Characterization of N-Terminal Asparagine Deamidation and Clipping of a Monoclonal Antibody

Author:

Zhen Jing1ORCID,Lee Jennifer1,Wang Yueyang1,McLaughlin Lena1,Yang Fei1,Li Zhengjian1ORCID,Wang Jihong1ORCID

Affiliation:

1. Department of Analytical Sciences, U.S. Technical & Biologics Development, Horizon Therapeutics, Rockville, MD 20850, USA

Abstract

This study presents a novel degradation pathway of a human immunoglobulin G (IgG) molecule featuring a light chain N-terminal asparagine. We thoroughly characterize this pathway and investigate its charge profiles using cation exchange chromatography (CEX) and capillary isoelectric focusing (cIEF). Beyond the well-documented asparagine deamidation into isoaspartic acid, aspartic acid, and succinimide intermediate, a previously unreported clipping degradation pathway is uncovered. This newly identified clipped N-terminal IgG variant exhibits a delayed elution in CEX, categorized as a “basic variant”, while retaining the same main peak isoelectric point (pI) in cIEF. The influence of temperature and pH on N-terminal asparagine stability is assessed across various stressed conditions. A notable correlation between deamidation percentage and clipped products is established, suggesting a potential hydrolytic chemical reaction underlying the clipping process. Furthermore, the impact of N-terminal asparagine modifications on potency is evaluated through ELISA binding assays, revealing minimal effects on binding affinity. Sequence alignment reveals homology to a human IgG with the germline gene from Immunoglobulin Lambda Variable 6-57 (IGLV6-57), which has implications for amyloid light-chain (AL) amyloidosis. This discovery of the N-terminal clipping degradation pathway contributes to our understanding of immunoglobulin light chain misfolding and amyloid fibril deposition under physiological conditions.

Funder

Horizon Therapeutics

Publisher

MDPI AG

Subject

Drug Discovery,Immunology,Immunology and Allergy

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