Investigating interactions between phospholipase B-Like 2 and antibodies during Protein A chromatography

• Published in: Journal of Chromatography A Vol. 1438; pp. 31 - 38
• Main Authors: Tran, Benjamin, Grosskopf, Vanessa, Wang, Xiangdan, Yang, Jihong, Walker, Don, Yu, Christopher, McDonald, Paul
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 18.03.2016
• Subjects: Animals; Antibodies; Antibodies, Monoclonal - isolation & purification; Antibodies, Monoclonal - metabolism; Chinese hamster ovary cell proteins (CHOP); CHO Cells; Chromatography; Cricetinae; Cricetulus; ELISA; Host cell proteins; Immunoglobulin G - metabolism; Lysophospholipase - metabolism; Phospholipase; Phospholipase B-Like 2 (PLBL2); Plasmons; Protein A; Protein A chromatography; Proteins; Purification; Staphylococcal Protein A - chemistry; Therapeutic antibody; Host cell proteins; Chinese hamster ovary cell proteins (CHOP); Therapeutic antibody; Protein A chromatography; Phospholipase B-Like 2 (PLBL2)
• ISSN: 0021-9673; 1873-3778
• DOI: 10.1016/j.chroma.2016.01.047

•PLBL2 levels in the Protein A pool do not always correlate with total host-cell protein levels.•PLBL2 co-elution during Protein A is highly dependent on antibody titer and PLBL2 titer.•Antibody load density and intermediate wash conditions can modulate PLBL2 levels in the Protein-A pool.•PLBL2 interacts preferentially with IgG4 compared to IgG1 subclass antibodies.•Interaction of PLBL2 ant antibodies is multivalent and likely located in the hinge and/or the CDRs. Purification processes for therapeutic antibodies typically exploit multiple and orthogonal chromatography steps in order to remove impurities, such as host-cell proteins. While the majority of host-cell proteins are cleared through purification processes, individual host-cell proteins such as Phospholipase B-like 2 (PLBL2) are more challenging to remove and can persist into the final purification pool even after multiple chromatography steps. With packed-bed chromatography runs using host-cell protein ELISAs and mass spectrometry analysis, we demonstrated that different therapeutic antibodies interact to varying degrees with host-cell proteins in general, and PLBL2 specifically. We then used a high-throughput Protein A chromatography method to further examine the interaction between our antibodies and PLBL2. Our results showed that the co-elution of PLBL2 during Protein A chromatography is highly dependent on the individual antibody and PLBL2 concentration in the chromatographic load. Process parameters such as antibody resin load density and pre-elution wash conditions also influence the levels of PLBL2 in the Protein A eluate. Furthermore, using surface plasmon resonance, we demonstrated that there is a preference for PLBL2 to interact with IgG4 subclass antibodies compared to IgG1 antibodies.