Compliance-ready LC-UV-MS-based monitoring of antibody quality attributes using a single quadrupole mass spectrometer

Importance of the topic

Therapeutic monoclonal antibodies require rigorous quality monitoring throughout development and manufacturing to ensure safety, efficacy, and regulatory compliance. Conventional high-resolution mass spectrometry methods offer detailed peptide mapping but can be costly and complex for routine QC labs. Integrating ultraviolet (UV) detection with a single quadrupole mass spectrometer provides a compliance-ready, targeted workflow that confirms peak identity by mass-to-charge ratio and retention time, while simplifying sample preparation and data evaluation.

Objectives and study overview

This study describes a streamlined liquid chromatography–UV–single quadrupole mass spectrometry (LC-UV-SQMS) workflow for targeted monitoring of post-translational modifications (PTMs) on the IgG1 antibody adalimumab. Key aims include demonstrating automated sample digestion, establishing system suitability and intelligent run control checks, and evaluating reproducibility of glycopeptide quantification under cGMP-compatible conditions.

Methodology and Instrumentation

Sample preparation employed a SMART Digest Trypsin Kit with magnetic beads on a KingFisher Duo Prime system, using a two-step digest (74 °C for 15 min, then 40 °C for 30 min) and quenching with formic acid. Chromatographic separation used a Thermo Scientific Hypersil GOLD Peptide column (150 × 2.1 mm, 1.9 µm) on a Vanquish Flex UHPLC with UV detection at 214 nm and mobile phases of water/formic acid and acetonitrile/formic acid. MS detection utilized an ISQ EM single quadrupole mass spectrometer operating in SIM mode, covering m/z 10–2,000 with component-based acquisition windows for 15 peptide standards (PRTC) and 12 adalimumab glycopeptides.

Použitá instrumentace

• Vanquish Flex UHPLC system with Binary Pump, Split Sampler FT, Column Compartment, and Variable Wavelength Detector
• ISQ EM single quadrupole mass spectrometer (HESI, positive ion mode)
• Thermo Scientific SMART Digest Trypsin Kit and KingFisher Duo Prime Purification System
• Hypersil GOLD Peptide column (150 × 2.1 mm, 1.9 µm)
• Chromeleon 7.3.2 Chromatography Data System with SST/IRC functionality

Main Results and Discussion

Automated system suitability tests (19 criteria) were embedded in the CDS method and executed in real time, ensuring chromatographic resolution, peak asymmetry, retention time precision, and carry-over limits. All criteria passed without manual intervention. UV chromatograms of adalimumab digests showed complex profiles, but SIM-MS detection reliably confirmed glycopeptide identities. Relative quantification of glycan variants (A2 core structures with varying galactose, mannose, and fucose) achieved percent relative standard deviations ≤9.4% for low-abundance species (<5%) and ≤3.2% for major forms (>5%) across three independent digests.

Benefits and Practical Applications

• Compliance-ready workflow with 21 CFR 11 traceability and eSignature support
• Rapid, automated sample digestion and data processing increase throughput
• Mass confirmation via SQMS enhances selectivity for co-eluting or low-abundance peptides
• Targeted quantitation of critical quality attributes suitable for QC laboratories

Future Trends and Potential Applications

• Extension of single-quad targeted workflows to broader multi‐attribute methods (MAM)
• Integration of real-time decision tools and AI-driven QC checks
• Adaptation to other biotherapeutic classes (fusion proteins, ADCs)
• High-throughput screening for process control and comparability studies

Conclusion

The LC-UV-SQMS workflow described provides a robust, streamlined solution for targeted peptide monitoring in monoclonal antibody quality control. Automated digestion, SST/IRC checks, and combined UV/MS detection deliver reliable identity confirmation and reproducible quantification of glycopeptide variants under cGMP-compatible conditions, enhancing productivity and compliance in QC laboratories.

Reference

1. Gucinski AC, Boyne MT. Evaluation of intact mass spectrometry for the quantitative analysis of protein therapeutics. Anal Chem. 2012;84(18):8045–8051.
2. Jefferis R. Posttranslational modifications and the immunogenicity of biotherapeutics. J Immunol Res. 2016;1–15.
3. Millan‐Martin S, Jakes C, Carillo S, Bones J. Multi‐attribute method (MAM) to assess analytical comparability of adalimumab biosimilars. J Pharm Biomed Anal. 2023;234:115543.