title: Correlations between changes in conformational dynamics and physical stability in a mutant IgG1 mAb engineered for extended serum half-life.
abstract: This study compares the local conformational dynamics and physical stability of an IgG1 mAb (mAb-A) with its corresponding YTE (M255Y/S257T/T259E) mutant (mAb-E), which was engineered for extended half-life in vivo. Structural dynamics was measured using hydrogen/deuterium (H/D) exchange mass spectrometry while protein stability was measured with differential scanning calorimetry (DSC) and size exclusion chromatography (SEC). The YTE mutation induced differences in H/D exchange kinetics at both pH 6.0 and 7.4. Segments covering the YTE mutation sites and the FcRn binding epitopes showed either subtle or no observable differences in local flexibility. Surprisingly, several adjacent segments in the CH2 and distant segments in the VH, CH1, and VL domains had significantly increased flexibility in the YTE mutant. Most notable among the observed differences is increased flexibility of the 244-254 segment of the CH2 domain, where increased flexibility has been shown previously to correlate with decreased conformational stability and increased aggregation propensity in other IgG1 mAbs (e.g., presence of destabilizing additives as well as upon de-glycosylation or methionine oxidation). DSC analysis showed decreases in both thermal onset (Tonset) and unfolding (Tm1) temperatures of 7°C and 6.7°C, respectively, for the CH2 domain of the YTE mutant. In addition, mAb-E aggregated faster than mAb-A under accelerated stability conditions as measured by SEC analysis. Hence, the relatively lower physical stability of the YTE mutant correlates with increased local flexibility of the 244-254 segment, providing a site-directed mutant example that this segment of the CH2 domain is an aggregation hot spot in IgG1 mAbs.
authors.affiliation: a Department of Pharmaceutical Chemistry ; University of Kansas ; Lawrence , KS USA.
authors.forename: Ranajoy
authors.initials: R
authors.lastname: Majumdar
authors.forename: Reza
authors.initials: R
authors.lastname: Esfandiary
authors.forename: Steven M
authors.initials: SM
authors.lastname: Bishop
authors.forename: Hardeep S
authors.initials: HS
authors.lastname: Samra
authors.forename: C Russell
authors.initials: CR
authors.lastname: Middaugh
authors.forename: David B
authors.initials: DB
authors.lastname: Volkin
authors.forename: David D
authors.initials: DD
authors.lastname: Weis
mesh_terms: D019943:Amino Acid Substitution; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics*; D006207:Half-Life; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics*; D007700:Kinetics; D020125:Mutation, Missense; D010084:Oxidation-Reduction; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D044967:Serum / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G
keywords: CH1-CH3, constant domains 1–3, respectively, of the heavy chain of a monoclonal antibody; DSC, differential scanning calorimetry; Fab, antigen binding fragment of a monoclonal antibody; Fc, crystallizable fragment of a monoclonal antibody; HC, heavy chain of a monoclonal antibody; HX-MS, hydrogen/deuterium exchange mass spectrometry; LC, light chain of a monoclonal antibody; VH/VL, variable domain of the heavy/light chain of a monoclonal antibody; YTE mutation; aggregation; differential scanning calorimetry; flexibility; hydrogen/deuterium exchange; immunoglobulin G1; mass spectrometry; monoclonal antibody; stability