Impact of antibody disulfide bond reduction during harvest on downstream processing and product stability

Therapeutic monoclonal antibodies (mAbs) are primarily produced in mammalian cell culture systems, which can be susceptible to disulfide bond reduction during harvest. This reduction, which is dependent on cell line and cell culture process, has been correlated with the release of reducing components from the cells and depletion of dissolved oxygen before, during and after harvest. Consequently, these factors can lead to disulfide reduction during storage at room temperature prior to initial purification via Protein A chromatography.

This work seeks to understand the impact of disulfide reduction on product quality and on the downstream unit operations. mAb disulfide bond reduction appears to be reversible as the redox potential changes during the purification, however, we observed that the levels of fragment and aggregate increase during the low pH treatment proportional to the amount of free thiol present. The increase of such product related impurities impact the process yield and in process intermediate stability. Furthermore, the drug substance produced from clarified culture with disulfide reduction clearly showed a higher susceptibility to temperature induced changes on stability, potentially indicating incomplete assembly. In addition, for several mAb cases where disulfide bond reduction has been observed, we compared it with DTT induced mAb disulfide reduction. This comparison showed a different mAb sensitivity order to reduction by DTT than the observed at harvest indicating a potential different mechanism for disulfide reduction.