondary path that contains the oxygen probe assembly and its associated tubing plus one sample port The EC contains the Expansion Chamber harvest tubing factor tubing check valves two access ports for sampling or inoculation and the BRX volume outside the hollow fibers known as the ECS Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 3 5 System Description 65 69 i m EE h Note S G9 EC RETURN oE Figure 3 4 Schematic diagram of ACUSYST MAXIMIZER flowpath Intracapilla
124. for the ACUSYST MAXIMIZER Page 7 15 Troubleshooting ee eS Figure 7 24 Alarm Screen 33 This alarm means the air temperature at the internal heater box has reached 65 C This condition activates the over temperature relay to shut off the heater but its fan continues running to try to cool the system The Monitor screen will show gt to the right of the temperature value until the heater box incubator temperature cools down The heater re starts only after the heater box temperature falls to 45 C If the ambient temperature is within specified values Appendix A and the problem occurs the instrument likely requires repair service Note The incubator temperature mentioned above is measured internally with a separate temperature sensor It should not be confused with the media flowpath temperature that is measured with the cable connected to the incubator bulkhead jack Figure 7 25 Alarm Screen 34 This alarm means the instrument has experienced a power outage whether intentional or unintentional Datal shows the number of hours and data2 shows the number of minutes that the instrument was without power P N 700037 000 Rev H Biovest International Inc Page 7 16 Operations Manual for the ACUSYST MAXIMIZER Troubleshooting SS EE EEE Figure 7 26 Alarm Screen 35 This alarm means the main program could not communicate with one of the secondary programs that was then followed by an electronic reset that did restore c
125. from the MAXIMIZER to an IBM compatible computer or a serial printer The MAXIMIZER sends the same information to the parallel and serial ports An advantage of using a serial printer rather than a parallel printer is that a serial printer may be placed an extended distance from the instrument for remote printing outside the cleanroom environment for instance One advantage of using a computer over a printer is that unlike printouts a computer can capture the printout as data This allows the run data to be collected without using particulate generating paper Other advantages of using a computer are selective reviewal graphical analysis and archiving of run data and remote monitoring of instrument operation Biovest International offers ACULINK DATA LOG a custom hardware and software solution that runs on a PC computer to log run data and provide the benefits listed above For computer information about the ACULINK DATA LOG system contact Biovest International s Account Services Department There are four modes available for parallel and serial port operation These different modes allow the MAXIMIZER to adapt to the requirements and equipment of the production facility The differences between the four modes are described below Refer to Figure 4 3 and 4 4 for details of configuring the desired mode in the MAXIMIZER instrument The factory default configuration of the MAXIMIZER is parallel printer mode at a data transmission speed
126. g steps 3 8 P N 700037 000 Rev H Biovest International Inc Page 6 24 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures ee 14 When the instrument Pressurization Test passes press ENTER to display the following screen Figure 6 16 Final Pressuirzation Test Screen 15 Ignore the clamp instruction because it is irrelevant when pressure testing the instrument 16 Press ENTER to return to the Active Setup Menu screen Figure 6 12 17 Disconnect the Tygon gassing line from the IC and EC bulkhead ports Set it aside for possible use later in the run Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 25 Operating Procedures SS CULTUREWARE SETUP PROCEDURES pH Probe Selection The ACUSYST MAXIMIZER automatically controls pH of the circulating cell culture medium when an in line pH probe is in use Biovest offers two types of pH probes for this purpose One type is reusable The other type is disposable The two types of pH probes are not distinguished by their functionality in controlling pH Rather they are distinguished by the expertise time testing documentation and troubleshooting that are required during their use The reusable pH probe requires validation assembly autoclave sterilization and other procedures to prepare it during run startup Expertise is needed to ensure these steps are properly performed so the probe functions well and is not
127. gure 2 2 Major Components of the Integration Circuit red and Expansion Circuit yellow Integration Chamber Temperature Probe Circulation Pump Dissolved Oxygen Probe 3 Gas Exchanger Expansion Chamber pH Probe Q Volume Sensor Bioreactors volume inside hollow fibers is IC volume surrounding hollow fibers is EC Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 2 3 System Theory De The Integration Circuit The Integration Circuit IC is the part of the flowpath which coincides with the inside of the fibers in the BRX It includes E the circulation pump E the Integration Chamber where media and factors are added to the system and waste is removed the GEX oxygenator the IC flowpath tubing the ICS of the BRX s the probes used for monitoring pH DO and temperature of the system Note Figure 2 2 has been simplified and does not show the DO probe because it is part of a secondary IC Circuit See Figure 3 4 for a more detailed diagram When the circulation pump is on pressure is created in the IC which forces media to flow through the circuit and into the ECS from the ICS This half of the flowpath is basically the same as 1n a conventional hollow fiber system If left to circulate this way the Starling Effect would create nutrient and waste gradients which would not permit efficient large scale production P N 700037 000 Rev H Biovest International
128. has been growing for many days it may continue to expand when IC medium is unsupplemented P N 700037 000 Rev H Biovest International Inc Page 6 101 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures EE Serum free Media Experience with serum free media varies widely A culture can be started with serum supplemented medium then weaned onto serum free medium or it can be started with serum free medium Another technique is to add serum free medium only to the EC Regardless of the technique the important point to consider is that the large molecular weight components of serum free medium must be added directly to the EC Hybridomas grown in serum free medium may not remain in the BRX if the protein content of the serum free medium is too low In this case cycling and harvesting conditions should be modified to avoid excessive loss of cells P N 700037 000 Rev H Biovest International Inc Page 6 102 Operations Manual for the ACUSYST MAXIMIZER Troubleshooting See SOFTWARE If you have a problem that appears to be related to the software verify the following items before notifying Account Services 1 Are Override Controls in use and are they performing as expected to supersede Process Control Have you properly removed an override or is it still active 2 Determine which revision level of firmware your instrument is currently using This may be done by checking the PROMS list to do this press SETUP th
129. he first calibration for each run After the first calibration 1s completed pressing CALIBRATE displays the screen in Figure 6 56 Figure 6 52 Main Menu Probe Calibration Screen before Sampling 3 Remove and discard the pre sample volume from the cultureware 4 Replace the pre sample syringe with the analysis sample syringe Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 65 Operating Procedures Ce nna 5 Press 1 Take Sameil2 to display the following screen Figure 6 53 Probe Calibration Screen 2 6 To abort the calibration either before or after removing the sample a Press ENTER b The screen in Figure 6 52 re appears to re start calibration if desired c Press MONITOR to leave the calibration screen and discontinue calibration 7 To continue with the calibration a Remove the analysis sample b Immediately press START to store the current probe readings and advance the screen _ c The elapsed time for samp ie bakt is when START was pressed This also is logged by tt the AT or AGU ane DATA LOG if in use c Go to step 8 only if you pressed START Figure 6 54 Main Menu Calibration Screen after Sampling 8 Immediately analyze the sample offline for pH or DO 9 Press 2 Enter Hew Lilies to display the following screen Figure 6 55 Probe Calibration Screen for Data Entry P N 700037 000 Rev H Biovest International Inc Page 6 66 Operations M
130. hen either of these functions components is exhausted Monitoring growth and productivity parameters will give an indication of the need to add serum Cultures that show a drop in metabolic activity may require additional serum to recover A syringe can be used to inject a quantity of pure serum into the EC m However as a culture matures it becomes less dependent on the serum To reduce the serum content of the ECS two procedures may be used The factor bottle may be replaced with one that has less serum or the factor pump rate may be lowered These changes should be performed gradually to avoid sudden changes in environmental conditions Metabolism and productivity should be monitored to determine the effect of lowering the serum content fa culture does not begin expanding and consuming nutrients for many days after inoculation an option to encourage their growth is to add growth supplement to the IC medium at the same concentration as what is in the EC medium or switch to serum free media in the IC 1f it is the EC medium and FBS is not in use Continue this until the culture has expanded for one to two weeks Change directly to unsupplemented IC medium at that time or 1f necessary periodically reduce the concentration in steps of growth supplements in the IC medium This scenario can eliminate a dialysis loss of low molecular weight components from the ECS that was causing the culture to remain in lag phase Once the culture
131. hese steps for the other tubing line Ensure the latches of both probe case tubing lines face upwards CAUTION The pH and DO probe case tubing lines and their connection methods to the probe cases will be leak free without using cable ties We recommend to NOT add cable ties to this tubing Adding cable ties can actually cause leaks to occur electrical cap reusable electrical ca pH probe j probe case threaded connection locking ring q DO probe probe case upper section probe case upper section large o ring probe case probe case tele ae lower section lower section latch o ring e higher port 7 ower port small o a 2 Figure 6 19 Components of DO probe case Figure 6 20 Components of pH probe case assembly assembly Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 31 Operating Procedures 7 SS Probe Case Assembly Pressure Tests It 1s important to pressure test the pH and DO probe case assemblies to ensure they do not leak They are tested one at a time and either probe assembly may be tested first Successful pressure tests for these probe assemblies ensure that cell culture medium will not leak out at a later time The pressure test instructions do not apply to the disposable pH probe The instructions for pressure testing the DO probe and reusable pH probe are 1 Before proceeding ensure the Instrument Pressurization Test
132. ii Table of Contents C Appendix B Ordering Information Instrument and Accessories 66 dcac45408S4Sso eo bnSeb60 perce an doowand eee B 1 Cultureware Options cee ee eee e teens B 1 Sterile pH Probe ItemMs 0 ccc ec eet ee eee eee eee nnenes B 1 Reusable pH Probe Items eee eee teen ee eees B 1 Dissolved Oxygen Probe Items eee eee e eee eees B 1 Miscellaneous Sterile Optional Items 0 ee ee eee B 2 Reusable Glass Bottles and Polypropylene Cap Assemblies B 2 Cap Assembly for Plastic Bottles ce ee eens B 3 Miscellaneous Spare Pats lt eciccandacaoneeesesenneeun dacadaedonaneens B 3 SEVICE S era bas od eee ee oe epee eee eG eee eR eee ees B 3 Appendix C Relocation Preparing the MAXIMIZER 0 eee eee ee eee eens C 1 Instrument Lifting InstructiONS 0 0 ce ee eens C 2 ARO We MOVO ns onto ee ee eee aes Gee SSS Hee Geo eeennn ane es C 2 Appendix D Metabolic Calculations GIUGOSe UPAKO vee eee eens Gone aeeseeedeses ETETEAREN EEEE ANERE D 2 Lactate PROGUCTION sac aaa524ecoe tenterrnkand needa dede dade eedaae an eae hae ae D 2 Glutamine Uptake cc eee ee ee ee eee eee e eee e ene D 3 Ammonia Production s 5220008 0800208 6o5e5asoeekaeaaeaaoesoen es eesacanes D 3 Oxygen Uptake ssc554 4e45 ge nenenesy sn oaeeneeead bahay obese 2005955444058 05 D 4 Product Formation ge tcccaccw yoo oeeanaunneagadadaagecaseooeneeEEeenneeseee D 4 Appendix E
133. ilibrated so calibrate the pH probe Note The volume of media within the flowpath and particularly the Integration Chamber may be very low when Active Process Control starts This is normal This may result in air bubbles circulating through the IC circuit which is undesirable but does not cause damage If this occurs use the Pumps Override Control to set the Circulation Pump rate to 0 ml min and the Media Pump rate to 800 ml hr When the Outflow pump begins drawing medium out of the top of the Integration Chamber clear the Circulation Pump and Media Pump overrides Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 61 Operating Procedures SS MAINTENANCE PROCEDURES Sampling This part of Operating Procedures describes various procedures that are used during the pre inoculation and post inoculation phases of a culture The intent is to present them only once as a group so as to be easier to locate in the future The procedure on the following page is used to remove samples of medium from the flowpath These samples can be from either fluid circuit m IC for metabolite pH or DO analysis or probe calibration m EC for various product analyses Details specific to calibrating the pH and DO probes are provided in the next section Probe Calibration Post BRX IC samples may be used to calculate metabolic uptake rates and production rates See Appendix D for the formulae in th
134. ils on pH control CO gas supply properly set to deliver 15 1 ps1 m Gas override active and setting the gas number outside the range m GEX Out clamp closed Correct amount of bicarbonate added to the medium m Medium should not include HEPES m pH probe functioning properly See Section 6 pH Probe Evaluation Also has the pH probe been calibrated regularly m Ifa base solution is attached to Factor 1 does its addition to the culture cause changes in pH that are too great or too small Ifso you may need to replace this solution with one that has a lower or higher concentration m pH cable attached to the probe and bulkhead securely m pH probe may require preventative maintenance Refer to the manufacturer s instructions that are provided with the probe Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 7 23 Troubleshooting eee SSS mr Product Secretion Failure of a culture to attain a desired level of productivity may be caused by a variety of factors The following is a list of suggestions to consider if you find yourself in this situation Due to the many unknowns in cell culture it is not a comprehensive list but Account Services personnel are prepared to discuss any problem you may have Could there be problems with the product assay m Are there problems with storage of harvested medium Temperature length of storage and pH should be considered m Dras
135. in the pump head Is its pump tubing segment leaking Is its pump tubing segment kinked inside the pump head m Any unintended pump overrides still active particularly Media Pump Is the Media supply empty Its clamp closed Its vent filter plugged Its tubing male femal Luer connection loose Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 7 9 Troubleshooting E eS Figure 7 11 Alarm Screen 17 This alarm means the printer is unable to print for a variety of reasons Verify the following items to try to correct the problem Printer offline or deselected Printer out of paper Printer paper jammed Printing head jammed or cannot move on its track Figure 7 12 Alarm Screen 18 This alarm means the printer is not turned on or the printer cable is not fully connected to the printer and or instrument Note More than one printer alarm usually occurs at the same time To minimize unnecessary printer alarms disable the printer SETUP screen before adjusting the printer functions or paper supply Figure 7 13 Alarm Screen 19 This alarm means attempts to send log information to the printer have failed The printer may need service P N 700037 000 Rev H Biovest International Inc Page 7 10 Operations Manual for the ACUSYST MAXIMIZER Troubleshooting Figure 7 14 Alarm Screen 20 This alarm means the monitor board or possibly the control board is faulty This prob
136. ing and operating the MAXIMIZER It is a guide to the operations and features of the system and provides guidelines for laboratory procedures sometimes necessary in the preparation or operation of the instrument Complete information on the following tasks is included to aid your work from start to finish E how to assemble and inoculate the cultureware E how to program the microprocessor for growth and harvest conditions E how to maintain and support the culture E how to dismantle the cultureware at the end of the run The manual is organized into six sections plus a Glossary five appendices and this introduction The sections are organized in the order in which procedures or knowledge is necessary although some cross referencing may be required Read completely through each procedure before starting To ensure efficiency and reduce the risk of contaminating the system make sure you have all necessary supplies before beginning the procedure Familiarize yourself with the basics of Biovest s hollow fiber design by reading Section 2 System Theory Then use Sections 3 and 4 to learn the components of the instrument and cultureware The milestones table in Section 6 gives you an idea of how to plan for laboratory preparations and needs of the instrument Use it to prevent unnecessary delays in setup and to adjust process controls before inoculation Throughout the text key terms will be in bold type when they are introduced and defined
137. ior to installing the probes in their cases which occurs before pressure testing and autoclaving the probe case assemblies Contact Account Services if you have questions about a probe or cable Note Skip steps 3 and 4 for the sterile disposable pH probe see page 6 24 pH Probe Selection and Appendix B Ordering Information P N 700037 000 Rev H Biovest International Inc Page 6 16 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures ea System Tests These tests and the one titled Pressurization Test should always be performed before probes are tested and a new cultureware is opened and put into use Plug in the MAXIMIZER and turn on its power switch at the rear of instrument The LCD should show If E in the upper left corner If not press HOLD then 2 Press SET UP to display the Sustem T Figure 6 3 System Tests Menu Screen When SET UP was pressed above the Lagging Initersal screen may have been displayed instead Its use is described later in this chapter Figure 6 4 Logging Interval Screen Press ENTER to display the S2ien Testa menu if necessary Note Repetitively pressing ENTER cycles between the 32ien Tests Inberual screens Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 17 Operating Procedures ee Note Perform each of the System Tests consecutively by pressing 1 6 After each test passes press ENTER If a tes
138. is sometimes called a transmembrane method of inoculation Depending on the pore size and total surface area of the BRX s this method may cause a temporary pressure buildup back pressure within the ECS If the EC CHAMBER RETURN clamp is opened immediately after this type of inoculation the back pressure will cause a short burst of media to flow from the ECS of the BRX s to the Expansion Chamber To avoid displacing cells in this manner keep the Expansion Chamber clamps shut until process control has begun manually vent the Integration Chamber to zero room pressure and then open the Expansion Chamber clamps Replace the vent line to the Integration Chamber after a few seconds P N 700037 000 Rev H Biovest International Inc Page 6 96 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures SSS m Seeding a BRX more than once may be considered an option in the following situations low inoculum density and or viability poor initial growth re use of the flowpath Ideally the first situation should be avoided but time constraints or other factors may take precedence over preparing a suitable inoculum In the other two situations it may be best to flush the cells out of the BRXs before re use although cleaning a BRX that was densely populated is usually difficult and may not be recommended because of manufacturing regulations involving the desired product Poor initial growth in the MAXIMIZER culture may be attributed to many c
139. item 1 should not be confused with the red HOLD key They perform different functions Pressing HOLD stops all process control and override activities The EC Cycle Hold override interrupts only the current Rise or Fall phase of the EC Cycling process When the Hold override is chosen the level of medium in the Expansion Chamber is maintained at the level when the Hold override became active The level will not change until the Hold override is cleared The Low override item 2 works similarly to the Hold override except that the Low override first forces the level in the Expansion Chamber down to its low volume 108 ml then the level is maintained at this level until the Low override is cleared Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 5 21 User Interface _ E E EEEeeay The Drain override item 3 is only shown when the Idle mode 1s active This override is provided for use afte the culture run is terminated When the Drain override is chosen pressure is applied to the Expansion Chamber to force all of the media out of the EC Chamber and down the Cell Removal line to collect the product into syringes If collecting the product is unnecessary leave the Cell Removal clamp closed and the media is then pushed into the IC circuit This override merely drains the flowpath before cleanup operations at the end of a run Refer to Section 6 End of Cul
140. ive culture run press 2 The following changes occur m ILE state is activated Elapsed time resets to lili Hi Process control settings are reset to the default values Active overrides stop and these settings are reset to the default values All six items listed above stop After pressing 2 alarm messages remain in queue and must be reset manually Incubator temp control continues to function before and after pressing 1 or 2 NOTE Special use of HOLD during the Fill Flush procedure and Pressurization test is described in chapter 6 Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 5 25 User Interface awl ZT ALARM Key When the Alarm LED is off no alarm is stored in the alarm queue In this situation pressing ALARM displays the following screen Figure 5 23 Alarm Screen No Alarm When the Alarm LED is on and the alarm screen is not displayed an alarm s is stored in the alarm queue In this situation pressing ALARM displays a screen specific to one type of alarm An example alarm screen Figure 5 24 Alarm Screen Example Alarmt in Queue Press or to review however many alarm messages are stored in the queue Alarms occur when a probes in the cultureware or instrument measure a value that is out of range or b instrument hardware circuitry malfunctions MARMER hits aiga 33 distinct alarms most of which are active only during HOC and lid T Mo modes When an alarm
141. l sources of a leak are a Clamp was opened that should have been closed b Tubing is not centered in a closed clamp particularly F1 F2 F3 Harvest Factor Prime Inoc EC Sample c Lower and upper sections of the probe cases weren t tightened don t over tighten them d Tab of pH IN LO connector or pH OUT HI connector was not fully pressed in while connecting it on to the pH probe case port fitting which damaged the o ring or pushed it out of place e Loose male female Luer connection f Loose male Luer plug g Cable ties were installed on tubing that connect to DO or pH probe case port fittings h The instrument itself if it did not successfully pass the instrument Pressurization Test described earlier in this chapter Perform this test if necessary 11 After each attempt to fix the leak repeat the Pressurization Test a If necessary press ALARM b Use or to locate the Pressure dros detected alarm c Press ENTER to restore the alarm d Press START then SET UP e Press ENTER if necessary to return to the Active Setup Menu screen Figure 6 27 f Repeat the instrument Pressurization Test using steps 2 6 Note If you cannot find the leak after a few attempts using reasonable efforts contact Biovest s Account Services group before proceeding Keep the cultureware intact and sterile to not jeopardize the product warranty or incur re sterilization expense and delay P N 700037 000 Rev H Biovest In
142. l you to do so 1 Press START then SET UP if necessary to display the following screen Figure 6 35 Screen to Begin Fill Flush Procedure 2 Press 2 to begin the Fill amp Flush procedure P N 700037 000 Rev H Biovest International Inc Page 6 50 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures er eee 3 Open or close the clamps as instructed on the next four screens shown below to prepare the cultureware for Fill Flush Make certain that closed clamps are centered over the tubing and completely pinch it Even if you have recently finished the cultureware pressurization test make certain these clamps are adjusted properly Press ENTER when instructed Figure 6 36 Instructions Screens 1 4 for Fill Flush Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 51 Operating Procedures a 4 The fifth screen tells you to load the medium pump head Follow the instructions in Appendix E to do this step Attach the loaded pump head to the mounting position labeled MEDIA When this is done aseptically attach the medium pump tubing to the bottle or bag of medium that will be used to flush the flowpath Then close the Outflow clamp Figure 6 37 Instructions Screen 5 for Fill Flush Note The Media and Outflow pump tubing segments must be loaded into size 7016 peristaltic pump heads Factor and Harvest pump tubing segments must be loaded into size 7014 peristal
143. lastic bellows assembly that is part of the flowpath module It is mounted inside the incubator with two metal screws and a threaded plastic knob Keypad and LCD To the right of the incubator is the control panel for the microprocessor It consists of a touch sensitive keypad and a liquid crystal display LCD Separate buttons are used to activate the MONITOR screen CALIBRATE function ALARM listing SETUP functions PROCESS CONTROL parameters and the INOCULATE procedure In addition a row of buttons is available to control certain OVERRIDE functions The four arrow buttons are used to move the blinking cursor when it 1s accessible and the numeric section of the keypad is used to ENTER or CLEAR values when accessible fields are shown in the LCD The START and HOLD buttons at the lower left are used for special situations Details on keypad operation are given in Section 5 User Interface Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 3 3 System Description Sse Electrical and Gassing Features The rear of the instrument contains several features necessary for the operation of the instrument Figure 3 3 The power cord 1s attached to the lower left near the on off rocker switch Immediately to the right of the power switch is an access panel for the fuse An alarm with volume adjustment ring is located in the lower center of the instrument A 36 pin parallel port is available for attach
144. leak the test fails see bottom screen of Figure 6 14 Go to step 9 Figure 6 14 Pressurization Test Passed Screen and Pressure Test Failed Screen Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 23 Operating Procedures a 9 Ifthe instrument Pressurization Test fails press ALARM to silence the audible alarm and deactivate the external alarm relay 10 A screen similar to Figure 6 15 appears Figure 6 15 Pressuirzation Test Failure Alarm Screen 11 The leak must be located and fixed before proceeding with the run Potential sources of a leak are a Tightness of gassing line in the IC and EC bulkhead ports b Connection of the gassing line s tubing on its male Luer fittings c Damage to the gassing line s male Luer fittings or tubing d Damage to the instrument s bulkhead port female Luer fittings e The instrument has an internal leak 12 For 11 d and e contact Biovest s Account Services department For 11 a c repair or replace the parts and repeat the Pressurization Test 13 After each attempt to fix the leak repeat the Pressurization Test to confirm the leak has been fixed a If necessary press ALARM b Use or to locate the Pressure dros deLected alarm c Press ENTER to restore the alarm d Press START then SET UP e Press ENTER if necessary to return to the Active Setup Menu screen Figure 6 12 f Repeat the instrument Pressurization Test usin
145. lected flush medium Figure 6 47 Instructions Screen 12 for Fill Flush Note Using Outflow and Media pumps is necessary at this point You must decide when if Factor and Harvest pumps are needed If so and they are attached to their respective bottles open their clamps now to allow their pumping later on 19 The next screen informs you that Fill Flush is finished Pressing enter causes the next screen to appear Figure 6 49 Figure 6 48 Instructions Screen 13 for Fill Flush P N 700037 000 Rev H Biovest International Inc Page 6 58 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures rr eee 20 The instructions in the next screen Figure 6 49 mean that you need to temporarily leave this Fill Flush screen to go into the Process Control screens to m Change the default pH setpoint to what is best for your particular cell line This ensures the Pre inoculation phase to follow adjusts the cell culture medium to what is necessary for the inoculum m Start E Sae by changing the default Fe 1 s a from 7 BE BE to Gengi Starting EC Cycling speeds pH viii ontogeny the ealtareware The remaining EC Cycling defaults are OK F8 13 i5 a Press PROCESS CONTROL b Fress ENTER until Leoirk appears c amp nter the pH that eoi to mid log phase growth for the pending inoculum if known Otherwise 7 1 7 3 often works for many cell lines d Press ENTER until EC Ceg ime appears e Change DELAY time to 8 88 BE m S
146. lem likely requires repair service Figure 7 15 Alarm Screen 21 This alarm only occurs when the Pressurization Test fails and not during an active culture run It indicates there is a leak large enough to cause a rapid loss of pressure This may happen during the pressurization test of the instrument probe case assemblies or cultureware If EC leaked the second number is gt 0 If IC leaked the third number is gt 0 Verify that all connections are properly sealed on probe case assemblies Gassing lines are firmly connected to the IC and EC bulkhead ports and the probe case assemblies or cultureware If tubing extension lines or othe items were added to the cultureware do not include them in a repeat of the Pressurization Test If the test then passes the integrity tubing extension lines or other items should be checked Remove cable ties added to the pH and DO probe case assemblies and repeat the Pressurization Test Re check the tightness of the male Luer plugs on the cultureware and repeat the Pressurization Test If the instrument Pressurization Test fails the instrument likely needs repair Service Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 7 11 Troubleshooting er eS Figure 7 16 Alarm Screen 22 This alarm means the instrument s user interface buffer is unable to send characters to the printer Verify the following possible causes Printer power on Prin
147. libration Cycling Osmolarity Offgassing Cytotoxicity testing Sterility testing Serum pre treatment of the ECS Inoculation Procedure Post inoculation Procedures End of Culture Procedures Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 9 Operating Procedures SS Ss BIOLOGICAL SETUP PROCEDURES Cell Line Characterization Successful mammalian cell culture production requires the capability of precise process control The MAXIMIZER is equipped with a process control system which provides you with complete control of the cell environment A good process control strategy will allow cells to grow to a high density quickly and then shift control parameters to keep the cells in a stable environment with production at its peak It also allows you to vary conditions if required to maximize that production Thus a successful process control strategy depends on a thorough knowledge of the characteristics of your cell line There are many possible methods for characterizing cells but they all should establish specific values for optimum pH m choice of medium m serum dependence m glucose and lactate levels m product yield m cell viability These established values and other information are used to obtain target values for the process control strategy Before beginning production a full characterization of your cell line should be completed All cell lines differ in their
148. limits set in Process Control Verify the following items to try to correct the problem Bulkhead IN port connected to GEX IN vent filter port Wet GEX Vent filter GEX Out clamp open Gas lines to and from the GEX pinched or plugged Off gas port at the instrument s back panel not obstructed or plugged DO cable properly connected to the probe and bulkhead jack DO probe A D reading within acceptable range See Section 6 DO Probe Evaluation Calibration of the DO probe fixes problem Calibration procedure being properly performed Circulation pump turning smoothly Bubbles in pH probe case Integration Chamber vent filter wet and thereby creating an over pressurized system P N 700037 000 Rev H Biovest International Inc Page 6 Operations Manual for the ACUSYST MAXIMIZER Troubleshooting IIE IE ES EES ESD Figure 7 9 Alarm Screen 15 This alarm means the Expansion Chamber is approaching its maximum volume Take immediate action 1 Inspect the IC and EC chambers 2 Press HOLD if either chamber is about to or has flooded 3 If either chamber s filter is about to become wet a Close IC Vent clamp if the IC vent filter is in jeopardy b Put a temporary clamp on the tubing between the Expansion Chamber and its vent filter if the EC vent filter is in jeopardy c Close EC RETURN clamp if the EC vent filter is in jeopardy c Prevent these filters from getting wet If this happens the filt
149. ment of the printer cable optional An air inlet and an exhaust fan which cool the system electronics are located in the lower right and upper left respectively Mounting holes for the coalescent gassing filters are identified with a diagram on the lower right One of these filters will be attached to the internal air compressor while the other filter will be attached to an external supply of 100 carbon dioxide CO2 Two fittings are provided for a backup air supply and an outgassing connection both optional 14 3 13 reverent a GOD canes PP ne gt 6 7 8 9 1011 Figure 3 3 Rear view of ACUSYST MAXIMIZER showing the main features 1 exhaust fan 2 alarm 3 serial interface port 4 on off switch 5 power cord outlet 6 fuse 7 parallel printer port 8 alarm relay 9 offgassing port 10 air filter 11 backup air port 12 carbon dioxide filter 13 inlet fan 14 label for gas filter assembly P N 700037 000 Rev H Biovest International Inc Page 3 4 Operations Manual for the ACUSYST MAXIMIZER System Description eee CULTUREWARE COMPONENTS The disposable part of the cultureware is the flowpath module which consists of a plastic tray that holds the BRX gas exchange cartridge bellows type circulation pump two vessels called Integration Chamber and Expansion Chamber plus associated tubing clamps and vent filters The module is self contained and pre sterilized The reusable parts o
150. mode The serial printer may be placed at a remote location outside the production room for instance for convenience or other purposes Serial computer mode is primarily meant for only using the optional ACULINK DATA LOG system The serial computer mode does however allow the use of a parallel printer The serial port will override any parallel printer failures after a short timeout Restricted mode allows simultaneous printing from both parallel and serial printers A parallel printer may be used in serial printer mode The serial port will override any parallel printer failures after a short timeout P N 700037 000 Rev H Biovest International Inc Page 4 6 Operations Manual for the ACUSYST MAXIMIZER Instrument Setup ey ACULINK DATA LoG SETUP OPTIONAL Biovest International optionally offers ACULINK DATA LOG a custom hardware and software solution that runs on a PC computer to electronically collect and store run data rather than printing this information as described above Advantages of using ACULINK DATA LOG include m saving space in the clean room m eliminating particulate generating paper in the clean room m faster simpler review of log data m archiving run log data m faster simpler monitoring or instrument operation m improving reliable documentation of log data Contact Biovest International s Account Services Department for further information about ACULINK DATA LOG Biovest International Inc P N
151. most common method of operating the Harvest Pump To achieve this the ir and fic 1 times must be set to the same non zero time If the fr and Sx 1e times remain at the default of 8 H the Harvest Pump will not turn on even when the fiz s time is elapsed and the F414 is Allowed parameters Delay 0 00 00 999 23 59 in increments of 1 minute Rate 0 200 ml hr in increments of 1 ml see page A 4 On 0 00 00 999 23 59 in increments of 1 minute On time must be lt Cycle time m Cycle 0 00 00 999 23 59 in increments of 1 minute Figure 5 15 Continuous Harvest Control Screen Note The use of Cycle time in this screen should not be confused with and is unrelated to EC Cycling parameters described on page 5 12 P N 700037 000 Rev H Biovest International Inc Page 5 16 Operations Manual for the ACUSYST MAXIMIZER User Interface _ _ _ _ _ EEeEe ay Continuous Batch Harvesting When this selection is made the screen in figure 5 16 will be displayed by pressing ENTER Delay has elapsed this harvesting method becomes active The Harvest Pump then will run at a pre programmed high flow rate to remove the user defined ic lume each time the Expansion Chamber volume is near its lowest level during the EC Cycling s Rise and Fall process Product concentration is highest when the Expansion Chamber volume 1s at its lowest level as compared to when this volume is at its highest
152. n serum mg ml conc of glucose in factor mg ml medium rate Gm glucose rate Gg factor rate Gf serum rate Gs The calculation of lactate production is based on the following equation LPR where LPR Lactate Lactate P N 700037 000 Rev H Page D 2 r Lactate e Lactate l e Lactate Production Rate mg h previous lactate reading mg ml current lactate reading mg ml Biovest International Inc Operations Manual for the ACUSYST MAXIMIZER Appendix D Metabolic Calculations E GLUTAMINE UPTAKE The calculation of glutamine uptake is based on the following equation Glut r Glucose e Glut r Glut GItUR l e where GItUR Glutamine Uptake Rate mmol h Gltm glutamine concentration in medium mmol ml Gltf glutamine concentration in factor mmol ml Glutamine medium rate Gltm factor rate Gltf mmol h Glutamine previous glutamine reading mmol ml Glutamine current glutamine reading mmol ml AMMONIA PRODUCTION The calculation of ammonia production is based on the following equation ape r Amm e Amm l e where APR Ammonia Production Rate mmol h Amm previous ammonia reading mmol ml Amm current ammonia reading mmol ml Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page D 3 Appendix D Metabolic Calculations al OXYGEN UPTAKE Some investigators pr
153. nal medium to dilute lactic acid See chapter 5 Factor 1 F1 Base Addition Pump and Medium Pump Rates Rate for further information about Base Addition and Medium Dilution Gassing s variable gas mixtures are assigned relative Gas Numbers that range from 0 15 Only 1 14 are used to control pH during an active culture run See Figure 6 1 Gas 1 is the most potent for raising pH Gas 14 is the most potent for lowering pH Gas 15 1s used by service personnel only Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 1 Operating Procedures In FUN THO mode Gas s 1 14 automatically change as necessary to respond to changing culture conditions and maintain pH at its setpoint In POLE SETUP and IHG modes Gas 0 is used GAS RELATIVE AIR PERCENT PERCENT NUMBER FLOW RATE AIR CO 0 OOnN DOOR WDNDN CO OoODnnarWODWDODDO00 O 0 T 6 5 4 3 2 1 T 6 5 4 3 2 1 0 Figure 6 1 CO Concentration and Air Flow Rates Note Gas 15 is used only by service personnel User Defined pH Control Parameters The following parameters are set in their Process Control screens pH setpoint Allowed range of gas numbers Medium pump rate for Medium Dilution Base F1 pump enabled disabled for Base Addition Base volume to be pumped before start of Medium Dilution Automatic gassing system control and pH control can be disabled by overriding Gas Media Pump speed
154. next sampling If you use caps on sample ports replace it now and disinfect the junction of the cap and sample port If you use LAVs do not leave a syringe connected to the LAV Wipe it as previously mentioned and reconnect the dust cover If you used a septum remove the syringe plus needle and wipe any drops of medium from the septum s surface 8 Seal the analysis syringe with its cap or the needle guard 9 While performing the above tasks perform the software steps needed for calibration see Probe Calibration procedure below Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 63 Operating Procedures ee 10 If this sample is used for pH or DO calibration proceed immediately to the offline pH meter or blood gas analyzer or equivalent and obtain the desired value Then return to the MAXIMIZER and complete the software steps in the calibration sequence If this sample is used for other metabolites store it appropriately before analysis Note To avoid inaccurate readings due to changes in pH or DO inside the syringe you should prepare the offline meter or analyzer in advance of removing the sample and measure pH or DO immediately after the sample has been withdrawn Samples that are used for other metabolites or for product analysis should be stored appropriately to avoid degradation before the desired analyte can be measured P N 700037 000 Rev H Biovest International Inc
155. ng Procedures rr ee OPTIMIZATION PROCEDURES The following suggestions are offered to users to improve the quality of cultures grown in the MAXIMIZER Because each cell line responds individually to culture conditions it is not possible to specify protocols that will work in all situations However this information will give you some ideas to consider Experience with using these suggestions is usually the best method of instruction Biovest International s Account Services personnel are always available to assist you in optimization and will be happy to answer your questions as they arise Inoculation Several factors related to the inoculum influence the growth of the culture in the flowpath These include the number of cells per BRX their viability and the stage of growth when the inoculum is prepared m A higher inoculum population will shorten the period between inoculation and indication of significant metabolism Although this may seem to be an advantage the user must be aware of the practical concerns in preparing a higher density inoculum lt How many static culture vessels are needed e What volume of cell suspension can be processed safely How much time is required m Anchorage dependent cells may require a higher inoculum density than suspension cells Re suspending them in conditioned medium rather than fresh medium may improve the initial growth period by providing the cell specific growth factors secreted by th
156. ns a failure of the chip on the control board and it must be replaced by service personnel When the clock fails all operations related to elapsed time will not function e g process control circulation Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 7 3 Troubleshooting Figure 7 4 Alarm Screen 10 This alarm means the media temperature is outside of the limits set in Process Control This may be due to recent activity inside the incubator a fault in the heating system neither of which is user serviceable Users can check the following items to try to correct this problem m Incubator door fully latched on its pin Is the gasket around the door completely sealing the incubator against air leaks m Sufficient clearance at the rear of the instrument for air to circulate through the inlet and outlet fans m Temperature cable fully connected in the cultureware and is the insulation covering the sensor tip Temperature probe fully connected in its bulkhead jack Room temperature within the operating specifications 18 26 C Does another temperature probe fix the problem Figure 7 5 Alarm Screen 11 This alarm means the pressure in the Expansion Chamber is gt 280 mmHg Verify that the chamber is not flooded and its gassing line is attached properly This problem likely requires repair service P N 700037 000 Rev H Biovest International Inc Page 4 Operations Manual
157. nual for the ACUSYST MAXIMIZER Operating Procedures SS 14 When the BRX s have been inoculated replace the last empty syringe that had cells with the 10 ml syringe containing cell free medium 15 Open the INOC EC SAMPLE clamp 16 Inject the medium to flush any residual cells in the tubing up and into the BRX s 17 Close the INOC EC SAMPLE clamp 18 Press ENTER to advance to the following screen 19 For the instruction kiese Line IMUC GHP hm ol ame close the clamp labeled INOC EC SAMPLE 20 Replace the flush syringe with a new sterile syringe not the green plug which is no longer provided as an accessory with the cultureware 21 Press ENTER to display the following screen This also causes the Outflow pump to stop Note Do not open the EC RETURN clamp at this time EC Cycling is inactive at this time so this clamp does not need to be open and it is safe to leave this clamp closed The EC RETURN clamp will be opened when EC Cycling is activated see EC Cycling information on page 6 95 Hitachi Lh beri lias Lite by connecting the reusable Tygon tubing to the IC bulkhead port Figure 6 62 Instructions Screen 6 for Inoculation 22 Pressing ENTER displays the following screen which briefly appears to indicate the inoculation procedure 1s finished Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 85 Operating Procedures re Figure 6 63 Completion Sc
158. number and manufacturing number of your cultureware the conditions applied from Fill Flush to the removal of cytotoxicity samples and the specific cytotoxicity results Sterility Testing Optional At the same time that you test for cytotoxicity the system should be tested for sterility to be sure that you have not introduced bacteria or fungi into the system before inoculating it with your cell line Draw samples from each of the ports used for cytotoxicity testing and use the medium quality control procedures used previously see Medium Quality Control Because sterility tests generally required several days to be completed this test is used to double check the sterility of the system If aseptic technique has been used throughout the system should still be sterile This test 1s simply a confirmation of that fact P N 700037 000 Rev H Biovest International Inc Page 6 76 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures er ee Serum Pre treatment of the ECS For cultures using serum supplemented media serum is added to the ECS at the end of the pre inoculation phase to prepare the ECS with the same serum concentration as in static culture This creates an environment similar to that in static culture and minimizes the stress incurred from inoculating into a serum free environment Pre treatment of the fibers can be done any time before inoculation however it is most effective if done between four hours and twen
159. ny one time see caution note below OVERRIDE To display the series of diagnostics screens press START then the Diagnostics key an unlabeled key Diagnostics screens may be displayed at any time during the run so the caution note below does not apply to the diagnostics screens Lastly START also is used during the calibration procedure according to the onscreen instructions CAUTION Be careful about pressing START See caution note on page 5 7 If START is accidentally pressed press MONITOR or CLEAR before pressing another key P N 700037 000 Rev H Biovest International Inc Page 5 2 Operations Manual for the ACUSYST MAXIMIZER User Interface R MENU KEY OPERATIONS MONITOR Key Pressing MONITOR displays the main screen that shows most of the parameters that are automatically monitored and or controlled see Figure 5 2 Figure 5 2 MONITOR screen The Monitor screen shows which of the four modes of operation FELE SE TUF IHOR and RUMP Iho 1s currently active and for how long the mode of operation has been active as e n by the elapsed time Elapse time is represented as days hours minutes with maximum Aups of 999 days 23 hours and 59 minutes Elapsed time resets to 68 H4 whenever a new mode of operation is actived Elapsed time EPEE TE Scad culture time not the time of day or date The remaining information on the Monitor screen is shown for all four modes of operation and represents currently monitore
160. o count down to 8 8 In this way Timed Batch harvesting generally occurs less frequently than Continuous Batch harvesting although both batch methods remove fluid when the Expansion Chamber volume is near its low level Allowed parameters m Delay 0 00 00 999 23 59 in increments of 1 minute Volume 0 35 ml in increments of 1 ml Cycle 0 00 00 999 23 59 in increments of 1 minute Figure 5 17 Timed Batch Harvest Control Screen Note EC Cycling s timing and Timed Batch Cycle time are distinct from one another and they operate independently from one another P N 700037 000 Rev H Biovest International Inc Page 5 18 Operations Manual for the ACUSYST MAXIMIZER User Interface ee een Override Controls In the lower left portion of the keypad four keys are grouped vertically under the title Override Controls PUMPS GAS E C CYCLE and TEMPERATURE They may be used anytime in Idle Setup Inoculate or Running mode Overrides must be manually activated and cannot be set to automatically turn on or off in advance Once active overrides remain in effect despite whatever activity the Setup Inoculate or Running modes require until the override is manually cleared cancelled For this reason use overrides carefully until you are very familar with the operation of the MAXIMIZER and be sure to clear them as soon as possible Pumps Press PUMPS to display the following screen Figure 5 18 Pumps Override Cont
161. o that the serum concentration is the same as that in static culture 6 All of the flasks should have identical medium make ups before proceeding to the next step 7 Flush the flasks with 5 10 CO in air 8 Inoculate each flask with 5 x 10 cells with a viability of at least 85 9 Incubate in a 37 C CO incubator for two days 10 Observe the flasks daily for cell number and viability Include a count from the day that the flasks were seeded 11 Test results may be determined in two ways First a quantitative result may be obtained by counting cell number and cell viability in all of the flasks and recording the results If there are no significant differences based on customer pre determined values the system is ready to be inoculated Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 75 Operating Procedures ee Second flasks may be observed carefully under a phase microscope and their appearance assessed in comparison to the controls If the cell appearance in the sample flasks are as good as those of the controls or better the system passes the test and is ready for inoculation If the test results confirm cytotoxicity do not inoculate the flowpath module Continue to add medium to the system with EC Cycling in effect m Re examine the flowpath for stagnant BRX or pinched lines Contact Biovest International s Account Services Be prepared with the lot
162. of 9600 baud P N 700037 000 Rev H Biovest International Inc Page 4 4 Operations Manual for the ACUSYST MAXIMIZER Instrument Setup Caution Contact Biovest International s Account Services Department if another mode of operation is necessary You risk instrument damage and or personal injury by changing the rocker switch settings Figure 4 3 Serial interface board settings for printer and computer Rocker Switch Settings Switch 1 Switch 2 Baud Rate Figure 4 4 Serial interface board settings for baud rate Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 4 5 Instrument Setup E To use a serial printer configure its communications for 8 data bits no parity and 1 stop bit and to use XON XOFF flow control The serial port uses only transmit receive and ground signals Figure 4 5 The printer must be compatible with the printer s control characters Pin 2 RECEIVE Cinput Pin 3 TRANSMIT Coutput Pin 7 Signal Ground Figure 4 5 Location of main pin functions on serial interface port Parallel Port and Serial Port Modes of Operation Parallel printer mode is primarily meant for only parallel printing but the serial port is active for use by the optional ACULINK DATA LOG system see serial computer mode Serial printer mode allows simultaneous printing from both parallel and serial printers A parallel printer must be used in serial printer
163. ological Setup Procedures ccc eee eee e eee e eens 6 10 Cell Line Characterization 6220444624 0 etcteraaeaetanoooeS eee hedaee aa 6 10 Sterilizing Bottles and Cap Assemblies 0 cece eee ee eens 6 11 Medium Preparation 0 ccc eee ee eee e eee e eens 6 13 Sterilizing Medium 0 cc ee eee eee eens 6 13 Medium Quality Control 0 0 eee eee teen eens 6 14 Scale up for Inoculation 2acedeceeucccdeceedcae ce eeceeeaeeaanaanaees 6 14 Instrument Setup Procedures cece ekendudd cheer ess nes card don steer eaane ss 6 15 Prepare UUES 22244 4cedadasadednaeceseetbhbe ch eoaesereeu de dene 6 15 Prepare Data Logging Equipment ccc eee eee eens 6 15 System Checkout a sccacecvekcecesenatdads d atadect desGaeseeaade5 6 16 Cultureware Setup Procedures cece tenet nett eee eens 6 26 DH Probe SOlOCCUON sa044 4244 2450646 parse recaeaeareeeeeotndeces ees 6 26 DF Probe EVaMdtlOl 6 seacaaedaudoacexc44eeneseeeeone eee eunaacrers 6 27 DO Probe Evaluation 0 ccc ccc eee eee ee ee ee eens 6 28 Probe Case Assemblies 0 ccc ccc ee eee eee ee eeennenes 6 30 Probe Case Assembly Pressure Tests 0 0 0 ccc eee t eee 6 32 Sterilization Of PlODES 4 222220 8685006 ganda Reese SRS eee Hada Speers 6 36 Cultureware Sterile Assembly 0 0c ccc cee eee eee eens 6 37 Cultureware Installation 0 0 0 ccc ee eee eens 6 42 CUIRURE Ware LADClS otic e534 ta5 te teeed Renee eeaeaaedaeeeoi
164. ommunications Datal indicates which processor caused the error If this alarm occurs very infrequently it may not be symptomatic of a problem If it occurs more frequently than that the instrument should be serviced Figure 7 27 Alarm Screen 36 This alarm means the main program could not communicate with one of the secondary programs that was then followed by an electronic reset that did not restore communications Datal indicates which processor caused the error If this alarm occurs very infrequently t may not be symptomatic of a problem If it occurs more frequently than that the instrument should be serviced Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 7 17 Troubleshooting eee SSS em DIAGNOSTICS SCREENS Some of the uses of these four screens are explained in Section 5 and 6 Although these screens were meant for service personnel to check on the internal system status you may be asked to refer to certain information during discussions with Account Services REMINDER To view this series of screens press START then the hidden key empty space between the HOLD key and the Advance through the series by pressing ENTER P N 700037 000 Rev H Biovest International Inc Page 7 18 Operations Manual for the ACUSYST MAXIMIZER Troubleshooting Figure 7 28 Diagnostics Screen 0 The screen shown in Figure 7 28 is called Screen 0 and you have been taught how to use
165. onal Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page C 1 Appendix C Relocation E Instrument Lifting Instructions The Instrument is quite heavy Two people working together should be used to lift and carry the instrument Failure to follow these instructions could result in injury to the person s lifting or carrying the instrument or damage to the instrument Handle with care Do not lift the instrument by grasping the door or gas filters Grip sides and back of instrument only After the move m Have the instrument s internal system serviced by a qualified technician This will ensure that transporting the MAXIMIZER has not damaged electrical or mechanical components Be certain the electrical circuit in the new room will not be overloaded by the installation of the MAXIMIZER and that it contains the desired backup power facility Note Contact Biovest International s Account Services Department for additional instructions if relocation requires shipping the MAXIMIZER over extended distances Biovest International recommends shipping the MAXIMIZER in specially designed packaging only P N 700037 000 Rev H Biovest International Inc Page C 2 Operations Manual for the ACUSYST MAXIMIZER Appendix D Metabolic Calculations ees a This section details calculations to determine uptake rates for glucose glutamine and oxygen and the production rates of lactate ammonia and secreted product
166. onditions that can be avoided before inoculation e Cytotoxicity should be assessed before cells are added to the BRXs The procedure for this type of evaluation is described in Cytotoxicity Testing e Characteristics of cells in static culture will give some indication of how fast they will appear to grow in MAXIMIZER cultures Cells that exhibit relatively slow growth in static culture will respond similarly in perfusion systems e Problems associated with microbial contamination may be avoided by using antibiotic free media in static culture and by routinely testing stock cultures for mycoplasma and unwanted viruses Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 97 Operating Procedures 7 SS el Circulation Rate EC Cycling As a culture grows cells consume more of the oxygen dissolved in the surrounding medium and therefore need a greater delivery to maintain the same level of available oxygen By increasing the circulation pump rates the flowpath is capable of delivering more dissolved oxygen from the IC saturated with oxygen by the gas exchanger to the cell mass If cultures require dissolved oxygen for growth or production it is best to increase the circulation pump rate gradually and allow 1 2 hours before measuring any changes in dissolved oxygen EC Cycling should be activated when the cell culture shows signs of steady metabolic growth a period of two to thr
167. ons on the upper right wall Figure 3 2 These are used in conjunction with cultureware to monitor and control various parameters of the culture pH pO2 TEMP Figure 3 2 Bulkhead connections in the incubator Upper row connections are for electrical cables Lower row connections are for gassing lines To prevent over heating the temperature probe supplied with the MAXIMIZER should be connected to its bulkhead connection whenever the instrument is on and the incubator door is closed In the center of the incubator floor is the mounting bracket for the circulation pump Tubing can be routed in and out of the incubator by placing it in the tubing raceway panel below the door P N 700037 000 Rev H Biovest International Inc Page 3 2 Operations Manual for the ACUSYST MAXIMIZER System Description oe ee The MAXIMIZER is shipped with 4 pieces of Tygon tubing with color coded Luer Lok collars They are designed to connect various parts of the flowpath module to bulkhead ports inside the incubator These are reusable and should not be discarded Pump Modules Six mounting brackets are located below the tubing raceway panel These are labeled for their respective peristaltic pump heads Outflow Medium Factor 1 Factor 2 Factor 3 Harvest Two size 7016 and four size 7014 pump needs are included with the MAXIMIZER Each pump head is packaged with a set of four mounting screws and a pump key The Circulation pump is a p
168. orthwhile Use the EC Cycling Override 1 Put clamps on the IC inlet and outlet tubing of the BRX s to prevent unwanted IC medium from also being collected Aseptically connect the F3 tubing to a Harvest collection container 3 Remove the F3 tubing from the pump head Close these clamps a INOCULATE EC SAMPLE b HARVEST c FACTOR PRIME d CELL REMOVAL e Lower CHECK VALVE BYPASS 5 Open these clamps a FACTOR 3 b EC RETURN c Upper CHECK VALVE BYPASS Override EC Cycling to Drain Collect supernatant from BRX s and proceed when the ECS is empty Close the upper CHECK VALVE BYPASS clamp Open the lower CHECK VALVE BYPASS clamp 10 Collect supernatant from Expansion Chamber Proceed when it s empty 11 Press HOLD then 2 12 Close all clamps and process the supernatant lt p o o P N 700037 000 Rev H Biovest International Inc Page 6 90 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures ee ee Use the Factor 3 Pump 1 Put clamps on the IC inlet and outlet tubing of the BRX s to prevent unwanted IC medium from also being collected Remove the F3 pump segment from the pump head 3 Reverse load the F3 pump segment in the pump head in the orientation the Harvest pump segment is loaded Aseptically connect the F3 tubing to a Harvest collection container Disconnect the reusable Tygon gassing line from the vent filter on the Expansion Chamber 6 Close these clamps a INOCULATE EC SAMPLE b HARVEST
169. ovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 55 Operating Procedures er entering the EC chamber completely close the EC Return clamp and the lower Check Valve Bypass clamp If the problem persists contact Biovest 13 The first phase of Fill Flush is complete 14 The results of the instructions in the screen Figure 6 43 a Opening the EC RETURN clamp and closing the HARVEST clamp diverts the flow of cell culture medium from exiting the Harvest line to entering the Expansion Chamber This is the first time the Expansion Chamber should become wet b Pressing ENTER causes the Medium pump to automatically turn on and run at 4320 ml hr Figure 6 43 Instructions Screen 9 for Fill Flush 15 The meaning of the instructions in the following screen Figure 6 44 is Press ENTER when you see that the Expansion Chamber is approximately half full This stops the Media Pump and changes the screen This step only takes about 1 5 2 minutes CAUTION You must decide when to stop the pump It will NOT automatically stop Do NOT become distracted during this brief step Otherwise the Expansion Chamber could flood and leak cell culture medium possibly ruining the cultureware and damaging the instrument Figure 6 44 Duty Screen 2 for Fill Flush P N 700037 000 Rev H Biovest International Inc Page 6 56 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures rr eee 1
170. p 1 2 4 9 4 15 5 17 5 55 Appendix E Rev C 6 95 CELLEX BIOSCIENCES Inc Name Change all pages updated Rev D 7 96 Revised pages 11 vi Al A3 amp C2 Rev E 5 03 Updated for name change to Biovest International Inc Rev F 3 10 Udated for new pH probe options Rev G 7 13 Updated for new GEX and conversion to PDF Rev H 3 14 Updated inoculation procedure s clamping instructions P N 700037 000 Rev H Biovest International Inc ji Operations Manual for the ACUSYST MAXIMIZER Table of Contents T Introduction ABOUT THIS MANUAL ccccecwsooua idid EEEE ERA 1 1 SERVICE AND SUPPORT 00 ccc cece eee eee eee eee e ee eeeneeneees 1 2 VOCHOICdl SCIVICES ce tena enn eueeneeeseetewatee ee eeadeaedaaee ee eueaees 1 2 Customer Service ecsra 5G Sed amp Bb here aeek at ie aw dean a Se eee Oe ware aw 1 2 FICIC SORVICCs amp sagt ben pees er ee ee co Gea ee ee eaw a4 EE 1 2 Sales SUP OOM po os Sebo deny eben dona ondeneeedeessteeeee eee es 1 2 System Theory Hollow Fiber Technology cc cece cee eee eee eee e nent eens 2 2 ACUSYST Technology Difference cc ee eee eee eee eens 2 3 The Integration Circuit 0 ce ee eee eens 2 4 Fie ExDaNSION CCults lt 6 once deuasdene se ea gaataaueceoesenety4e4esees 2 5 SOLE Eai estes ees eee oe a geen Sa EE E See eee 2 5 System Description Hardware Components cc eee eee eee eee e eee e eee nnes 3 1 Instr ment CADINCl at ae o4 e6006046 so Hataeee eee ti ae SARS
171. perating Procedures SSS 8 The correct route of Fill Flush is described below and shown in Fig 6 41 m Medium bottle to Medium pump MP MP to top of IC chamber Bottom of IC chamber to Circulation Pump CP m CP to GEX to pH probe m pH probe to bottom IC inlet of BRX s m Top IC outlet of BRX to POST BRX sample line m Sample line to EC FLUSH IN line m EC FLUSH IN to bottom ECS inlet of BRX s m Top EC outlet of BR X s to Harvest line m Harvest line to Waste collection bottle Note Flush medium may flow from the BRX IC to the BRX EC directly through the fiber wall rather than via the EC Flush In line which is acceptable lt _e eer SO p 4 a i E t ma m ee FACTOR 3 e e e m ei 00 Xo met B Flush Media Figure 6 41 Route of media flow during initial Flush P N 700037 000 Rev H Biovest International Inc Page 6 54 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures e ee 9 When you are sure the Fill Flush route is free from obstructions in the previous step press ENTER 10 The screen shown in Figure 6 42 appears Also the Medium pump automatically turns on and runs at 4320 ml hr to flush the wetting agent out of the hollow fiber
172. ponents A separate part of Section 6 Contamination will discuss the details of handling cultureware affected by contamination Shutdown Procedures Perrform the following steps to terminate the active culture run 1 IfACULINK DATA LOG is in use export the log and save it Shut down the computer and turn off its power 2 Press HOLD then 2 Figure 6 65 Selection Screen for HOLD key 3 The instrument sends Process Aborted to the printer if Logging is enabled ACULINK DATA LOG does not receive the Process Aborted log entry If a printer is in use retain the printout Turn off power to the printer Close the CO supply to the instrument Backup Air is optional If it is in use close this supply to the instrument 7 eo a Turn off power to the instrument to minimize wear and tear to its components Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 89 Operating Procedures EE Recovery of Harvest After the run is terminated and the Circulation Pump speed is 0 ml min you may if desired manually empty the Expansion Chamber and BRX s to collect their remaining supernatant These are just two methods to do this Other methods also are possible such as using multiple syringes connected to various ports The effectiveness of any method depends on the accumulated cell mass If product titer was low when the run was terminated this harvest recovery process is not likely w
173. r suspension cells or by selecting anchorage dependent cells at the appropriate typically 50 70 confluency in static culture To shorten the lag time of growth in the BRX a minimum of 2 x 108 viable cells should be prepared for each bioreactor in the cultureware The steps used to collect cells for inoculation should ensure the highest possible percent viability Resuspend the cells to 50 ml per BRX using a large bore 15 gauge needle and the 60 ml syringes provided in the accessories package Conditioned medium will probably provide the least stressful environment Fill a small 10 ml syringe with cell free medium to flush the tubing after cells are injected into the BRX Proceed immediately to the MAXIMIZER Even though the Outflow pump is running during the inoculation procedure if you use inoculum volumes greater than 50 ml per BRX you will run the risk of flooding the Integration Chamber Watch this chamber s volume and do NOT allow it to fill faster than the Outflow Pump can remove volume If the application has the potential to release a biohazardous agent contact Biovest s Account Services department P N 700037 000 Rev H Biovest International Inc Page 6 82 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures rr eee 1 Press START then INOCULATE All of the pumps and logging stop and the software reminds you to make any necessary changes to Process Control The most important change in Process Cont
174. rating Procedures a 1 rinse the probe case pieces with water and clean with tissue culture grade detergent and rinse with deionized water j save both probe cases for the next run k store the pH probe so its tip remains wetted in storage solution 1 store the DO probe preferably vertically with the tip at the bottom Notes 1 After a culture has been terminated do not autoclave sterilize the pH and oxygen electrodes until they are prepared immediately before the next run Excessive autoclave cycles will shorten their lifespans and or reduce their effectiveness If the flowpath is contaminated or a biohazardous agent may be present contact Biovest s Account Services department 2 Do not forget to remove the temperature probe from the cultureware Autoclaving it will damage it m Properly dispose of the contents of all bottles that were attached to the flowpath Remove the cap assemblies and any labels from the bottles and clean as described in Sterilizing Bottles and Cap Assemblies m Clean the incubator and cabinet of the MAXIMIZER with a disinfectant or mild soap solution Be careful to not allow disinfectant liquids to wick behind the keypad or enter the instrument in other locations Avoid spilling any solutions on the circulation pump rotor in the bulkhead connections and in the incubator circulation vent P N 700037 000 Rev H Biovest International Inc Page 6 94 Operations Manual for the ACUSYST MAXIMIZER Operati
175. rature probe to the non invasive in line fitting in the large diameter tubing near the middle of the BRX s a Note the fitting has two small plastic knobs b Note the temperature probe s connector has two curved channels c Align the channels openings with the small plastic knobs d While pushing the tip of the probe into the fitting rotate the temperature probe connector turn to lock the probe in the fitting e Push the temperature probe s insulation over the fitting 3 Set aside the two metal screws and the white plastic threaded cap for the circulation pump Do not skip this step It can prevent cultureware damage 4 Carefully place the cultureware into the incubator The upper tray sits behind the metal ciculation pump bracket Do not pinch any tubing between the tray and incubator Allow the long tubing lines to hang in front of the instrument Do NOT load any pump heads at this time 5 Connect pH DO temperature and EC ultrasonics cables to their respective bulkhead connections Connect Tygon gassing lines to their color coded bulkhead fittings then attach them to the IC EC and GEX IN filters 6 Simultaneously slide the upper and lower portions of the cultureware s bellows pump onto the metal bracket and crank pin If necessary grasp the black hexagonal part of the bellows pump while rotating the black flat arm of this pump to align it with the crank pin Do not loosen the bellows pump assembly 7 Fully thr
176. re medium or any other liquid does NOT enter the cabinet via the opening for the circulation pump motor P N 700337 000 Rev H Biovest International Inc Page F 2 Operations Manual for the ACUSYST MAXIMIZER Appendix F Maintenance E E Acusyste INSTRUMENT SERVICE Biovest offers the following validation and defined maintenance services to facilitate the use of your ACUSYST system in a Quality System Regulation environment These services also help to keep its operation as trouble free as possible and to minimize unplanned downtime and disruptions to manufacturing operations Installation Qualification Operational Qualification IQ OQ validation service is optional with a new instrument purchase Validation can be performed before the instrument s first use at a later time and whenever it is relocated IQ OQ includes Calibration Calibration Biovest International specifies annual calibration of all ACUSYST systems Adherence to the schedule facilitates consistent optimal manufacturing Calibration returns the instrument s electrical and mechanical systems to Biovest s factory standards Preventive Maintenance Biovest International specifies a five year interval for preventive maintenance on all ACUSYST systems The purpose of preventive maintenance is to minimize the risk that the system s operation will fail and prematurely end a production run which could further delay future production scheduling Preventive m
177. reen for Inoculation 23 The following screen appears and Process Control automatically activates before ENTER is pressed Figure 6 64 Final Screen for Inoculation 24 Press ENTER to leave this last Inoculation screen 25 The Monitor screen appears The following actions have automatically occurred m Elapsed time has reset to H H Hi m HOC has ended and EHHHIHG is the active mode of operation m Active Process Control is active Active Process Control LED is on so logging alarms setpoint control and other activities are now active 26 Press ALARM to silence the audible alarm and deactivate the external alarm relay Alarms normally occur such as temperature pH and DO immediately when mode LHHHI HE mode begins Review the alarms stored in the queue to ensure unusual alarms are not present 27 Press MONITOR and watch temperature pH and DO readings After approximately 20 minutes they will be equilibrated and likely within the 28 Press ALARM Individually restore each stored alarm by using arrow keys to locate each parameter that is in its alarm range and pressing ENTER to restore its alarm Press MONITOR P N 700037 000 Rev H Biovest International Inc Page 6 86 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures ee ee 29 You may wish to m record the time of day that corresponds to the new elapsed time of 0 00 00 to know when the inoculation was completed m take time zero IC sample of medium to
178. ring rings should be removed from the bottles and washed separately 3 Replace the pouring rings on all bottles Put the caps on all bottles leaving them loose enough to allow steam to enter Cover these caps with foil or other autoclavable covering 4 Put the two male Luer plugs in their respective holes IN and OUT ports on the cap assemblies Install a 0 2 micron Acrodisc filter onto each cap assembly These are supplied as part of the Bottle and Cap Startup package See Appendix B Wrap the cap assemblies with foil or other autoclavable covering Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 11 Operating Procedures a 5 Place all prepared bottles and cap assemblies in the autoclave 6 Autoclave at 121 C at 15 psi maximum for approximately 30 minutes or at the conditions determined during your autoclave validation 7 Remove the bottles and cap assemblies from the autoclave and allow them to cool Tighten caps when completely cool Do not remove the covering 8 Store bottles in a dust free cabinet until you are ready to use them Bottles may be stored sterilized for six weeks before they need to be re sterilized It is a good idea to sterilize extra bottles so that they will be available as needed This can save time during your production run Note Each bottle is equipped with a removable plastic pouring ring Although it may be autoclaved at the conditions describ
179. rizes the probe The required period of time to fully polarize the probe can vary Two hours is the minimum suggested time The best strategy is to allow the probe to polarize overnight before proceeding 9 Press START then HIDDEN KEY to display the diagnostic screens if necessary 10 Press ENTER until the following screen appears 1 column to the right of 2 Standard reading from probe mmHg m 2 column to the right of 2 A D reading from probe Figure 6 18 Diagnostics Screen 0 11 A functional DO probe s A D reading when its tip is exposed to air is 65 130 at 37 C Do not use the probe 1f the value is outside this range 12 If the A D reading is unacceptable disassemble the probe Use a new membrane cartridge and or a new bottle of electrolyte P N 700037 000 Rev H Biovest International Inc Page 6 28 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures sr eS Notes 1 Do NOT touch the membrane cartridge that is at the tip of the DO probe 2 Polarization time is an important step in this procedure If the A D count is Slowly changing wait additional time until it has reasonably stabilized before determining if the probe is working within specification 3 Ensure the electrolyte solution has not expired 4 See Appendix B Ordering Information for replacement kits of membrane cartridges and electrolyte Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMI
180. rly over many years of service The replacement parts are provided in a Re membrane Kit see Ordering Information in Appendix B Usage information is in DO Probe Evaluation in chapter 6 Operating Procedures Other considerations for pH and DO probes E Aggressive cleaning of the reusable pH probe and the DO probe is generally not necessary Rinsing these probes with water at the end of the production run is usually sufficient Store them at room temperature Do not store them in a room with a high relative humidity Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page F 1 Appendix F Maintenance BEE PUMP HEADS Pump heads routinely accumulate small pieces of tubing debris during production runs This debris must be cleaned at the end of each production run Pump heads require two white teflon washers They wear out over time Replace them 1f they are worn or missing see Ordering Information in Appendix B Pump heads typically last for many years of service However if the metal roller shows signs of wear or doesn t revolve freely in the pump head replace the pump head see Ordering Information in Appendix B CLEANING Use a gentle detergent solution to clean the cabinet Do NOT spray liquid on the electrical connections and keypad and cabinet openings to prevent liquid from entering into the cabinet Clean any cell culture medium spill in the incubator Use caution to ensu
181. rol Screen The abbreviations used here are the same as the ones used on the Monitor screen page 5 3 To activate a Pump rate override use f to move the cursor until it is in the field with the desired pump rate value press the desired number keys the desired pump speed and press ENTER Overrides are noted by the display of an asterisk m To the left of the pump label as shown for F3 pump in figure 5 18 m To the right of the pump label as shown for F3 pump in figure 5 1 The Pumps LED does not turn on when PUMPS is pressed when no pump override is already active The LED turns on only when a Pump override speed is selected and ENTER is pressed The Pumps LED remains lit until all pump overrides have been cleared To clear a Pump override press PUMPS use f to move the cursor until it is on the desired asterisk not the pump rate value then press CLEAR The pump may stop or turn depending on whether Inoculate or Running is active and what their process control settings are at this time Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 5 19 User Interface EE a Gas Press GAS to display the following screen Figure 5 19 Gas Override Control Screen The Gas shown in this screen is a relative value and the current one in use to control pH It represents a variable mixture of air and CO being delivered to the GEX see chapter 6 pH Control When R
182. rol is to set the EC Cycling Delay to a future time to ensure it does not activate immediately after completing the inoculation procedure which could cause the inoculum to flow into the Expansion Chamber Figure 6 57 Instructions Screen 1 for Inoculation CAUTION EC Cycling typically should not activate until approximately two to three days after inoculation for most applications When using low or protein free medium in the ECS longer delays may provide better results by giving the culture more time to become established before the start of EC Cycling Note Perform the inoculation with minimum delays to prevent pH changing in the flowpath as a result of the stopped pumps 2 Press PROCESS CONTROL 3 Press ENTER until EC scl irre appears 4 Change Heilsa to i i Aa 5 Confirm the remaining Process Control settings are appropriate for a newly inoculated culture Refer to the settings on page 6 80 81 6 Press START then INOCULATE to return to the previous screen Fig 6 57 7 Press ENTER to display the following screen Figure 6 58 Instructions Screen 2 for Inoculation 8 EC pressure rises to lower Expansion Chamber volume to its low setting 108 ml The screen shows this excess volume being removed Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 83 Operating Procedures ey 9 When the excess volume has been removed the Outflow pump stops and the screen mre eo the
183. ry Circuit shown with red tubing Extracapillary Circuit shown with yellow ish tubing Biovest offers four flowpath options a single 1 5 m or 2 1 m bioreactor not shown in the figure above or double 1 5 m or 2 1 m bioreactors shown in the figure above See Appendix A for further specifications The circulation pump moves media through the IC while the cycling process causes media to flow through the EC and between the IC and EC Depending on which IC sample clamp is open IC medium flows through the DO probe from a site located upstream or downstream PRE or POST from the BRX Peristaltic pump tubing is made of PharMed which is a highly durable material that will last throughout each run The section of tubing that contains the DO probe and IC sampling port is made of medical grade Tygon to minimize errors in measuring DO P N 700037 000 Rev H Biovest International Inc Page 3 6 Operations Manual for the ACUSYST MAXIMIZER System Description oe ee An ultrasonic level sensor is attached under the Expansion Chamber to assist in the regulation of the cycling process by monitoring changes in fluid volume in the chamber The drop tube in the Integration Chamber is connected to the Outflow pump which runs continuously to remove excess media The silicone membrane oxygenator that serves as the primary pH control device Regardless of the gas mixture used for pH control the oxygenator provides near saturation levels of dissolv
184. ry Space The portion of the flowpath that includes the outside of the fibers in the bioreactors the Expansion Chamber and the associated tubing The space in the bioreactor outside of the hollow fibers where cells are grown part of the Expansion Circuit Can also be used to refer to the volume in the bioreactor where the cells are grown A supplement or addition to a growth medium This may be serum insulin transferrin or other components typically added to the ECS or ICS P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER G 1 Glossary Flowpath Gas Exchange Cartridge GEX Harvest Hollow Fibers IC ICS Integration Chamber Integration Circuit Intracapillary Space ICS LCD Medium Dilution MWCO Outflow P N 700037 000 Rev H G 2 All components of the EC and IC see Cultureware The cartridge in the cultureware which is responsible for the introduction of gases CO and O2 into the media Also known as an oxygenator See Gas Exchange Cartridge The process of collecting media containing cell product or The media which contains the cell product The thousands of porous hydrophilic tubes within a bioreactor They permit exchange of nutrients and wastes and serve as growth substrate for cultured cells See Integration Circuit See Intracapillary Space The media reservoir for the Integration Circuit The portion of the flowpath which includes the inside of the fibers
185. s ccc senene erreren 6 87 End of Culture Procedures cece ee ee eee e eee eees 6 88 Recovery OF anVGst lt 4 s 2cacacecence cuse eases EENEN A 6 89 Removal of Cultureware Components 000 cece eee teens 6 91 Optimization Procedures 0 ccc ccc ete cece ene eee eseeeecernes 6 94 IMOGCUIAUION 2 4 cauaveds bee deneeeee gaeaaecoessaakadeee oe suede sees 6 94 Circulation Rate saoeee anes coneeeese Tei eE E A ee eeae ss 6 97 EC CVGUNG erates oresaeeeeeeeeseneweeeatue se eeeeeeeeuce terrae een h 6 97 Harvesting lt 4 6 6cr4 wo Genera nen Gu ee SO Reo eee Om Nae ee ee eee oe 6 98 Serum Requirements 0 ccc eee nren rennan renner enne 6 99 Serum free Media uneneen eenaa 6 100 P N 700037 000 Rev H Biovest International Inc vi Operations Manual for the ACUSYST MAXIMIZER Table of Contents T Troubleshooting SOFTWARE sesede e e r EE AAEE RRAN 7 1 e EANN op oa baee ease ee oss eesaeaadaemeneeuaeaee EE 7 1 Diagnostics SCVECIIS 4 ance chew eee se SeGe GROSS SE 5550 AEN ee ee 4 7 18 Problems with Biological Performance ccc cee eee eee eee eee eees 7 23 OF CONUClageae oes sae eesaaeeaseeercnaeeeeneeheeeneees ee enaaasesas 7 23 Prod ct SCCICUON so odad c dcdawweweudadananddeceaegeenanee bananas oe es 7 24 CONTANMMALION geaue te s3e3e4 seen eae een eer ee eeaen ene ne weeee ners 7 25 Hardware and Cultureware ccc ereenn runnner 7 26 PUINDS sc2022 5 5000 oo eR Ad eee eee EEE e
186. s below its ie es m Base Addition is is disabled current as is the LO value and pH drops below its setpoint When Medium Dilution is activated the result is the mela l speed automatically increases from the 221 rate up to the Dilution rate to dilute Lactate an acid in the EAE in an attempt to E pH te to its setpoint Medium Dilution continues until pH reaches its setpoint and the current a2 gt the LG value coun Dilution then and the MP speed automatically deeras from the i lut iorn rate to the Hass 1 rate Only Gassing continues to be used to control oH If oH H drops below its eisai again the whole cascade of using Base Addition and Medium Dilution repeats as described above When Medium Dilution is disabled the MP speed remains at the F221 rate so no extra medium is used to dilute lactate Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 5 Operating Procedures 7 SS SS Oxygen Delivery Regardless of the gas mixture number used for pH control see above the GEX provides near saturation levels of dissolved oxygen DO in the medium before it reaches the BRX s As a culture matures cells will consume oxygen from the surrounding medium and produce a lower DO value downstream from the BRX s The cultureware design allows you to monitor the DO of medium either before or after it is pumped to the BRX s The difference between these values is used to calculate an
187. s in the BRX s Il Fresas ENTER te terminate dedbtial Flush means press ENTER to ip the A MP after the ouble fee saleco of at least 8 L 2 BRXs or 6L 1 BRX of flush medium into the container connected to the Harvest tubing line This step requires about 1 5 2 hours using default MP speed 12 Fress HOLL to rigiii means D N HOLD HA one of the following occurs m Flow route of medium is not according to the schematic Fig 6 41 m Cell culure media leaks from the cultureware m One of the following three Notes O i isa ig aa b Correct the problem then press 1 to bo guppreri screen not shown Notes 1 If the route of medium flow is different from what is illustrated or medium leaks from the cultureware press HOLD to stop the procedure temporarily After the problem has been resolved resume Fill Flush procedure by selecting pressing 1 to Continue current activity 2 During this phase of the Fill Flush procedure it is CRITICAL that the level of medium in the IC chamber does NOT continue to rise and fill the IC chamber If the medium continues to rise beyond 5 cm 2 of medium ensure that the IC vent clamp is completely closed If the problem continues there may be a blockage downstream of the IC Chamber If you cannot resolve this press the HOLD key and contact Biovest 3 During this phase of the Fill Flush procedure the EC chamber receives NO Fill Flush medium and should remain DRY If Fill Flush medium is Bi
188. se 600100 072 m vee AH CS feet 1 8 M B Remove A Tubing Extension Line _ Remove B Connect this end to cultureware Connect LAV or syringe to this end c In line media filters minimize the risk of a contaminant entering the flowpath and typically are used for MEDIA and F2 because these deliver nutrient fluids into the flowpath Use the in line filter listed in Appendix B not shown at the blue circled location or add your own filter assembly at the end of an individual tubing line S 2 fi Remove male luer sterile port covers Add LAVs and or tubing extension lines to these tubing lines as listed in a and b above 6 The flowpath is now completely assembled and ready to be installed in the MAXIMIZER incubator Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 41 Operating Procedures E Cultureware Installation To install the cultureware you will need 3 Tygon gassing lines red green and blue Luers pH probe cable DO probe cable and temperature probe that were supplied with the MAXIMIZER The black collared Tygon gassing line for off gassing may be needed see Pre inoculation Procedures 1 Connect pH and DO cables to their respective probes Save the electrical caps for future autoclave cycles and for probe storage 2 Connect the tempe
189. shed among the bundle of hollow fibers When this strategy is followed increase the transfer volume in step wise amounts of 15 35 ml every 2 3 days until the maximum suggested transfer volume of 70 ml is in use unless metabolic activity worsens In such cases increase the transfer volume more slowly Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 99 Operating Procedures E Harvesting m For secreted products that show feedback inhibition the user should determine the threshold level to monitor before starting the harvest pump at a time different from the default value Samples analyzed from the flowpath will provide the necessary information in this case m After the harvest pump has been started product will be collected and measured for content Depending on the type of product secreted by the cells this measurement may be in units of concentration or activity The harvest rate may be gradually increased until the product levels reach a satisfactory concentration activity or until the daily yield reaches a maximum amount or until the harvest volume becomes difficult to process The previous discussion on feedback inhibition should also be considered when pump rates are changed mE Acomparison of secreted product from the harvest bottle and the sample port will undoubtedly show different levels concentration or activity of harvested material This happens because the harvest
190. ss several screens to enter or review the settings that define how the MAXIMIZER operates when in 1 2 mode The various screens include information on pH control pump rates and pump schedules alarm limits temperature setpoint and EC Cycling parameters These parameters have specific default values but they may be changed at any time during a culture run Pressing PROCESS CONTROL displays whichever of the series of the Process Control screens was last viewed This also causes the Process Control LED in the gray area of the key to turn on Repetitively press ENTER to scroll through the series of Process Control screens To exit from any of these screens press MONITOR Chapter 6 provides a detailed explanation of pH control oxygenation and basic strategy implemented in a culture run P N 700037 000 Rev H Biovest International Inc Page 5 8 Operations Manual for the ACUSYST MAXIMIZER User Interface _ _ _ _ _ EEEee ey pH Setpoint and Gas Numbers The screen below shows the current sH begini that process control will attempt to maintain This setpoint ideally is specific to the mid log phase of growth that is specific to the cell line being actively cultured This value may be changed anytime during the active culture run Acceptable values are in the range of 6 00 8 00 pH units in increments of 0 01 units Figure 5 6 pH and Gas Number Screen This screen also allows you to limit the Lf
191. st This program applies air pressure at the EC and IC bulkhead ports and anything connected to them such as pH or DO probe assemblies or the entire cultureware then measures any detectable reduction as a sign of leakage The test is performed several times per culture run to confirm the MAXIMIZER instrument probe assemblies and cultureware are leak free Fill Flush Test This program is used to fill the cultureware and rinse the wetting agent from the hollow fibers in the bioreactor cartridge in preparation for the pre inoculation phase CAUTION During an active culture run the Monitor screen shows HLHHIHGS as the run mode and the LED for Active Process Control is turned on During an active run do NOT press START then SET UP because this will stop the active run and display the screen shown in figure 5 5 However if this is accidentally done press START then PROCESS CONTROL to restart the active run Process Control settings will be Peraga if the backup battery is still good but elapsed time will reset to 22 22 which would affect process control parameters that include a delay time feature Press PROCESS CONTROL and review all screens to ensure their settings return the instrument s operation to what it was prior to accidentally deactivating the run Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 5 7 User Interface EEN PROCESS CONTROL Key Press PROCESS CONTROL to acce
192. t fails contact Biovest s Account Services before proceeding with the run a Press 1 b LCD displays a 4x40 pattern of rectangles b All pixels of each rectangle should be black and easy to see c Cursor flashes in the lower right corner d If all pixels are black the test passed e Press ENTER Figure 6 5 LCD Test Screen a Press 2 b LCD displays AlarM Test b Audible alarm turns on for several seconds c Audible alarm turns off d If the audible alarm makes noise the test passed This is the sound that occurs whenever an alarm happens during a run e Press ENTER Figure 6 6 Alarm Test Screen The volume of the audible alarm can be adjusted somewhat by turning its plastic cover to open or close the openings built into the cover Repeat the Alarm Test by pressing 2 as often as necessary to allow you to adjust this volume The audible alarm is mounted at the rear of the instrument Refer to Figure 3 3 for further details P N 700037 000 Rev H Biovest International Inc Page 6 18 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures c If all 13 red LEDs turn on the test passed d Press ENTER Figure 6 7 LED Test Screen 43 keema Test a Press 4 b LCD displays Kesrad Testi c Cursor flashes to gt the then of this prompt d Sequentially press 1 0 and the decimal key e LCD updates to show each number and decimal at the top of the screen and in the lower right hand corner f S
193. t is in good working order Note The air filter must be attached to the rear of the MAXIMIZER see Figure 3 3 at this time pH pO2 TEMP Figure 6 11 Bulkhead preparation for internal system pressure test 1 Use the reusable black collared Tygon gassing line to interconnect the IC and EC bulkhead ports inside the incubator Figure 6 11 This seals the internal gassing system from leaks through the IC and EC ports 2 Press START then SET UP The LCD displays the following screen Figure 6 12 Active Setup Menu Screen 3 Press 1 to start the Pressurization Test 4 Press ENTER to begin the test 5 Instructions for testing a cultureware are displayed in a series of five screens These screens instuctions are not relevant for pressure testing the instrument itself Ignore the instructions to open or close various clamps in these screens P N 700037 000 Rev H Biovest International Inc Page 6 22 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures SSS 6 Repetitively press ENTER to advance past these five screens until the top screen of Figure 6 13 appears 7 The LCD updates to show the three automated stages of the Pressurization Test PRESSURTSTHO STABILIZE and TESTING Figure 6 13 Pressurization Test Duty Screens 8 The Pressurization Test automatically finishes within 90 seconds m If there was no leak the test passes see top screen of Figure 6 14 Go to step 14 m If there was a
194. ter online or selected Printer paper empty Another reason the printer is offline Printer cable fully connected to the printer and MAXIMIZER If the alarm persists call Account Services Figure 7 17 Alarm Screen 23 This alarm means the printer is unable to receive information while it is in the process of printing Verify the following possible causes Printer power on Printer online or selected Printer paper empty Another reason the printer is offline Printer cable fully connected to the printer and MAXIMIZER Printing head jammed or cannot move on its track If the alarm persists call Account Services P N 700037 000 Rev H Biovest International Inc Page 7 12 Operations Manual for the ACUSYST MAXIMIZER Troubleshooting Figure 7 18 Alarm Screen 24 This alarm means the calibration value entered data2 was zero or the sampled A D count datal equaled zero Ensure DO calibration is properly performed Figure 7 19 Alarm Screen 25 This alarm means the gas mixture number 1 14 is outside of the gas limits set in Process Control Verify the following items to try to correct the problem m pH probe measuring accurately If so you may have to change the gas limit setting in Process Control m CO gas supply properly set to deliver 151 psi m Gas override active and setting the gas number outside the range m Calibration of the pH probe fixes problem m Calibration procedure being properly perform
195. ternational Inc Page 6 48 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures SSS 12 When the cultureware Pressurization Test passes press ENTER to display the following screen Figure 6 34 Final Pressuirzation Test Screen 13 Open the GEX OUT clamp 14 Press ENTER to return to the Active Setup Menu screen Figure 6 27 15 After the cultureware passes the pressurization test proceed with the Fill Flush procedure CAUTION Do not proceed to Fill Flush unless the culureware passes the Pressurization Test Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 49 Operating Procedures Fill Flush Procedure After the flowpath has been installed and tested for leaks the cultureware can be primed with sterile medium Notes 1 This procedure requires sterile medium and a waste collection bottle large enough to contain this medium A single BRX cultureware 600058 xxx Should be flushed by collecting at least 6 L A double BRX cultureware 600068 xxx should be flushed by collecting at least 8 L When this procedure is completed you will also need sterile medium and factor solutions as well as one sterile waste bottle to begin the Pre inoculation phase of cultureware setup 2 This procedure operates only with the medium pump head loaded Do NOT load any other pump heads until the procedure is completed and the onscreen instructions tel
196. that is taped to the tray and between the two clear chambers Save this for use with the DO probe case assembly When using the reusable pH probe remove the autoclaved packaging materials and insert the probe case into the right hand black clip on the front of the cultureware The tubing lines connected to the probe case should point to the left and right Go to step 3 When using the disposable pH probe disinfect the e outside of its sterile bag and place it in Reusable pH Probe Holder the laminar flow hood S e reusable pH probe holder and place it in the laminar flow hood S Grooves for Groovetaor Insert the reusable pH probe holder into the DO probe s eee right hand black clip on the front of the PAGE CUIS black clip cultureware illustrated at right Ensure the N small black clip on the reusable pH probe holder is facing forward and positioned below the round section held by the cultureware s clip Disposable pH Probe Latch Remove and discard the packaging for the Assembly disposable pH probe assembly Locking Ring Press the disposable pH probe case into the reusable pH probe holder s black clip and position it as shown at right Biovest International Inc Operations Manual for the ACUSYST MAXIMIZER Operating Procedures 3 Connect the cultureware tubing to the pH probe assembly These steps apply to both the disposable and reusable pH probe assemblies a The pH probe assembly replaces the cultur
197. the default parameters for cycling see below pH Calibration During the pre inoculation phase the pH probe should be calibrated at least once to enable the system to establish the desired pH setpoint If calibration is not performed the software does not have an accurate basis for reading pH from the probe and media components could be adversely affected by extreme pH created by an incorrectly monitored probe Calibration at this time may also be helpful in detecting a probe that did not survive the autoclave conditions A replacement can therefore be installed before the culture begins Details on the calibration procedure are given in Maintenance Procedures DO Calibration Although the system does not control dissolved oxygen levels in the media calibrating the DO probe during the pre inoculation phase permits the software to monitor DO readings accurately from the probe This may prove helpful in detecting a probe that did not survive the autoclave conditions A replacement can therefore be installed before the culture begins Details on the calibration procedure are given in Maintenance Procedures EC Cycling The conditions necessary to create EC cycling should be tested in the pre inoculation phase To do this you will have to change the default delay time in Process Control to zero To measure the actual Rise and Fall times set the printer log time to a convenient interval using SETUP not Process Control Biovest Internation
198. the BRX s and the Expansion Chamber If you are using the double BRX flowpath be sure that both BRXs are cycling properly but confirming there is no stationary air bubble in either of the two tubing lines that connect to the lower EC side ports Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 79 Operating Procedures SSS aaa Process Control Summary during Pre Inoculation Phase Single Bioreactor Flowpath 600058 xxx for either bioreactor size pH setpoint 7 2 7 1 7 3 or the value determined from the cell line characterization Temp setpoint 37 0 C Alarms pH Low 7 10 High 7 50 DO Low 100 High 200 mmHg Temp Low 36 5 High 37 5 a Circulation pump 250 500 ml min Media pump Basal 25 75 ml hr Dilution 0 ml hr Base F1 Disabled EC Cycling Delay 0 00 00 DDD HH MM Up 15 minutes Dn 15 minutes Transfer Vol 70 ml Serum Control F3 Rate 0 ml hr Delay 0 00 00 DDD HH MM ON 0 00 00 DDD HH MM Cycle 0 00 00 DDD HH MM F2 Control F2 Rate 0 ml hr Delay 0 00 00 DDD HH MM ON 0 00 00 DDD HH MM Cycle 0 00 00 DDD HH MM Harvest None option 4 P N 700037 000 Rev H Biovest International Inc Page 6 80 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures SS Double Bioreactor Flowpath 600068 xxx for either bioreactor size pH setpoint 7 2 1 1 7 3 or the value determined from the cell line characterization Temp setpoint 37 0 C Alarms pH Low 7 10 High 7
199. tic changes in productivity suggest a significant change has occurred in some aspect of process control Changing the harvest rate or serum concentration too severely are common sources of this type of problem Replacing serum supplemented medium with serum free or similar medium too suddenly is another m Changes in medium or serum lots might affect productivity due to differences in quality You may need to examine the medium preparation process and or quality control procedures m Changes in productivity may be seen immediately after cells are removed from the Expansion Chamber The protocol used in this procedure may have to be altered m Harvest strategy may have to be changed to accommodate the nature of the cells production process For example batch harvesting may need to be compared to continuous harvesting In addition some metabolic parameter pH DO may need to be changed to enhance production P N 700037 000 Rev H Biovest International Inc Page 7 24 Operations Manual for the ACUSYST MAXIMIZER Troubleshooting SSS Contamination Sources of contamination Termination of a culture run due to contamination 1s always a humbling experience It is important to recognize the most likely source s of such a problem because it they must be avoided in future runs Sometimes diagnosing the cause of contamination is simplified by observing whether the problem 1s restricted to the ICS or ECS loop Refer to any records of autocl
200. tic pump heads 5 The sixth screen tells you to attach the Harvest line aseptically to the collection bottle This bottle should be large enough to contain the medium intended for flushing When this is done disconnect the IC tubing line from its bulkhead connection This step acts as a safety measure against medium leaking through the vent filter and flowing inside the instrument Figure 6 38 Instructions Screen 6 for Fill Flush P N 700037 000 Rev H Biovest International Inc Page 6 52 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures ee 6 The next screen tells you to close the two EC Check Valve Bypass clamps After that open the Medium and Harvest line clamps for the inlet and outlet lines during the Fill Flush procedure Figure 6 39 Instructions Screen 7 for Fill Flush 7 The next screen tells you to inspect the flowpath before the Medium pump is started Carefully follow the tubing route from the Medium bottle to the Harvest bottle When the route meets another piece of tubing be certain that the appropriate clamps are shut to force medium to flow in only one direction Verify that closed clamps are centered over their tubing and completely occlude the tubing Be certain that tubing is not pinched between the cultureware tray and the incubator walls Figure 6 40 Instructions Screen 8 for Fill Flush Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 53 O
201. ture Procedures before using this feature Note If cell buildup is excessive completely draining the EC Chamber using the EC Cycle override may not work Any medium remaining in the EC Chamber may be removed by using the Cell Removal line even though product collection is unnecessary The Cycle override item 4 activates the EC Cycling Rise Fall process using the parameters currently set This allows EC Cycling to function even during the Idle Inoculate or Setup mode when EC Cycling is normally disabled In these modes EC Cycling occurs using the default parameters 70 ml 15 minute Rise 15 minute Fall If Running mode is active the Cycle override uses these same default parameters when the Elapsed Time is less than the EC Cycling Delay time Otherwise the Process Control parameters are used when the Elapsed Time is greater than the EC Cycling Delay time P N 700037 000 Rev H Biovest International Inc Page 5 22 Operations Manual for the ACUSYST MAXIMIZER User Interface _ _ _ _ _ _ EEE EEoxooe ay Temperature Press TEMPERATURE to display the following screen Figure 5 21 Temperature Override Control Screen The temperature below Current is the current temperature that is measured from the temperature probe that is installed in a fitting in the cultureware in the incubator The temperature below Set Point is the current control value in effect This
202. ty four hours before the inoculation Because Biovest International s standard cultureware uses BRXs with a MWCO of 60 000 daltons media components with molecular weights lower than this may or may or may not freely exchange between the ECS and ICS An important consideration in the pre treatment procedure is whether the components in the growth supplemented EC media are smaller than 60 000 daltons If they are it is possible they may pass to the IC This dialysis can cause no or slow growth of the inoculum in which case the IC medium also can be supplemented The volume of serum required for pre treatment depends on the concentration of serum used routinely in static culture and it depends on the cultureware volume which varies by bioreactor size and the number of bioreactor in the cultureware Refer to Appendix A for system and ECS volumes in MAXIMIZER cultureware Notes 1 For other types of ECS pre treatment contact Account Services 2 The serum pre treatment protocol outlined here is optional and may be replaced with other pre treatment procedures depending on the type of medium and or cells you are using Consult Account Services if you have any questions Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 77 Operating Procedures ee To perform the pre treatment procedure follow the steps outlined below Refer to the procedures described in Sampling 1 Aseptically
203. uggest a a Circulation Rate of 250 ml min for 1 BRX or 2 BRX cultureware during first few hours of pending Pre Inoculation Phase CAUTION Do NOT exceed 500 ml min Circulation Rate when using cultureware with 1 BRX at any time Figure 6 49 Instructions Screen 1 for Pre inoculation Phase Notes In addition to entering the desired pH setpoint calibrate pH approximately 30 minutes after starting the Pre inoculation phase step 24 below when the on line pH will have reasonably equilibrated This ensures accurate pH control Calibration is described below in Maintenance Procedures 21 If ACULINK DATA LOG or a printer is connected enable them and enter the desired Logging Interval by pressing a SET UP b ENTER if necessary to display Loesing Inberual c Number keys for the desired logging frequency d ENTER Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 59 Operating Procedures a 21 When the changes to Process Control and Logging Interval are complete press START then SET UP to return to the screen shown in Figure 6 49 22 Press ENTER to advance to the next screen which 1s provided as a safety feature If you have performed the Fill Flush procedure with the incubator door open close the door to activate the incubator heater Figure 6 50 Instructions Screen 2 for Pre inoculation Phase 23 Press ENTER to proceed to the last screen in the Fill Flush procedure Figure 6 51
204. unning or Inoculate is active and the Gas 1s not overriden no asterisk is displayed the Gas automatically changes with the demands of the pH control strategy and set point Manually overriding the Gas results in disabling automatic pH control The gas mixture remains constant because the Gas remains constant To override the Gas press GAS use the number keys to enter a number from 1 14 and press ENTER An asterisk appears as shown above and the Gas LED turns on to indicate a gas override is active Now even if automatic pH control wants to vary the Gas to maintain pH at the desired setpoint the Gas will not change Why control pH manually via a gas override If the pH probe in the cultureware has malfunctioned mid run automatic pH control will not work Rather than aseptically replacing the probe enter a gas override wait 20 minutes asep tically sample IC medium assay pH off line and adjust the Gas up or down if necessary to achieve the desired pH Repeat these steps every few days If a gas override 1s a number that 1s outside of the range set in Process Control an alarm occurs but this gas override is allowed After the override is cleared the gas returns to the range set in Process Control Notes 1 Using gas overrides inhibits pH control and generally should not be used long term if the pH probe is properly functioning 2 The Monitor screen does not indicate a gas override is active You must r
205. urement of the sample Contact Account Services for details 2 Ifa DO probe is in the cultureware but there is no offline equipment to measure DO with the PRE BRX DO clamp open calibrate DO to 140 mmHg weekly After the calibration is complete close the PRE BRX DO clamp and open the POST BRX DO clamp It is more important to monitor post BRX DO When it drops below 100 mmHg increase the speed of the CP by 100 ml min or more unless it is at the maximum of 500 ml min single BRX or 1000 ml min double BRX Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 67 Operating Procedures 7 SS SS Replacing Bottles The attachment of medium outflow factor and harvest bottles is a necessary pre inoculation and maintenance procedure Use this procedure any time you have to connect bottles to the system Use sterile gloves and aseptic technique during bottle replacement Work smoothly and quickly Procedure 1 Prepare the appropriate bottle with RESCuU cap assembly to exchange with the bottle presently attached to the system 2 Cover the connection between the media line and the cap assembly or sterile protection of the presently used bottle with sterile gauze saturated with disinfectant 3 Sterilize the connection by soaking it for several minutes 4 Meanwhile cover the appropriate connecting port see below of the new bottle with sterile gauze saturated with disinfectant
206. ware Sterile Assembly see on page 6 37 e remove the temporary clamps f drain the probe case assemblies g remove both probe case assemblies and set them aside h coil the long tubing lines and place them on top of the culutreware 1 seal the biohazard bag and dispose of it as a biohazard 15 Clean the pump heads They likely will have small pieces of pump tubing in their grooves and other places They also may have media spills in them 16 Each pump head requires two white teflon washers Replacements must be installed before the next run See Appendix B Ordering Information 17 Once the pump heads are clean and reassembled store them or reattach them with the mounting screws 18 Reconnect to the bulkhead jacks the reusable pH cable the DO cable if in use the temperature probe and the four gassing lines 19 Clean the reusable pH probe and the DO probe case assemblies a remove the probes from their cases b do not touch the tip of the DO probe c briefly rinse the probes with water d put the pH probe in its storage bottle with fresh storage solution see Appendix B e screw the electrical cap on the pH probe f put the protective cap on the tip of the DO probe and screw on its electrical cap g disassemble the probe cases h discard the o rings 3 for pH 1 for DO cultureware includes new ones Biovest International Inc P N 700037 000 Rev H Operations Manual for the ACUSYST MAXIMIZER Page 6 93 Ope
207. was successful see page 6 21 If that test fails the Probe Case Pressurization Tests will fail 2 Remove the sterility protector small tubing with white plug to expose the female Luer fitting from both probe case tubing lines of the DO and pH probe assemblies 3 Connect the reusable IC and EC gassing lines supplied with the instrument to the IC and EC bulkhead ports inside the incubator Figure 6 21 O O pH pO2 TEMP probe case Figure 6 21 Bulkhead Attachments for Probe Case Pressurization 4 Connect the male Luer fittings of the two gassing lines to the female Luer fittings of the probe case tubing lines from step 2 Press START then SET UP Press 1 to start the Pressurization Test Press ENTER to begin the test oe Se Instructions for testing a cultureware are displayed in a series of five screens These screens instuctions are not relevant for pressure testing the probe case assemblies Ignore the instructions to open or close various clamps in these screens 9 Repetitively press ENTER to advance past these five screens P N 700037 000 Rev H Biovest International Inc Page 6 32 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures rr eee 10 The LCD updates to show the three automated stages of the Pressurization Test FRESSURTZINMG STAHEILIZE and TESTING Figure 6 22 Pressurization Test Duty Screens 11 The Pressurization Test automatically finishes within 90 seconds
208. with phosphate buffered saline or bicarbonate buffered saline solution until the liquid and electrode are in contact Be certain the sterility protectors tubing with white plugs cover the ends of the probe case tubing Loosen the probe cases turn to allow for thermal expansion in the autoclave Be certain the electrical connection at the top of each probe is covered with its screw cap Put the probe case assemblies in the autoclave so the tubing is facing upward at an angle and is not pinched shut Use validated sterility indicators Run autoclave at 121 C 55 minutes slow liquid cycle exhaust Carefully transfer the sterile probe case assemblies to a laminar flow hood If liquid has accumulated inside the probe case tubing allow it to drain into the probe case before attaching the assembly to the flowpath module Allow the probe case assemblies to cool to room temperature in a laminar flow hood Re tighten the probe case bodies by hand after they have cooled Note The sterility protectors contain porous white plugs that cannot survive more than one autoclave cycle If more than one sterilization cycle must be performed the sterility protectors must be replaced or the ends of the tubing must be covered Contact Account Services for details P N 700037 000 Rev H Biovest International Inc Page 6 36 Operations Manual for the ACUSYST MAXIMIZER Operating Procedures rr ee Cultureware Sterile Assembly After the probe case assembli

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