Simple, Robust, High Quality Intact Mass Analysis—A Biosimilars Case Study

Importance of the Topic

The rapid expansion of biosimilars and monoclonal antibody therapeutics requires analytical workflows that deliver high-resolution structural information in a fast, reliable manner. Intact mass analysis provides a consolidated view of critical quality attributes—glycosylation profiles, amino-terminal processing, lysine clipping and aggregation—that underpin efficacy and safety. By minimizing sample preparation and accelerating data acquisition, advanced intact MS techniques support both development and routine quality control of these complex biopharmaceuticals.

Objectives and Study Overview

This case study describes the implementation of a native and denaturing intact mass analysis platform at the National Institute for Bioprocessing Research and Training (NIBRT). The goal was to demonstrate a streamlined workflow for comparing originator monoclonal antibodies and candidate biosimilars, correlating mass shifts with post-translational modifications and ensuring batch-to-batch consistency.

Methodology and Applied Instrumentation

The workflow integrates high-performance chromatography with Orbitrap mass spectrometry under both denaturing and native conditions. Sample separation is performed on size-exclusion and reverse-phase columns, followed by high-resolution MS detection. Deconvolution algorithms generate mass profiles for rapid comparison of proteoforms.

• Vanquish™ Flex UHPLC system with SmartInject technology
• MAbPac™ SEC-1 and RP Thermo Scientific™ chromatographic columns
• Thermo Scientific™ Q Exactive™ Plus Hybrid Quadrupole-Orbitrap with BioPharma Option
• Thermo Scientific™ Chromeleon™ CDS software
• BioPharma Finder™ Software 3.0

Main Results and Discussion

Switching from denaturing to native LC-MS reduced the average charge state distribution and extended m/z envelopes above 5,000. High mass range (HMR) mode on the Q Exactive Plus enabled clear separation of native charge envelopes, improving sensitivity for large proteoforms. Case data illustrate distinctions in glycoform abundance and N-terminal pyroglutamate formation between an originator mAb and its biosimilar, all within a 1-hour analysis cycle.

Benefits and Practical Applications

This intact mass solution offers:

• Minimal sample preparation with rapid turnaround
• High spectral resolution for confident PTM mapping
• Native analysis to preserve noncovalent assemblies and detect aggregation
• Automated deconvolution and sequence-based annotation for streamlined data processing

Future Trends and Opportunities

Emerging strategies in intact MS include higher-throughput multiplexing, integration with ion mobility, and enhanced software AI-driven deconvolution. These developments will further refine biosimilarity assessments, expedite biosimilar approval pathways, and support real-time process control in manufacturing.

Conclusion

The combination of robust UHPLC, Orbitrap-based native MS, and advanced data analysis delivers a powerful platform for intact mass characterization of monoclonal antibodies and biosimilars. This approach meets industry demands for speed, accuracy and depth of structural information.

Reference

Thermo Fisher Scientific application note CS72666-EN, 2018