Efficient Non-Reduced mAb Subunit LC-MS Analysis to Screen for Modifications of Unpaired Cysteines in Innovator and Biosimilars

Importance of the Topic

Liquid chromatography–mass spectrometry (LC-MS) is essential for characterizing modifications that influence monoclonal antibody (mAb) stability and bioactivity. Unpaired cysteine residues in the complementarity-determining regions can undergo cysteinylation or glutathionylation, potentially altering therapeutic efficacy and aggregation behavior. Rapid and reliable detection of these low-level variants is critical, particularly in biosimilar development where demonstrating similarity to innovator products is required.

Aims and Study Overview

This study aimed to develop and validate a streamlined, high-throughput subunit LC-MS workflow under non-reducing conditions to detect and quantify unpaired cysteine modifications in innovator and biosimilar IgG1 mAbs. Collaboration between Similis Bio and Waters focused on a five-minute enzymatic digestion and five-minute LC-MS analysis to support large sample sets during biosimilar process development.

Methodology and Instrumentation

The workflow combined limited enzymatic digestion at non-reducing conditions using FabRICATOR (IdeS enzyme) with fast UPLC-TOF analysis on the BioAccord LC-MS System. Key steps:

• Non-reduced subunit preparation: 5 min digestion at 37 °C with FabRICATOR, followed by dilution and direct LC-MS injection.
• LC conditions: ACQUITY Premier UPLC C4 column, 0.1% formic acid mobile phases, 5-min gradient, 0.4 mL/min flow.
• MS conditions: ACQUITY RDa TOF in positive ESI mode (400-7000 m/z), automatic data capture, diverts to waste outside target windows.
• Data analysis: Intact Mass App in waters_connect for deconvolution, mass matching (±20 ppm), and relative abundance calculation of unmodified and modified subunits.
• Site confirmation: Non-reduced peptide mapping with RapiZyme Trypsin, targeted DDA on Xevo G3 QTof to localize cysteinylation to the expected LC residue.

Main Results and Discussion

The non-reducing FabRICATOR digestion produced two main subunits: Fc (~25 kDa) and (Fd′+LC)2 (~100 kDa). Deconvoluted spectra revealed +119 Da (cysteinylation) and +305 Da (glutathionylation) variants on the Fab fragment. Innovator reference showed ~5% total cysteine modification, whereas biosimilar candidates ranged from 16% to 63%. Glycation (2–11%) and oxidation were also measured. The workflow provided confident quantitation down to ~0.5% variant abundance. Peptide mapping confirmed the unpaired cysteine site by matching y-ion ladders in targeted MS/MS spectra.

Benefits and Practical Applications

• Five-minute digestion and LC-MS analysis for rapid data turnaround.
• High specificity for unpaired cysteine modifications under non-reducing conditions.
• Automated processing and reporting using waters_connect and Intact Mass App, enabling near-real-time results.
• Scalable to large sample sets, supporting biosimilar process development and QC environments.

Future Trends and Applications

Integration of this rapid subunit LC-MS approach into GMP-compliant workflows may streamline release testing of mAb therapeutics. Advancements in high-resolution MS and improved data analytics will further enhance sensitivity for low-level variants. Combining non-reduced subunit analysis with orthogonal methods, such as intact mAb assays and advanced peptide mapping, could provide comprehensive quality attribute monitoring for next-generation biopharmaceuticals.

Conclusion

A five-minute non-reducing FabRICATOR subunit LC-MS workflow effectively screens unpaired cysteine modifications in mAbs, offering high throughput, precision, and compatibility with regulatory requirements. This method supports rapid decision-making in biosimilar development and can be adapted for broader mAb quality assessments.

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