Intact Protein Mass Spectrometry of Cell Culture Harvest Guides Cell Line Development for Trispecific Antibodies

IgG-like multispecific antibodies with asymmetric constructs have become widely used formats for therapeutic applications in recent years. Correct assembly of the subunits in this class of therapeutics is a critical quality attribute (CQA) with direct impact on biological activity. Therefore, early drug development efforts such as clone selection during cell line development must be guided by information on potential chain mispairing to enable timely decision making and risk mitigation. Here we describe a high-throughput analytical platform based on denaturing size-exclusion ultraperformance liquid chromatography (UPLC) coupled with intact protein mass spectrometry for profiling of mispairing and other product-related impurities, including half antibodies. This method can be performed directly on the clarified cell culture harvest fluid without the need for Protein A purification or other sample preparations and provides unbiased information on the product quality of the clones and the effect of growth conditions in a fast and cost-effective manner. Screening large numbers of clones expressing different trispecific antibody (tsAb) constructs revealed that although chain mispairing primarily depends on the antibody sequence and structure, it is also a characteristic of the clone. In addition, different growth conditions may affect the type and distribution of half antibodies and mispaired species impurities but not the quality ranking of the clones.