title: Investigating interactions between phospholipase B-Like 2 and antibodies during Protein A chromatography.
abstract: 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.
authors.affiliation: Benjamin Tran, Purification Development, Genentech, 1 DNA Way, South San Francisco, CA 94080, United States; Pharma Technical Development, Genentech, Inc., United States. Electronic address: tran.benjamin@gene.com.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Tran
authors.affiliation: Pharma Technical Development, Genentech, Inc., United States.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Grosskopf
authors.affiliation: Development Sciences, Genentech, Inc., United States.
authors.forename: Xiangdan
authors.initials: X
authors.lastname: Wang
authors.affiliation: Development Sciences, Genentech, Inc., United States.
authors.forename: Jihong
authors.initials: J
authors.lastname: Yang
authors.affiliation: Pharma Technical Development, Genentech, Inc., United States.
authors.forename: Don
authors.initials: D
authors.lastname: Walker
authors.affiliation: Pharma Technical Development, Genentech, Inc., United States.
authors.forename: Christopher
authors.initials: C
authors.lastname: Yu
authors.affiliation: Pharma Technical Development, Genentech, Inc., United States.
authors.forename: Paul
authors.initials: P
authors.lastname: McDonald
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification* / Q000378:metabolism*; D016466:CHO Cells; D002845:Chromatography; D006224:Cricetinae; D003412:Cricetulus; D007074:Immunoglobulin G / Q000378:metabolism; D008245:Lysophospholipase / Q000378:metabolism*; D013205:Staphylococcal Protein A / Q000737:chemistry*
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D013205:Staphylococcal Protein A; D008245:Lysophospholipase
keywords: Chinese hamster ovary cell proteins (CHOP); Host cell proteins; Phospholipase B-Like 2 (PLBL2); Protein A chromatography; Therapeutic antibody