mAb

production & purification: frequently asked questions 1. Media requirements a. Sample media formulations are listed below. Because serum (FBS) s not used, certain elements will need to be added to the media, such as transferrin (binds iron), insulin, albumin. b. Same media is used throughout the process. c. If you choose to make the media, you will need a media prep room. 2. Cho cell growth a. These cells will be adapted for growth in suspension and do not need to adhere to a solid substrate (ie, microcarrier) b. The cells have very strict growth requirements and will grow slowly, have poor production or die if not treated properly. For instance, the cells will not grow well at densities less than 10^5/ ml and will not grow to densities higher than 10^7/ ml. c. At all times, the temperature will need to be maintained at 37 C with 5% CO2 to provide buffering capability with bicarbonate in the media and adequate dissolved oxygen. In the larger steel reactors, this will require heat removal. d. Upon thawing, the 1 ml frozen stock will be inoculated into 10 mls media (not all of the cells survive freezing and thawing). 3. Seed train vs production reactor a. In the seed train, the priority is to expand the cell mass by maintaining a healthy cell population in the exponential growth phase. As soon as they reach the end of the exponential phase, dilute them into a larger volume of fresh media. The cells will be secreting antibody during this time, but that will not be harvested during seed train production. Early seed train reactors can include T-flasks, roller bottles, wave bioreactors (disposables), etc. These reactors are operated as batch or fed-batch (to replace consumed nutrients) there is no outlet because this would result in cell loss. b. In the steel production bioreactor, the priority is to use the cell mass to maximize yield of antibody. We are no longer concerned with propagating a healthy cell population. Thus, cells will be seeded into the reactor, allowed to grow until waste products accumulate and the cells begin to die. The process is terminated when a sufficient number of cells have dies that the live cells are no longer producing high levels of antibody. Note, these larger reactors will require steaming in place to sterilize prior to use.

CELL CULTURE TECHNIQUES

GENERAL RULES FOR MANIPULATING ANIMAL CELLS A. Never use the sample bottle of medium or buffer for two different cell lines. B. Never handle two different cell lines in the hood simultaneously. C. After the manipulation of cells, the hood must be cleaned (remove the medium designated for the first cell line being handled) before introducing the materials (medium, flasks, tyrpsin, etc.) for the second cell line. D. The passge of cells must be recorded. The split ratio of each passage must not be changed except under special circumstances. When it is changed, it must be recorded. E. UV lamp must be left on overnight in the hood. F. Contaminated culture flasks must be removed from the incubator immediately, autoclaved and disposed of properly. G. Spent medium must not be left on the bench. It must either frozen for later analysis or be disposed of properly after cultivation. H. The glass cultureware, after removing the medium, must be cleaned promptly. The glassware should be stored clean and dry. It must not be stored with water or PBS as its content. Microrganisms may glow when liquid is present. GENERAL RULES FOR MEDIA PREPARATION UPDATED 2/22/02 A. The sterile room will be used for sterile operations only and will be kept as uncluttered as possible. It will not, in any way, be used as a storage room. B. During media dispensing only things which are essential to the operation should be placed in the hood area. This is to minimize the disruption of sterile air flow in the most critical areas. C. Face mask, gloves, and clean lab coat will always be worn during media preparation. D. Bottles should always be wiped with a solution of Wescodyne and left for approximately 15 minutes prior to the addition of serum to the medium. (Wescodyne was once available through U-Stores, but apparently is not anymore. Iodine can be used instead) E. At the end of each work day, make sure the sterile room is cleaned up, check to insure that the floors are being frequently wet mopped with a disinfectant solution. Lights in the laminar flow hood room should be turned off and the door closed. F. In the daytime the door to the laminar flow hood room may be kept open but the door to 260B should also be kept closed. G. Once each month check for sterility within the hood by placing open blood agar plates in the hood for one (1) hour. Incubate and observe for colony formation. if the hood is working properly, only an occasional colony should be observed. H. Filter used in media preperation must be kept in a secure location. A contaminated filter can ruin an entire batch of media, filters must be stored where the user can be confident that the packaging will not be comprimised.

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PREPARATION OF PHOSPHATE BUFFERED SALINE (WITHOUT MG OR CA)

UPDATED 2/22/02

Materials NaC1 KC1 Na2HPO4 KH2PO4

1X g/L 8.0 0.20 2 0.4

mM 136 2.68 14.1 29.4

10X g/L 80 2.0 20 4.0

mM 1360 26.8 141 294

Making 10 L of 1X solution: A. Dissolve compounds in 7.0 liters of double distilled water. B. Add to 10 L use volumetric flask or graduated cylinder. C. Check and adjust pH to 7.2 - 7.3 for 1 x solution; D. Dispense 1X solution in bottles and autoclave at 250F for 30 minutes (liquid mode). 10X stock solution is not sterilized and stored at room temperature. Making 1 L of 10X solution: A. Dissolve compounds in 700 mL distilled H2O B. Add to 1 L using volumetric flask or graduated cylinder C. Do not adjust pH. Store at room temperature. Adjust pH and sterilize when diluted to working concentration. Making 1 L of 1X solution from 10X stock: 1. Add about 800 mL DI water to graduated cylinder (will help absorb heat produced during dilution) 2. Add 100 mL 10X stock solution to graduated cylinder. 3. Transfer to beaker with stir bar. 3. pH the solution to 7.2 4. Return solution to graduated cylinder and add appropriate volume of DI water to bring total volume to 1 liter. 5. Transfer solution to bottle. If sterile PBS is desired, autoclave the PBS in the bottle with cap loosened and covered in tin foil, liquid cycle.

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PREPARATION OF 1 X CELL CULTURE BASAL MEDIUM UPDATED 2/22/02 A. GENERAL Medium will usually be prepared in 20 to 50 liter lots and dispensed in 500 ml bottles (450 ml/bottle), 20 bottles per case. Each case will be clearly labeled as to date, lot number and type of media. Bottles should be sterilized and covered with a piece of heavy duty tin foil. Caps will be sterilized separately in baskets. After preparation, filtration and dispensing, the medium should be left at room temperature for 2 days and supplemented with serum if no contamination is detected. Supplemented media will be frozen at -20C and will not be used until sterility testing is complete (this medium will be designated for the maintenance of the cells). Filters used in media preparation must be stored by the user in a secure location in order to ensure the sterile packaging has not been compromised. B. Materials Required 1. Powdered medium. 2. Double distilled water collected in a 200-liter polypropylene tank. 3. Polypropylene mixing paddle. 4. Pall #3001 ultrapore filter; 293 mm Millipore filter holder set up or Millipore Twin - 90 can be used as a back-up system. 5. Variable speed peristaltic pump (Randolph 610). 6. 500 ml bottles, 20 per case. 7. Sodium bicarbonate, penicillin, streptomycin. 8. Source of compressed CO2 for pH adjustment, or HCL (for REMI). C. Steps in Preparation 1. Preparation of Equipment a. Sterile filter should be obtained from a secure location where the sterile packaging has not been compromised. b. Bottles: Bottles are sterilized in large boxes (24 bottles per case) in autoclave ("A" or "B" autoclaves). Dry cycle (30 minutes + 30 minutes dry). For the average 150-liter lot, 15 cases are required. Label all cases clearly, i.e., date, type of medium, etc. c. Water: Milli Q water is used as the source for the Bellco Still. The water is collected in the 20 liter (or 50 liter) polypropylene tank in which the media is to be prepared. 2. Preparation of Medium a. Add the appropriate amount of powdered medium to the water slowly and with stirring to prevent clumping. Add sodium bicarbonate. i) Add half of the quantity of the water to the container. ii) Add the appropriate amount of powdered medium to the water. Add medium slowly. iii) Stir continuously to prevent clumping.
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i) Adjust pH of DMEM to 6.8-6.9 by adding 1 N HCl into the medium using a Pasteur pipette. ii) RPMI medium requires pH adjustment to 7.2 by the addition of HC1. c. i) Place hose from pump all the way to bottom of the tank. Attach tubing from CO2 tank and allow a slow but steady stream of CO2 to blanket the medium. Place lid on tank. ii) When adding 1 N HC1 to RPMI, use small amounts and mix well. d. Attach media bell to the ring stand in the sterile room and carefully remove foil and wrapping. e. When initially pumping media through the tubing and filter unit, leave the top screw valve loose to permit the release of any air which may be trapped in the unit. Once the media starts to flow out, seal off the valve. Return the first four bottles of filtered media to the polypropylene tank and stir thoroughly. This will wash out any residual water in the hoses and filter from the autoclaving (this step hasnt been seen in the lab). f. Fill bottles using a flow rate of 4 to 6 liters/minute. A 500-ml bottle should be filled to the 450-ml level using a "guide" bottle filled to that volume. No bottle should be filled past the level where the bottle narrows, as this will cause cracking during freezing. g. All bottles should be flamed before and after filling, and caps should be placed on tightly. h. Remove the first, middle, and last bottle of each lot and use for sterility tests. (incubation at 37C for 14 days.) i. Allow the entire lot to remain at room temperature for two days. If preliminary results of incubated samples indicate that the batch is sterile, and individual bottles checks substantiate sterility, the entire lot should be stored at -20C until supplementation, or supplement immediately and freeze.(we just store william E in cold room) 3. Use of Millipore Twin-90 Filter a. The Pall #3001 filter is the primary media production filter. b. The Twin-90 should only be used for back-up purposes. This filter is package pre-sterilized and does not require a media bell for dispensing. c. The 293-mm Millipore Filtration Assembly should be used (non-sterilized) as a pre-filter when the Twin-90 is used for filtration of batches of media in excess of 125 liters. 4. Optional Use of 293 mm Millipore Filtration Assembly a. The 293-mm filter may be utilized as a pre-filtration unit with the Pall #3001 filters. b. Information concerning the assembly of the 293-mm filter may be obtained from the files in the office or in the general information notebook in the media production room.

b.

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IMX MEDIA UPDATED 2/22/02 Cells: Chinese Hamster Ovary Cells (CHO)
Component IMX 1.2 NaCl Na Bicarbonate Soy Peptone Glucose Trace Elements 1000xA Trace Elements 1000xB Trace Elements 1000xC Methotrexate L-Glutamine Penicillin G Streptomycin Intralipid Vendor Cat # Storage Mixing 13.252 g 4g 1.2 g 5g 2.0 g 1 ml 1 ml 1 ml 100 ml 32 ml 0.0593 g 0.1 g 100 ml Stock Conc. N/A N/A N/A N/A N/A N/A N/A N/A 1.5 mM 200 mM N/A N/A 20% fat emulsion Conc. In Media N/A ~68.4 mM ~14.3 mM N/A ~36.1 mM (w/ IMX 1.2) N/A N/A N/A ~0.15 mM 6.4 mM ~0.17 mM ~68.6 M N/A

JRH Biosciences 571004-50L 4C Sigma S5886 Inorganic Cabinet Sigma S4019 Organic Cabinet EM Science 1.07212.0500 Amino acid cabinet Sigma Cellgro Cellgro Cellgro Sigma Gibco BRL Sigma Sigma Sigma G5146 99-182-LI 99-175-Cl 99-176-LI M8407 25030-081 PEN-NA S6501 I141 Organic Cabinet 4C 4C 4C minus 20C minus 20C 4C 4C 4C

Procedure: 1. Sterilize 2 500 ml bottles 2. Add above components (except intralipid) into a clean 1L erlenmeyer flask 3. Add < 1 L of DI/UF water 4. Gently invert and swirl so as to avoid bubbles 5. Adjust pH to 7.0 6. Adjust to final volume (1L) 7. Filter sterilize into 500 ml bottles using a 0.22 mm filter 8. Add intralipid (50 ml for each 500 ml bottle) & gently mix 9. Perform sterility test 10. Store at 4C until use

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POOLED SUPPLEMENTS UPDATED 2/22/02 Component Albumin Linoleic Acid EGF Sigma Cat # A-2058 L-5900 E-4127 Mixing 5g 50mg/25 mL H2O 0.1mg/5mL PBS 100mg/(8m PBS+2mL EtOH) 100mg/5mL H2O 2mg/10mL PBS 1mg/10mL H20 Vol in Pooled Supplements 5g 25 mL 2.5 mL 0.392 mL 3 mL 0.2 mL 0.210 mL Concentration Conc. In Media 50 mg/mL 500 g/mL 15 g/mL 150 ng/mL 0.5 g/mL 39.2 g/mL 0.6 mg/mL 0.4 g/mL 0.21 g/mL 5 ng/mL 392 ng/mL or 1M 6 g/mL 4 ng/mL 2.1 ng/mL

Dexamethasone D-1756 Transferrin Glucagon Na2SeO3 T-1147 G-3157 S-5261

Materials: 100 mL bottle Balance, weighing dish/paper Sterile pipets Magnetic Stir bar Magnetic Stir plate Syringe and syringe filter 20 or 40 Falcon Tubes Procedure: 1. Add Albumin to sterile 100 mL bottle 2. Add 5 mL DI water to each of 5 bottles of Linoleic Acid 3. Mix together and add 25 mL DI water (mix until Albumin dissolves) 4. Add remaining 5 compounds 5. Add 43.3 mL DI water to bring final volume to 100 mL 6. Filter sterilize (use pre-filter) and aliquot into 2.5 or 5 mL sized aliquots. Use sterile falcon tubes (which can withstand freezing) 7. Store at -200 C

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TRACE ELEMENTS UPDATED 2/22/02 Component: Media CuSO4*5H20 Na2SeO3 ZnSO4*7H20 Sigma Cat #: Mixing: C-8027 S-5261 Z-0251 0.125g/50 mL water 0.015g/50 mL water .065 mg/50mL water Concentration: Concentration in 10 mM 300 g/mL 5 M 0.1 M 3 ng/mL 50 pM

Materials: Small Beaker Magnetic Stir Bar Magnetic Stir Plate Balance, weighing dish/paper Syringe and Syringe Filter 10 sterile 15 mL centrifuge tubes Procedure: Add the CuSO4*5H20 to a small beaker with 50 mL of water and a stir bar. Stir solution, CuSO4*5H20 will take a while to enter dissolve. Once dissolved, add Na2SeO3 and ZnSO4*7H20. Sterilize using a syringe filter. Aliquot into approximately 5 mL size aliquots in sterile centrifuge tubes. Date and label, store at 40C.

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THAWING CELL FROM DEEP FREEZE STATE UPDATED 2/22/02 Materials: 1. pipettes, sterile 2. centrifuge tubes, 15ml, sterile 3. media, warmed to 37C 4. same size T-flask from which cells originally came 5. trypan blue dye, stock solution Procedure: 1. Remove cryotube from liquid nitrogen and record it as removed in Liquid Nitrogen Database. 2. Thaw IMMEDIATELY by holding cryotube in 37C water bath in such a way that the juncture of cap and tube is not submerged in the water. 3. Using a pipette remove suspension from vial, note volume withdrawn, and place in a centrifuge tube. 4. Add l ml media for each l ml thawed cell suspension S-L-O-W-L-Y drop by drop. You will see a nonhomogeneous layer of medium on top. Thump to mix until homogeneous. Let centrifuge tube containing cells and media sit in 37C water bath for 5 minutes. 5. SLOWLY but not dropwise add enough media to double the current volume. Thump, gently invert to mix. Let sit in 37C water bath for 5 minutes. 6. Add enough media to again double the new volume (you have now diluted the cell suspension 8-10 times). Invert to mix. 7. Centrifuge at 700-800 rpm, 5 minutes, room temperature. 8. Suck off the supernatant media. 9. Replace the 8-10 ml you just sucked off with fresh media. Shoot the media in to break up cell clumps. 10. Suck in and out as few times as possible to break up major lumps, without creating air bubbles, until the suspension is homogeneous. 11. Transfer the cell suspension into the T-flask (spinner). *Steps 12 & 13 are applicable only when there is enough volume. 12. Remove a measured amount of cell suspension from the T-flask, dilute with the same amount of Trypan Blue dye (1:2 dilution). 13. Using the hemocytometer count the cells for a viable cell count. Count both the dead (dark blue) and the live (colorless). If 60-80% of the cells are viable that reflects on a good freeze technique. 14. Place T-flask in 37C CO2 incubator, loosen cap. 15. Incubate overnight. 16. Repeat viable counts, in the case of non-anchorage dependent cells, every 24 hours. 17. For anchorage dependent cells, change medium to remove residual DMSO and dead cells on the second day.

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b) place T-flask in incubator for 2-3 minutes, cap loosened c) at the end of 2-3 minutes to check if cells are coming off. Slap the side of the flask--cells should slide off. If cells are not coming off, incubate longer d) add medium containing serum to stop reaction e) shear with a pipette to mix SPLITTING CELLS FROM T-FLASKS UPDATED 2/22/02 Procedure: 1. Add enough medium to stop reaction (minimum is twice the volume of trypsin) plus enough volume to split comfortably. For example: for a 1:4 split add 7ml to the already existing l ml of trypsin, then remove 2ml of the mixed cell suspension to each T-flask the same size as the original. 2. Shear gently for the purpose of dispersing cells in newly added media. Do not suck up all of the volume or release the entire volume at once or else you will get bubbles and subsequent protein degeneration. 3. Remove appropriate amount from parent flask for each daughter flask. For example: Take 2ml out of the total of 8ml for each of 4 flasks for a 1:4 dilution as in example above. 4. Label flask with split ratio, cell line name, passage number, media, date, initials, return flask to incubator and loosen caps. *All unlabeled T-flasks will be discarded. The responsible person will be disciplined.

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