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Alternatives to Monoclonal Antibody Production (Proceedings)

Monoclonal Antibody Production in Gas-Permeable Tissue Culture Bags Using Serum-free Media

J.R. Heidel and B.V. Stang
College of Veterinary Medicine
 Oregon State University
 Corvallis, OR 97331-4802

Cultivation of hybridoma cells in flexible, gas-permeable (G-P) tissue culture bags is not novel, but their use with serum-free media has not been explored. We measured the growth dynamics and monoclonal antibody yield in serum-free media by four different hybridomas using these bags.

Materials and Methods

Four hybridomas, 290 (anti-blue tongue virus, IgG2a; Heidner et al., 1988); D5 (anti-Aeromonas salmonicida, IgG2b; Rockey et al., 1991); 4D3 (anti-Renibacterium salmoninarum, IgG1; Wiens and Kaattari 1989); and E5 (anti-infectious pancreatic necrosis virus, IgG1; Caswell-Reno et al., 1989), were grown to a density of 8 X 105to 1.5 X 106 cells/ml (90 to 100% viability) in 25 ml Hybridoma Serum-Free Media (SFM; Life Technologies) supplemented with 2 mM L-glutamine in 75 cm2 vented tissue culture flasks (Corning). The cells were then seeded into G-P bags (Lifecell Tissue Culture Flask, Baxter Biotech North America) at 0.6 X 105 to 2 X 105 cells/ml, in 465 mls of SFM, via a 60 cc syringe barrel (Sherwood Medical) attached to the open female port. Vented 75 cm2 rigid tissue culture flasks were seeded for comparative analysis. All cultures were incubated at 37°C in a humidified 5% (v/v) CO2 atmosphere for 3 to 5 weeks. A sampling site coupler (Baxter Biotech North America) inserted into a bag's injection port allowed periodic sampling for cell counts and antibody assay.

At the end of incubation, media and cells were collected by opening the bag's male port and allowing the media to drain from the bag. Cells were removed by centrifugation (6,000 x g for 15 min) and the supernatant was concentrated using dialysis tubing (6-8,000 MWCO; Spectrum Medical Industries) covered with polyethylene glycol (Sigma). The final 30-50 ml was further concentrated and desalted into phosphate buffered saline (pH 7.0) with a Centriprep-30 concentrator (Amicon). Antibodies were purified using Affi-Gel Protein-A agarose MAPS II chromatography kits (Bio-Rad) according to provided instructions. Antibody isotype and concentration were determined by a sandwich ELISA, using the following standards: mouse IgG1 k, IgG2a k and IgG2b k (Sigma). Monoclonal antibody activity was assayed as previously described, with minor modifications (Caswell-Reno et al. 1989; Arkoosh and Kaattari, 1990; Reddington et al., 1991; Rockey et al., 1991).

Results

The results for hybridoma 290 are typical, although antibody yield (?g/ml) was hybridoma dependent (Table 1). Live-cell concentrations in G-P bags demonstrated an initial period of rapid growth, and maximum mean live-cell concentrations (0.7 ? 0.3 X 106 live-cells/ml) were reached within 4 days of culture. Viable cell numbers diminished slowly to 0.1 X 106 cells/ml at approximately 25 days. Compared to rigid flasks, live cell concentrations in G-P bags peaked at similar times, but maximum cell densities were more than two-fold lower in bags. However, live cell concentrations decreased more rapidly in flasks, with no viable cells present in rigid flask cultures after Day 11. In contrast, live cells were till found in G-P bags after 30 days.

Antibody concentrations in G-P bags increased rapidly during the first week of culture and reached and maintained maximum concentrations after approximately 15 days. Gas-permeable bags took 5 to 10 days longer to produce maximum concentrations of IgG than flasks, but yielded more IgG per milliliter of supernatant than flasks (201.4 µg/ml vs 154.8 µg/ml respectively).

Discussion

When compared to traditional tissue culture flasks, G-P bags produced more total antibody while utilizing less incubator space and labor. Although flasks achieved maximum levels of antibody earlier than G-P bags, concentrations of IgG/ml tended to be higher in gas-permeable bags. In addition, gas exchange constraints limit the volume of cell suspension that can be placed into flasks (25 ml vs 465 ml; flask vs bag), significantly limiting total yield. To achieve the same total yield of antibody as one 1L G-P bag, eighteen 75 cm

2

tissue culture flasks would have to be seeded and incubated.

Although the media of choice may be hybridoma dependent, consistent results were obtained using serum-free media with these hybridomas. As expected, antibodies grown in serum-free media were easier to purify and concentrate as they did not contain contaminating proteins (fetal bovine serum). The hybridomas grew well in serum-free media without prior adaptation. Serum-free media resulted in better antibody production when compared to the same cells grown in RPMI supplemented with FBS.

We suggest routine planting of G-P bags at a concentration of approximately 1.00 X 105 cells per ml in serum-free media. Seeding at this density allows reasonable numbers of stock cultures (2-3 75-cm2 flasks per bag) to be grown, and results in maximal antibody production within two to three weeks. Planting cells at densities as high as 3 X 106 cells/ml results in higher antibody concentrations, but this is accompanied by increased difficulty in growing sufficient numbers of viable seed cells in rigid flasks, together with increased need for incubator space.

The use of flexible, gas-permeable tissue culture bags is a reasonable alternative to the use of ascitic tumors for monoclonal antibody production. Although adaptable to use with automated seeding and media transfer systems, we were able to manually seed G-P bags easily and inexpensively using a syringe barrel and gravity flow. Materials required for gas-permeable bag culture are commercially available, as are complete kits. The gas-permeable property of the bags allows the use of standard laboratory incubators (5% CO2 v/v, 37°C). In addition, as the bags are essentially a closed system after inoculation, risk of bacterial contamination is reduced. In this study, no antibiotics were used in the growth media and contamination was rare even in bags allowed to incubate more than 40 days and sampled on a daily basis.

In addition to these technical considerations, the G-P bags can provide an economic advantage. At our university, the cost for ascites-derived antibodies is a minimum of $6.50/mg, and can easily range from $10.00 to $20.00/mg. Antibody produced in the G-P bags costs $0.42 to $1.76/mg.

a DiagXotics Inc., Wilton, CT
Acknowledgment: This project was funded by grants from the Alternatives Research and Development Foundation, Eden Prairie, MN.

References

Arkoosh, M.R. and Kaattari, S.L. (1990) Quantitation of fish antibody to a specific antigen by an enzyme-linked immunosorbent assay (ELISA). In: Techniques in Fish Immunology, (eds. Stolen, J.S., Fletcher, T.C., Anderson, D.P., Roberson, B.S., and Van Muiswinkel, W.B.), SOS Publications, Fair Haven, NJ, pp 15-24.

Caswell-Reno, P., Lipipun, V., Reno, P.W. and Nicholson, B.L. (1989) Use of a group-reactive and other monoclonal antibodies in an enzyme immunodot assay for identification and presumptive serotyping of aquatic birnaviruses. J. Clin. Micro. 27: 1924-1929.

Heidner, H.W., MacLachlan, N.J., Fuller, F.J., Richards, R.G. and Whetter,L.E. (1988) Bluetongue virus genome remains stable throughout prolonged infection of cattle. J. Gen. Virol. 69: 2629-2636.

Rockey, D.D., Dungan, C.F., Lunder, T., and Rohovec, J.S. (1991) Monoclonal antibodies against Aeromonas salmonicida lipopolysaccharide identify differences among strains. Dis. Aquat. Org. 10: 115-120.

Wiens, G.D. and Kaattari, S.L. (1989) Monoclonal antibody analysis of common surface proteins (s) of Renibacterium salmoninarum. Fish Pathol. 24: 1-7.

Table I

Comparison of maximum antibody production (µg/ml; Mean ± SD) of hybridoma cultures planted in gas-permeable bags and 75 cm2 rigid tissue culture flasks. All bags and flasks seeded at densities between 0.6 X 105 and 2 X 105 live cells per ml in SFM. Number in parentheses refers to number of replicates.

Hybridoma290D54D3E5
Bag201.4 ± 63.7 (6)65.2 ± 14.5 (5)32.8 ± 2.6 (3)82.9 ± 73.0 (3)
Flask154.8 ± 45.8 (4)39.0 ± 6.8 (3)48.3 ± 7.6 (2)16.2 ± 1.3 (2)

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