title: Trimerization Dictates Solution Opalescence of a Monoclonal Antibody.
abstract: Opalescence, sometimes observed in antibody solutions, is thought to be mediated by light scattering of soluble oligomers or insoluble particulates. However, mechanistic features, such as stoichiometry and self-association affinity of oligomeric species related to opalescence, are poorly understood. Here, opalescence behavior of a monoclonal antibody (mAb-1) solution was studied over a wide range of solution conditions including different protein concentrations, pH, and in the presence or absence of salt. Hydrodynamic and thermodynamic properties of mAb-1 solutions were studied by analytical ultracentrifugation and dynamic light scattering. Opalescence in mAb-1 solutions is pH and concentration dependent. The degree of opalescence correlates with reversible monomer-trimer equilibrium detected by analytical ultracentrifugation. Increased trimer formation corresponds to increased opalescence in mAb-1 solutions at higher pH and protein concentrations. Addition of NaCl shifts this equilibrium toward monomer and reduces solution opalescence. This study demonstrates that opalescence in mAb-1 solutions does not arise from the light scattering of monomer or random molecular self-associations but is strongly correlated with a specific self-association stoichiometry and affinity. Importantly, at pH 5.5 (far below isoelectric point of mAb-1), the solution is not opalescent and with nonideal behavior. This study also dissects several parameters to describe the hydrodynamic and thermodynamic nonideality.
authors.affiliation: Regeneron Pharmaceuticals, Formulation Development Group, 777 Old Saw Mill River Road, Tarrytown, New York 10591. Electronic address: tengchieh.yang@regeneron.com.
authors.forename: Teng-Chieh
authors.initials: TC
authors.lastname: Yang
authors.affiliation: Pharmaceutical Research and Development, Pfizer, 700 Chesterfield Parkway West, Chesterfield, Missouri 63017.
authors.forename: Alex Jacob
authors.initials: AJ
authors.lastname: Langford
authors.affiliation: Pharmaceutical Research and Development, Pfizer, 700 Chesterfield Parkway West, Chesterfield, Missouri 63017.
authors.forename: Sandeep
authors.initials: S
authors.lastname: Kumar
authors.affiliation: Pharmaceutical Research and Development, Pfizer, 700 Chesterfield Parkway West, Chesterfield, Missouri 63017.
authors.forename: John Carl
authors.initials: JC
authors.lastname: Ruesch
authors.affiliation: Bayer Pharmaceuticals, 800 Dwight Way, Berkeley, California 94710. Electronic address: wei.wang9@bayer.com.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002626:Chemistry, Pharmaceutical; D004355:Drug Stability; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000737:chemistry*; D000068897:Iridescence; D008956:Models, Chemical; D055503:Protein Multimerization; D012542:Scattering, Radiation; D012965:Sodium Chloride / Q000737:chemistry; D012995:Solubility; D012996:Solutions; D055672:Static Electricity; D013816:Thermodynamics
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D012996:Solutions; D012965:Sodium Chloride
keywords: analytical ultracentrifugation; dynamic light scattering; monoclonal antibody; nonideality; opalescence; self-association