Probing Conformational Diversity of Fc Domains in Aggregation-Prone Monoclonal Antibodies

• Published in: Pharmaceutical research Vol. 35; no. 11; pp. 220 - 12
• Main Authors: Majumder, Subhabrata, Jones, Michael T., Kimmel, Michael, Alphonse Ignatius, Arun
• Format: Journal Article
• Language: English
• Published: New York Springer US 01.11.2018; Springer; Springer Nature B.V
• Subjects: Amino acids; Analysis; Biochemistry; Biomedical and Life Sciences; Biomedical Engineering and Bioengineering; Biomedicine; DNA fingerprinting; Fc receptors; Fingerprinting; Fragmentation; Fragments; Glycosylation; Health aspects; Heat measurement; Homology; Investigations; Medical Law; Melt temperature; Monoclonal antibodies; Mutation; NMR; Nuclear magnetic resonance; Pharmacology/Toxicology; Pharmacy; Proteins; Research Paper; Shelf life; Storage stability; Fc domain; differential scanning calorimetry; nuclear magnetic resonance spectroscopy; storage stability; aggregation; Fab domain; molecular fingerprinting; monoclonal antibodies
• DOI: 10.1007/s11095-018-2500-8
• ISSN: 0724-8741

Purpose Fc domains are an integral component of monoclonal antibodies (mAbs) and Fc-based fusion proteins. Engineering mutations in the Fc domain is a common approach to achieve desired effector function and clinical efficacy of therapeutic mAbs. It remains debatable, however, whether molecular engineering either by changing glycosylation patterns or by amino acid mutation in Fc domain could impact the higher order structure of Fc domain potentially leading to increased aggregation propensities in mAbs. Methods Here, we use NMR fingerprinting analysis of Fc domains, generated from selected Pfizer mAbs with similar glycosylation patterns, to address this question. Specifically, we use high resolution 2D [ 13 C- 1 H] NMR spectra of Fc fragments, which fingerprints methyl sidechain bearing residues, to probe the correlation of higher order structure with the storage stability of mAbs. Thermal calorimetric studies were also performed to assess the stability of mAb fragments. Results Unlike NMR fingerprinting, thermal melting temperature as obtained from calorimetric studies for the intact mAbs and fragments (Fc and Fab), did not reveal any correlation with the aggregation propensities of mAbs. Despite >97% sequence homology, NMR data suggests that higher order structure of Fc domains could be dynamic and may result in unique conformation(s) in solution. Conclusion The overall glycosylation pattern of these mAbs being similar, these conformation(s) could be linked to the inherent plasticity of the Fc domain, and may act as early transients to the overall aggregation of mAbs.