| Since Kohler and Milstein firstly developed the monoclonal antibody with cellular fusion technology in the 1970's, it has raised great interests for its important functions and broad applications in clinical diagnosis, treatment, diseases prevention and protein purificaton. As mouse monoclonal antibody is a strong immunogen for human beings, gene-engineering technology has been adopted to modify the structure of monoclonal antibody in order to reduce its immunogenicity. Singal-chain antibody, chimeric antibody and humanized antibody are developed in recent year. As the most potent immunosuppressing agent, anti-CD3 McAb is routinely used for treatment of accute rejection and GVHD. Humanized modification of mouse anti-CD3 antibody may provide a potent immunosuppressin agent with low immunogenicity and minimum side-effect for transplantation patients.At the present, serum-free mediums are commonly used in culturing cells in vitro because of avoiding frequent change of medium, simplifing the down stream process of production, and being cost effective. But target cells cultured in serum-free mediums were sensitive to alteration of culture conditions, especially the lack of amino acid. As culture conditions vary to cells for their proliferation and protein expression, it is very important to explore the optional culture system for different cells.We investigated the concentrations of lactic acid and ammonia in our culture system. The results indicated that the concentration of lactic acid should be less than 500mg/dl and that of ammonia should be lower than 60mmol/L. Amino acid is regarded as one of the key factors for the cell growth and protein synthesis. Butyrate sodium can modulate mammalian cell growth and proliferation, and regulate gene expresson which induce protein synthesise. In addition, iron(III) citrate, sodium pyruvate and ethanolamine also play a role in the cellular proliferation and protein expression.We successfully established a simple and sensitive immunoassay for the analysis of the recombinant humanized anti-CD3 antibody. The intraday precision wasevaluated from the relative standard deviation analysis of the quality controls samples and RSD ranged from 3% to 9%. The percent inaccuracy for the quality controls was from 91% to 110%. The linearity of the assay was from 3.125~100ng/ml. This technique provided a necessary guarantee for culture condition optimization.Butyrate sodium, iron(III) citrate, sodium pyruvate may also have effects on cell growth and protein production. The results showed that butyrate sodium at the concentration of 2mmol/L can duplicate the quantity of antibody production nearly 2 times. This became more notable when iron (III) citrate was also added (the quantity of cell expression was nearly 3 times). These results indicated that butyrate sodium and iron (III) citrate have a syngeneic effect on protein expression. Comparison of ingredient in fresh and spend fluids provides a useful window to the metabolic requirements of the cell. Individual addition of amino acids, which was rapidly taken up in the culture, does not lead to the enhancement of cell expansion or protein expression, while adding simultaneously these amino acid such as serine, leucine, cysteine, isoleucine, valine, glutamic acid, were found to effectively enhance cell proliferation and protein production. The results of SDS-PAGE and IEF indicated that butyrate sodium, iron(III) citrate, Sodium pyruvate and amino acid have no significant effects on protein glycosylation.Finally, we add butyrate sodium, iron(III) citrate, sodium pyruvate and amino acid into the cell culture medium in a optimized concentration and to investigate the combinatory effects of the components on cultured cells. The results indicated that cell density and humanized antibody production were markedly increased in this optimized medium.This work is a preliminary exploration in optimization of serum-free culture condition for culturing mammalian cell, which may provide important information for large scale prod...
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