ReadyToProcess columns - GE Healthcare Life Sciences
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1. A loading of more than 100 gram antibody liter chromatography media could be used in this step Using Capto adhere it is possible to replace the two post Protein A steps with a single polishing step and end up with a two step process If necessary Capto adhere can also be used in combination with Capto Q in a three step process Capto Q is also run in non binding flowthrough mode and loading media with more than 100 gram antibody liter is possible Cell culture Cell removal MabSelect SuRe Virus inactivation amp filtration CIEX Capto S AIEX Capto Q Capto adhere AIEX Capto Q Capto adhere Pool for final filtration UF DF Fig 3 2 Media toolbox for mAB purification An alternative intermediate step is cation exchange chromatography using Capto S At optimized conditions binding capacity exceeding 100 gram antibody liter has been demonstrated 22 RTP MabSelect SuRe Columns User Manual 28 9195 81 Edition AB Media characteristics MabSelect SuRe 3 3 4 Process development It is recommended to use small scale columns for example Tricorn and or XK columns for initial process development and evaluation of the chromatographic conditions Choose a residence time that fulfills your requirements for dynamic binding capacity and nominal fluid velocity The nominal fluid velocity must be compatible with the pressure flow characteristics of the ReadyToProcess columns Column packing information for small scale col2. 1000 1 Polyethylene glycol PEG 5000 1 Potassium phosphate 1M 1 propanol 5 2 propanol 30 SDS 1 Sodium acetate 1M Sodium chloride 4M Sodium citrate 1M Sodium sulfate 1M Sodium hydroxide 1M Sorbitol 1M Sucrose 1M RTP MabSelect SuRe Columns User Manual 28 9195 81 Edition AB 17 2 Technical specifications and characteristics 2 4 Chemical resistance of column parts Chemical Concentration Triton X 100 1 Tween 20 1 Tween 80 1 Urea 8M 1 The column is intended to be used for up to 5 purification cycles For the listed concentrations and exposure times the temperature difference between the fluid running through the column and the ambient temperature in the room should never be greater than 20 C 18 RTP MabSelect SuRe Columns User Manual 28 9195 81 Edition AB Media characteristics MabSelect SuRe 3 3 Media characteristics MabSelect SuRe 3 1 General description MabSelect SuRe is an alkali tolerant protein A derived medium for capturing monoclonal antibodies from large feed volumes by packed bed chromatography It is characterized by e high dynamic binding capacity DBC which reduces process time and amount of medium used a high flow agarose matrix which allows processing of large volumes of feed 3 2 Properties The protein A derived MabSelect SuRe ligand is produced in Escherichia coli Fermentation and subsequent purification are performed in the ab
3. 16 The effects of a chemical will generally be more severe at higher temperatures Note also that the combined effects of agents have not been taken into account in this table In general the ReadyToProcess columns are not compatible with long time exposure to organic solvents Table 2 3 Chemical resistance of the external surfaces of ReadyToProcess columns for exposure times up to a maximum of 24 hours Chemical Concentration Ethanol 70 2 Propanol 70 Acetone 2 5 WARNING The columns are not intended for use in a potentially explosive atmosphere or for handling flammable liquids Table 2 4 Chemical resistance of the wetted parts of empty ReadyToProcess columns Chemical Concentration Acetone 2 5 Acetic acid 1M Aqueous buffers pH 2 12 03M Arginine 2M Ammonium sulfate 4M Benzyl alcohol longtime exposure 2 DTE DTT dithioerythritol dithiothreitol 100 mM EDTA ethylenediaminetetraacetic acid 100 mM Ethanol longtime exposure 20 RTP MabSelect SuRe Columns User Manual 28 9195 81 Edition AB Technical specifications and characteristics 2 Chemical Concentration Ethylene glycol 50 Formaldehyde 0 1 Glycerol 10 Glycine 2M Guanidine hydrochloride 6M Imidazole 1M Mercaptoethanol 20 mM Phosphoric acid 0 1M Pluronic F 68 1 Polyethylene glycol PEG 500 5 Polyethylene glycol PEG
4. example RTP MabSelect Sure will at the same time as they are equilibrated to buffer also be adjusted to the temperature of the buffer If the columns are stored after use they should be stored in a solution preventing microbial growth for example 20 ethanol The inlets and outlets must be sealed by blind flanges CAUTION Handle the column carefully to retain its performance RTP MabSelect SuRe Columns User Manual 28 9195 81 Edition AB Operation of the column 5 5 Operation of the column Note It is recommended that you read the entire User Manual before using the columns ReadyToProcess columns are supplied as complete units that can be connected directly to BioProcess chromatography systems for example AKTAprocess chromatography systems AKTApilot is only applicable for 2 5 L columns within a limited liquid velocity range 5 1 Installation 5 1 1 Removing the packaging from the column The column is supplied in double bags The outer bag is removed first for example with a pair of scissors and the column in the unbroken inner bag is moved to the intended clean environment for example a cleanroom where the inner plastic bag is removed The column can now be moved to the system for example placed on an optional cart suitable for this purpose for installation and use WARNING Do not remove the tie rods disconnect or release the CPC sealing cap Do not remove the welded tubes attached to the column in
5. listed in B 4 Related literature on page 36 600 5 500 D fo 25LF 20L Velocity cm h S N 3 fo 3 fo r 1 o 0 5 1 15 2 Pressure Bar Fig 3 1 Pressure flow profile for RTP MabSelect SuRe 2 5 and 20 water run in room temperature 20 C Table 3 1 Characteristics of MabSelect SuRe Composition Rigid highly cross linked agarose Average particle size d50v um 85 Ligand Alkali tolerant protein A derived E coli Coupling chemistry Epoxy Dynamic binding ca pacity Approx 30 mg human lgG mI medium Chemical stability Stable in all aqueous buffers commonly used in Protein A chromatography pH working range 3 12 Regeneration of column 0 1 0 5 M NaOH 20 RTP MabSelect SuRe Columns User Manual 28 9195 81 Edition AB Media characteristics MabSelect SuRe 3 Temperature stability CC 4 to 40 Storage temperature C to 8 Maximum pressure drop over 2 packed bed bar Storage solution RTP MabSelect 20 ethanol SuRe columns 1 d50v is the medium particle size of the cumulative volume distribution 2 Determined at 10 breakthrough by frontal analysis at a liquid phase velocity of 250 cm h in a column with a bed height of 10 cm i e resi dence time is 2 4 min 3 See MabSelect SuRe Instruction 4 Recommended long term storage conditions 4 to 8 C 3 3 Recommended Antibo
6. ready for immediate use when delivered The test results are presented in the Certificate of Analysis which is enclosed in the Extended Product documentation for each column 2 2 Technical specifications Table 2 1 General specifications of the ReadyToProcess columns 2 5L 10L 20L Inner diameter mm 126 251 359 Inner cross section cm 124 495 1012 Column volume L 2 5 9 9 20 2 Packed bed height mm 200 200 200 Mechanical compression factor 15 15 15 Outer height mm 378 388 407 Outer diameter incl lid mm 195 342 484 Weight kg 6 25 50 Inlet TC25 connectors tubing i d 63mm 95mm 12 7 mm 0 25 0 375 0 5 Outlet TC25 connectors tubing i d 63mm 95mm 12 7 mm 0 25 0 375 0 5 Ambient temperature CC 4 30 4 30 4 30 Liquid temperature C 4 40 4 40 4 40 RTP MabSelect SuRe Columns User Manual 28 9195 81 Edition AB 13 2 Technical specifications and characteristics 2 3 Materials 2 5L 10L 20L Maximum liquid pressure bar 4 4 4 see Table 3 1 on page 20 for media specific limits Estimated shelf life years 1 1 1 1 The mechanical compression factor does not include flow compression which may vary depending on the medium and the size of the column 2 The temperature difference between the fluid running through the col umn and the ambient temperature in the room should never be greater than 20 C 3 While th
7. 0 100ml 17 5444 01 1L 17 5444 03 5L 17 5444 04 captoQ 25m 17 5316 10 500ml 17 5316 01 5L 17 5316 04 10L 17 5316 05 Capos f 25m 17 5441 10 100ml 17 5441 01 1L 17 5441 03 5L 17 5441 04 Phenyl Sepharose 6 Fast Flow ow sub 25ml 17 0965 10 200m 17 0965 05 1L 17 0965 03 5L 17 0965 04 RTP MabSelect SuRe Columns User Manual 28 9195 81 Edition AB 35 B Ordering information B 4 Related literature B 4 Related literature Product Title Code No Datafile ReadyToProcess columns 28 9159 87 Datafile MabSelect SuRe 11 0011 65 Instructions MabSelect SuRe 11 0026 01 Datafile Capto adhere 28 9078 88 Instructions Capto adhere 28 9064 05 Datafile Capto Q and Capto S 11 0025 76 Instructions Capto Q and Capto S 28 4074 52 Datafile Phenyl Sepharose 6 Fast Flow 18 1020 53 low sub Instructions Phenyl Sepharose 6 Fast Flow 71 5002 39 low sub Handbook Process chromatography A guide 18 1121 56 to optimization scale up and validation Application note Efficiency test of ReadyToProcess 28 9198 21 columns Application note Purification of a monoclonal 28 9198 56 antibody using ReadyToProcess columns 36 RTP MabSelect SuRe Columns User Manual 28 9195 81 Edition AB Ordering information B This page intentionally left blank RTP MabSelect SuRe Columns User Manual 28 9195 81 Edition AB 37 B
8. Columns User Manual 28 9195 81 Edition AB 33 B Ordering information B 1 ReadyToProcess columns Appendix B Ordering information B 1 ReadyToProcess columns Product Column Code No size L RTP MabSelect SuRe 2 5 25 28 9017 17 RTP MabSelect Sure 10 10 28 9017 18 RTP MabSelect Sure 20 20 28 9017 19 RTP Capto adhere 2 5 25 28 9017 14 RTP Capto adhere 10 10 28 9017 15 RTP Capto adhere 20 20 28 9017 16 RTP Capto Q 2 5 25 28 9017 23 RTP Capto Q 10 10 28 9017 24 RTP Capto Q 20 20 28 9017 25 RTP Capto S 2 5 2 5 28 9017 29 RTP Capto S 10 10 28 9017 30 RTP Capto S 20 20 28 9017 31 RTP Phenyl Sepharose 6 FF low sub 2 5 25 28 9017 35 RTP Phenyl Sepharose 6 FF low sub 10 10 28 9017 36 RTP Phenyl Sepharose 6 FF low sub 20 20 28 9017 37 B 2 Empty lab scale columns Product Packsize Code No XK 16 40 column 1 18 8774 01 includes tube end piece and adapter XK 16 adapter 1 18 8778 01 Tricorn 10 50 column 10 mm i d 1 18 1163 14 Tricorn 10 200 column 10 mm i d 1 18 1163 17 1 To achieve a 20 cm bed height an extra XK16 adapter must be used This adapter is ordered separately 34 RTP MabSelect SuRe Columns User Manual 28 9195 81 Edition AB Ordering information B B 3 Bulk media Product Packsize Code No ml L MabSelect SuRe 25 ml 17 5438 01 200ml 17 5438 02 1L 17 5438 03 5L 17 5438 04 Captoadhere 2m 17 5444 1
9. Do not remove the welded tubes attached to the column inlet and outlet until the column is ready to be connected to a system and used See 5 1 Installation on page 25 for instructions WARNING Exceeding the pressure limit may cause the tie rods to break To avoid risk of possible injury do not remove the top protective rim from the column The purpose of this rim is to keep the top part of the tie rods in place in case of a breakage WARNING The columns are not intended for use in a potentially explosive atmosphere or for handling flammable liquids in areas defined as zone 0 to 2 according to EN 60079 10 2002 WARNING Wash the column before disconnecting it from a system after use to ensure that no hazardous chemicals for example halogen containing salt buffers are left in the column prior to disposal gt gt gt PPP gt RTP MabSelect SuRe Columns User Manual 28 9195 81 Edition AB 11 1 Introduction 1 7 Safety precautions WARNING Ensure that there is no residual pressure in the column before any tubing is disconnected 12 RTP MabSelect SuRe Columns User Manual 28 9195 81 Edition AB Technical specifications and characteristics 2 2 Technical specifications and characteristics 2 1 Column testing Each ReadyToProcess column is packed and efficiency tested sanitized and sampled for testing endotoxin lt 0 25 EU ml and microbiological growth CFU lt 10 100 ml The columns are
10. GE Healthcare ReadyToProcess columns MabSelect SuRe User Manual Important user information All users must read this entire manual to fully understand the safe use of ReadyToProcess columns Safety notices This manual contains warnings and cautions concerning the safe use of the products See definitions below WARNING The WARNING symbol and notice highlight instructions that must be followed to avoid personal injury Do not proceed until all stated conditions are met and clearly understood CAUTION The CAUTION notice highlights instructions that must be followed to avoid damage to the product or other equipment Do not proceed until all stated conditions are met and clearly understood Note A Note is used to indicate information that is important for trouble free and optimal use of the product Contents Contents Introduction wetness 1 1 General description oo esssesssssessssssssssssssssssssssssssssesssstesssse 1 2 Intended use of the columns 1 3 Scope of this User Manual eesssssssssssssssssssssssssssssssssee 14 Handling the column UPON arrival sssri 7 1 5 Identifying the main components of your column 9 1 6 Labelling of the column 1 7 Safety precautions u csssssssssssssssssesssssssssmessssssssssssees Technical specifications and characteristics 13 2 1 Column testing eee seesssssesssssssssnsesseeneessenseseeenenceennnceeneneeee 13 2 2 Technical specificati
11. Ordering information B 4 Related literature This page intentionally left blank 38 RTP MabSelect SuRe Columns User Manual 28 9195 81 Edition AB For contact information for your local office please visit _ www gelifesciences com contacts www gelifesciences com BioProcess GE Healthcare Bio Sciences AB Bjorkgatan 30 751 84 Uppsala Sweden imagination at work GE imagination at work and GE monogram are trademarks of General Electric Company BioProcess Capto MabSelect SuRe ReadyToProcess Sepharose Tricorn AKTApilot and AKTAprocess are trademarks of GE Healthcare companies Separating viral particles with Capto Q products may require a icense under United States patent number 6537793 B2 and equivalent patents and patent applications in other countries owned by Centelion SAS Such a license is not included with the purchase of Capto Q but is included with the purchase of Capto ViralQ products With the purchase of Capto ViralQ the customer is granted a free imited license under US patent 6537793 B2 and equivalent patents and patent applications in other countries owned by Centelion SAS o separate viral particles solely through use of the product purchased The Tricorn column and components are protected by US design patents USD500856 USD506261 USD500555 USD495060 and their equivalents in other countries All third party trademarks are the property of their respective owners 2007 General Electric Company Al
12. ambient temperature where it is used Perform the efficiency test again For more information see 5 2 Clearance of storage solution and equilibration of the column on page 27 Efficiency test result is not the same as on the certificate The test is performed ona different instrument than the one used in the production The plate number can also vary as much as 10 between runs If the variation is within acceptance limits according to the certificate the column is OK to use RTP MabSelect SuRe Columns User Manual 28 9195 81 Edition AB Appendix A Flow conversion tables Flow conversion tables A The following tables show flow rates and retention times at different liquid velocities for the different sizes of ReadyToProcess columns Al 25Lcolumn Liquid velocity Flow rate Flow rate Retention time cm h ml min L h min 100 208 12 48 12 200 415 24 90 6 300 623 37 38 4 400 831 49 86 3 500 1038 62 28 25 A 2 10Lcolumn Liquid velocity Flow rate Flow rate Retention time cm h ml min L h min 100 824 49 44 12 200 1649 98 94 6 300 2473 148 38 4 400 3297 197 82 3 500 4122 247 32 2 5 A 3 20L column Liquid velocity Flow rate Flow rate Retention time cm h ml min L h min 100 1686 101 16 12 200 3372 202 32 6 300 5059 303 54 4 400 6745 404 70 3 500 8431 505 86 25 RTP MabSelect SuRe
13. consists of the following parts as illustrated below in figure 1 2 gt Plug Tie rod Column label see figure 1 3 Top lid Inlet TC connection marked Bottom Inlet CPC sealing cap on top of the packing valve Outlet TC connection marked Top Outlet Protective rim Product label Column tube Bottom lid a7 roonmoonow Fig 1 2 ReadyToProcess column 20L 1 6 Labelling of the column The ReadyToProcess columns are labeled with product name code number lot number referring to a series of columns produced at a specific time ID number identifying the individual column and estimated expiry date as illustrated in figure 1 3 below The bar code GS1 128 a total of 30 digits contains an application identifier 90 the code number digits 3 to 10 a second application identifier 10 lot number digits 13 to 20 a third application identifier 21 and the ID number digits 23 to 30 RTP MabSelect SuRe Columns User Manual 28 9195 81 Edition AB 9 1 Introduction 1 7 Safety precautions 10 GE Healthcare 6G O RTP 28 9017 19 MabSelect SuRe 20 I column uma R10 Flommoable UUM NE DONUA UUU Fig 1 3 ReadyToProcess column label example 1 7 Safety precautions The columns must be used in accordance with the safety instructions and technical specifications given in this User Manual and in the separate Safety Instructions leaflet to avoid personal injury or possible damage to equipment T
14. dy purification scheme Large scale purification of monoclonal antibodies mAbs usually consists of three chromatographic steps Initially the mAb is captured using Protein A affinity chromatography giving a product with a high purity typically 99 This product is then further polished by for example cationic and anionic exchange chromatography In the chromatography media toolbox for mAb purification offered by GE Healthcare Fig 3 2 MabSelect SuRe is recommended for the Protein A capture step due to its alkaline stability enhanced protease resistance and generic elution profile The MabSelect SuRe step has a binding capacity for media with approximately 30 gram antibody liter at 2 4 min residence time QB 10 The MabSelect SuRe step is often run repeatedly A typical purification of a produced batch could consist of four to five repeated runs on the MabSelect SuRe column and one run on the second and third step respectively The media of choice for the intermediate step and polishing of mAbs is Capto adhere It is a multimodal strong anion exchanger which offers a different selectivity compared to traditional ion exchangers Capto RTP MabSelect SuRe Columns User Manual 28 9195 81 Edition AB 21 3 Media characteristics MabSelect SuRe 3 3 Recommended Antibody purification scheme adhere is designed for selective removal of protein A leakage aggregates host cell proteins nucleic acids and viruses when used in flowthrough mode
15. e maximum liquid pressure stated depends on the pressure rat ing of the column restrictions for the maximum pressure drop over the column depend on the packed chromatography medium in order to ensure bed stability The pressure drop between inlet and outlet of the ReadyToProcess column should therefore never exceed the specified maximum pressure drop listed in Table 3 1 on page 20 See Fig 3 1 Pressure flow profile for RTP MabSelect SuRe 2 5 and 20 water run in room temperature 20 C on page 20 for a typical curve 2 3 Materials The polymer materials used to manufacture ReadyToProcess columns have been chosen for their biological and chemical compatibility with the samples buffers and solutions used during operation and during sanitization procedures The materials meet the USP United States Pharmacopoeia class VI requirements according to USP 88 gt Biological Reactivity Tests In Vivo The material is of non animal origin or has been produced under manufacturing conditions complying with EMEA 410 01 guidance The columns are designed to comply with hygienic requirements Table 2 2 below lists the wetted materials 14 RTP MabSelect SuRe Columns User Manual 28 9195 81 Edition AB Technical specifications and characteristics 2 Table 2 2 List of wetted materials in ReadyToProcess columns Trade name Material Column part PP Polypropylene Column tube lids TC connections support nets support screens strea
16. er columns manually Refer to Table 2 1 General specifications of the ReadyToProcess columns on page 13 for approximate column weights 2 Checkthe contents against the packing list 3 Without breaking the plastic bags check the column for signs of damage that may have occurred during transportation If there is any damage please contact your local GE Healthcare representative 4 Moveittoa suitable place for keeping until it is time for the column to be used Refer to section 5 1 Installation on page 25 for the subsequent steps to perform Refer to chapter 4 Storage of the column on page 24 if the column is to be stored for more than a limited time before usage WARNING Place the column where it is protected from possible damage and on an even surface where it cannot be unbalanced and fall to cause personal injury or damage CAUTION To avoid contamination do not break the outer plastic bag or disturb the welded pieces of tubing mounted on the inlet and outlet TC connections of the column See section 5 1 Installation on page 25 Copies of the column User Manual and the Safety Instructions leaflet are enclosed with the column Extended Product documentation specific for each individual column is also included in a separate folder RTP MabSelect SuRe Columns User Manual 28 9195 81 Edition AB Introduction 1 15 Identifying the main components of your column AReadyToProcess column
17. g to the column Top outlet At this stage air in the tubing will not be a problem since the column will be run in upflow mode later on 4 Before connecting the Bottom Inlet start the pump at a low liquid velocity for example 50 cm h with the system valves set to column inline and upflow to prime the system tubing that will be connected to the column Bottom Inlet 5 Openthe column TC connector marked Bottom Inlet and remove the welded piece of tubing Save also this piece to be used for sealing the column before disposal If there is air in the Bottom Inlet tubing on the column use a squirt bottle and fill the tubing for example with running buffer or 20 ethanol RTP MabSelect SuRe Columns User Manual 28 9195 81 Edition AB Operation of the column 5 6 After spraying both the TC connectors with or dipping them in for example 70 ethanol connect the system column inlet tubing to the column Bottom Inlet 7 Turn off the pump or proceed to remove the storage solution 5 2 Clearance of storage solution and equilibration of the column A ReadyToProcess column is delivered packed with chromatography media in a storage solution consisting of 20 ethanol The column needs to be washed to clear it from the storage solution before starting the purification process We recommend washing the column with 6 column volumes CV running buffer low salt containing buffers or H O ata con
18. his User Manual does not guarantee user safety it is the responsibility of the user s employer to ensure safe operation and provide appropriate training Information given in this User Manual is a suggested best working practice and shall in no way take precedence over individual responsibilities or local regulations Great effort has been made to design and manufacture the various parts of the equipment so that it will comply with all applicable safety aspects for this type of equipment During the operation and during other work with a column it is always each individual s responsibility to consider Their own and others personal safety e The safety of the equipment through correct use in accordance with the descriptions and instructions given in this User Manual RTP MabSelect SuRe Columns User Manual 28 9195 81 Edition AB Introduction 1 WARNING When using hazardous chemicals take all suitable protective measures such as wearing protective glasses and gloves resistant to the chemicals used Follow local regulations and instructions for safe operation and maintenance of the system WARNING To avoid risk of possible personal injury and damage to the column and other equipment never use the column without an appropriate pressure alarm WARNING Never exceed the maximum rated pressure of the column WARNING Do not disconnect or release the CPC sealing cap remove the tie rods or the protective rim
19. l rights reserved First published J une 2007 All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them A copy of these terms and conditions is available on request Contact your local GE Healthcare representative for the most current information GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Healthcare UK Ltd Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK GE Healthcare Bio Sciences Corp 800 Centennial Avenue P O Box 1327 Piscataway NJ 08855 1327 USA GE Healthcare Bio Sciences KK Sanken Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan 28 9195 81 AB 10 2007
20. let and outlet until the column is ready to be connected gt break To avoid risk of possible injury do not remove the top protective rim from the column The purpose of this rim is to keep the top part of the tie rods in place in case of a breakage A WARNING Exceeding the pressure limit may cause the tie rods to RTP MabSelect SuRe Columns User Manual 28 9195 81 Edition AB 25 5 Operation of the column 5 1 26 Installation A 5 1 2 Connecting the column to a BioProcess system WARNING Read about the operation of the BioProcess system before connecting the column CAUTION Ensure that as little air as possible is introduced when the system tubing is connected to avoid damaging the packed bed Note The column will be connected and run in upflow mode Note Forlarge columns i e 10 Land 20 L an outlet tubing diameter of 10 mm is recommended An outlet tubing diameter of 6 mm is recommended for smaller columns 1 By using the system pump prime the system tubing with the running buffer see 5 2 Clearance of storage solution and equilibration of the column below 2 Open the top TC connection of the column marked Top Outlet and remove the welded piece of tubing Gave the welded piece of tubing which will be used later to seal the column before disposal 3 After spraying the TC connector with or dipping it in for example 70 ethanol connect the system column outlet tubin
21. lumn before disposal 1 Using the system pump empty the column from liquid by pumping air through it Turn off the pump 2 Use the system indicators to verify that all pressure is released 3 Release any remaining pressure in the column by setting the system valves to column inline and opening a system outlet 4 Disconnect the column from the system 5 Seal the TC connectors using the welded pieces of tubing that were mounted on the column at the delivery The column is now ready for disposal 5 4 Disposal of the column Make sure to always follow the local regulations for handling and disposal The columns are designed to be disposed intact as combustible material RTP MabSelect SuRe Columns User Manual 28 9195 81 Edition AB Evaluation of the packed bed optional 6 6 Evaluation of the packed bed optional 6 1 Evaluation before delivery The ReadyToProcess column has been efficiency tested with a liquid velocity of 100 cm h at the production facility using a system comparable with a standard BioProcess system to achieve representative and comparable data The stability of the packed ReadyToProcess column has been verified by transport testing For more information see the Datafile for ReadyToProcess columns 6 2 Efficiency and A testing Astandard method of expressing the efficiency of a packed column is in terms of number of theoretical plates meter N m and the asymmetry factor A These values are easil
22. m stoppers hose connections PEEK Polyetheretherketone Plug filter holder media packing valve Tygon 2275 Polyolefin Hose inlet tubing welded tubing for inlet outlet protection FPM Fluorocarbon rubber O rings EPDM Ethylenepropylenediene TC gaskets 2 4 Chemical resistance of column parts ReadyToProcess columns and the chromatography media are resistant to chemical agents used in protein recovery including agents commonly contained in crude broth buffer solutions for adsorption elution and washing and to solutions effective in cleaning and storage Table 2 3 below lists the resistance of the outer surface of the column for chemicals and Table 2 4 below lists the resistance of the wetted parts The concentrations and exposure times listed are typical for the intended use of the ReadyToProcess columns Note Chemical resistance data has been collected from several published sources not from individual tests on column components It should be used only as a guide Note Table 2 3 and Table 2 4 list the chemical resistance of the column and not the chromatography media For the chemical resistance of the chromatography media see chapter 3 Media characteristics MabSelect SuRe on page 19 and the bulk chromatography media datafile RTP MabSelect SuRe Columns User Manual 28 9195 81 Edition AB 15 2 Technical specifications and characteristics 2 4 Chemical resistance of column parts
23. milarly the contribution related to molecular diffusion of the tracer will be constant providing that specific test conditions such as temperature are constant Therefore the packing quality can be evaluated at conditions where mass transfer contribution is not optimized i e minimized Consequently performing efficiency tests at higher velocity than the normally recommended range will provide sufficient data to correctly assess column integrity if the other contributions to the overall dispersions are known Figure 6 2 below shows efficiency tests of a ReadyToProcess 20 L column at 30 and 100 cm h liquid velocity respectively Velocity 30 cm h N m 5432 Asymmetry 1 13 Velocity 100 cm h N m 3635 Asymmetry 1 08 15000 16000 17000 18000 19000 20000 ml Fig 6 2 ReadyToProcess column efficiency test N m theoretical plates per meter packed bed RTP MabSelect SuRe Columns User Manual 28 9195 81 Edition AB 31 7 Troubleshooting 7 Troubleshooting 32 Problem Action Comment Air has entered into the column During installation or during run Apply a low flow e g 100 cm h over the column for 6 CV High backpressure The column is Perform clogged during rerun regeneration of the column e Filter the sample before it is applied Efficiency testresults Buffer and column Ensure that the are not acceptable do not have the column has the same temperature same temperature as the buffer and the
24. ned for the purification of biomolecules and specifically suited for production of material for clinical phase and phase Il studies Depending of the scale of operations they can also be used for full scale manufacturing as well as preclinical studies Primarily ReadyToProcess columns are intended for use together with BioProcess chromatography systems for example AKTAprocess The 2 5 LReadyToProcess columns may also be used with AKTApilot systems within a limited liquid velocity range When the column has been used it shall be disposed of as described in 5 4 Disposal of the column on page 28 The ReadyToProcess columns cannot be repacked WARNING ReadyToProcess columns must not be used for any other purpose than the intended purposes described above WARNING The columns are not intended for use in a potentially explosive atmosphere or for handling flammable liquids in areas defined as zone 0 to 2 according to EN 60079 10 2002 gt gt 6 RTP MabSelect SuRe Columns User Manual 28 9195 81 Edition AB Introduction 1 13 Scope of this User Manual This User Manual is designed as a general introduction and guide to ReadyToProcess column operation Specifically the manual covers Chapter 1 General introduction including actions to perform when the column is delivered safety precautions and labeling Chapter 2 Technical specifications including principles of column function column specificatio
25. ns and chemical resistance Chapter 3 Characteristics of media including descriptions storage conditions and process development Chapter 4 Storage of the column Chapter 5 Operations including installation preparation of a column for use preparations before a run disconnecting the column and disposal Chapter 6 Optional evaluation of the packed bed efficiency testing Chapter 7 Troubleshooting Appendix A Tables listing liquid velocities flow rates for the dif ferent column sizes Appendix B Ordering information 14 Handling the column upon arrival The column is delivered in a cardboard container Follow the steps described below after the cardboard packaging is opened 1 Without breaking the plastic bags lift the column using the handles on the top lid and remove it from the cardboard container Place the column on a cart ora similar device Note The large ReadyToProcess columns are packed with an expanded ethylene insert that is easily removed before the outer package is removed leaving the column accessible on the pallet RTP MabSelect SuRe Columns User Manual 28 9195 81 Edition AB 7 1 Introduction 14 Handling the column upon arrival A WARNING Appropriate transportation and lifting devices must be used when handling the larger columns for example carts telphers or pallet lifters All lifting must be performed in accordance with local regulations Two to four persons are required to lift the larg
26. ons sssssssssssrssssrssissrisssrsssrss 13 2 3 Materials sssssssssissssisrsssrsrsssisrnssrsrssseerssnrerssnenrnnnnersanenrnanans 14 24 Chemical resistance of column parts sses 15 Media characteristics MabSelect SuRe s19 3 1 General description 3 2 Properties 3 3 Recommended Antibody purification scheme 21 3 4 Process development sssrinin 23 3 5 Regeneration of the column between purification Cycles sssssssssssssrsissrrssssrrssssrrsnssrinssranssnnnsnrnnnns 23 Storage Of the column sess 24 Operation of the column aressssssssssssssssssssrsrsrsessssesees 29 51 IMStealleatio ta cease tesstecstsssezsccceezectececccevenceennccecceceatnpavettnie 5 1 1 Removing the packaging from the column 5 1 2 Connecting the column to a BioProcess system 26 5 2 Clearance of storage solution and equilibration of the COLUMN wesc 27 5 3 Disconnecting the column prior to disposal 28 5 4 Disposal of the COIUMIN wucssccsccssssssssssssssssssssssssssssrsssssesssees 28 Evaluation of the packed bed optional 29 6 1 Evaluation before delivery sssri 29 RTP MabSelect SuRe Columns User Manual 28 9195 81 Edition AB Contents 6 2 Efficiency and AS testing sssri 29 6 3 Efficiency testing with a high liquid velocity 30 7 Troubleshooting eT F Appendix A Flow conversion tables 33 A1 2 5 L COLUMN sssi 33 A2 LOL COLUMN wee eeeesssssssssssssssssssssssssssssssssssstsssssssssss
27. sence of animal products The ligand has been specially engineered to create an affinity medium with enhanced alkali stability and high binding capacity for IgG The specificity of binding to the Fc region of IgG is similar to that of conventional Protein A and provides excellent purification in one step Additional features of MabSelect SuRe are a generic elution profile for different monoclonal antibodies and an enhanced protease resistance Alkali tolerance high capacity and low ligand leakage plus the rigid base matrix make MabSelect SuRe ideal for the purification of monoclonal antibodies for clinical applications The pressure drop over the packed bed depends on the applied liquid velocity as well as the viscosity and temperature of the liquid In order to ensure stability of the packed bed a maximum pressure drop of 2 bar shall not be exceeded Typically a liquid velocity of 350 cm h results in a pressure drop of less than 2 bar when using the largest column 20 L with aqueous solutions at room temperature Pressure flow curves for RTP MabSelect Sure columns are shown in Fig 3 1 for operation with water at room temperature 20 C RTP MabSelect SuRe Columns User Manual 28 9195 81 Edition AB 19 3 Media characteristics MabSelect SuRe 3 2 Properties The characteristics of the medium are summarized in Table 3 1 For further information about MabSelect SuRe please see the bulk chromatography media Datafile or the Instruction both
28. stant liquid velocity of 100 cm h By following the washing procedure the chromatography media is cleared from storage solution The wash can in many cases be performed with the desired running buffer low salt containing buffers to save time equilibration of the column The wash will also adjust the temperature of the column to the running temperature important as an example for RTP MabSelect Sure columns which are recommended to be stored at 4 to 8 C Note Itis not recommended to temperature adjust the column only by moving it into a room temperature environment Tests have shown that temperature adjustment of a 20 L ReadyToProcess column by moving it into a room temperature environment could take several days Note Since they are less effective and consequently will be more expensive to use buffers containing high concentrations of salt are not recommended for removal of storage solutions RTP MabSelect SuRe Columns User Manual 28 9195 81 Edition AB 27 5 Operation of the column 5 3 Disconnecting the column prior to disposal A A 28 5 3 Disconnecting the column prior to disposal WARNING Wash the column before disconnection to ensure that no hazardous chemicals for example halogen containing salt buffers are left in the column prior to disposal WARNING Ensure that there is no residual pressure in the column before any tubing is disconnected Follow the steps below to disconnect the co
29. tography A guide to optimization scale up and validation For more information about efficiency testing see the application note Efficiency test of ReadyToProcess columns Ordering information is listed in B 4 Related literature on page 36 6 3 Efficiency testing with a high liquid velocity Zone broadening in a chromatography column is related to hydrodynamic dispersion molecular diffusion and mass transfer resistance Because the extent of the hydrodynamic dispersion will depend on column packing quality a typical protocol for assessing quality of column packing requires performing the quality test efficiency test at conditions where the contributions from mass transfer resistance and molecular diffusion could be minimized These specific conditions depend on many factors including type of mobile phase and tracer chosen for the efficiency test temperature at which the test is performed and type of chromatography medium especially its particle size distribution Considering that GE Healthcare chromatography media are manufactured according to high quality standards and within a very tight technical specification it can be expected that the contribution from intraparticle mass transfer resistance to the overall dispersion in RTP MabSelect SuRe Columns User Manual 28 9195 81 Edition AB Evaluation of the packed bed optional 6 the column will be constant and in effect scalable for a given chromatography medium Si
30. tsssssssssset 33 A3 QOL COLUMN ssis 33 Appendix B Ordering information d4 B 1 ReadyToProcess COLUMNS oie 34 B 2 Empty lab scale columns B3 BUIK media ssis B A Related literature sssi RTP MabSelect SuRe Columns User Manual 28 9195 81 Edition AB Introduction 1 1 Introduction 11 General description ReadyToProcess columns are part of the ReadyToProcess platform from GE Healthcare including chromatography and filtration products as well as other downstream solutions ReadyToProcess columns are pre packed pre qualified by efficiency testing and pre sanitized chromatography columns They are available with a range of BioProcess media such as MabSelect SuRe Capto Q Capto S Capto adhere and Phenyl Sepharose 6 Fast Flow low sub and in the following sizes e 2 5L d 126 mm bed height 200 mm e 10L d 251 mm bed height 200 mm e 20L d 359 mm bed height 200 mm All wetted parts of the ReadyToProcess columns are of USP class VI with all components traceable to their production batches The columns are pre packed in cleanroom class ISO 8 environment The columns are pre sanitized and tested for endotoxin as well as microbiological growth and released according to specifications Fig 1 1 ReadyToProcess columns RTP MabSelect SuRe Columns User Manual 28 9195 81 Edition AB 5 1 Introduction 1 2 Intended use of the columns 12 Intended use of the columns ReadyToProcess columns are desig
31. umns is available in the bulk chromatography media instruction See appendix B 2 Empty lab scale columns on page 34 for order information for small scale columns or contact your local GE Healthcare representative 3 5 Regeneration of the column between purifi cation cycles Regeneration is the removal of very tightly bound precipitated or denatured substances from the purification system If such contaminants are allowed to accumulate they may affect the chromatographic properties of the column reduce the capacity and potentially contaminate product in subsequent runs If the fouling is severe it may block the column increase the pressure drop over the column and as a consequence limit the applicable liquid velocity It has been verified that the column can be regenerated and reloaded five times RTP MabSelect SuRe Columns User Manual 28 9195 81 Edition AB 23 4 Storage of the column 4 Storage of the column 24 ReadyToProcess columns are supplied with 20 ethanol as storage solution All columns except RTP MabSelect SuRe are preferably stored at room temperature but can also be stored in a cold room storage temperature 4 to 30 C The RTP MabSelect SuRe columns shall be stored at 4 to 48 C Before using the column it must be equilibrated with a running buffer or equivalent as described in section 5 2 Clearance of storage solution and equilibration of the column on page 27 Columns stored in a cold room for
32. y determined by applying a tracer such as acetone 2 solution in 20 ethanol to the column using 20 ethanol as eluent The values are calculated as follows N 5 54 x ValWn assuming a Gaussian peak where N number of theoretical plates Vr peak retention elution volume or time Wp peak width at half height expressed in the same units as Vp 10 peak height ajb Fig 6 1 Typical test chromatogram showing asymmetry factor As value calcu lations RTP MabSelect SuRe Columns User Manual 28 9195 81 Edition AB 29 6 Evaluation of the packed bed optional 6 3 Efficiency testing with a high liquid velocity 30 Asymmetry factor A b a where a is partial peak width measured at 10 of the peak height for the leading part of the peak Fig 6 1 and b is partial peak width measured at 10 of the peak height for the tailing part of the peak The calculated plate number will vary depending on the test conditions and it should therefore be used as a reference value only Keep conditions and equipment constant so that results are comparable Changes in solute solvent eluent sample volume liquid velocity cm h liquid pathway temperature etc will influence the results If an acceptance limit is defined in relation to column performance the column plate number can be used as part of the acceptance criteria for column use For more information of the principles for efficiency testing in general see Process chroma
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