title: Effects of temperature and osmolytes on competing degradation routes for an IgG1 antibody.
abstract: Addition of excipients is a common strategy to slow protein aggregation during storage. Excipient effects on the mechanism(s) and temperature (T) dependence of aggregation for a monoclonal antibody solution were tested using size-exclusion chromatography, differential scanning calorimetry (DSC), temperature scanning monomer loss (TSML), and laser light scattering; previous work in buffer-only conditions had shown non-Arrhenius behavior and implicated Fab and/or CH 3 unfolding as a key step in aggregation. Excipients included citrate, amino acid salts (histidine-HCl, arginine-HCl), and polyols (mannitol and glycerol). DSC and TSML showed that Fab, rather than CH 3, unfolding corresponded with the onset of aggregation for each condition. Isothermal incubation at 56.5°C, 40°C, and 2°C-8°C resulted in aggregation, while fragmentation occurred readily at only 40°C. The primary effect of the different excipients appeared to be preferential accumulation/exclusion, affecting the concentrations of partially unfolded monomer key intermediates. In addition, aggregation rates were clearly non-Arrhenius, causing aggregation to dominate over fragmentation at high and low T, and making long-term stability predictions problematic based on commonly employed 40°C conditions. Possible reasons for non-Arrhenius behavior include a strong T-dependence of the Fab unfolding enthalpy and/or a switch from Fab-mediated to Fc-mediated aggregation as one moves from high to low T.
authors.affiliation: Biopharmaceutical Research and Development, GlaxoSmithKline, King of Prussia, Pennsylvania, 19406; Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, Delaware, 19716.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Roberts
authors.forename: Douglas P
authors.initials: DP
authors.lastname: Nesta
authors.forename: Nayoung
authors.initials: N
authors.lastname: Kim
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002152:Calorimetry, Differential Scanning / Q000379:methods; D004355:Drug Stability; D005079:Excipients / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry*; D009994:Osmolar Concentration; D059748:Proteolysis; D013696:Temperature; D013816:Thermodynamics
chemical_list: D000911:Antibodies, Monoclonal; D005079:Excipients; D007074:Immunoglobulin G
keywords: excipients; protein aggregation; protein formulation; stability; thermal analysis