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Alternatives to Monoclonal Antibody Production (Proceedings)

Practical Applications and Comparison of Ascites and In Vitro Methods for MAB Production

Norman C. Peterson, DVM, MS and Jennifer E. Peavey
Department of Pathology & Laboratory Medicine
 University of Pennsylvania
 Philadelphia, PA 19004

Economic, technical, legislative, and ethical issues influence decisions relevant to alternatives to the use of animals in biomedical research. Since the development of hybridoma technology, the production of ascites in mice has been a popular method for the generation of high concentrations of monoclonal antibodies . However, the availability of several in vitro methods which can be performed in most laboratories makes alternative means to monoclonal antibody production an attractive option. In order to evaluate the practicality of using in vitro techniques, three tissue culture methods and ascites production were compared on the basis of yield, material costs, and time commitment. The results presented demonstrate that the time and material costs to produce monoclonal antibody 7.16.4 by ascites in irradiated mice was similar to that of tissue culture in standard plastic flasks and hollow fiber cartridge bioreactor. Tissue culture in gas permeable bags was slightly less expensive than these methods in terms of cost and time. When ascites production in irradiated mice was compared to plastic tissue culture flasks for monoclonal antibody 225, the in vitro method was approximately five times more expensive than ascites production. The examples presented in this report outline factors which need to be considered and evaluated when making choices among monoclonal antibody production methods. Furthermore, these results imply that in vitro technologies for the production of monoclonal antibodies can be adapted to most conventional laboratories in order to provide sufficient resources for the most commonly performed experimental protocols.

This study was initiated when our laboratory required gram quantities of monoclonal antibody (mAb) 7.16.4 for another experimental protocol. In order to efficiently produce this amount, three different in vitro methods were used, and the results were compared to previous data we had obtained for mAb production by ascites induction in mice. Smaller quantities of mAb 225 and 528 were also produced and are included for comparison. Hybridoma cell-lines vary greatly in the amount of mAbs they produce and how they respond to different culture environments(1). Although this analysis was somewhat limited, our results with in vitro techniques for mAb production were favorable, and we believe the approaches and principles exemplified here can be applied to other laboratory settings with similar outcomes.

Data from 27 irradiated Balb/c mice inoculated with 7.16.4 hybridomas and 5 mice inoculated with 225 hybridomas revealed that each mouse produced an average mAb yield of 2.28mg and 4.53mg, respectively. These results are lower than some reports which list average yields of 10-20mg per mouse(2)(3)(4) This may be partially due to lack of ascites accumulation in a significant percentage of mice and several mice were low producers. These mice were included in our estimates to accurately reflect animal use for this approach. Additionally, our IACUC guidelines for monoclonal antibody production were followed in that only two ascites harvests were taken. Some earlier studies report as many as seven taps with mice remaining in ascites production protocols until death (2). These and similar aggressive approaches, although less humane, may result in higher mAb yields.

In order to fairly compare in vivo to in vitro techniques the following parameters should be established: (1) the total number of mice inoculated with hybridoma cells to produce the mAbs (2) the number of taps that were taken and the extent to which the ascites was allowed to progress (were IACUC guidelines followed?) (3) the method of mAb quantification.

The simplest approach to producing mAbs in vitro is to adapt the hybridoma cell lines to serum-free media(5). MAbs can then be directly purified from the culture media by affinity chromotography with protein G(6). The concentration of mAb 7.16.4, 225, and 528 in the media supernatant taken from cultures grown in a standard 225 cm2 plastic tissue culture flask ranged from 2.85-9.38 µg/ml. Although this is about 100 fold more dilute than the ascites mAb concentrations, affinity purification of 300 ml volumes yielded significant quantities of high quality, concentrated antibodies. This demonstrates that limitations imposed by methods which produce mAbs at low concentrations can be over-come by affinity chromotography. If further concentration of the product is desired, the elutant can be concentrated by centrifugal filtration concentration devices.

Hybridoma cells can also be grown in gas permeable bags (Lifecell) (Baxter, Fenwell Div., Deerfield, IL). The increased availability of dissolved oxygen permits these cells to grow at higher density than when grown in plastic tissue culture flasks and this results in increased concentrations of secreted mAb in the media supernatant (7). The mAb concentration of tissue culture supernatants from hybridomas cultured in gas permeable bags were 70% (mAb 7.16.4) and 24% (mAb 225) higher than supernatants from conventionally cultured hybridomas.

The third in vitro method we analyzed for the production of mAb 7.16.4 was the hollow fiber cartridge (HFC) bioreactor (Cellco, Germantown, MD). This method produced the highest concentration of mAb 7.16.4 (0.45 mg/ml) of all the in vitro methods. The disadvantage of using the HFC bioreactor to produce mAbs is the relatively large initial investment in the system ($800-$1200). Additionally, cells grown at high concentrations in the HFC are vulnerable to rapid changes in lactic acid and this metabolite should be monitored daily during at least the first few weeks of operation. The initial investment and technical familiarity required to optimize mAb production from the HFC bioreactor make it more suitable for large scale production (>100mg).

Although the concentration of the mAbs produced by each of the methods described did not affect our ability to obtain good quality affinity purified mAbs, the concentration of the supernatants influences the material costs and time necessary for processing. In order to compare the material costs of each method, we used the data presented above to estimate amount of materials necessary to produce 100mg of mAb 7.16.4 and mAb 225 by in vivo and in vitro approaches. Estimates of material costs necessary to produce 100mg of 7.16.4 were similar for ascites, tissue culture flask, and HFC bioreactor methods. The gas permeable bags were the least expensive by a small margin. This was likely attributed to 70% gain in mAb 7.16.4 concentration which resulted in less media being expended. If athymic mice were used instead of irradiated mice, ascites would have been the most costly method to produce mAb 7.16.4.

MAb 225 yields from ascites were greater than mAb 7.16.4, however 225 hybridomas did not grow as well in the serum free culture media. These differences were reflected in a five fold higher material cost to produce mAb225 by tissue culture than ascites. The 24% gain in mAb 225 concentration was not significant enough to off-set the slightly higher price of the gas permeable bags, and, hence, the material costs of culturing 225 hybridomas in plastic flasks or gas permeable bags was similar. The time commitment and labor cost profiles to produce 100mg of each mAb paralleled those of the material costs. This was not surprising as both the material and labor costs of the in vitro methods were highly influenced by the amount of media which was purchased and processed.

In summary, we found that in vitro methods could produce sufficient quantities of mAb 7.16, 528 and 225 to meet our laboratory needs. Additional costs were not incurred to produce mAb 7.16.4 by in vitro methods, whereas, the tissue culture approach to producing mAb 225 was five times more expensive. Although the production results of other hybridomas in other laboratories is likely to vary, this paper and many others demonstrates that in vitro techniques are a practical means of producing mAbs(8)(9)(10)(11)(12). As the scientific community becomes more aware of these approaches and the resources available, in vitro techniques will likely replace animals as the standard approach to producing mAbs.

(Information presented in this report has been submitted in greater detail for publication in Contemporary Topics in Laboratory Animal Science - 10/27/97)

References

  1. Tarleton, R. and Beyer, A. (1991). Medium-scale production and purification of monoclonal antibodies in protein-free medium. BioTechniques. 11(5): 590-593.
  2. Brodeur, B., P, T. and Y, L. (1984). Parameters affecting ascites tumour formation in mice and monoclonal antibody production. J Immunol Methods. 71(2): 265-72.
  3. Mueller, U., Hawes, C. and Jones, W. (1986). Monoclonal antibody production by hybridoma growth in Freund's adjuvant primed mice. J. Immunol. Methods. 87(2): 193-196.
  4. Hoogenraad, N., Helman, T. and Hoogenraad, J. (1983). The effect of pre-injection of mice with pristane on ascites tumor formation and monoclonal antibody production. J Immunol. Methods. 61(3): 317-20.
  5. Hoover, C. and Martin, R. (1990). Antibody production and growth of mouse hybridoma cells in nutridoma media supplements. BioTechniques. 8(1): 76-82.
  6. Akerstrom, B., Brodin, T., Reis, K. and Bjorck, L. (1985). Protein G: a powerful tool for binding and detection of monoclonal and polyclonal antibodies. J. Immunol. 135: 2589-2592.
  7. Miller, W., Wilke, C. and Blanch, H. (1987). Effects of dissolved oxygen concentration on hybridoma growth and metabolism in continous culture. J Cell Physiology. 132(3): 524-30.
  8. Evans, T. and Miller, R. (1988). Large-scale production of murine monoclonal antibodies using hollow fiber bioreactors. BioTechniques. 6: 762-767.
  9. Falkenberg, F., Weichert, H. and Krane, M. (1995). In vitro production of monoclonal antibodies in high concentration in a new and easy to handle modular minifermentor. J. Immunol. Methods. 179(1): 13-29.
  10. Federspiel, G., McCullough, K. C. and Kihm, U. (1991). Hybridoma antibody production in vitro in type II serum-free medium using Nutridoma-SP supplements. comparison with in vivo methods. Journal of Immunological Methods. 145(1-2): 213-221.
  11. Jackson, L. R., Trudel, L. J., Fox, J. G. and Lipman, N. S. (1996). Evaluation of hollow fiber bioreactors as an alternative to murine ascites production for small scale monoclonal antibody production. J. Immunol. Methods. 189: 217-231.
  12. Peterson, N. and Peavey, J. (1998). Practical applications of in vitro monoclonal antibody production. Contemporary Topics Lab Animal Sci. : (submitted for publication)

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