![]() 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. 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. The combination of different analytical tools, mass spectrometry and computational modeling provide a general strategy on how to study residual HCP for biotherapeutics development. To the best of our knowledge, HEXB is the first residual HCP reported to have impact on the glycan profile of a formulated drug product. A risk assessment was conducted by evaluating the in silico immunogenicity risk and the impact on product quality. An improved bioprocess was developed to significantly reduce HEXB levels in the final drug substance. A potential interaction between HEXB and mAb‐1 was initially observed from the analysis of process intermediates by proteomics among several mAbs and later supported by computational modeling. Subsequently, enzymatic and targeted multiple reaction monitoring (MRM) MS assays were developed to support process and product characterization. HEXB was identified by liquid chromatography–mass spectrometry (LC‐MS) based proteomics approach to be enriched in the impacted stability batches from mAb‐1. The root cause for this instability was attributed to Hexosaminidase B (HEXB), an enzyme known to remove terminal N‐acetylglucosamine (GlcNAc). However, significant N‐glycan degradation was observed for one mAb under accelerated and stressed stability conditions. One product quality attribute not expected to be stability indicating is the N‐glycan heterogeneity profile. In this study, product quality attributes were tracked for several mAbs under the intended storage and accelerated stability conditions. Host cell proteins (HCPs) are process‐related impurities derived from host organisms, which need to be controlled to ensure adequate product quality and safety. In addition, strategies that can minimize the impact of these two factors are briefly discussed. This article reviews the two major mechanisms that contribute to copurification of CHO HCPs, namely leaching from chromatin heteroaggregates and nonspecific HCP-mAb association. The relative difficulty of HCP clearance is case dependent and in certain cases a small population of HCPs can persist throughout the downstream process. Although Protein A affinity chromatography alone can typically remove more than 90% of HCPs in the clarified culture harvest, in many cases reducing HCPs in the final drug product to an acceptable level (e.g., <100 ppm) can still be a challenging task. HCPs are highly heterogeneous in nature, differing in molecular weight, isoelectric point and hydrophobicity, and some of them can be difficult to remove. For CHO expressed monoclonal antibodies (mAbs), host cell proteins (HCPs) represent a major class of process-related impurities and their removal is a key focus of downstream process development.
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