Antibodies from cell culture

Clausen, A. M., Subramanian, A., and Carr, P. W., Purification of monoclonal antibodies from cell culture supernatants using a modified zirconia-based cation-exchange support, /. Chromatogr. A, 831, 63, 1999. 

Lutkemeyer, D., Bretschneider, M., Buntemeyer, H., and Lehmann, J. (1993). Membrane chromatography for rapid purification of recombinant antithrombin III and monoclonal antibodies from cell culture supernatant. J. Chromatogr. 639, 57-66. 

Bukovsky, J., and Kennett, R. H. (1987). Simple and rapid purification of monoclonal antibodies from cell culture supernatants and ascites fluids by hydroxylapatite chromatography on analytical and preparative scales. Hybridoma 6, 219-228. 

Necina, R., Amatschek, K., and Jungbauer, A. (1998). Capture of human monoclonal antibodies from cell culture supernatant by ion exchange media exhibiting high charge density. Biotechnol. Bioeng. 60, 689-698. 

Bukovsky, J., and Kennet, R. (1987). Simple and Rapid Purification of Monoclonal Antibodies from Cell Culture Supernatants and Ascites Fluids by Hydroxyl Apatite Chromatography on Analytical and Preparative Scales, Hybridoma 6 219-228. 

Fig. 5 Purification of monoclonal antibody from cell culture supernatant by centrifugal precipitation chromatography.

Vaccines and monoclonal antibodies from cell cultures... 

A. General description Abciximab is the Fab fragment of the chimeric human-murine monoclonal antibody 7E3. It binds to the glycoprotein (gp) Ilb/IIIa receptor of human platelets and inhibits platelet aggregation. The chimeric antibody is produced by continuous perfusion in mammahan cell-culture. The 48kDa F b fragment is purified from cell-culture supernatant. 

Figure S Projections for antibody concentrations obtained from cell culture.

IgG from several species (mouse, rabbit, goat, and human), Aviscumin, research antibody in cell culture media 2003 A, B, C Factor 100-1000 (IPCR 0.1-0.01 amol [50-500 fg/mL] IgG, 40 pg/ mL Aviscumin in serum, 100 pg/ mL research antibody) Adler et al. [62], real-time IPCR (Section 2.2.3), internal competitor (Section 2.3.6), commercial antibody-DNA conjugates, AbiPrism 7000 sequence detector (Applied Biosystems)... 

In more practical terms, titers may vary from 1 100 to 1 2000 for polyclonal antisera, from 1 10 to 1 1,000 for monoclonal antibodies in cell culture supernatants, and up to 1 1,000,000 for monoclonal antibodies in ascites fluid. These dilutions may likely be... 

Purification of humanized monoclonal antibodies has also been published using nickel affinity chromatography.190 Directly after filtration of feed stock, antibodies were adsorbed and elution was achieved by a descending pH gradient from 7.5 to 4.25. Purity obtained was about 90% and the fraction did not contain free albumin nor free light chains. Recovery was also reported as greater than 9Q% from ascitic fluids as well as from cell culture supernatants. 

Ostlund, C., Borwell, P., and Malm, B. (1987). Process-scale purification from cell culture supernatants Monoclonal antibodies. Dev. Biol. Stand. 66, 367-375. 

Immobilized cells are also used in biotechnology in the production of protein molecules. For example, entrapped hybridoma cells have been used for the production of monoclonal antibodies which are secreted into the microcapsules. This allows for easier collection of the antibodies compared to growing the hybridoma cells directly in the culture medium. The microcapsules are easily separated from the culture medium and broken to collect the antibodies. Isolation of the antibodies from the culture medium involves numerous purification steps, and product is lost during each of these steps to an extent which depends on the efficiency of the process. Live vaccines have been encapsulated. For example. Bacillus Calmette Guerin has been encapsulated in an alginate polylysine-alginate system. 

Antibodies are a powerful and essential tool in scientific laboratories being used in an array of applications such as immuno-histochemistry, immunobloting, immunoprecipitation and enzyme-linked immunosorbent assays (ELISA). The different sources for antibodies include polyclonal antisera from immunized animals and monoclonal antibodies from cells in culture or from ascites in animals. Both polyclonal and monoclonal antibodies have their advantages, and or disadvantages, but in general the production of monoclonal antibodies is more time consuming and requires tissue culture facilities and skills. The use of either monoclonal or polyclonal antibodies in some of the applications may require that the antibody is in a purified form. They can be purified by a variety of methods described in the next few chapters. The availability of commercially available kits primarily designed for the purification of IgG and IgM classes of antibodies derived from all common animal species should also be mentioned. 

Antibodies have become useful, versatile reagents and essential tools in the scientific laboratory. The different sources of antibodies include polyclonal antisera from immunized animals and monoclonal antibodies from cells in culture or from ascites in animals. 

The most common sources of impurities are derived from the fermentation process and would include such factors as host cell protein or DNA, or components from cell culture growth media. In addition, endogenous retroviruses from hybrido-mas in monoclonal antibody production can also be present as impurities, all of which should be removed and tested for in the final product. [1], These impurities can be present as lipopolysaccharides, oligonucleotides, and leachates from containers. The most common tests that are conducted for the reference standard and production batches are shown in Table 9.2. 

Two types of antibodies can be distinguished monoclonal and polyclonal antibodies. Polyclonal antibodies are essentially a mixture of antibodies as produced by a host upon injection of an antigen. They can bind to several epitopes on the antigen. Monoclonal antibodies on the other hand bind to only one particular epitope on the antigen. They are more specific and reproducible and thus usually preferred for analytical assays. Such monoclonal antibodies have to be produced from cell cultures. 

One major advantage of monoclonal antibodies from plants is the potential low cost of large-scale production. There are commercial companies (such as EPIcyte Pharmaceutical Inc) who are planning clinical dials for plant-produced secretory antibodies for human therapy. These so-called plantibodies can be produced at an estimated cost of 0.01 to 0.1/mg as opposed to 1 to 5/mg for production from cell culture processing of animal-derived hybridomas. The cost of microbial fermentation is lower than that of mammalian cell culture but bacteria lack the ability for efficient multimeric protein assembly and of any post-translational modification. A further potential advantage of the plantibodies is delivery by consumption of plant tissue and thus avoiding any need of purification. These possibilities are particularly applicable in certain cases such as the previously shown ability of a plant-produced... 

IIS Yang,)., Lu, C., Stasny, B., Henley,)., Guinto, W, Gonzalez, C. et al. (2007) Fed-batch bioreactor process scale-up from 3-L to 2,500-L scale for monoclonal antibody production from cell culture. Biotechnol. Bioeng., 98 (1), 141-154. 

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