Effects of Glycan Structure on the Stability and Receptor Binding of an IgG4-Fc

• Published in: Journal of pharmaceutical sciences Vol. 109; no. 1; pp. 677 - 689
• Main Authors: Kang, Huan, Larson, Nicholas R., White, Derek R., Middaugh, C. Russell, Tolbert, Thomas, Schöneich, Christian
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.01.2020
• Subjects: Antibody Affinity; conformation; differential scanning calorimetry; Drug Stability; Glycosylation; IgG4; Immunoglobulin Fc Fragments - chemistry; Immunoglobulin Fc Fragments - metabolism; Immunoglobulin Fc Fragments - radiation effects; Immunoglobulin G - chemistry; Immunoglobulin G - metabolism; Immunoglobulin G - radiation effects; intrinsic fluorescence; Kinetics; Light; mass spectrometry; monoclonal antibodies; Pharmaceutical Preparations - chemistry; Pharmaceutical Preparations - metabolism; Pharmaceutical Preparations - radiation effects; Photolysis; photostability; Polysaccharides - chemistry; Polysaccharides - metabolism; Polysaccharides - radiation effects; Protein Binding; Protein Conformation; Protein Stability; Receptors, IgG - metabolism; Temperature; differential scanning calorimetry; intrinsic fluorescence; glycosylation; IgG4; mass spectrometry; conformation; Fc; photostability; monoclonal antibodies
• ISSN: 0022-3549; 1520-6017
• DOI: 10.1016/j.xphs.2019.10.036

A series of well-defined N-glycosylated IgG4-Fc variants were utilized to investigate the effect of glycan structure on their physicochemical properties (conformational stability and photostability) and interactions with an Fc γ receptor IIIA (FcγRIIIA). High mannose (HM, GlcNAc2Man(8+n) [n = 0-4]), Man5 (GlcNAc2Man5), GlcNAc1, and N297Q IgG4-Fc were prepared in good quality. The physical stability of these IgG4-Fc variants was examined with differential scanning calorimetry and intrinsic fluorescence spectroscopy. Photostability was assessed after photoirradiation between 295 and 340 nm (λ max = 305 nm), and HPLC-MS/MS analysis of specific products was performed. The size of glycans at Asn297 affects the yields of light-induced Tyr side-chain fragmentation products, where the yields decreased in the following order: N297Q > GlcNAc1 > Man5 > HM. These yields correlate with the thermal stability of the glycoforms. The HM and Man5 glycoforms display increased affinity for FcγRIIIA by at least 14.7-fold compared with GlcNAc1 IgG4-Fc. The affinities measured for the HM and Man5 IgG4-Fc (0.39-0.52 μM) are similar to those measured for fucosylated IgG1. Dependent on the mechanisms of action of IgG4 therapeutics, such glycoforms may need to be carefully monitored. The nonglycosylated N297Q IgG4-Fc did not present measurable affinity to FcγRIIIA.