700446-000C HF Primer™ User Manual
Contents
1. Remove the HF Primer from its sealed package and discard the white foam wrapping and blue tape Set the coil of tubing on either side of the rack Mount the clear plastic clip that holds the hollow fiber bioreactor and gas exchange cartridge on to the stainless steel rack as shown in Figure 7 Set the media reservoir on either the left or right hand side of the rack 00 OU O ao Ensure all seven plastic pinch clamps are open Contact Biovest s Account Services department if a clamp is closed Media Reservoir s Media Clamp Media Reservoir s IC Return Clamp Harvest Clamp IC Bioreactor BRX Out pO Clamp Post Bioreactor Sample Port Clamp EC Factor 1 Clamp Fill Flush Clamp O Ensure all Luer fittings are not loose Do not over tighten them They can be difficult to open when necessary Bottle Cap In Port Bottle Cap Out Port Pre Bioreactor Port UVOVDODOU Post Bioreactor Port Vent Filter attached to the bottle cap Figure 7 Lower GEX Port Do not over tighten to prevent cracking this Slide the clip s lower edge behind the metal bar here OO000O0 polycarbonate port 1 Aseptically replace the sterility protectors male Luer fitting with the short piece of tubing and the white plug inside this tubing from the Post Bioreactor and the Pre Bioreactor sample ports with the provided sterile Luer plugs O The HF Primer is ready for the Fill amp Flush procedure HF Primer User Manual Pag2. HF Primer User Manual Innovation Leader Since 1981 Biovest International Copyright Notice O 2009 Biovest International Inc All rights reserved No part of this document may be reproduced in any form without the prior written consent of Biovest International Inc Biovest International Inc 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 Inc assumes no responsibility for any errors that may appear in this document Trademark Acknowledgements HF Primer is a trademark of Biovest International Inc Masterflex and Easy Load are a registered trademark of Cole Parmer Instrument Company Luer and Luer Lok are trademarks of Becton Dickinson and Co Tygon is a registered trademark of Norton Performance Plastics PharMed is a registered trademark of Saint Gobain BD Cell MAb Media is a trademark of Becton Dickinson Inc Document Number 700446 000 Revision A Initial release Revision B Update instructions Revision C Update sample volume instructions WARNING Operating the HF Primer requires the use of pump motors and pump heads It is important to refer to the user manuals provided by the manufacturer of the pump motor and pump head to ensure they are used in a safe and proper manner CAUTION Wherever a caution statem
3. may not correlate to when the product is at its highest concentration Page 28 of 36 HF Primer User Manual Biovest International Procedure for Harvesting amp Adding Complete Media CAUTION Harvesting and adding complete media must always be done aseptically However it is VOU JUUUODOOU possible to perform this procedure while the HF Primer remains in the CO incubator To reduce the risk of contamination this procedure should be done within a laminar flow hood to minimize the chance of contamination go to the next step If sampling is done when the HF Primer remains in the CO incubator the circulation and gassing pumps can continue to operate Contact Biovest s Account Services for further information Turn off the circulation and gassing pump motor s and unload the pump heads Close the Media and IC Return clamps This prevents loss of fluid from the bioreactor Disinfect and place the HF Primer in the laminar flow hood Review the schematic in Figure 5 in the Extracapillary Circuit EC section of the manual Disinfect the Pre Bioreactor Sample Port and Post Bioreactor Sample Port Attach a syringe containing 20 mL of pre warmed fresh complete medium to the Pre Bioreactor Sample Port Attach an empty 20 mL syringe to the Post Bioreactor Sample Port Open the EC Factor 1 Harvest and Post Bioreactor Sample Port clamps Inject complete medium into the Pre Bioreactor Sample Port at the same rate as removing
4. a general protocol that works with your similar but new cell lines even if it s not ideal for any one line specifically When a cell line is cultured several times then it is possible to Following are areas to consider if optimization is pertinent to your application It may be helpful to discuss your goals and cell lines with Biovest s Account Services for assistance with optimization See the Cell Line Characterization worksheet at the end of this user manual Optimization Tips O Consistently passage the T flask roller bottle spinner scale up culture to maintain mid log phase growth Inconsistent scale up conditions can lead to long term differences in the HF Primer culture Determine the conditions that correspond to mid log phase growth in static culture and use this information in developing the culturing strategy for the growth phase of the HF Primer production pH Concentration of glucose and or lactate and other metabolites depending on cell line Use the pH and metabolite information determined above for the HF Primer culture Add or change the basal medium in the Media Reservoir to maintain this pH glucose concentration lactate concentration etc Maintaining these parameters during the growth phase of the HF Primer culture shorten post inoculation lag phase and minimize time until the bioreactor becomes confluent Once the HF Primer bioreactor is confluent continued growth isn t the main goal A
5. cell culture The Pre BRX Sample Port connects to either the IC Circuit or the EC Circuit depending on how the clamps near this port are set but it is intended to primarily connect to the EC Circuit When the EC FACTOR 1 clamp is open and the Fill Flush clamp is closed the Pre BRX Sample Port connects to the EC Factor 1 bioreactor port Note some applications use protein free medium which means the IC and EC media are identical Because there are no large molecular weight additives in protein free media secreted protein is the only protein present in the bioreactor IC BRX Media IC Return OUT PO Post BRX Clamp Clamp Clamp Clamp Harvest Sample Supernatant This sample location is primarily used to harvest supernatant and at the beginning of the run to inject the inoculum It also may be used to periodically collect small volumes for product analysis Harvest O Cimo Harvest AV CAC Y 0 00 Circulation 7 The Post BRX Sample Pump oH l Port onih Fill Flush EC Factor Complete Media ort connects to either eine Clamp Clamp Addition the IC Circuit or the Pump Gas Exchange EC Circuit depending Cartridge on how the clamps near this port are set Figure 6 When the Harvest clamp is open and the IC BRX OUT pO clamp is closed the Post BRX Sample Port is used to harvest supernatant Page 10 of 36 HF Primer User Manual re Biovest International Equipment and Supplies Required Equipme
6. pump s to drive medium circulation at 300 mL min 375 rpm The gassing pump head also will operate at 375 rpm Allow the HF Primer s pH and temperature to equilibrate which will take longer if the medium in the media reservoir was not pre warmed UC Proceed to the Inoculation procedure only after pH and temperature are stable HF Primer User Manual Page 19 of 36 Biovest International Inoculation Inoculation is the result of scaling up cells using conventional cell culture methods Passaging of the flask culture during scale up should be performed consistently when the culture is in mid log phase growth Maintain this passaging routine for several passages before beginning the inoculation procedure below This passaging routine generally achieves maximum cell viability Care should be taken to ensure maximum viability of the inoculum Poor viability at the time of 2 inoculation can lead to extended lag phase and continue to have a long term impact on the hollow fiber culture Prepare 2 x 108 viable cells The inoculum culture should have been maintained in mid log phase of growth for several passages and have a viability greater than 90 If a significantly higher number of cells are inoculated a larger initial volume of basal medium in the media reservoir may be beneficial Perform the following steps in a laminar flow hood and follow standard procedures for disinfecting items entering the hood and handling
7. recorded at values recorded at maximum maximum growth rate product concentration Lactate conc mg dL TD 1 Page 32 of 36 HF Primerm User Manual Biovest International Cell Line Characterization Data Sheet Cell Line Product Date Sample Time A H ur lot Cell Count Percent Total Density Glucose Lactate Product p P live dead Viability x10 cells mL mg dL Concen Comments Control Flask HF Primer User Manual Page 33 of 36 Production Batch Biovest International HF Primer Production Metabolic Data Record Cell Line Product o O N OO A A W N D D BB MB NN NO D a ES st st a 1 a O oa A wo N O O O N O oOo A O N oO Date Time Pre Brx pH Post Brx DO Metabolites Metabolic Activity Gle Lac mg dL mg d GUR L mg hr LPR mg hr IC Media Volume Added EC Media Volume Added Harvest Volume Prod Conc Comments Inoc via cell Page 34 of 36 Page HF Primer User Manual Biovest International HF Primer Production Metabolic Data Record Production Batch Cell Line Product Metabolites Metabolic Activity IC EC Media Media Gle Lac GUR LPR Volume Volume Harvest Prod Comments mg dL jmg dL mg hr mg hr Added Added Volume Conc Day Pre Brx Post N Date Time pH Brx DO Page of HF Primer
8. somewhat lower pH lower glucose concentration higher lactate etc discourage continued culture expansion and result in greater protein secretion Media Reservoir changes occur when the media is somewhat more spent than during the growth phase A Cell Line Characterization protocol is provided to highlight this worthwhile effort O Evaluate several basal media Remember that RPMI is generally not a good cell culture medium for use in the HF Primer Troubleshooting Points O Low pH In static culture allow a flask to go into the death phase Determine PH of the medium in this flask just prior to the decline in viability Do not let the medium in HF Primer to decrease to this pH Use a larger medium bottle or change the medium more frequently When the bioreactor is full of culture often the culture is metabolizing high amounts of glucose into lactate The resulting lactate concentration an acid source drives pH down The CO concentration in the incubator also is an acid source driving pH lower Reduce the incubator s CO concentration in steps until the pH of the medium in the HF Primer s Media Reservoir rises to the desired value Page 30 of 36 HF Primer User Manual Biovest International If reducing the incubator s CO concentration isn t possible adjust the concentration of Sodium bicarbonate in the basal medium to achieve the desired pH using the following formula pH 6 38 log 30 53 N
9. visually determining pH is sufficient but perhaps not ideal Glucose can be measured using diabetic test strips See the section Basal Media Changes for further information or contact Biovest s Account Services for further information O Ifrun to run consistency or optimal production yield is desired monitoring pH and metabolites should be performed O Monitoring dissolved oxygen concentration is optional and rarely necessary However DO data may be collected from the same sample of medium used to assay pH and metabolites when these data need to be monitored Procedure Calibrate the pH meter and DO meter first then proceed with removing the sample for analysis This sequence minimizes 1 the time between withdrawing the sample and measuring its pH and or DO and 2 the drift of the sample s pH and or dissolved oxygen concentration Measure the sample s pH and DO first as they can change rapidly Then measure the sample s metabolite concentration s NOTE Sampling must always be done aseptically However it is possible to perform this procedure while the HF Primer remains in the CO incubator To reduce the risk of contamination sampling the HF Primer should be done within a laminar flow hood to minimize the chance of contamination go to the next step If sampling is done when the HF Primer remains in the CO incubator the circulation and gassing pumps can continue to operate Contact Biovest s Account Services for f
10. User Manual Page 35 of 36 o a o A te oe Biovest International Biovest is the leader in large scale perfusion technology We provide a range of hollow fiber systems for a variety of production needs Fete E RATIO S Perfusion Done Right FDA Accepted Technology Linear Scale up Grams to Kilograms Suspension and Adherent Cell Lines Pioneered Single Use Disposables in the 1980 s High Density Perfusion Culture Page 36 of 36 HF Primerm User Manual
11. aHCO3 CO2 Example when cell culture medium contains NaHCO at3 g L and the CO incubator is set for 5 CO pH would be approximately 7 64 This calculation does not take into account the culture s metabolism Remember that the cell culture produces variable amounts of lactate an acid source which will reduce pH in the medium At the start of the run lactate production is low and increases Adjusting the Sodium bicarbonate concentration for high lactate production rates may be necessary Option add additional buffers such as HEPES up to 15 mM Note mixing pH buffers can complicate pH control O Poor Growth Long Lag Phase Some cell lines are sensitive to the concentration of certain uncharacterized low molecular weight components in serum These components are not formulated in the basal medium Therefore these components dialyze away from the ECS and their concentration becomes limiting to the cell culture This results in very slow initial growth or culture death To prevent this problem the simple solution is to add serum to the IC circuit the Media Reservoir before inoculation at the same concentration as the EC In these cases supplementation of the IC circuit is often only necessary for the first ten to fourteen days After that time use just basal medium when changing media in the Media Reservoir Increase the cell number in the inoculum from 2 x 108 up to 5 x 108 viable cells Small inoculum volumes lead to high inoc
12. actor 1 Harvest and Post BRX Sample Port clamps Inject the complete medium while pulling the displaced medium into the empty syringe at the same Close all clamps except the Media and IC Return clamps Replace the filled syringe with a new empty 60 mL syringe Discard the filled syringe when it is the first one filled after the flushing procedure Otherwise save and handle the filled syringe in an appropriate manner because it contains usable harvest supernatant o Page 18 of 36 The HF Primer is ready for the next procedure placement into the CO incubator HF Primer User Manual Biovest International Placing the HF Primer into the CO incubator The HF Primer operates within the incubator for the cell culture medium Periodically the HF Primer the same reasons as flask cultures The incubator will be returned to the laminar flow hood to provides the HF Primer with temperature control replenish the media reservoir with fresh basal Additionally the incubator s air CO gas mixture medium inject fresh complete medium in the ECS maintains pH control and provides oxygenation of and withdraw harvest supernatant Position the HF Primer the rack with bioreactor clip and media reservoir inside the incubator Route the pump tubing out of the incubator and secure it to prevent it from being pinched when incubator door is closed Ensure that the pump is as close to the incubator as possible to prevent condensation 1 P
13. ajor components of the Intracapillary Circuit are Media Reservoir The Media Reservoir contains up to 2L of basal cell culture medium meaning medium that is not supplemented with growth factors such as FBS or other high molecular weight factors The culture consumes the nutrients in this feed medium so it is periodically replaced A suggested replacement frequency is provided as a guideline Optionally you may alter the media change frequency to what best suits your cell line This frequency controls nutrient and metabolic waste concentrations More frequent changes stimulates growth Less frequent changes stimulate production which is done once the bioreactor is confluent Circulation Pump The Circulation Pump provides a high flow rate of medium through the IC to provide sufficient pH control and oxygenation due to the function of the gas exchange cartridge The Circulation Pump CP is a peristaltic positive displacement pump that is self priming A single CP flow rate is specified for the duration of the run This rate is set at the highest rate necessary to ensure sufficient oxygenation when the cell culture is at its maximum density It is unnecessary to start the CP slow and increase its rate over time to correlate with the culture s growth rate The CP flow rate creates no shear or stress on the cell culture Gassing Pump The Gassing Pump pulls a source of ee gases p the co OUTIPO Post BRX as Exchange m Car
14. and will ensure oxygen is not limiting and pH control is adequate low Over time product concentration increases and becomes the primary reason that complete media and supernatant volumes are exchanged When serum is being used as the growth supplement many researchers observe that serum concentration can be decreased in steps with a corresponding increase in production secretion rate O Remember that the first addition of complete media should occur before inoculation Doing so allows the cells to move from static culture to hollow fiber culture with as little change to their environment as possible O Pre warm complete media before injection into the ECS to prevent the culture experiencing a temperature shock bi Experimenting with various protocols frequency and exchanged volume for complete media addition and supernatant collection can yield greater product secretion rates General guidelines for Harvesting amp Complete Media Beginning exchange volumes should be 20 mL D Add complete media and remove supernatant three times a week As the culture expands increase the exchange volume up to a maximum of 50 mL Check product concentration several days after each increase of the ECS exchange volume Calculate total protein yield concentration x volume Continue increasing the exchange volume once every several days until the maximum secretion rate is achieved Note that the maximum secretion rate
15. anted from the bottle and discarded Filling the HF Primer s integral 2L bottle referred to as the media reservoir O Oooo O Pre warm the bottles of sterile cell culture medium or PBS if they ve been stored at 4 C Warm the bottles until they are at least at room temperature Pre warming them to 37 C is not necessary Wipe them dry disinfect them and put them into the laminar flow hood Loosen the cap of the media reservoir by holding the cap and rotating the bottle clockwise Loosen the caps of the bottles containing the sterile cell culture medium or PBS With one hand lift the cap of the media reservoir only far enough to expose the bottle s opening It is unnecessary to lift the cap so high that its internally connected tubing comes out of the bottle With another hand remove the cap of the first bottle of cell culture medium of PBS and pour the contents into the media reservoir It is OK for the medium being poured to run down the tubing lines inside the media reservoir culture medium or PBS tubing lines Close the Post Bioreactor Sample Port Clamp Close the EC Factor 1 Clamp OO OU Oo Page 14 of 36 Continue pouring the contents of additional bottles until the media reservoir contains 2L of cell Tighten the cap of the media reservoir by holding the cap and rotating the bottle counterclockwise Set the media reservoir into the square opening of the stainless steel rack Be careful not to tangle the HF P
16. d and ready for immediate use Fill the Media Reservoir with basal medium to begin run startup This eliminates time consuming cleaning amp sterilization steps necessary with other in vitro cell culture equipment or the use and sacrifice of mice in Ascites production The HF Primer s bioreactor cartridge can be used to culture both suspension and anchorage dependent cells to very high cell densities The bioreactor is part of the disposable flowpath see figure 1 which is maintained in a CO incubator The HF Primer maintains cells in an optimal environment one that mimics the mammalian body In fact we use the analogy of a body to describe how the HF Primer functions Cells are grown in the EC space surrounding the outside of the hollow fibers the capillaries of the bioreactor The cell culture is supported by the other components Cell culture medium the blood of the system flows through the inside of the hollow fibers to carry fresh nutrients and oxygen to the cells while carrying away cell waste products such as lactate ammonia and CO The Circulation Pump acts as the heart to circulate medium through the IC Circuit The Gassing Pump and Gas Exchange Cartridge act as the lungs to create respiration for the culture and provide oxygenation and PH maintenance As cells secrete protein it is concentrated within a fluid circuit EC that is separate from the feed media and metabolic waste IC Concentrated produc
17. de of the bioreactor ss 18 Placing the HF Primer into the CO nO eo an ada Ta iiaiai iate 19 ale ond ja aja RED e Rar ee o RIR RD RS NC AA A P RR di 20 e RP DS DO Sa 22 CR a nurerrnret rere eet rererenettei pe recite eer re 23 Bet ac ene te IATA EAN a enschede E te 24 Monitor pH metabolites and dissolved oxygen DO of the cell culture medium 25 Dei PR RR O REP O RR RREO RR PNR RR 27 ED PS On 28 Faves tino amd L mp da walele tials ope bd dia duo 28 Procedure lor Harvesting A Gene COPIE nue 29 MAUR OR Tp i DORE da a a nn on 30 D ds 31 Cell Line Characterization oro 32 AF PME A PME Neon Lala ROBOT Sd dd ed no monde 34 HF Primerm User Manual Page 3 of 36 ey Biovest International About this Manual This User Manual will guide you through the concepts and proper steps to effectively use the HF Primer including set up inoculation and operation There are several hyperlinks within this User Manual Excluding graphics hyperlinks are indicated by blue text Items in the Table of Contents are hyperlinks to the corresponding section in the manual The footers on each page are hyperlinks back to the Table of Contents Other hyperlinks are provided to speed navigation between specific topics within the manual Blue text in figures are not hyperlinks This user manual applies to using the HF Primer part number 600297 205 see Ordering Information below Support If you have any technical questions after reading this User Manual plea
18. e 13 of 36 Biovest International Fill amp Flush Hollow fibers are stored with a wetting agent to maintain their physical integrity This wetting agent must be removed before inoculation is performed The wetting agent is removed by flushing the product with two liters of sterile basal cell culture medium Using cell culture medium is preferable because it is saves time and aseptic manipulations An alternative to using cell culture medium is to use 2 L of 1x sterile PBS to flush the product Using PBS may be preferred if an expensive cell culture medium will be used during the run However while the usage of PBS may save some money compared with using cell culture medium using PBS adds time and complication to run startup The PBS must be prepared sterilized and removed from the product before replacement with the chosen cell culture medium These tasks take time and increase the number of aseptic manipulations that must be performed correctly Do not skip the flushing procedure because the wetting agent is cytotoxic After two liters of media have flushed the hollow fibers the product will be non cytotoxic It is most convenient to perform the flushing procedure in a laminar flow hood at room temperature The flush volume circulates via the size 16 pump head and pump motor from the integral media reservoir through the hollow fibers and back to the bottle After circulating for a period of time the flush volume is dec
19. e IC BRX OUT pO clamp is open and the Harvest clamp is closed the Pre BRX Sample Port samples IC BRX OUT pO gt media HF Primer User Manual Page 9 of 36 Raio Biovest International Extracapillary Circuit EC The Extracapillary Circuit is the fluid circuit of the components and tubing that connect to the cell side of the hollow fiber membrane within the bioreactor and is shown in yellow in Figure 6 The functions of the components of the Extracapillary Circuit are Bioreactor Extracapillary Space The extracapillary space ECS within the bioreactor consists of the space surrounding the hollow fibers and is separated from the lumen IC by the porous hollow fiber membrane see Figure 4 Within the ECS are the cells high molecular weight growth factors and cell secreted proteins They are too large to pass through these pores and are retained within the ECS EC Factor 1 Sample This sample location is primarily used to inject fresh complete media each time supernatant is harvested Complete media is either basal medium supplemented with growth factor such as FBS or it is a serum free media In either case complete media contains large molecular weight supplements too large to fit through the fiber pores For this reason complete media must be added directly to the ECS in order for the supplements to reach the cell culture Adding complete media to the Media Reservoir will trap the supplements within the IC they won t reach the
20. ent is used the documentation needs to be consulted in order to find out the nature of the potential hazard and any action which may need to be taken Failure to do so may result in injury or damage to the system All equipment is for indoor use only Biovest International Inc 8500 Evergreen Boulevard Minneapolis MN 55433 USA Telephone 763 786 0302 Toll free in the U S 800 325 1112 Telefax 763 786 0915 Email accountservices biovest com Page 2 of 36 HF Primer User Manual E o Biovest International Table of Contents CRE DD EENT ANE E AE RINE ete N IT AN Tn ITEE NS ee oe eo ee ner ee 4 RO nn eee rate 4 Bar sa Daea a a EBENE A AE Teer ee E A Ont A NE A EA E T 4 E T E A A E AA T AE 5 Hollow Fiber ronda di 6 E e a U a e E E A E D A E AT EAN E E 6 e a e e a E esa A A E TE E ai 8 E o sa a1 0H ge een ene ee E A AT ET A N O T RR E T 8 a ed di D 8 a T ed do nl us 8 Ceci a Ro 9 a ern 9 RUGAS E E e E A RR RR centre hee ree 9 1G bie Ugg 61 EEEN AA AAEN e ami 9 a nee een en ee ern ee A A eee eee ee 10 RC Mier a OE SUS Og ii 10 EE ee de AE 10 ed CS id RD ri DO D ie 10 Re LRO RS le be pes tot dde ei uno noie 11 RE SD O O iene en E 11 PR men einer i ree eRe inte etn 12 a ai remanent eer a 13 e gp 14 Pie be HF Famer s E CICLO io a e aee PO NES ap O 14 Loading the HF Primer on to the circulation pump head and motor 15 RE ini Ne RP NIRO RR EE SPA ADRIENNE RR RR RR odes 16 Injecting complete medium into the cell si
21. f 36 Required Supplies O Basal cell culture medium IC Media Suggest 4 5 e L Glucose gt 6 mM Glutamine 3 6 g L NaHCO and no other pH bufferin 88 8 8 P 8 agent DMEM F12 1 1 works well for murine hybridomas and CHO cell lines Medium consumption can be difficult to predict The HF Primer supports approximately 2 5 x 10 cells which naturally will consume a lot of basal medium Depending on cell line stability the run will last weeks or months so prepare to have enough basal medium Alternative basal media media such as BD Cell MAb Media are consumed in significantly smaller volumes than classic basal medium formulations Although these proprietary media have a higher per liter cost the benefits are very infrequent media changes which minimizes handling time and contamination risk UC RPMI is generally not recommended for use in hollow fiber cell culture productions Complete Media EC Media Complete media is cell culture medium containing high molecular weight supplements UC Add the necessary amount of growth supplement FBS for example into the basal medium of choice Alternative complete media a proprietary serum free medium formulation also works well in place of serum supplemented media When using serum free media as the complete media using classic basal medium for the IC Media often works well and reduces media costs For further information contact Biovest s Account Services Routine sta
22. f media components for the inoculum Place the media reservoir into the rack OO Open the Media and IC Return clamps Page 16 of 36 HF Primer User Manual re Biovest International 1 Close the Fill Flush clamp It usually will NOT be opened for the remainder of the run 1 Close the IC BRX OUT pO clamp CU Close the HARVEST clamp The HF Primer is ready for the next procedure Injecting Complete Medium CAUTION When moving the HF Primer it is best to keep the media reservoir in the rack to keep it at the same level as the bioreactor and gas exchanger This prevents fluids from draining out of the bioreactor which would be a problem once it contains a cell culture If necessary close the Media and IC Return clamps to prevent this problem from occurring Be careful to open these clamps before removing media from the sample ports or turning on the circulation pump HF Primerm User Manual Page 17 of 36 Biovest International Injecting complete medium into the cell side of the bioreactor Complete medium is generally a mixture of basal medium and growth supplements e g fetal bovine serum Alternatively various media vendors chemically defined serum free medium can be used instead of serum supplemented medium Regardless of what is being used for complete medium what distinguishes it from basal medium is the presence of high molecular weight components that enhance the growth of the culture or secret
23. he flush volume for at least one hour The resulting flow will flush the inside IC and outside EC spaces within the bioreactor NOTE If PBS was used after the minimum one hour flushing period it must be removed and replaced with the chosen basal cell culture medium At least 500 mL of basal cell culture medium should be circulated for at least one hour to sufficiently dilute the PBS Discard this volume and add 250 mL of basal medium see below Turn the pump motor off open the Easy Load pump head and remove the circulation size 16 pump segment Close the Media and IC Return clamps Lift the media reservoir from the rack and set it down on whichever side is easier With the HF Primer still in the laminar flow hood loosen the media reservoir s cap by holding it and rotating the bottle clockwise Lift the cap enough to be able to decant the flush medium or the 500 mL of PBS cell culture medium into a sterile container It is unnecessary for the cap s internally connected tubing to fully come out of the bottle O ddad ad Loosely screw the cap on the media reservoir Oo Pre warm disinfect and bring into the laminar flow hood in the manner previously done another bottle of sterile basal cell culture medium UC Pour 250 mL of basal medium into the media reservoir and tighten the media reservoir s cap Putting more medium than this into the media reservoir at the start of the run can cause a detrimental dilution o
24. hollow fiber internal volume and membrane permeability provide the ability to 1 deliver low molecular weight nutrients and O to the cells 2 collect metabolic waste products and CO Fill Flush This tubing line interconnects the IC and EC circuits and is primarily used only during run startup to divert a portion of the Fill Flush media from the IC Circuit through the ECS of the bioreactor to flush the outside of the hollow fibers The Fill Flush clamp is open during Fill Flush and then typically remains closed during the long term culture phase of the run to prevent loss of the secreted product into the IC Circuit In the event during the culturing phase of the run a pre bioreactor sample of IC medium is desired the operator would supply an additional clamp to temporarily close the EC tubing leading to the bioreactor EC port to enable the withdrawal the desired volume of IC medium through the Fill Flush tubing line IC Brx Out pO gt Sample This sample location is primarily used to monitor pH and the concentration of low molecular weight metabolites such glucose and or lactate Dissolved oxygen or other low molecular weight components also may be assayed at this sample port These tests are optional but can help you understand the condition of the cell culture and adjust feed rates etc when necessary The Post BRX Sample Port connects to either the IC Circuit or the EC Circuit depending on how the clamps near this port are set When th
25. ime O Whenever moving the HF Primer it is best to keep the media reservoir in the rack to keep it at the same level as the bioreactor and gas exchanger This prevents fluid from draining out of the bioreactor which would be a problem once it contains a cell culture If necessary close the Media and IC Return clamps to prevent this problem from occurring Be careful to open these clamps before turning on the medium circulation pump O Record the volume of added media to the metabolic data record Page 22 of 36 HF Primer User Manual Biovest International Basal Medium Change Procedure g O o UOUOU OO OOOO a OOO Turn off the circulation and gassing pump motor s and unload the pump heads Close the Media and IC Return clamps This prevents disruptions in volumes within the bioreactor It also prevents air from entering into and medium from draining out of the Media and IC Return tubing when the cap is lifted to empty the media reservoir and add fresh media into it Disinfect and place the HF Primer in the laminar flow hood Lift the media reservoir from the rack and place it on whichever side is easier Disinfect the media reservoir bottle cap and the tubing connected to it Loosen the media reservoir s cap by holding it and rotating the bottle clockwise Lift the cap enough to be able to decant the spent medium into a sterile container It is unnecessary for the cap s internally connected tubing to fully come
26. ion Worksheet Cell Line Conditions Medium Lot H Supplements Serum Other Determine the minimum sample volume required for measuring pH and metabolite and product concentrations Use this value to calculate the volume and number of flasks required for characterization Suggestion when using T flasks determine the number of days that will transpire from inoculation through 48 hours of declining viability Set up this number of flasks plus one for a cell free control When recording sampling data on the following page sacrifice a flask for each sampling which will eliminate the necessity for aseptic technique Suspension Culture Results Seed ____ spinnerflasks or ____T flasks with 0 1 x 10 cells ml Collect a minimum sample volume daily and record data on the back of this sheet Data required for recommended characterization are as follows pH viability total cell density and glucose lactate and product concentrations Additional metabolite data may be useful Collect data for an additional 48 hours after viability begins to fall Graph these data against time and determine the following results Maximum product concentration Maximum viable density x 10 cells ml Doubling time at mid log phase Baseline Setpoints Use the setpoints below as a baseline strategy for entering process control parameters for the AcuSysre or AutovaxID production run Growth Phase Setpoints Production Phase Setpoints values
27. ion of the desired protein Because hollow fiber technology inherently uses a semi permeable membrane that has a very low molecular weight cut off the high molecular weight supplements in complete medium are mostly too large to pass through the hollow fibers pores This is a distinct benefit and greatly reduces the amount of supplement that is necessary compared to conventional cell culture methods Whereas basal medium flows through the IC circuit non cell side and readily exchanges with the EC space cell side and is metabolized by the culture complete medium must be directly injected into the EC space to achieve delivery of the high molecular weight components to the culture It is important that the first injection of complete medium occur at least several hours to one day before inoculation to allow the supplements to fully mix within the ECS Do not rely on the inoculation procedure to serve as the first complete medium injection NOTE media are injected into the Pre Bioreactor port and removed from the Post Bioreactor port to prevent leaving air bubbles in the ECS Prevent the injection of air into the ECS Close the IC BRX Out pO clamp rate OOO OOOOOUOO Disinfect the Pre BRX Sample Port and Post BRX Sample Port Prepare a syringe with 50 mL of pre warmed complete medium Connect the filled syringe to the Pre BRX Sample Port Connect an empty 60 mL syringe to the Post BRX Sample Port Open the EC F
28. is the rate that is necessary when the cell culture has filled the bioreactor Because the cell culture is on the opposite side of the hollow fiber from the circulation rate the Setting the Circulation Pump Speed culture experiences no shear stress from a higher than necessary circulation rate earlier in the production run If you prefer to start slow prior to inoculation set the circulation rate to 50 75 mL min After the culture has been expanding for a week based on visual observation or metabolic data increase circulation rate to 200 mL min If DO is being monitored these data can be a guideline to setting the circulation rate Maintain the post bioreactor DO concentration above 100 mmHg Os O Ifyou have a motor that directly displays the pump rate in mL min for a size 16 pump segment programming the pump to achieve the desired circulation rate is simple Either set the pump for 50 or 200 mL min O Ifyou have a motor with adjustable speed but it does not display the pump rate in mL min you then adjust motor speed based on its revolutions per minute rotational speed The flow rate for size 16 tubing is 0 8 mL pump head revolution Set the motor s speed accordingly For 50 mL min circulation rate set the motor s speed for 63 revolutions per minute For 200 mL min circulation rate set the motor s speed for 250 revolutions per minute O For information on how to load the size 16 pump segment i
29. items within the hood Decant the supernatant into a sterile container sterile Draw the 15 mL inoculum into a syringe Prepare a 3 mL syringe with fresh medium Ensure the Fill Flush clamp is closed Open the EC Factor 1 clamp Open the Post Bioreactor Sample Port clamp into the empty syringe at the bottom port OO OOOOUOOOOOO0O adadad Concentrate the scale up culture via centrifugation using routine methods and supplies Resuspend the cell pellet in 15 mL of conditioned medium and keep the remaining supernatant Disinfect the HF Primer and bring it into the laminar flow hood See Caution note on next page Disinfect the Post Bioreactor and Pre Bioreactor Sample Ports Remove the male Luer plug from the Post Bioreactor Sample Port and connect the inoculum syringe Attach an empty syringe gt 15 mL in size to the bettem Pre Bioreactor Sample Port Close the IC Bioreactor Out clamp and open Harvest clamp Inject the inoculum into the top port while simultaneously withdrawing medium at the same rate Close Harvest and Post Bioreactor Sample Port clamps Slowly inject the medium collected in the syringe at the bottom port back into the bioreactor Note the fluid will be filtering through the hollow fiber membranes so it is normal to feel some pressure on the syringe and it will take a few moments to be complete Close the EC Factor 1 clamp Oo of fresh medium Replace the empty inoculum syringe connected to Po
30. lace the pump motor and head as close to the incubator as possible Load the circulation pump segment to deliver flow in the direction indicated by the label Load the size 14 gassing pump segment yellow tubing marked 06508 14 PharMed into another pump head to deliver flow in the direction indicated by the label gases flow out of the incubator NOTE The white plug in the end of the gassing tubing line does not need to be removed as it is porous and will breathe UC Ifthe pump motor is rated to drive two interconnected pumps heads mount the gassing pump head to the circulation pump head with the circulation pump segment using the hardware supplied by the pump head manufacturer If the pump motor is rated to drive only one pump head mount the gassing pump head to a second pump motor Pump motors often can be stacked to save space Consult the pump motor s user manual for information CU With the pump s turned off set them to their lowest speed rpm CAUTION Turn on the pump s and slowly increase their speed until the pump heads begin to turn slowly Ensure cell culture medium and incubator gases are flowing in the correct directions The two pump heads should rotate at the same speed If two pump motors are in use set both motors to the same the speed revolutions per minute Although the pump heads will turn at the same rate the larger tubing medium circulation will have a higher flow rate Slowly increase the
31. nge with a new 1 mL syringe The 1 mL syringe will function as a sterile port cover until the next sampling It will then be used to flush the tubing line Express the sample volume into a conical tube and screw on the cap Close the Media and IC Return clamps Return the HF Primerto the incubator Open the Media and IC Return clamps Load the circulation pump segment into its pump head Be careful that the orientation of the pump segment results in cell culture medium being pumped out of the media reservoir via the Media line Load the gassing pump segment into its pump head Be careful that the orientation of the pump segment results in incubator gases being pumped out of the incubator and into room air Start the pump motors and ensure pumping happens per the two previous steps OO O OOUOO OO Assay the sample s pH and metabolite and DO concentrations Discard the sample and record the data Page 26 of 36 HF Primer User Manual Biovest International Circulation Rate The Circulation Pump creates the circulation rate within the IC Circuit Circulation rate affects oxygenation and pH control While oxygenation and pH are important for a healthy culture it is easy to determine the circulation rate to use The circulation rate does not need to be adjusted during the production run so simply set the circulation rate at the beginning of the run to the maximum rate and leave it at that speed 200 mL min The maximum rate
32. nt C Reusable rack for the HF Primer sold separately O Peristaltic pump motor s at least 0 250 rpm either Option A or B Option A One pump motor capable of driving two interconnected pump heads Regarding the motor order either a or b and also order c a quantity one of Cole Parmer s Precision Standard Drive part number EW 07520 60 for 90 to 130 VAC 60Hz b quantity one of Cole Parmer s Precision Standard Drive part number EW 07520 67 for 190 to 260 VAC 50Hz c 400114 000 set of double length pump head screws Purchase these from Biovest Option B Two single pump motors each motor driving one pump head contact Biovest s Account Services for options O Peristaltic pump heads 1 One Masterflex Easy Load pump head for the size 16 circulation pump segment Jj One Masterflex Easy Load pump head for the size 14 gassing pump segment UC Regarding the two pump heads order quantity two of Cole Parmer s Easy Load II Pump Heads for Precision Tubing part number EW 77200 60 as of the writing of this user manual NOTE the motor and pump head suggestions are relevant to operating a single HF Primer If multiple HF Primers are simultaneously in use there are other motor and pump head options that may be preferable contact Biovest s Account Services O Space in a humidified 37 C CO incubator Width 4 7 11 9 cm UC Depth 8 20 3 cm DO Height 12 9 32 8 cm HF Primer User Manual Page 11 o
33. nto the pump head see Loading the HF Primer on to the circulation pump head and motor NOTE Masterflex tubing is designed to tolerate the rigor of the pump head The tubing does not need to be rotated over time to a fresh segment of Masterflex tubing CAUTION Only operate the PharMed pump segments in the pump heads Do not operate another type of tubing in the pump heads or it likely will be damaged quickly leading to a leak HF Primer User Manual Page 27 of 36 Biovest International Gassing Rate The Gassing Pump creates the gassing rate The gassing rate in combination with the gas exchange cartridge affects oxygenation and pH control While oxygenation and pH are important for a healthy culture it is easy to determine the gassing rate to use Harvesting and Complete Media Addition Many times during the course of the production run fresh complete media is needed by the culture and supernatant is removed to collect product Complete media and supernatant are exchanged in equal volumes to maintain the fluid volume in the bioreactor Once the culture has come out of lag phase and has been growing for three to seven days the cells likely will benefit from additional growth supplement so an equal volume of supernatant is removed even though product concentration is Important Points Simply set the gassing rate to the same rate as the circulation rate The gas exchange cartridge has a very large surface area
34. off mwco from approximately 10 39 KDa These pores allow only small molecules to freely move across the fiber membrane Bioreactor Case Hollow Fiber Bioreactor Figure 2 Fluid Dynamics Two critical functions for cell growth and production are the supply of fresh nutrients and the removal of waste products This essential exchange occurs across the hollow fiber membrane within the bioreactor see Figure 4 The bioreactor contains thousands of hollow fibers which in a simple sense function as a single membrane The membrane has pores with a molecular weight cutoff mwco range from approximately 10 39 KDa This mwco allows the exchange diffusion of basal media nutrients and Oz and metabolic wastes and CO This mwco range does not allow passage across the fiber membrane of the cells and most added growth supplements and secreted proteins Figure 3 Page 6 of 36 HF Primer User Manual Biovest International The benefits of the fibers semi permeability are O Secreted product is concentrated and is not diluted regardless of the volume of feed media used O The large volume of feed media consumed during the run is product free and discarded as waste Therefore supernatant volumes for purification remain very small Nutrient concentrations can be manually controlled to either stimulate growth of the culture or secretion of product Diffusion driven exchange of low molecular weight components is assisted by
35. out of the bottle Early in the run when the media reservoir has not yet been running with 2L of media simply add fresh medium to the bottle rather than first emptying it See Figure 8 Paa y y Day 4 7 Day 7 and on to Day 1 2 Add Media Add Media Add Media Change Media Figure 8 Set the cap on the media reservoir Disinfect the bottles of fresh media and loosen their caps Lift the media reservoir cap up several inches and away from being directly over the opening of the media reservoir Remove the cap from the pre warmed bottle of fresh basal media and pour it into the media reservoir Add more basal until the media reservoir contains the desired volume Set the cap on the media reservoir and tighten it by holding the cap and rotating the bottle counterclockwise Place the media reservoir in the rack Return the HF Primer to the incubator Open the Media and IC Return clamps Load the circulation pump segment into its pump head Be careful that the orientation of the pump segment results in cell culture medium being pumped out of the media reservoir via the Media line Load the gassing pump segment into its pump head Be careful that the orientation of the pump segment results in incubator gases being pumped out of the incubator and into room air Start the pump motors and ensure pumping happens per the two previous steps HF Primer User Manual Page 23 of 36 Biovest International Sampling Sampling is optional b
36. rculation pump maintains oxygenation and it is necessary for pH control to the cell culture Minimize the amount of time the HF Primer is out of the incubator to minimize temperature changes O Disinfect the HF Primer and miscellaneous supplies that enter the laminar flow hood and be very careful about aseptic handling while working in the hood The HF Primer can operate for months so preventing contamination allows it to be used for a long time Whenever moving the HF Primer it is best to keep the media reservoir in the rack to keep it at the same level as the bioreactor and gas exchanger This prevents fluid from draining out of the bioreactor which would be a problem once it contains a cell culture If necessary close the Media and IC Return clamps to prevent this problem from occurring Be careful to open these clamps before turning on the medium circulation pump NOTE Luer connections can be made with the HF Primer inside the CO incubator if aseptic technique is used For more precaution against contamination antibiotics can be added to the medium Production Metabolic Data Record O Metabolic data from sampling assays can be logged in the provided table Page 24 of 36 HF Primer User Manual Biovest International Monitor pH metabolites and dissolved oxygen DO of the cell culture medium O Monitoring pH and metabolites is optional If the cell culture medium in the Media Reservoir contains phenol red
37. rimer User Manual Biovest International Loading the HF Primer on to the circulation pump head and motor 1 Locate the size 16 pump segment which is the yellow tubing marked 06508 16 PharMed This pump segment creates the circulating flow of cell culture medium through the flowpath of the HF Primer NOTE PharMed tubing is specially made to withstand the mechanical rigor of the pump head CAUTION Do NOT operate the off white silicone tubing in the pump head because it will quickly be damaged causing cell culture medium to leak Place the pump motor and pump head on a bench next to the laminar flow hood or on a cart in front of the hood Mount the Easy Load pump head on to the pump motor as described in the user manual provided by their manufacturer Observe the label near the size 16 pump head It shows the direction in which the cell culture medium flush medium PBS must flow Ooo oO ad Determine how to operate the pump motor and note the direction the Easy Load pump head rotates Some motors rotate in only one direction Other motors have a switch to change the direction of rotation If you are using a standard L S pump head rather than an Easy Load pump head contact Biovest s Account Services department for assistance UC Open the Easy Load pump head and load the size 16 pump segment the circulation pump segment into the pump head in the correct orientation left to right or right to left so the pump head ro
38. se contact Account Services for assistance Account Services also provides customer service for placing orders Ordering Information O HF Primerm the complete pre assembled pre sterilized single use hollow fiber cultureware Its ordering part number is 600297 205 Rack for the HF Primer This rack is stainless steel and reusable Its ordering part number is 103021 000 Note the rack is sold separately from the HF Primer Set of reusable double length pump head screws 400114 000 We have other items that may simplify using the HF Primer or make using it more adapted to the layout of your laboratory or equipment Contact Biovest s Account Services for assistance Page 4 of 36 HF Primer User Manual Raio Biovest International Overview The HF Primer is a low cost hollow fiber cell culture system capable of producing small quantities of highly concentrated monoclonal antibodies and other secreted proteins The HF Primer is much simpler to use than conventional static culture methods or Ascites production This allows the user to focus on the science rather than protein production The HF Primer also is an inexpensive method to affordably evaluate new cell lines or various media formulations in perfusion technology If necessary development in the HF Primer can be scaled up to a range of larger hollow fiber systems provided by Biovest International Inc The HF Primer is supplied fully pre assembled and pre sterilize
39. st Bioreactor Sample Port with the 3 mL syringe Open Harvest and Post Bioreactor sample clamps Page 20 of 36 HF Primer User Manual Biovest International Inject the 3 mL fresh medium into the top port gt J Close the following clamps UC Post Bioreactor Sample Port UC Harvest 1 IC Bioreactor Out 1 EC Factor 1 Leave the empty syringes connected to both sample ports They can be used the next time sampling will be done This minimizes aseptic connections and handling o Optional some cell lines are benefitted by adding the saved conditioned medium from the fourth inoculation step to the media reservoir Other cell lines grow well with 100 fresh basal medium in the media reservoir If conditioned medium will be added to the media reservoir reduce the amount of fresh basal medium in the media reservoir by the volume of conditioned medium that will be added Return the HF Primer to the CO incubator and load the circulation and gassing pump heads Ensure the Media and IC Return clamps are open Set the circulation pump to 50 ml min 62 5 rpm with the gassing pump running at the same rpm Allow the system to run gt O 0O O O The HF Primer should now be monitored either visually or by sampling media and performing assays off line If the inoculum number was 2 x 108 cells the first thing you likely will have to do is add basal media on day 2 Day 0 is the day of inoculation If the inoculum number
40. supernatant from the Post Bioreactor Sample Port CAUTION At all times be careful to not introduce air bubbles into the ECS If this happens remove the OO O OOOOO air bubbles using the syringe connected to the Post Bioreactor Sample Port Close the EC Factor 1 Harvest and Post Bioreactor Sample Port clamps Replace the filled 20 mL syringe on the Post Bioreactor Sample Port with a new sterile 1 mL syringe Return the HF Primer to the incubator Open the Media and IC Return clamps Load the circulation pump segment into its pump head Be careful that the orientation of the pump segment results in cell culture medium being pumped out of the media reservoir via the Media line Load the gassing pump segment into its pump head Be careful that the orientation of the pump segment results in incubator gases being pumped out of the incubator and into room air Start the pump motors and ensure pumping happens per the two previous steps Log the harvest volume that was removed and volume of complete media that was added in the metabolic data record below HF Primer User Manual Page 29 of 36 Biovest International Optimization Tips amp Troubleshooting Optimization is optional Generalized protocols optimize the process and increase yield while often successfully yield a good amount of protein reducing media consumption When new cells lines routinely are being cultured once or twice only optimization in a sense is finding
41. t is harvested and fresh growth supplements are added in small volumes using syringes HF Primer Design Overview Supernatant Harvest With these general concepts in mind the mt detailed information on the following pages will give you a better understanding of how and why the HF Primer works Hollow Fiber Bioreactor Media mH Complete o Reservoir Media Addition Circulation ump Gassing IC Circuit Pump Gas Exchange CAMES EC Circuit Figure 1 HF Primerm User Manual Page 5 of 36 to te re Biovest International Hollow Fiber Technology The core of all hollow fiber based mammalian cell culture EC Space systems is the hollow fiber bioreactor BRX The BRX is a plastic cylindrical housing containing several thousand hollow fibers that are attached at each end of the cylinder see Figure 2 IC Space Hollow fibers contained within a cylinder create two separate fluid volumes within the bioreactor The volume within the fibers is called the Intracapillary Space or ICS The volume surrounding the fibers is called the Extracapillary Space or ECS The ICS and ECS are connected to one another only via the small pores within the hollow fiber see Figure 3 The fibers provide a substrate upon and between which the cells grow The permeability of the fiber membrane permits the exchange of nutrients and wastes The pores have a range of molecular weight cut
42. tates in the direction of the label near the size 16 pump segment tubing Ensure the Post Bioreactor Sample Port Clamp and the EC Factor 1 Clamp are closed and all other clamps are open HF Primer User Manual Page 15 of 36 Biovest International Flushing the HF Primer Turn the pump motor s speed to the lowest setting plug in the motor turn the motor s power switch on and slowly increase the speed setting until the pump head begins to turn slowly 1 Look at the media reservoir and confirm fluid soon is coming out its cap s Out port If not the pump motor may be pumping in the wrong direction through the size 16 pump segment If necessary stop the pump motor and re orient the pump segment UC Increase the pump rate to 50 mL min 62 5 rpm to drive the flush volume through the flowpath and back to the media reservoir CU Observe the flowpath for any leaks If there is a leak turn the pump motor off and clean the spill Do not directly spray isopropanol or ethanol on to the clear polycarbonate plastics The cooling effect from the rapid evaporation can cause stress cracks Instead use these alcohols to wet a sterile gauze and wipe the area clean or use a sterile prep pad to wipe the area CU When the flush volume has been circulating for several minutes without leaking increase the circulation rate to 200 mL min 250 rpm See Setting the Circulation Pump Speed for further information CU Continue circulating t
43. the circulation pump as it forces basal medium through the inside of the hollow fibers As basal medium enters the inside of the fibers there is a slightly higher pressure in the ICS than ECS This pressure difference forces medium through the pores of the hollow fiber and into the ECS near the proximal end entrance of the BRX carrying with it basal medium nutrients and O2 As medium flows along ICS and seeps into the ECS the pressure within the ICS continues to drop Near the distal end of the BRX there is a slightly higher pressure in the ECS than ICS This pressure difference forces medium through the pores of the hollow fiber and back into the ICS carrying with it the low molecular weight metabolic wastes and CO from the cell culture Due to the flow of medium along the length of the ECS growth supplements such as FBS and secreted proteins can be at higher concentration than at proximal end This process is known as the Starling Effect Harvest Complete Medium Cells grow to tissue density in ECS Oxygen Figure 4 HF Primer User Manual Page 7 of 36 One of thousands of semi permeable hollow fiber membranes Basal Medium Nutrients amp HD Biovest International Intracapillary Circuit IC The Intracapillary Circuit is the fluid circuit of the components and tubing that connect to the non cell side of the hollow fiber membrane within the bioreactor and is shown in red in Figure 5 The functions of the m
44. tic cell culture equipment amp supplies to produce a scale up inoculum of 2x10 viable cells for inoculation UC Sterile syringes of various sizes such as 1 mL 5 mL 30 mL and 60 mL You may find other sizes are useful too Syringes are used to inject complete media and the inoculum Syringes also are used to remove IC medium samples for pH and metabolite analyses and EC medium to collect supernatant There are various methods and equipment for analyzing these samples The chosen methods and equipment determine the necessary sample sizes Optional Equipment A A A A pH meter Glucose or lactate assay kit Glutamine assay kit useful if the cell line doesn t use glucose as the main energy source Dissolved oxygen meter unnecessary for routine productions using the HF Primer Page 12 of 36 HF Primerm User Manual LD Biovest International Unpacking amp Setup The unpacking and setup steps ensure the product remains sterile and is leak free and ready for use It is important to ensure the clamps are open on arrival and that the fittings are not loose 1 Remove the sterile sealed packages containing the HF Primer and male Luer plugs from the shipping carton Disinfect the outside of the sealed packages and put them into a laminar flow i hood Disinfect the reusable stainless steel rack for the HF Primer and put it into the laminar flow hood Slide the clip s U shaped top over the metal bar here
45. tridge Post Bioreactor The source of the gases is generally the CO incubator a high percentage of CO in air Under some circumstances the source may be room air a low Media Reservoir X E IC Sample AV UA U0 Y 0 00 Page 8 of 36 f CO Circulation percentage o 2 Pump x Fil Flush EC Factor Pre Bioreactor am am Gassing i P Sample Pump Gas Exchange Cartridge Figure 5 HF Primer User Manual Biovest International Gas Exchange Cartridge The Gas Exchange Cartridge GEX oxygenates the circulating cell culture medium The GEX also decreases or increases pH of the circulating cell culture medium When CO incubator gases flow through the GEX the cell culture medium is oxygenated and pH decreases When room air flows through the GEX the cell culture medium is oxygenated and pH increases The GEX is a membrane based device that separates two compartments the gas side and the cell culture medium side Gas blue in the figure flows on one side of the membrane while culture medium flows on the other side The membrane is permeable to gas transfer allowing bubble free exchange of CO and oxygen from air Bioreactor BRX Lumen The internal volume of the semi permeable hollow fibers within the bioreactor is part of the IC and is referred to as the Intracapillary Space ICS See Figure 2 The ICS is on the non cell side of the hollow fiber membrane The
46. ulum density This can negatively impact some cell lines throughout the HF Primer production Even if the cells aren t themselves impacted low inoculum volumes can lead to poor distribution of the inoculum in the bioreactor which can lead to poorer growth and filling of the bioreactor O Harvest Optimization To optimize harvest strategy increase the frequency and volume of harvest until you no longer see an increase in protein production Although rare the secretion rate of the protein of interest can be up or down regulated by the concentration of the protein itself Adjust the harvest strategy accordingly For protein free applications more frequent harvesting can minimize the potential for degradation or alteration of the secreted protein due to background culture lysis in the bioreactor When using serum supplementation decrease its concentration in steps once the bioreactor is full At this time less serum often leads to increased protein secretion rates Reference D 0000 IC Volume is 110 mL plus the volume of media in the Media Reservoir EC Volume is 50 mL Pump rate calculation mL min pump head rpm Circulation 0 8 Gassing 0 22 Space 4 7 Wide x 8 Deep x 12 9 High 11 9 cm W x 20 3 cm D x 32 8 cm H Weight 2 Ibs 0 9 kg plus the volume of media in the Media Reservoir HF Primerm User Manual Page 31 of 36 Date Pe a e Biovest International Cell Line Characterizat
47. urther information Turn off the circulation and gassing pump motor s and unload the pump heads Oo Close the Media and IC Return clamps This prevents loss of fluid from the bioreactor It also prevents air from entering into and medium from draining out of the Media and IC Return tubing when the cap is lifted to empty the media reservoir and add fresh media into it Disinfect and place the HF Primer in the laminar flow hood Review the schematic in Figure 5 in the Intracapillary Circuit IC section of the manual Close the Harvest clamp if necessary Open the IC BRX OUT pO clamp Disinfect the Post BRX Sample Port Connect a 1 mL syringe to the Post BRX Sample Port unless it is already connected from the previous sampling Open the Media and IC Return clamps Open the Post BRX Sample Port clamp and fill the syringe Close the Post BRX Sample Port clamp Replace the 1 mL syringe with a 3 to 5 mL syringe Discard the 1 mL syringe because it contains stagnant medium that is not relevant O UOUU dadadada Open the Post BRX Sample Port clamp and draw 2 to 4 mL into the syringe HF Primer User Manual Page 25 of 36 CAUTION Sample volumes greater than 2 to 4 mL may drain liquid volume from the bioreactor and result in the accumulation of air If larger sample volumes are necessary for the desired assay s proceed with caution Close the Post BRX Sample Port and IC BRX OUT pO clamps Replace the 3 to 5 mL syri
48. ut suggested in order to respond to the needs of the large number of cells that the HF Primer can culture approximately 2 5 x 108 Sampling does not require a lot of time yet can provide the following benefits improved product yield economical use of media and successful culture expansion Sampling typically is done more often during the growth phase of the run when the culture is dividing and filling the bioreactor The expanding culture needs ever greater amounts of fresh media etc Sampling frequency during the growth phase is often performed every one to two days During the production phase the cell number is relatively static which leads to Important Points a fairly consistent media consumption rate etc Therefore during the production phase sampling frequency is lower than during the growth phase When the same cell line is cultured repeatedly sample data and experience from these runs can be used to create a general production strategy This strategy can then be repeated with reduced sampling requirements throughout the run IC samples are used to monitor the culture s metabolic activity in order to know when to change the media in the Media Reservoir EC samples are used to monitor the product secretion rate in order to optimize the harvesting frequency and volume removed O Ifsampling will be done in a laminar flow hood minimize the amount of time the circulation pump is not running Like your heart the ci
49. was higher the culture might need more media on day 1 Additionally you periodically will need to UC add fresh basal medium perform sampling of IC or EC media 1 add fresh complete media amp remove harvest NOTE Luer connections can be made with the HF Primer inside the CO incubator if aseptic technique is used For more precaution against contamination antibiotics can be added to the medium CAUTION When disinfecting the HF Primer use caution to ensure disinfecting chemicals such as Isopropanol do not enter the gas side of the GEX HF Primerm User Manual Page 21 of 36 Biovest International Basal Medium Changes Basal medium needs to be changed periodically and the frequency will vary by cell line Even when using high glucose basal media the consumption rate can reach 1 L day because the HF Primer supports a very high number of cells Because the media reservoir initially operates at less than its full volume the first several times that fresh medium is necessary simply add fresh medium to the media reservoir rather than emptying it and adding fresh medium Once the media reservoir contains 2L it should be completely emptied and refilled when fresh media is necessary There are two general ways to determine when to change the medium in the media reservoir 1 observing the color of the cell culture medium when it contains phenol red or 2 aseptically Important Points sampling medium using a s
50. yringe and measuring the concentration of glucose or lactate Visual observations are an easy but not the best method and can lead to inconsistent results This is OK if you do not need to optimize production Alternatively if optimizing production is desired monitoring metabolite concentration s ensures consistent results which can be especially useful if the process eventually will scale up to larger hollow fiber production systems There are several options for measuring metabolites Glucose can be measured using diabetic test strips There are reagent based kits that measure glucose or lactate Also there are a variety of instruments that measure these metabolites but they can be expensive at the scale of the HF Primer Contact Account Services for more information about these options O Minimize the amount of time the circulation pump is not running Like your heart the circulation pump maintains oxygenation and it is necessary for pH control A the cell culture Minimize the amount of time the HF Primer is out of the incubator to prevent temperature changes to O Pre warm new media to 37 C to prevent a temperature shock to the cell culture Disinfect the HF Primer bottles of fresh media and miscellaneous supplies that enter the laminar flow hood and be very careful about aseptic handling while working in the hood The HF Primer can operate for months so preventing contamination allows it to be used for a long t
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