Antibody disulfide bond reduction during process development: Insights using a scale-down model process

Abstract

Formation of mAb free thiols linked to redox potential shift 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 (IgG1κ). Different companies have reported the presence of fragmented IgG1 antibodies in the clarified cell culture fluid (CCCF) at manufacturing scale. For instance it was demonstrated that enzymes of the thioredoxin system released during the decline phase of the culture were responsible for the interchain disulfide bond reduction [1]. However, our observations were different as the majority of the interchain disulfide bonds were still oxidized in the CCCF. The massive fragmentation of interchain disulfide bonds was observed only after pilot scale protein A purification step. This study presents our understanding of the antibody reduction process and the insights obtained using scale-down models. Printed by This study demonstrated that the massive increase in mAb reduction can be replicated experimentally in small-scale when the antibody was subjected to protein denaturing conditions. We concluded from these experiments that intradomain free thiols can reduce interchain disulfide bonds by disulfide scrambling when these are exposed to denaturing conditions during SDS-PAGE preparation or protein A elution at pilot scale. We also demonstrated that the accumulation of free thiols observed at the start of the decline phase, first inside the cells, then in the supernatant, led to the increase of free intradomain thiols in the mAb present in the CCCF at the end of the culture. One hypothesis is that these accumulated intracellular reductive forms were released in the supernatant as soon as the viability decreased, leading to a more reductive environment. The detailed mechanisms responsible for this intracellular change remain unclear. Cell lysate-induced free thiols under denaturing conditions lead to inter-chain mAb reduction Lysis of cells + incubation at 37°C Small scale purification Denaturing step at 70 °C +/-NEM