Biophysical Characterization and Stability of Modified IgG1 Antibodies with Different Hexamerization Propensities

J Pharm Sci. 2022 Jun;111(6):1587-1598. doi: 10.1016/j.xphs.2022.02.016. Epub 2022 Feb 27.

Abstract

The hexamerization of natural, human IgG antibodies after cell surface antigen binding can induce activation of the classical complement pathway. Mutations stimulating Fc domain-mediated hexamerization can potentiate complement activation and induce the clustering of cell surface receptors, a finding that was applied to different clinically investigated antibody therapeutics. Here, we biophysically characterized how increased self-association of IgG1 antibody variants with different hexamerization propensity may impact their developability, rather than functional properties. Self-Interaction Chromatography, Dynamic Light Scattering and PEG-induced precipitation showed that IgG variant self-association at neutral pH increased in the order wild type (WT) < E430G < E345K < E345R < E430G-E345R-S440Y, consistent with functional activity. Self-association was strongly pH-dependent, and single point mutants were fully monomeric at pH 5. Differential Scanning Calorimetry and Fluorimetry showed that mutation E430G decreased conformational stability. Interestingly, heat-induced unfolding facilitated by mutation E430G was reversible at 60°C, while a solvent-exposed hydrophobic mutation caused irreversible aggregation. Remarkably, neither increased dynamic self-association propensity at neutral pH nor decreased conformational stability substantially affected the stability of concentrated variants E430G or E345K during storage for two years at 2-8°C. We discuss how these findings may inform the design and development of IgG-based therapeutics.

Keywords: Biophysical properties; Conformational and Colloidal stability; Developability; HexaBody; Hexamerization; Reversible self-association.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Complement Activation*
  • Humans
  • Immunoglobulin G* / metabolism
  • Mutation
  • Protein Stability

Substances

  • Immunoglobulin G