title: The effect of a point mutation on the stability of IgG4 as monitored by analytical ultracentrifugation.
abstract: There is presently considerable interest in the state of aggregation and biophysical integrity of antibody preparations, and recent advances in the analysis of data from the analytical ultracentrifuge renders it a powerful probe of these stability phenomena, under both storage and freeze-thaw conditions. Solutions of a wild-type IgG4 antibody and a single amino acid hinge mutant IgG4m (serine residue 241 converted to proline) were exposed to different accelerated stress conditions, namely (i) elevated temperature storage for various periods (up to 59 days at 37 degrees C) or (ii) a series of freeze-thaw cycles (storage at -80 degrees C then incubation at 20 degrees C for 1 h under different conditions). Analysis using the nondisruptive probe of sedimentation velocity in the analytical ultracentrifuge indicated that for both antibodies the monomer was always the most common species present whatever storage regime had been used. Sedimentation coefficient distribution analysis showed that other higher oligomer species and half-antibodies were present, and appeared to be not in chemical equilibrium with each other. Solution heterogeneity was found to increase considerably with treatment for both native and hinge-mutant antibodies although the latter appeared to be more resistant to freeze-thaw-induced aggregation.
authors.affiliation: National Centre for Macromolecular Hydrodynamics, University of Nottingham, Sutton Bonington, LE12 5RD, UK.
authors.forename: Yanling
authors.initials: Y
authors.lastname: Lu
authors.forename: Stephen E
authors.initials: SE
authors.lastname: Harding
authors.forename: Arthur J
authors.initials: AJ
authors.lastname: Rowe
authors.forename: Kenneth G
authors.initials: KG
authors.lastname: Davis
authors.forename: Brendan
authors.initials: B
authors.lastname: Fish
authors.forename: Paul
authors.initials: P
authors.lastname: Varley
authors.forename: Chris
authors.initials: C
authors.lastname: Gee
authors.forename: Sandrine
authors.initials: S
authors.lastname: Mulot
mesh_terms: D004355:Drug Stability; D005615:Freezing; D007074:Immunoglobulin G / Q000737:chemistry*; D017354:Point Mutation; D011487:Protein Conformation; D014461:Ultracentrifugation
chemical_list: D007074:Immunoglobulin G