Elucidation of Proteoforms of Chinese Hamster Ovary (CHO) Phospholipase B‐Like 2 (PLBL2) Captured From a Monoclonal Antibody

Removing host cell proteins (HCPs) during biotherapeutic manufacturing is essential to ensure patient safety and supply continuity. Yet, this goal remains exceptionally challenging for some HCP species. For example, phospholipase B-like-2 (PLBL2) from Chinese hamster ovary (CHO) cells plagues process engineers by evading typical purification strategies. Separation challenges for CHO PLBL2 and other HCPs are due in large part to a dearth of detailed biochemical characterization for HCPs, such as their proteoforms or variants. Herein, we present as a case study the elucidation of proteoforms of PLBL2, which was endogenously expressed in CHO alongside pembrolizumab (a monoclonal IgG4 antibody) and inadvertently co-purified following a typical downstream process. Using site-specifically modified polyclonal anti-CHO PLBL2 antibodies immobilized onto a solid support, CHO PLBL2 was captured from pembrolizumab, enriched, and recovered, enabling deep characterization by mass spectrometry and other techniques. These analyses revealed a detailed understanding of CHO PLBL2's proteoforms, including charge variants, N-linked and O-linked glycosylation, phosphorylation, and cleavage to afford multiple molecular weight isoforms. To the best of our knowledge, this is the first published, deep characterization of a challenging CHO HCP. Armed with a greater understanding of the impurities that plague purification processes, we can design new, targeted strategies to ensure their removal and safeguard both patient safety and biotherapeutic supplies. While our work centered on CHO PLBL2, our antibody immobilization, affinity enrichment, and characterization approach should be broadly applicable to enable the examination of innumerable challenging HCPs from modalities across the increasingly diverse bioprocessing landscape.