Comparing biosimilars using intact mass analysis under denaturing and native conditions

Significance of the topic

The structural complexity and microheterogeneity of monoclonal antibodies demand robust intact mass analysis to guarantee biosimilar quality, safety, and efficacy. Combining denaturing and native mass spectrometry delivers complementary information on glycosylation, charge variants, and higher-order structure without extensive sample modifications.

Objectives and study overview

This study compared an originator infliximab drug product (DP1), a commercial biosimilar (DP2), and an in-house CHO-expressed biosimilar using high-resolution accurate-mass (HRAM) spectrometry under both denaturing and native conditions. The goal was to evaluate workflows that are rapid, reproducible, and minimize artefacts in biosimilar characterization.

Instrumentation

• Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer with BioPharma Option and extended mass range
• Vanquish Flex Binary UHPLC system with SmartInject capability
• MAbPac RP column (2.1 × 50 mm, 4 µm) for denaturing RPLC
• MAbPac SEC-1 column (4.0 × 300 mm, 5 µm, 300 Å) for native SEC
• HiTrap Protein A column, Amicon 10 kDa filters, Nanodrop 2000 spectrophotometer
• BioPharma Finder 3.0 software with ReSpect™ algorithm

Methodology

Samples were prepared at 3 mg/mL. Denaturing analysis used RPLC with 0.1% formic acid in water and acetonitrile gradient at 0.3 mL/min, 70 °C. Native analysis employed isocratic 50 mM ammonium acetate, 0.3 mL/min, 30 °C. MS source parameters were optimized for each mode; full-scan data were acquired at 35 000 resolution. Deconvolution and variant assignment used sliding-window ReSpect processing in BioPharma Finder.

Main results and discussion

Denaturing MS revealed that DP1 primarily carried fucosylated G0F/G0F glycoforms (27.7%) with both C-terminal lysines intact (21.6%) and one-lysine loss (12.0%). DP2 exhibited higher lysine truncation (54.7%), increased terminal galactose (41.5%), and reduced high-mannose (0%). The in-house biosimilar showed dominant double-lysine loss (88.8%) and elevated high-mannose glycoforms (12.1%). Native MS charge envelopes above m/z 5000 confirmed these distributions with high spectral resolution. Minor discrepancies between denaturing and native quantitation were within analytical variance.

Benefits and practical applications

• Single-instrument workflow for denatured and native intact mass analysis
• Minimal sample preparation reduces artificial modifications
• High mass resolution allows detection of low-abundance variants
• Rapid profiling supports biosimilar comparability and QC release testing

Future trends and prospects

Advances are expected in automated data processing, Orbitrap instruments with extended mass range for large biotherapeutics and complexes, integration with functional assays, and higher throughput UHPLC-MS platforms to accelerate biosimilar development and regulatory approval.

Conclusion

The Q Exactive Plus with BioPharma Option, coupled to Vanquish Flex UHPLC and BioPharma Finder software, enables fast, reproducible intact mass characterization of originator and biosimilar mAbs under both denaturing and native conditions. This approach provides comprehensive quality assessment with minimal sample artefacts.

References

1. Pisupati K, et al. A Multidimensional Analytical Comparison of Remicade and the Biosimilar Remsima. Analytical Chemistry. 2017;89(9):4838–4846.
2. Lee C, et al. Glycosylation Profile and Biological Activity of Remicade® Compared with Flixabi® and Remsima®. mAbs. 2017;9(5):968–977.
3. Tassi M, et al. Advances in Native HPLC and Intact Mass Spectrometry for Biopharmaceutical Characterization. Journal of Separation Science. 2018;41(1):125–144.
4. Millán Martín S, et al. Automated High-Throughput Peptide Mapping for Infliximab Comparability. Thermo Scientific Application Note 21849. 2018.
5. Raju TS, Jordan RE. Galactosylation Variations in Marketed Therapeutic Antibodies. mAbs. 2012;4(4):385–391.
6. Bowden TA, et al. Chemical and Structural Analysis of an Antibody Folding Intermediate Trapped during Glycan Biosynthesis. J Am Chem Soc. 2012;134(45):17554–17563.
7. Celltrion. CT-P13 (infliximab biosimilar) Briefing Document for the Arthritis Advisory Committee. FDA. 2016.