期刊
JOURNAL OF PHARMACEUTICAL SCIENCES
卷 111, 期 6, 页码 1587-1598出版社
ELSEVIER SCIENCE INC
DOI: 10.1016/j.xphs.2022.02.016
关键词
Reversible self-association; Hexamerization; Developability; Biophysical properties; Conformational and Colloidal stability; HexaBody
资金
- Netherlands Organization for Scientific Research (NWO) [184.034.019]
- NWO Satin Grant [731.017.202]
This study biophysically characterized the impact of self-association in IgG antibody variants, finding a correlation between increased self-association propensity and functional activity. The mutation E430G decreased conformational stability, but had no substantial effect on storage stability. These findings provide insights for the design and development of IgG-based therapeutics.
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 degrees 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 degrees C. We discuss how these findings may inform the design and development of IgG-based therapeutics. (C) 2022 The Authors. Published by Elsevier Inc. on behalf of American Pharmacists Association.
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