Dialysis-based bioreactor systems for the production of monoclonal antibodies—alternatives to ascites production in mice (original) (raw)
2002, Journal of Immunological Methods
Two commercially available bioreactor systems, CELLine and miniPERMk, were evaluated for their ability to support the production of monoclonal antibody (mAb) from a variety of murine hybridoma cell lines. Production and purity of mAbs were compared between the two systems and with mouse ascites tumour fluid generation. The quality and purity of the mAb generated by each method was analysed on SDS-PAGE gels and the antibody immunoreactivity in each case was quantified by indirect ELISA tests. The relative benefits of conventional growth medium (Dulbecco's modified Eagle's media, DMEM) and serum-free medium (hybridoma serum-free media, H-SFM) using the miniPERMk system were also analysed, in terms of the amount of antibody produced, cell concentration and specific antibody titre. In all cases, the CELLine units tested gave higher protein concentrations compared to the miniPERMk system under the same conditions (means and 95% confidence limits are 4.2 F 0.8 and 2.1 F 0.8 mg/ml, respectively), yet the miniPERMk system yielded greater total amounts over a similar culture period (428.7 F 243.3 mg compared to 183.3 F 100.9 mg in the CL-350 CELLine unit). When defined by specific ELISA titre, both bioreactor systems yielded mAb levels that compared favourably with those derived from ascites. In addition, SDS-PAGE analysis indicated that the bioreactor antibody product was relatively free of contaminating protein, whereas ascites tumour fluid preparations displayed significant levels of extraneous protein. This study has shown that both bioreactor systems are acceptable in vitro alternatives to the in vivo production of mAbs in mice.
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Journal of Immunological Methods, 1985
A single-step method for purification of mouse monoclonal antibodies directly from ascitic fluids using hydroxylapatite column chromatography is described. The procedure yields highly purified IgG or lgM antibodies. The purified immunoglobulin is essentially free of contaminating mouse albumin, transferrin, and other ascites proteins, as determined by SDS-polyacrylamide gel electrophoresis. Hydroxylapatite chromatography can also separate monoclonal IgG antibodies from contaminating IgG antibodies found in ascites fluid of animals that have been immunosuppressed prior to ascites induction. Furthermore, the evidence presented here suggests that some hybridomas of SP2/0 origin synthesize an extraneous light chain resulting in the secretion of hybrid antibody molecules.
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Production of monoclonal antibodies in serum free medium
Journal of Immunological Methods, 1980
We have succeeded in growing several established hybridoma cell lines (1116NS-19; l116NS-33a; ll16NS-52a; 1083-17-1A; 691-19-19) in a serum free medium supplemented with physiological concentrations of insulin (5 pg/ml) and transferrin (5 pg/ml). The hybridoma cells replicate in this medium and will secrete monoclonal antibodies in quantities comparable to those produced in the presence of serum. Adapted hybridoma cultures have been secreting antibodies in the supplemented serum free (SSF) medium for more than 3 months. Parental mouse myeloma P3x63Ag8 cells and non-secreting subclone 653 cells cannot survive longer than 6 days under the same conditions.
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Monoclonal antibodies (mAb) are important reagents used in biomedical research, in diagnosis of diseases, and in treatment of such diseases as infections and cancer. These antibodies are produced by cell lines or clones obtained from animals that have been immunized with the substance that is the subject of study. To produce the desired mAb, the cells must be grown in either of two ways: by injection into the abdominal cavity of a suitably prepared mouse or by tissue culturing cells in plastic flasks. Further processing of the mouse ascitic fluid and of the tissue culture supernatant might be required to obtain mAb with the required purity and concentration. The mouse method is generally familiar, well understood, and widely available in many laboratories; but the mice require careful watching to minimize the pain or distress that some cell lines induce by excessive accumulation of fluid (ascites) in the abdomen or by invasion of the viscera. The tissue-culture method would be widely adopted if it were as familiar and well understood as the mouse method and if it produced the required amount of antibody with every cell line; but culture methods have been expensive and time-consuming and often failed to produce the required amount of antibody without considerable skilled manipulation. However, culture methods are now becoming less expensive, more familiar, and more widely available
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Large-scale production of monoclonal antibodies in suspension culture
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