Structural characterization of the therapeutic antibody and biosimilar product with hydrogen deuterium exchange mass spectrometry

Importance of the Topic

The structural characterization of monoclonal antibodies and their biosimilars is critical for ensuring therapeutic efficacy and regulatory compliance.
Hydrogen deuterium exchange mass spectrometry (HDX-MS) provides residue-level insights into protein conformation and dynamics under native conditions.
This approach addresses limitations of traditional techniques by requiring less sample and accommodating high molecular weight proteins.

Objectives and Study Overview

This study aimed to demonstrate a fully automated bottom-up HDX-MS workflow to compare the conformations of the therapeutic antibody trastuzumab and its biosimilar.
The workflow integrates automated sample handling, online digestion, liquid chromatography separation, and high-resolution mass spectrometry.
Key goals included achieving high sequence coverage, quantifying deuterium uptake across multiple time points, and evaluating conformational differences with statistical rigor.

Methodology and Instrumentation

Trastuzumab and biosimilar samples at 30 µM were diluted into deuterated buffer and incubated at 10 °C for six labeling time points (30 s to 4 h).
Deuteration was quenched with equal volume of cold quench buffer and online digested at 15 °C using a mixed-bed protease column.
Peptides were trapped, desalinated, and separated on C18 columns with a gradient of 3–95% acetonitrile in 0.1% formic acid.
Data were acquired on an Orbitrap Exploris 480 mass spectrometer in data-dependent acquisition mode, collecting full MS scans for triplicate HDX analysis and HCD MS2 for peptide identification.
Data processing involved BioPharma Finder 3.2 for protection factor calculation and HDExaminer 2.5 for deuterium uptake analysis.

Used Instrumentation

• Orbitrap Exploris 480 mass spectrometer
• H/D-X PAL automated HDX sampler (Trajan)
• UltiMate NCS-3500RS LC system
• Acclaim PepMap 100 and PepMap 300 C18 columns
• Mixed-bed immobilized protease (pepsin/fungal protease XIII)
• BioPharma Finder 3.2 and HDExaminer 2.5 software

Key Results and Discussion

Over 800 unique peptides covering >98% of both light and heavy chains were identified.
Deuterium uptake profiles derived from 86 light-chain and 264 heavy-chain peptides revealed solvent accessibility differences below 4% across most regions.
Residual plots highlighted minor deviations in glycosylated regions of the heavy chain, suggesting subtle conformational variation potentially due to glycoform heterogeneity.
Volcano plot analysis of 1548 (light chain) and 4752 (heavy chain) data points showed no statistically significant differences at 99% confidence.
Protection factor plots at single-residue resolution overlapped closely for both samples, confirming nearly identical conformations.

Benefits and Practical Applications

This automated HDX-MS workflow enables high-resolution conformational analysis with minimal manual intervention.
It supports biosimilarity assessment by providing quantitative, statistically robust comparison of antibody structures.
The approach accelerates quality control and streamlines regulatory submissions for biosimilar development.

Future Trends and Applications

Increasing automation and integration with complementary structural techniques will enhance throughput and data depth.
Advances in software algorithms will provide more precise protection factor mapping and dynamic modeling.
Expansion to other biotherapeutics, including antibody–drug conjugates and multi-domain proteins, will broaden HDX-MS utility in drug development.

Conclusion

The fully automated HDX-MS workflow utilizing the Orbitrap Exploris 480 system achieved comprehensive, high-resolution conformational comparison of trastuzumab and its biosimilar.
Statistical and protection factor analyses confirmed their structural equivalence, demonstrating the method’s suitability for biosimilarity assessments.

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