Elucidation of acid-induced unfolding and aggregation of human immunoglobulin IgG1 and IgG2 Fc

Abstract

Understanding the underlying mechanisms of Fc aggregation is an important prerequisite for developing stable and efficacious antibody-based therapeutics. In our study, high resolution two-dimensional nuclear magnetic resonance (NMR) was employed to probe structural changes in the IgG1 Fc. A series of 1H- 15N heteronuclear single-quantum correlation NMR spectra were collected between pH 2.5 and 4.7 to assess whether unfolding of C H2 domains precedes that of C H3 domains. The same pH range was subsequently screened in Fc aggregation experiments that utilized molecules of IgG1 and IgG2 subclasses with varying levels of C H2 glycosylation. In addition, differential scanning calorimetry data were collected over a pH range of 3-7 to assess changes in C H2 and C H3 thermostability. As a result, compelling evidence was gathered that emphasizes the importance of C H2 stability in determining the rate and extent of Fc aggregation. In particular, we found that Fc domains of the IgG1 subclass have a lower propensity to aggregate compared with those of the IgG2 subclass. Our data for glycosylated, partially deglycosylated, and fully deglycosylated molecules further revealed the criticality of C H2 glycans in modulating Fc aggregation. These findings provide important insights into the stability of Fc-based therapeutics and promote better understanding of their acid-induced aggregation process. © 2012 by The American Society for Biochemistry and Molecular Biology Inc.