Cathepsin D: Removal strategy on protein A chromatography, near real time monitoring and characterisation during monoclonal antibody production.

Monoclonal antibody (mAb) fragmentation is a well-known degradation pathway that results in product loss and can significantly impact product quality, efficacy, or even cause immunogenic reactions, thus potentially endangering patients' health. It is recognised that residual proteases present among host cell proteins (HCPs) such as those expressed by Chinese Hamster Ovary (CHO) can induce fragmentation, and failure of their complete removal during downstream processing could cause fragmentation during mAb production and in the final drug product. We identified, using a protease inhibitor screen, an aspartic protease that contributes to proteolytic fragmentation of partially purified mAbs in multiple projects. Subsequent LC-MS analysis indicated that cathepsin D, a typical aspartic protease, was responsible for the observed fragmentation of in-process samples. To address the issue, an alternative chromatography wash was implemented at the capture step and has been demonstrated to be an effective and scalable solution to mitigate the residual cathepsin D associated fragmentation risk. Furthermore, a near real time targeted mass spectrometry method has been developed to proactively monitor the presence of cathepsin D during upstream and downstream process. Our approach demonstrated an emerging HCP mitigation strategy through integrated upstream and downstream involvement and holds great promise for a range of future applications.