title: A comparison of biophysical characterization techniques in predicting monoclonal antibody stability.
abstract: With the rapid growth of biopharmaceutical product development, knowledge of therapeutic protein stability has become increasingly important. We evaluated assays that measure solution-mediated interactions and key molecular characteristics of 9 formulated monoclonal antibody (mAb) therapeutics, to predict their stability behavior. Colloidal interactions, self-association propensity and conformational stability were measured using effective surface charge via zeta potential, diffusion interaction parameter (kD) and differential scanning calorimetry (DSC), respectively. The molecular features of all 9 mAbs were compared to their stability at accelerated (25°C and 40°C) and long-term storage conditions (2-8°C) as measured by size exclusion chromatography. At accelerated storage conditions, the majority of the mAbs in this study degraded via fragmentation rather than aggregation. Our results show that colloidal stability, self-association propensity and conformational characteristics (exposed tryptophan) provide reasonable prediction of accelerated stability, with limited predictive value at 2-8°C stability. While no correlations to stability behavior were observed with onset-of-melting temperatures or domain unfolding temperatures, by DSC, melting of the Fab domain with the CH2 domain suggests lower stability at stressed conditions. The relevance of identifying appropriate biophysical assays based on the primary degradation pathways is discussed.
authors.affiliation: a Sterile Product and Analytical Development Group , Biologics & Vaccines, Merck & Co., Inc. , Kenilworth , NJ , USA.
authors.forename: Geetha
authors.initials: G
authors.lastname: Thiagarajan
authors.affiliation: a Sterile Product and Analytical Development Group , Biologics & Vaccines, Merck & Co., Inc. , Kenilworth , NJ , USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Semple
authors.affiliation: b Center for Advanced Biotechnology and Medicine, Robert Wood Johnson Medical School , Rutgers University , Piscataway , NJ, USA.
authors.forename: Jose K
authors.initials: JK
authors.lastname: James
authors.affiliation: a Sterile Product and Analytical Development Group , Biologics & Vaccines, Merck & Co., Inc. , Kenilworth , NJ , USA.
authors.forename: Jason K
authors.initials: JK
authors.lastname: Cheung
authors.affiliation: a Sterile Product and Analytical Development Group , Biologics & Vaccines, Merck & Co., Inc. , Kenilworth , NJ , USA.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Shameem
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002152:Calorimetry, Differential Scanning / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D003102:Colloids; D004355:Drug Stability; D007074:Immunoglobulin G / Q000737:chemistry*; D057216:Propensity Score; D066329:Protein Aggregates; D011487:Protein Conformation; D055550:Protein Stability; D013050:Spectrometry, Fluorescence / Q000379:methods; D055471:Thermal Diffusion; D014364:Tryptophan / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D003102:Colloids; D007074:Immunoglobulin G; D066329:Protein Aggregates; D014364:Tryptophan
keywords: Aggregation; Immunoglobulin; colloidal stability; diffusion interaction parameter; fragmentation; monoclonal antibody; thermal stability; zeta potential