Hybridoma cell lines, production

In 1975, the first successful production of MAbs was reported (44). By fusing normal antibody-producing cells with a B-ceU tumor (myeloma), hybridoma cell lines resulted which produced antibodies having a specificity to only one deterrninant on an antigen ie, all the antibodies produced from the cell line are identical. These studies resulted in a standard approach to MAb production. In this approach, the hybridoma cells are produced in large quantities in culture and screened to select specific clones producing the desired MAb using an appropriate assay. The selected clones are then expanded in culture (or in animals), the cells are collected, and the MAbs are extracted and purified. 

Figure 13.5 Outline of the production strategy of CEA-SCAN. The antibody-producing hybridoma cell line was originally obtained by standard methods of hybridoma generation. Spleen-derived murine B-lymphocytes were fused with murine myeloma calls. The resulting stable hybridomas were screened for the production of anti-CEA monoclonals. The clone chosen produces an IgG anti-CEA antibody. Note that the finished product outlined above is not radiolabelled. The freeze-dried antibody preparation (which has a shelf life of 2 years at 2-8 °C) is reconstituted immediately prior to its medical use. The reconstituting solution contains 99mTc, and is formulated to facilitate direct conjugation of the radiolabel to the antibody fragment...

Hybridomas are derived from the fusion of spleen and myeloma cells and produce monoclonal antibodies. Each hybridoma cell line produces an antibody with a unique specificity allowing the production of highly defined reagents that can be used in many branches of immunochemistry. 

For the production of mAbs, the cell line with the best binding to the targeted epitope of the antigen is chosen from several engineered hybridoma cell lines. The obtained species of antibodies is referred to as mAbs because they derive from one original B lymphocyte and thus they are all identical (clones). 

The reduced glycolytic rate and formation of lactic acid may result in a slower growth rate (Griffiths, 2000). Duval et al. (1992) cultured a mouse hybridoma cell line (VO 208) in batch/fed-batch cultures in a medium supplemented with fructose instead of glucose and demonstrated an increase of the lifespan of the culture and an enhancement in the antibody secretion. However, it has been reported that complete glucose substitution by other hexoses can alter product glycosylation (Paredes et al., 1999). 

Ozturk, S. S., and Palsson, B. O. (1990). Loss of antibody productivity during long-term cultivation of a hybridoma cell line in low serum and serum free media. Hybridoma 9, 167-175. 

Figure IV-5 Production of a hybridoma cell line that produces a monoclonal antibody against an antigen of interest.

Production of Hybridoma Cell Lines. Eight-week-old female BALB/c mice were injected three times intraperitoneally with 500 xg of human serum albumin (HSA) at 2-week intervals. The first injection was in complete Freund adjuvant (1 1, v/v), and the succeeding two injections were given in incomplete Freund adjuvant (1 1, v/v). The mice were allowed to rest for 30-60 days and injected intravenously with 50 xg of HSA. Four days later, spleen cells from these mice were fused with SP2/0-Agl4 myeloma cells by using polyethylene glycol, as previously described (38). Hy-bridomas of interest were cloned by limiting dilution. 

Racher AJ, Looby D Griffiths JB (1990) Studies on monoclonal antibody production by a hybridoma cell line (C1E3) immobilized in a fixed bed, porosphere system. Journal of Biotechnology 15 129-146. 

Seamans TC Hu WS (1990) Kinetics of growth and antibody production by a hybridoma cell line in a perfusion culture. Journal of Fermentation Bioengineering 70 241-245. 

Figure 5.3. The generation of hybridoma cell lines for monoclonal antibody production.

Rodenburg, C. M. Memaugh, R. Bilbao, G. Khazaeli, M. B., Production of a single chain anti-cea antibody from the hybridoma cell line t84.66 using a modified colony-lift selection procedure to detect antigen-positive scfv bacterial clones, Hybridoma 1998, 17, 1-8... 

Gramer, M.J. Britton, T.L. Antibody production by a hybridoma cell line at high cell density is... 

There were two obstacles that antibody therapies had to overcome to get into the clinic. First, a production method would be needed to produce and purify antibodies on a massive scale. This has largely been accomplished by creating hybridoma cell lines able to grow to high density, in serum-free conditions, and other necessities critical for commercial production. Second, non-human antibodies will illicit an inunune response regardless of the epitope. Therefore, antibodies needed to be produced by a rodent, but appear human to our inunune system. Molecular biology techniques were used to create chimeric and later fully humanized antibodies, the distinction is characterized by the ratio of mouse and human DNA. 

Rat antibody production test (RAP) a broad-screen test for known virases potentially present in test article (for Mab-expressing hybridoma cell line only). 

---