Structure Characterization and Differentiation of Biosimilar and Reference Products Using Unique Combination of Complementary Fragmentation Mechanisms

Significance of the Topic

High-resolution mass spectrometry is critical to ensure quality and safety of biosimilars by providing detailed protein characterization including post-translational modifications and sequence variants. Comprehensive comparability studies rely on sensitive workflows to detect subtle structural differences between biosimilar and reference products.

Objectives and Study Overview

• Develop a robust LC-MS/MS workflow for differentiating biosimilar and reference proteins.
• Apply complementary fragmentation (HCD-ETD) on an Orbitrap Fusion Tribrid to achieve comprehensive mapping of peptide sequences and glycosylation sites.
• Quantitatively compare post-translational modifications across products.

Methodology and Instrumentation

• Protein digestion: Trypsin after reduction and alkylation of reference TPA, I-TNK variant, and G-TNK biosimilar samples.
• Liquid chromatography: Nano-LC with EasySpray source and 50 cm C18 column, 70 min gradient, flow rate 250 nL/min.
• Mass spectrometry: Thermo Scientific Orbitrap Fusion Tribrid with EASY-ETD ion source.
• Fragmentation strategy: Data-dependent HCD top-10 scans with on-the-fly detection of glycan oxonium ions triggering companion ETD MS/MS on the same precursor.
• Data analysis: PepFinder 1.0 for peptide identification, PTM mapping, and relative quantification with 5 ppm mass tolerance.

Main Results and Discussion

• 100% sequence coverage achieved for all samples, enabling confident peptide identification.
• Glycosylation mapping revealed four glycosylation sites; quantitative profiles showed site- and sample-specific differences at N103, N117, N184, and N448.
• Glycoform analysis at N448 indicated conserved sialylated structures across samples, while N103 glycoform profiles differed between I-TNK and G-TNK.
• Additional PTMs identified include N-deamidation (12 sites), cysteine modifications, oxidation, formylation, and glycation with quantifiable relative abundances.
• Detection of low-abundance double oxidation at W406 and precise localization of deamidation sites demonstrated high sensitivity.

Benefits and Practical Applications

• The combined HCDpdETD approach enables simultaneous glycopeptide characterization with sequence and glycan structure information.
• Quantitative PTM profiling supports lot-to-lot comparison and meets regulatory comparability requirements.
• The workflow accelerates biosimilar development by providing comprehensive structural insights into minor product differences.

Future Trends and Applications

High-throughput implementations of complementary fragmentation on advanced mass spectrometers will further enhance PTM mapping in complex biotherapeutics.
Integration with advanced software and machine learning will improve detection of novel variants and streamline regulatory submissions.

Conclusion

The developed Orbitrap Fusion LC-MS/MS workflow coupled with PepFinder 1.0 enables comprehensive and quantitative comparison of biosimilar and reference protein products, achieving full sequence coverage and detailed mapping of glycosylation and other PTMs. This approach provides reliable support for biosimilar comparability and quality control.