Human plasma-derived media supplement supports antibody production by hybridoma cells

July 23, 2017 | Autor: John Tharakan | Categoría: Technology, Biological Sciences, Cell line, Fetal Bovine Serum, Growth rate
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J. Tiss. Cult. Meth. 14:39-44, 1992 © 1992 Tissue Culture Association 0271-8057/92 $01.50+0.00

HUMAN PLASMA-DERIVED MEDIA SUPPLEMENT S U P P O R T S ANTIBODY PRODUCTION BY HYBRIDOMA CELLS Janet M. Young, Da-Wei Zhang, John Tharakan, 1 Larry Jenkins, Roger Collins, and William N. Drohan

American Red Cross, Jerome H. Holland Laboratory, 15601 Crabbs Branch Way, Rockville, Maryland 20855

SUMMARY: A new, virus-inactivated, media supplement from human plasma (GEMS) has been developed which maintains hybridoma ceils at a low growth rate. The rate of antibody production per ceil by hybridoma cells growing in the presence of GEMS is at least equivalent to the rate of antibody production by the same cell lines growing in fetal bovine serum.

Key words:human plasma-derived media supplement; hybridoma; antibody production.

I.

INTRODUCTION

supplement supports growth and antibody production by hybridoma cells growing either in flasks or in a bioreactor.

Cohn fractionation uses ethanol precipitation in the presence of specific pH, ionic strength, temperature, and protein concentration for fractionation of plasma proteins (2), and has been the starting process for methods of purification of factors IX and X, protein C, antithrombin III, thrombin, and fibrin glue (1). Cohn fraction IV-4, containing c~- and fl-globulins, ceruloplasmin, and transferrin, results from precipitation of proteins with 40% ethanol at a pH between 4.48 and 5.42 (3). Although fraction IV-4 is currently a discard in the Cohn fractionation process of the above plasma proteins, the ability of this fraction to support growth of hybridoma cells has been described in the literature since the 1950s (5). Since approximately 1 × 106 liters of plasma are fractionated in the United States per year, one can estimate that approximately 52 000 liters of Cohn fraction IV-4 is produced at 50 mg/ml of protein. If used at 1 mg/ml to support cell growth, approximately 2.6 × 10 6 liters of media can be prepared from this fraction. A significant problem in the use of a human-derived protein for growth of cultured cells is the presence of viruses which may be transmitted by the human material. This concern is usually addressed by virus inactivation in the end product of the cultured cells, rather than by treatment of the media supplement used to promote cell growth (4-7). We now report the preparation of a human plasma-derived media supplement (GEMS), from Cohn fraction IV-4, which is pasteurized at 60 ° C for 10 h, a method that has been shown to inactivate contaminating human viruses. We also show that this pasteurized media

II.

MATERIALS

A. Equipment Taylor Warton model 17k liquid nitrogen Cryostorage system, Roberts Oxygen 1 Biological safety cabinet, NuAire model no. NU-425-600, class II, Mid-Atlantic Labz Mistral 3000i centrifuge, no. DO-257-339, CMS, Curtis Matheson 3 Steri-cuh water-jacketed, CO2 incubator, model no. 3033, Forma Scientific 4 Incubator, model no. 2010, VWR Scientific 5 Microscope, model TMS-F96, Nikon 6 Tricentric bioreactor, no. TSP120803, SETEC, Inc. 7 Portable pipet-aid, no. 205-336, Drummond Scientific s P- 1000 pipettor, Rainin 9 P-200 pipettor9 P-20 pipettor9 Multi pipettor (8 tips)9 Water bath, equatherm, 14 liter no. 213-132, CMS3 V-max Kinetic Microplate Reader, Molecular Devices 1° IBM XT personal computer, IBMn SOFT-max software, version 2.011° B. Culture media and chemicals Growth Enhancing Media Supplement (GEMS), derived from Cohn fraction IV-4 and pasteurized at 60 ° C for 10 h, PDP Labs lz RPMI 1640 medium no. 320-1875AJ, GIBCO13 Dulbecco's modified Eagle's medium (DMEM) medium no. 320-1965AJ 13 Fetal bovine serum (FBS), no. 0268-00, Armour14 Penicillin-streptomycin, solution, no. 600-5070AG 13

l Presentaddress: Departmentof ChemicalEngineering, HowardUniversity, 2300 6th Street, N.W., Washington, D.C. 20059 39

40

YOUNG Insulin-transferrin-sodium selenite supplement (ITS), no. 1074547, Boehringer Mannheim is Pryogen-free water, Milli-Q water system, no. ZP2011584, 115v/60HZ, Millipore 16 Trypan blue stain, 0.4%, no. 630-5250AG 13 70% ethanol Phosphate buffered saline (PBS), no. 310-4287AJ lz Cohn fraction IV-4, American Red Cross plasma fractionation process, ARC t7 Normal mouse IgG, no. 6011-0080, Organon TM Goat anti-mouse Ig, unlabeled, no. 1010-01, Southern Biotechnology 19 Bovine serum albumin (BSA), no. A-6793, Sigma2° Tween-20, no. P-1379, Sigma2° Goat anti-mouse Ig, alkaline phosphatase labeled, no. 1010-0419 p-Nitrophenylphosphate C. Supplies 96-well tissue culture plates, no. 256-7393 Tissue culture flasks, 75 cm2, no. 25111, Corning 21 Tissue culture flasks, 25 cm2, no. 2510621 1-ml pipets, no. 4012, Costar22 5-ml pipets, no. 405122 10-ml pipets, no. 410122 Yellow pipettor tips, no. RT209 Blue pipettor tips, no. RT2009 25-ml pipets, no. 42512~ 15-ml tubes, no. 2531921 50-ml tubes, no. 2533021 1.5 ml Eppendorf tubes, no. 72-690, Sarstedt 2s Hemacytometer, no. 02-671-5, Fischer zg 1.8 ml Nunc tubes, no. 36675-093 Millex-GV 0.22 #m filter unit, no. SLGV025LS 16

III.

PROCEDURE

A. Preparation of complete media and reagents 1. Heat inactivation of FBS a. The 500-ml bottle of FBS is thawed, and placed at 56 ° C for 30 rain. b. The bottle is placed in a nonfrost free freezer at - 2 0 ° for storage. 2. Preparation of ITS solution a. Dissolve 50 mg ITS powder in 5 ml sterile distilled water. b. Filter through a sterile filter into a 15-ml tube. c. Store at 4 ° C. d. ITS solution is added to medium just before using for cells at a concentration of 1/.tg of ITS/ml of medium. 3. Preparation of complete medium with FBS (RPMI 1640 + 5% FBS or DMEM medium + 10% FBS) To 500 ml of RPMI 1640 add the following: a. 5 ml of FBS (for 1%), 25 ml (for 5%) or 50 ml (for 10%) b. 5 ml of penicillin/streptomycin solution 4. Preparation of complete medium with GEMS To 500 ml of RPMI 1640 or DMEM add the following: a. 10 ml of a 50 mg/ml solution of GEMS for a final

concentration of 1 rng/ml or 1 ml of a 50 mg/ml solution of GEMS for a final concentration of 0.1 mg/ml or 0.1 ml of a 50 mg/mt solution of GEMS for a final concentration of 0.01 mg/ml. b. 5 ml of penicillin-streptomycin solution B. Growing cells in complete medium with FBS 1. Remove the Nunc tube containing the cells from liquid nitrogen storage (stored at 2 to 3 X 107 ceils per tube) and thaw the tube in a 37 ° C water bath. 2. With a P-IO00 pipettor, and autoclaved tips, remove the thawed cell suspension from the tube and place into a sterile 15-ml tube containing 10 ml of complete medium containing FBS. 3. Pellet the cells at 1000 rpm for 5 min in a Mistral 3000i centrifuge. 4. Pour off the media. 5. Resuspend the pelleted cells in 10 ml of complete medium containing FBS and ITS (1 #g/ml). Place into a 75-cm 2 flask. 6. The next day (16 to 24 h) cells are counted using a hemacytometer (8) and seeded at a density of 6 × t04 cells/ml. To seed the ceils, an aliquot is removed from the flask that contains the desired number of cells and placed into a new flask with complete medium at a total volume of 20 ml. C. Adapting ceils growing in complete medium containing FBS to grow in complete medium containing GEMS 1. Initial seeding density is 6 × 104 cells in 20 ml of medium, 90% of which is medium containing FBS and 10% is medium containing 1 mg/ml GEMS (90/10 medium). 2. Cells are transferred to a new flask with fresh 90/10 medium every 7 days, but counted every 2 to 3 days to monitor growth. 3. After cells are adapted to 90/10 medium as defined by growth to ~1 × 107 cells in 20 ml, they are seeded at 6 X 104 cells in 20 ml of medium containing 75% of complete medium containing FBS and 25% of complete medium containing 1 mg/ml GEMS (75/25 medium). 4. After cells are adapted to 75/25 medium as defined by growth to >1 )< 107 cells in 20 ml, they are seeded at 6 X 104 cells in 20 ml of medium containing 50% of complete medium containing FBS and 50% of complete medium containing 1 mg/ml GEMS (50/50 medium). 5. When cells have reached a density o f > l × 10 r ceils in 20 ml of 50/50 medium, they are seeded at 6 X 104 cells in 20 ml of medium containing 25% of complete medium containing FBS and 75% of complete medium containing 1 mg/ml GEMS (25/75 medium). 6. After cells are adapted to 25/75 medium, they are seeded at 6 )< 104 cells in 20 ml of medium containing 10% of complete medium containing FBS and 90% of complete medium containing 1 mg/ml GEMS (10/90 medium). 7. Cells are finally seeded into complete medium consisting of GEMS at 1 mg/ml with no added FBS. We have observed that the length of time required for

YOUNG adaptation of cells from growth in a serum supplement, such as FBS, to growth in GEMS takes approximately 1 mo., but is highly cell line dependent. The longest period is for adaptation from FBS to the 90/10 medium. Cells are counted every 2 to 3 days, and as soon as they have reached their maximum density they are seeded in the next higher concentration of GEMS. D. Enzyme linked immunoassay (ELISA) for IgG production 1. Prepare a diluted solution of goat-anti-mouse Ig (1 mg/ml stock) by adding 60 #1 of stock to 20 ml of PBS for a final concentration of 3 #g/ml. 2. Coat a 96-well plate with antibody by pipetting 100 #1 of diluted goat-anti-mouse Ig (no. 1) into wells of a 96-well plate. Seal top of plate with the plastic cover which comes with each package of plates, and keep the plate at 4 ° C overnight. Plates may be stored at 4 ° C for up to 1 mo. 3. The next day, keeping all samples on ice, prepare medium samples to be tested by performing serial dilutions of each sample into PBS/I% BSA as follows: 100 #1 into 900/.tl (hl0); 100 gl of 1:10 dilution into 900 #1 (1:100); and 200 #1 of the 1:100 dilution into 800 #1 (1:500). 4. To generate a standard curve, make an initial 1:200 dilution of a stock of normal mouse IgG (0.26 rag/ ml) by adding 5 #1 of the stock solution to 995 #1 of PBS. Dilute this solution (1.3 ~tg/ml) 1:2 serially by transferring 500 #1 to 500 #1 of PBS. This is repeated 9 additional times for a lowest concentration of 1.25 ng/ml. 5. Remove goat-anti-mouse Ig from the wells of the 96-well plate by inverting the plate and tapping inverted plate on paper towels. 6. Using an 8-tip multi-pipettor, add 200 #1 of PBS/

I H 5 B 7 Cell C u m u l a t i v e

41 Cumulative IgG P r o d u c t i o n by 1H5B7 Cells 50001 1 - 0 ~ tlss

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FIG. I. 1H5B7 hybridoma cellcumulative growth curve. A mouse hybridoma cellline producing antibody againsthuman factorIX, adapted to growth in GEMS, was seeded into 75-cm 2 flasksat a density of 6 × 104 cells/ml in 3 0 ml RPMI medium in the presence of either 1.0 (O - - - O), 0.1 (1"3- - - n ) , or 0.01 (xy _ _ _ V) m g / m l of GEMS. Medium was not changed during the 3 0 days, but atiquots were removed and assayed for cell growth and viability every 2 to 3 days. Cells were reseeded at the starting density every 7 days for 1 mo. A flask containing cells in 5 % FBS (O - - - 0 ) was also maintained as a control.

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FIG. 2. Cumulative IgG productionby IH5B7 cells.Cellsseeded in the flask described in the legend to Fig, l were also assayed for antibody production.Every 2 to 3 days medium was removed and debrispelletedby centrifugationat 1000 rpm for 5 rain in the Mistral 3000i centrifuge. Supernatantwas storedat -20 ° C untilassayed for the presence of mouse Ig by ELISA as described under Procedure. Symbols as for Fig. I.

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1% BSA to each well and incubate at 37 ° C for 20 min. Wash wells once with 200 #1 of PBS/I% BSA per well. Wash wells 3 times with 200 ~tl PBS/0.1% Tween 20 per well. Add 100 #1 of samples and standard curve to individual wells and incubate covered plate at 37 ° C for 2 h (capture step). Prepare a solution of alkaline phosphatase-conjugated goat anti-mouse Ig: add 10 #1 of stock solution (0.2 mg/ml) to 10 ml of PBS to give a 0.2 #g/ml solution. Remove samples and standard curve samples from wells by inverting plate and rinse wells 3 times with PBS/0.1% Tween 20. Add 100 #1 of diluted antibody-conjugate solution to each well and incubate covered plate at 37 ° C for 2 h (detection step). Prepare the substrate solution by dissolving 50 mg of p-nitrophenylphosphate in 50 ml of substrate buffer (0.1 M glycine; 1 mM MgC12; 1 mM ZnCI2; pH 10.4). Wash wells 3 times with PBS/0.1% Tween 20. Add 100 ttl of substrate solution to each well and incubate at room temperature for 15 min. After 15 min, or when the highest concentration of substrate is approximately 0.6 at o.d. 405 nm, read all samples in the plate reader.

DISCUSSION

The hybridoma cell line 1H5B7, which produces antibody against human Factor IX (9), was adapted to growth in RPMI medium containing GEMS from RPMI medium containing 5% FBS, as described under Procedure. Cell growth and antibody production by the adapted cells were

42

YOUNG then compared to nonadapted ceils growing in 5% FBS. Figure 1 shows a cumulative growth curve of the 1H5B7 ceil line, in the presence of different concentrations of GEMS or 5% FBS. Cells growing in FBS reached 1 × 106 ceUs/ml in a 4 to 5-day period, a density that led to a subsequent decrease in cell viability, requiring reseeding at a lower density. The ceils growing in GEMS grew at a much slower rate and did not go through a period of decreased viability as a result of too high a cell density. Figure 2 shows the cumulative human Ig production over 30-days. Although the cells growing in GEMS grew at a slower rate, they produced a similar amount of antibody on a per cell basis as the cells growing in the presence of FBS.

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Time (day) FIC. 4. 88.61 Cell growth curve and antibody production by 88.61 cells. A hybridoma cell line producing antibody against human protein C was seeded into 75-cm2 T flasks in DMEM medium in the presence of either 1.0 mg/ml GEMS ( © - - - ©) or 10% FBS ( @ - - - @) as described under the legend for Fig. 1. Medium was prepared for assay of mouse Ig as described under the legend for Fig. 2.

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Time ( d a y ) FIG. 3. 52.53 Cell growth curve and antibody production by 52.53 cells. A mouse hybridoma cell line producing antibody against a 41-kDa fragment of human factor VIII (amino acids 1974-2332) was seeded into 75-cm2 T flasks in RPMI medium in the presence of either 1.0 mg/ml GEMS (O - - - ©) or 5% FBS (@ - - - @) as described under the legend for Fig. 1. Mediumwas prepared for assay of mouse Ig as described under the legend for Fig. 2.

Cell growth and antibody production by two other hybridoma ceil lines are shown in Figs. 3 and 4. Cell line 52.53, producing antibody against a 41 kDa fragment of human Factor VIII (amino acids 1974-2332), was adapted to growth in GEMS as described in Procedure. Figure 3 shows that the GEMS-adapted 52.53 ceils grew at a slower rate than the nonadapted line growing in 5% FBS, but produce more than twice as much antibody per cell as the cells growing in FBS. A similar experiment was performed with the hybridoma cell line 88.61, producing anti-human protein C antibody. Ceil growth in the presence of DMEM containing GEMS was consistently low over a 21-day period as shown in Fig. 4, top. Antibody production by 88.61 cells growing in GEMS was similar to production by the nonadapted cell line growing in DMEM with 10% FBS (Fig. 4, bottom).

YOUNG

43

Concurrent G).ucose Consumptio= a n d IgG Production by 88.61 Cells in Bioreactor -12

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was equal to the rate of antibody production of the same cell line growing in medium containing FBS. GEMS was also found to support antibody production by the 88.61 hybridoma line growing in a SETEC bioreactor. The ability of GEMS to support a slower rate of cell growth as compared 1o the growth rate in medium containing FBS, while permitting the same rate of antibody production, could be useful for commercial production of antibody in bioreactors where a slow rate of growth is desirable. The lower protein concentration of medium with GEMS, especially at the lower concentrations (0.1 mg/ml) as compared to the protein concentration in medium with 5 % FBS (2.5 mg/ml), could also be advantageous in later protein purification steps. The use of a pasteurized product will eliminate potential viral contamination as shown by inactivation of encephalomyocarditis or sindbis viruses which were added to similarly prepared Cohn fraction IV-4 before heat treatment (data not shown).

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'rime (day) FIG. 5. Concurrent glucose consumption and lgG production by 88.61 cells in a bioreactor. A mouse hybridoma cell line, 88.61, producing antiprotein C antibody, was seeded in a SETEC hollow fil~r bioreactor at 7.6 × l0 s cells/ml in 3 ml of DMEM containing 1 mg/ml of GEMS. Medium containing GEMS was passed through the bioreactor at a flow rate of 50 ml/min. Cells were maintained in culture for 22 days with a total media volume of 300 ml. Every 3 to 4 days samples were removed for antibody (© - - - ©) and glucose (@ - - - @) analyses.

To determine the ability of GEMS to support cell growth and antibody production in a bioreactor that could be used for commercial production of antibody, 2.3 × 106 hybridoma cells already adapted to growth in GEMS (cell line 88.61) were seeded in a SETEC Tricentric Bioreactor. 7 This cell line is used for production of the anti-protein C antibody used for immunoaffinity purification of human protein C. Total antibody produced during the 22days was 4 rag, or 188 # g / m l . Figure 5 shows glucose depletion and antibody increase over the 22-day period. These results demonstrate that pasteurized Cohn fraction IV-4 (GEMS) can be used for growth and antibody production by hybridoma cells. For all of the cell lines tested, the growth rate of cells in GEMS was lower than the growth of the nonadapted cell line in medium containing FBS. Antibody production in the presence of GEMS

V.

REFERENCES

1. Clark, D. B.; Drohan,W. N.; Miekka,S. I., et al. Strategyfor purificationof coagulation factor concentrates. Ann. Clin. Lab. Set. 19:196-207; 1989. 2. Cohn, E. J.; Strong, L. E.; Hughes, W. L. Jr., et al. Preparation and properties of serum and plasma proteins. IV. A system for separationof the protein and lipoproteincomponentsof biologicaltissues and fluids. J. Am. Chem. Soc. 68:459-474; 1946. 3. Kistler,P.; Friedli,H. Ethanolprecipitation.In: Curling,J. M., ed. Methods of plasma protein fractionation.New York: AcademicPress; 1980:315. 4. MacLeod,A. J. The recoveryof proteins useful in animalcell culture from human plasma protein fractions. Dev. Biol. Stand. 66:562; 1987. 5. MacLeod, A. J. The use of plasma protein fractions as medium supplements for animal cell culture. Adv. Biochem. Eng. 37:56; 1988. 6. MacLeod,A. J.; Thomson,M. B. Use of plasma protein fractionsas serum substitutes for in vitro cell culture. Dev. Biol. Stand. 60:55-61; 1985. 7. Ng, P. K.; Dobkin, M. B. Cell growth media supplement. United Stales Patent 4,452,893; 1984. 8. Phillips,H. J. Dye exclusiontest for cell viability.In: Kruse, P. F.; Paterson, M. D., Jr., eds. Tissueculture methodsand applications.New York: Academic Press; 1973:406-408. 9. Tharakan,J.; Strickland,D.; Burgess, W., et al. Developmentof an immunoaff/nity process for factor IX purification. Vox. Sang. 58:21-29; 1990.

The authors thank Dr. Henryk Lubon for discussion and critical evaluation of the manuscript.

l Roberts Oxygen, Merrifietd,VA z Mid-AtlanticLaboratory EquipmentCompany,Germantown,MD 3 Curtis MathesonScientific,Inc., Jessup, MD 4 Forma Scientific,Marietta, OH s VWR Scientific,Bridgeport, NJ 6 Nikon, Garden City, NY 7 SeparationEquipmentTechnologies,Inc., Livermore,CA s DrummondScientific,Broomall, PA 9 Rainin, Woburn, MA w MolecularDevices, MenloPark, CA l~ IBM, Bohdan, Gaithersburg, MD 12Plasma Derivativesand ProductionLaboratories,Jerome H. Holland Lab, AmericanRed Cross, Rockville,MD

Is GIBCOLaboratories Life Technologies,Inc., Grand Island, NY 14Armour Laboratories, Inc., Tarrytown, NY is BoehringerMannheim,GmbH, Germany(Indianapolis,IN) 16MilliporeCorporation, Bedford, MA t~ Plasma Derivatives,Jerome H. Holland Lab, AmericanRed Cross, Rockville, MD is OrganonTeknike Corporation,Westchester, PA 19Southern BiotechnologyAssociation,Birmingham,AL 2oSigma ChemicalCorporation,St. Louis, MO ~l Coming Glass Works, Coming, NY 22 Costar, Cambridge, MA ~3Sarstedt, Newton,NC 2¢Fischer Scientific,Malvern,PA

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