Characterization of Cysteinylation and Trisulfide Bonds in a Recombinant Monoclonal Antibody

Abstract

A recombinant monoclonal antibody with trisulfide bonds and cysteinylation was thoroughly characterized in the current study. Trisulfide bonds and cysteinylation were first detected when the recombinant monoclonal antibody was analyzed by LC-MS to determine the molecular weights of the intact antibody and its F(ab')2 fragment generated from IdeS digestion. LC-MS analysis of nonreduced tryptic peptides indicated trisulfide bonds are associated with the interchain disulfide bonds of both A isoform and A/B isoform and cysteinylation is associated only with the A isoform. A low percentage of trisulfide bonds was detected in between the light chain and heavy chain disulfide bond of the A and A/B forms. While the majority of trisulfide bonds and cysteinylation is associated with the hinge region peptide that involves the four closely spaced cysteine residues of the heavy chain. The locations of trisulfide bond and cysteinylation were determined using a combination of Edman sequencing and LC-MS. In the A isoform, the major site of the trisulfide bond and cysteinylation is between the first disulfide bond in the hinge region. In the A/B isoform, the trisulfide was also located in between the disulfide bond that is formed by the second pair of cysteine residues.

A high-throughput hydrophilic interaction liquid chromatography coupled with a charged aerosol detector method to assess trisulfides in IgG1 monoclonal antibodies using tris(2-carboxyethyl)phosphine reaction products: Tris(2-carboxyethyl)phosphine-oxide and tris(2-carboxyethyl)phosphine-sulfide

“…More recently, trisulfides have been identified and characterized in recombinant monoclonal antibodies (mAbs) of all immunoglobulin G (IgG) sub-classes [4][5][6][7]. Using peptide mapping liquid chromatography mass spectrometry (LC-MS) for mAb characterization, mAb trisulfides have been found in the inter-chain disulfide bridges, predominantly in the heavy chain-light chain (HC-LC) bridges [5,[8][9][10]. Low levels of mAb trisulfides have been detected in the to other forms of analysis.…”

Subunit mass analysis for monitoring antibody oxidation

“…Specific digestion of mAbs with IdeS generates smaller subunits (∼25 kDa), which allows high-resolution mass analysis and easier data interpretation 25-30 . These IdeS-based approaches facilitated development of more sensitive and specific methods for detecting and monitoring quality attributes of mAbs, including identity, 15,31-33 glycosylation, 26,32,34-37 glycation, 38 oxidation, 15,16 C-terminal lysine 26,39 and cysteinylation 40 . Studies also explored IdeS as a useful tool for quick and robust characterization of drug-to-antibody ratios in antibody-drug conjugates 41,42 …”

Advanced multivariate data analysis to determine the root cause of trisulfide bond formation in a novel antibody–peptide fusion

“…Monitoring and controlling product-related variants are vital to ensure consistent drug safety and efficacy. Recently there have been numerous reports describing monoclonal antibody heterogeneities related to trisulfide bonds (TSBs), defined under cysteinerelated variants (Gu et al, 2010;Kita et al, 2016;Kshirsagar et al, 2012;Liu and May, 2012;Nielsen et al, 2011;Pristatsky et al, 2009). Trisulfides are typically formed through the insertion of a sulfur atom into a disulfide bond within the inter-chain linkages between the light and heavy chains of the antibody structure.…”