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

Significance of the Topic

Monoclonal antibodies (mAbs) are key therapeutics and their structural integrity is critical for safety and efficacy. Unpaired cysteine residues in the Fab region can lead to unwanted modifications, aggregation, and loss of biological activity, making rapid and sensitive monitoring of cysteinylation essential in biopharmaceutical development and quality control.

Study Objectives and Overview

The primary goal was to establish a high-throughput non-reduced subunit screening workflow to characterize free and modified cysteines in mAbs. The approach integrates IdeS (FabRICATOR) digestion under non-reducing conditions with a 5-minute LC-MS analysis to quantify unpaired cysteine modifications across innovator and biosimilar samples.

Used Instrumentation

• BioAccord LC-MS System with ACQUITY Premier BEH C4 column
• Xevo G3 QTof Mass Spectrometer with ACQUITY Premier CSH C18 column
• Data processing via INTAct and UNIFI apps within the waters_connect platform

Methodology

mAbs were digested with FabRICATOR (IdeS) without reducing agents, generating a linked (Fd'+LC)2 subunit (~100 kDa) and dissociated Fc fragments (~25 kDa). A rapid gradient (20–80% organic over 3 minutes) enabled separation and quantitation of free, cysteinylated, and glutathionylated species. Targeted non-reduced peptide mapping employed data-dependent acquisition to confirm the exact site of cysteinylation on the light chain unpaired cysteine.

Results and Discussion

Chromatographic profiles showed clear resolution of subunit species. Deconvolution revealed the innovator mAb had minimal cysteinylation (~4%) and glutathionylation (~1%), whereas biosimilar samples exhibited increasing modification levels (cysteinylation up to ~60%). Quantitation was reliable down to 0.5% abundance. Peptide mapping confirmed the modification site via characteristic fragment ions in the y9 ladder.

Benefits and Practical Applications

• Fast, 5-minute LC-MS workflow for high-throughput screening
• Avoids disulfide reduction to preserve modification information
• Quantitative assessment of unpaired cysteine modifications for process development and QC
• Compatibility with existing informatics for automated reporting

Future Trends and Applications

Future work may integrate this non-reduced subunit method into multi-attribute monitoring platforms, enable real-time analytics in bioprocessing, and leverage machine learning for automated spectral interpretation. Adaptations to diverse antibody formats and higher throughput platforms are anticipated.

Conclusion

The non-reduced FabRICATOR LC-MS subunit workflow delivers a robust, rapid, and sensitive method for detecting and quantifying unpaired cysteine modifications in mAbs, supporting efficient development and quality control of antibody therapeutics.

Reference

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