title: Molecular perspective of antibody aggregates and their adsorption on Protein A resin.
abstract: Antibody aggregate is a common issue in therapeutic antibodies, which may compromise product efficacy and cause adverse effects. Antibody aggregate level is normally controlled in bioprocessing by polishing steps after Protein A capture. This paper studied the Higher Order Structures (HOS) of antibody aggregates (dimer H1 and H2) and their adsorption on Protein A resin and thus elucidated the mechanism using Protein A capture for enhanced aggregate removal. The HOS of antibody aggregates and their complex with Protein A were characterized using HDX-MS combined with SEC-MALS, Protein Conformational Array (PCA), and molecular modeling. The aggregate size and Protein A binding ratio suggested that H2 has much more compact structure than H1. HDX-MS and PCA further revealed that H1 was formed by single Fab-Fab interaction while H2 formed by Fab-Fab and likely Fc-Fc interaction. On Protein A resin, both the molar binding ratio and the correlation between protein size and ligand distance support that each monomer can only bind one Protein A ligand, while each dimer can bind two ligands, thus resulting in stronger resin binding. Furthermore, dimer H2 binds stronger than dimer H1 due to its compact structure. By integrating biophysical analysis and molecular modeling with process development, this study revealed the antibody aggregate structures and the mechanism of aggregate removal using Protein A chromatography. It also provided a general strategy for in-depth product and process understanding in antibody and other biologics development.
authors.affiliation: Biologics Development, Global Manufacturing and Supply, Bristol-Myers Squibb Company, Devens, MA, USA. Electronic address: deqiang.yu@bms.com.
authors.forename: Deqiang
authors.initials: D
authors.lastname: Yu
authors.affiliation: Biologics Development, Global Manufacturing and Supply, Bristol-Myers Squibb Company, Devens, MA, USA.
authors.forename: Yuanli
authors.initials: Y
authors.lastname: Song
authors.affiliation: Bioanalytical and Discovery Analytical Sciences, Research and Development, Bristol-Myers Squibb Company, Princeton, NJ, USA.
authors.forename: Richard Y-C
authors.initials: RY
authors.lastname: Huang
authors.affiliation: Biologics Development, Global Manufacturing and Supply, Bristol-Myers Squibb Company, Devens, MA, USA.
authors.forename: Ryan K
authors.initials: RK
authors.lastname: Swanson
authors.affiliation: Biologics Development, Global Manufacturing and Supply, Bristol-Myers Squibb Company, Devens, MA, USA.
authors.forename: Zhijun
authors.initials: Z
authors.lastname: Tan
authors.affiliation: Biologics Development, Global Manufacturing and Supply, Bristol-Myers Squibb Company, Devens, MA, USA.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Schutsky
authors.affiliation: Biologics Development, Global Manufacturing and Supply, Bristol-Myers Squibb Company, Devens, MA, USA.
authors.forename: Angela
authors.initials: A
authors.lastname: Lewandowski
authors.affiliation: Bioanalytical and Discovery Analytical Sciences, Research and Development, Bristol-Myers Squibb Company, Princeton, NJ, USA.
authors.forename: Guodong
authors.initials: G
authors.lastname: Chen
authors.affiliation: Biologics Development, Global Manufacturing and Supply, Bristol-Myers Squibb Company, Devens, MA, USA.
authors.forename: Zheng Jian
authors.initials: ZJ
authors.lastname: Li
mesh_terms: D000327:Adsorption; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000302:isolation & purification*; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry; D008024:Ligands; D008958:Models, Molecular; D011108:Polymers; D066329:Protein Aggregates; D011487:Protein Conformation; D013205:Staphylococcal Protein A / Q000737:chemistry*
chemical_list: D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D008024:Ligands; D011108:Polymers; D066329:Protein Aggregates; D013205:Staphylococcal Protein A
keywords: Antibody aggregate; HDX-MS; Higher order structure; Molecular modelling; Protein A adsorption; Protein Conformational Array