title: Osmolyte Effects on Monoclonal Antibody Stability and Concentration-Dependent Protein Interactions with Water and Common Osmolytes.
abstract: Preferential interactions of proteins with water and osmolytes play a major role in controlling the thermodynamics of protein solutions. While changes in protein stability and shifts in phase behavior are often reported with the addition of osmolytes, the underlying protein interactions with water and/or osmolytes are typically inferred rather than measured directly. In this work, Kirkwood-Buff integrals for protein-water interactions (G12) and protein-osmolyte interactions (G23) were determined as a function of osmolyte concentration from density measurements of antistreptavidin immunoglobulin gamma-1 (AS-IgG1) in ternary aqueous solutions for a set of common neutral osmolytes: sucrose, trehalose, sorbitol, and poly(ethylene glycol) (PEG). For sucrose and PEG solutions, both protein-water and protein-osmolyte interactions depend strongly on osmolyte concentrations (c3). Strikingly, both osmolytes change from being preferentially excluded to preferentially accumulated with increasing c3. In contrast, sorbitol and trehalose solutions do not show large enough preferential interactions to be detected by densimetry. G12 and G23 values are used to estimate the transfer free energy for native AS-IgG1 (Δμ2N) and compared with existing models. AS-IgG1 unfolding via calorimetry shows a linear increase in midpoint temperatures as a function of trehalose, sucrose, and sorbitol concentrations, but the opposite behavior for PEG. Together, the results highlight limitations of existing models and common assumptions regarding the mechanisms of protein stabilization by osmolytes. Finally, PEG preferential interactions destabilize the Fab regions of AS-IgG1 more so than the CH2 or CH3 domains, illustrating preferential interactions can be specific to different protein domains.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Delaware , Newark, Delaware 19716, United States.
authors.forename: Gregory V
authors.initials: GV
authors.lastname: Barnett
authors.affiliation: Drug Product Development, Amgen Inc. , Seattle, Washington 98119, United States.
authors.forename: Vladimir I
authors.initials: VI
authors.lastname: Razinkov
authors.affiliation: Drug Product Development, Amgen Inc. , Seattle, Washington 98119, United States.
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Kerwin
authors.affiliation: Malvern Biosciences Inc., Columbia, Maryland 21046, United States.
authors.forename: Steven
authors.initials: S
authors.lastname: Blake
authors.affiliation: Malvern Biosciences Inc., Columbia, Maryland 21046, United States.
authors.forename: Wei
authors.initials: W
authors.lastname: Qi
authors.affiliation: School of Chemical Engineering and Analytical Science, The University of Manchester , Manchester, U.K.
authors.forename: Robin A
authors.initials: RA
authors.lastname: Curtis
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Delaware , Newark, Delaware 19716, United States.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Roberts
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry; D009994:Osmolar Concentration; D011092:Polyethylene Glycols / Q000737:chemistry; D055550:Protein Stability; D013012:Sorbitol / Q000737:chemistry; D013395:Sucrose / Q000737:chemistry; D013816:Thermodynamics; D014199:Trehalose / Q000737:chemistry; D014867:Water / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D014867:Water; D011092:Polyethylene Glycols; D013012:Sorbitol; D013395:Sucrose; D014199:Trehalose