Abstract
Antibody disulfide bond reduction during process development: Insights using a scale-down model process
Highlights
During the development of the production process for a monoclonal antibody, we observed a significant increase in the reduction of interchain disulfide bonds following pilot scale protein A purification of the produced antibody (IgG1).Different companies have reported the presence of fragmented IgG1 antibodies in the clarified cell culture fluid (CCCF) at manufacturing scale
Experimental approach First, the monoclonal antibody (mAb) fragmentation phenomenon was reproduced at small scale by incubation of mAb containing CCCF with cell lysate followed by SDS-PAGE with and without NEM (N-ethylmaleimide)
Oxidized antibodies were observed in the CCCF using non-reducing SDS PAGE with NEM, whereas without NEM, the interchain bonds of the antibodies were massively reduced
Summary
During the development of the production process for a monoclonal antibody (mAb), we observed a significant increase in the reduction of interchain disulfide bonds following pilot scale protein A purification of the produced antibody (IgG1).Different companies have reported the presence of fragmented IgG1 antibodies in the clarified cell culture fluid (CCCF) at manufacturing scale. It has been demonstrated [1] that enzymes of the thioredoxin system, released during the decline phase of the culture, were responsible for the interchain disulfide bond reduction. The massive fragmentation of interchain disulfide bonds occurred only after pilot scale protein A purification step.
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