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 (1)H-(15)N heteronuclear single-quantum correlation NMR spectra were collected between pH 2.5 and 4.7 to assess whether unfolding of C(H)2 domains precedes that of C(H)3 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(H)2 glycosylation. In addition, differential scanning calorimetry data were collected over a pH range of 3-7 to assess changes in C(H)2 and C(H)3 thermostability. As a result, compelling evidence was gathered that emphasizes the importance of C(H)2 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(H)2 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.
Highlights
Monoclonal antibodies and Fc fusion proteins contain an IgG Fc moiety, which is associated with various degradation processes, including aggregation
Effect of Acidic Conditions on IgG1 Fc Structure via nuclear magnetic resonance (NMR) Analysis— Fc is a relatively large protein (ϳ50 kDa), recent studies demonstrated that it is amenable to high resolu
High Resolution Structural Analysis of Fc Unfolding—Analysis of chemical shift perturbations via 1H-15N HSQC NMR is a powerful approach that allows a detailed understanding of protein structural changes
Summary
Monoclonal antibodies and Fc fusion proteins contain an IgG Fc moiety, which is associated with various degradation processes, including aggregation. Results: Fc unfolding is triggered by the protonation of acidic residues and depends on the IgG subclass and CH2 domain glycosylation. Significance: Understanding Fc aggregation is important for improving the quality of Fc-based therapeutics. Understanding the underlying mechanisms of Fc aggregation is an important prerequisite for developing stable and efficacious antibody-based therapeutics. The same pH range was subsequently screened in Fc aggregation experiments that utilized molecules of IgG1 and IgG2 subclasses with varying levels of CH2 glycosylation. Our data for glycosylated, partially deglycosylated, and fully deglycosylated molecules further revealed the criticality of CH2 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
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