title: 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 (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.
authors.affiliation: Drug Product Development, Amgen Inc., Seattle, Washington 98119, USA. rlatypov@amgen.com
authors.forename: Ramil F
authors.initials: RF
authors.lastname: Latypov
authors.forename: Sabine
authors.initials: S
authors.lastname: Hogan
authors.forename: Hollis
authors.initials: H
authors.lastname: Lau
authors.forename: Himanshu
authors.initials: H
authors.lastname: Gadgil
authors.forename: Dingjiang
authors.initials: D
authors.lastname: Liu
mesh_terms: D006031:Glycosylation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D017510:Protein Folding; D055550:Protein Stability; D017434:Protein Structure, Tertiary
chemical_list: D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G
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