Fully automated glycoform profiling and sequence validation of the NIST reference antibody

Significance of the Topic

This work addresses the critical need for rapid, reliable characterization of monoclonal antibodies in biopharmaceutical research, development and quality control. Detailed analysis of glycosylation patterns and primary sequence integrity is essential for innovator and biosimilar products to ensure safety, efficacy and regulatory compliance. Automation of mass spectrometry workflows accelerates data acquisition and interpretation, reducing manual intervention and potential for error.

Study Objectives and Overview

This study employs the NIST reference antibody as a model to demonstrate a fully automated platform, BioPharma Compass 2.0, combined with the maXis II ultra-high resolution QTOF mass spectrometer. Three complementary analytical approaches are integrated:

• Intact mass screening for rapid glycoform profiling and detection of processing variants.
• Subunit (middle-down) domain analysis for quantitation and localization of modifications at sub-ppm accuracy.
• Bottom-up peptide mapping for near-complete sequence validation and fragment coverage.

Instrumentation

The automated workflows rely on:

• BioPharma Compass 2.0 software for data acquisition and processing.
• Bruker maXis II UHR-QTOF mass spectrometer.
• Standard flow UHPLC systems with BEH C4 or C18+ columns (2.1×100 mm, 1.7–1.5 µm).
• Solvent A: 0.1 % formic acid in water; Solvent B: 0.1 % formic acid in acetonitrile.
• Online desalting gradient for intact mass; IdeS digestion and reduction for subunit analysis; tryptic digestion for bottom-up mapping.

Main Results and Discussion

Intact mass screening using a one-minute desalting gradient yielded high-confidence detection of major glycoforms (G0F, G1F, G2F), lysine clipping variants and low-abundance aglycon species with mass accuracy <2 Da. Subunit analysis resolved Fc/2, Fd and light chain domains to baseline, enabling precise monoisotopic mass assignment (<1 ppm) and quantitation of modifications such as glycation and pyroglutamylation. The G1F glycoform showed 91.2 % lysine clipping, illustrating the approach’s quantitative power. Bottom-up mapping achieved 99.5 % sequence coverage of the light chain in a single run, with 96.2 % MS/MS fragment coverage, confirming primary structure and potential sequence variants.

Benefits and Practical Applications of the Method

These integrated workflows deliver:

• Rapid screening of critical quality attributes in under 10 minutes for intact mass.
• Domain-specific localization and quantitation of post-translational modifications at sub-ppm accuracy.
• Comprehensive sequence validation in a single bottom-up experiment.
• Automated data processing with minimal user input, supporting 21 CFR Part 11 compliance.

Future Trends and Potential Applications

Emerging directions include further integration of orthogonal fragmentation techniques (ETD, MALDI), higher throughput UHPLC methods, and incorporation of machine learning for automated spectrum interpretation. Expanding regulatory toolkits and cloud-based data management will enhance comparability studies in biosimilar development and accelerate therapeutic antibody screening.

Conclusion

BioPharma Compass 2.0 paired with the maXis II platform enables fully automated, high-throughput characterization of monoclonal antibodies across intact, subunit and peptide levels. This unified solution provides rapid, accurate and in-depth insights into glycosylation profiles, sequence integrity and post-translational modifications, meeting the stringent demands of biopharmaceutical analysis.

References

• Formolo T et al. Determination of the NISTmAb Primary Structure. ACS Symposium Series 1201:1–62 (2015). DOI:10.1021/bk-2015-1201.ch001.
• Ayoub D et al. Correct primary structure assessment and extensive glycoprofiling of cetuximab by intact, middle-up, middle-down and bottom-up ESI and MALDI mass spectrometry. MAbs 5(5):699–710 (2013). DOI:10.4161/mabs.25423.