PEGS: HIGH THROUGHPUT CYSTEINYLATION SCREENING AT MAB SUBUNIT LEVEL USING LC-MS MONITORING WORKFLOW

Significance of the Topic

Monoclonal antibodies (mAbs) are critical biopharmaceutical therapeutics, and unpaired cysteine residues in the Fab region can undergo modifications such as cysteinylation and glutathionylation. These alterations may compromise structural integrity, promote aggregation, and reduce biological potency. Rapid and reliable monitoring of these modifications is essential for product quality, regulatory compliance, and the development of biosimilar candidates.

Objectives and Study Overview

The study aimed to establish a high-throughput LC-MS screening workflow at the mAb subunit level to quantify unpaired cysteine modifications. Innovator and multiple biosimilar samples were analyzed to assess the range of cysteinylation and glutathionylation levels and to demonstrate the method’s suitability for process and product comparability.

Methodology

A non-reducing digestion protocol using the enzyme FabRICATOR generated mAb subunits (Fc and (Fd’+LC)2) in five minutes. Subsequent five-minute LC-MS analysis was performed on a BioAccord system, with automated deconvolution and mass assignment via the Intact Mass App. Site confirmation of unpaired cysteine modifications in the innovator reference was achieved through targeted non-reduced peptide mapping using RapiZyme Trypsin and LC-MS/MS.

Used Instrumentation

• FabRICATOR enzyme for non-reducing digestion
• BioAccord LC-MS System with ACQUITY Premier UPLC
• 2.1 × 50 mm ACQUITY Premier BEH C4 column for subunit analysis
• Xevo G3 Mass Spectrometer coupled to ACQUITY Premier UPLC with 2.1 × 100 mm CSH C18 column for peptide mapping
• RapiZyme Trypsin for peptide digestion
• Waters_connect Informatics Platform with Intact Mass App for data processing

Key Results and Discussion

• Innovator mAb showed low levels of cysteinylation (4.2%) and glutathionylation (0.9%).
• Biosimilar samples exhibited a wider range of cysteinylation (10–60%) and glutathionylation (1.6–6.0%), reflecting process and cell line differences.
• UV and total ion chromatograms resolved Fc and (Fd’+LC)2 subunits, and deconvoluted spectra clearly identified modification mass shifts.
• Targeted peptide mapping confirmed the modification site on the unpaired cysteine via fragment ion analysis.

Benefits and Practical Applications

This rapid subunit LC-MS workflow delivers results in under ten minutes, enabling high-throughput screening for cysteine modifications. It supports quality control, comparability studies, and process development for both innovator and biosimilar mAbs. The automated data processing ensures compliance and streamlines reporting.

Future Trends and Applications

Advancements may include integration with automated sample preparation platforms, extension to additional post-translational modifications, predictive modeling for stability assessment, and application to alternative antibody formats. Enhanced data analytics and machine learning could further improve detection sensitivity and interpretation.

Conclusion

The optimized non-reduced subunit LC-MS workflow effectively quantifies unpaired cysteine modifications in mAbs, offering speed, robustness, and site-specific confirmation. It is readily adoptable for routine screening in biopharmaceutical development and quality assurance.

References

1. Liu H, May K. Disulfide bond structures of IgG molecules: structural variations, chemical modifications and possible impacts to stability and biological function. mAbs. 2012;4(1):17-23.
2. McSherry T, McSherry J, Ozaeta P, Longnecker K, Ramsay C, Fishpaugh J, Allen S. Cysteinylation of a monoclonal antibody leads to its inactivation. mAbs. 2016;8(4):718-725.
3. Banks DD, Gadgil HS, Pipes GD, Bondarenko PV, Hobbs V, Scavezze JL, Kim J, Jiang X, Mukku V, Dillon TM. Removal of Cysteinylation from an Unpaired Sulfhydryl in the Variable Region of a Recombinant Monoclonal IgG1 Antibody Improves Homogeneity, Stability, and Biological Activity. J Pharm Sci. 2008;97(2):775-790.