Disulfide Bond Identification of Biotherapeutic Proteins Using Various Fragmentation Techniques Available on an Orbitrap Fusion Tribrid Mass Spectrometer

Importance of the topic

Disulfide bonds are essential for correct folding and function of biotherapeutic proteins. They contribute to structural stability, biological activity, and immunogenicity. Regulatory agencies require comprehensive mapping of these linkages to ensure product safety and efficacy.

Objectives and overview

This study focuses on mapping disulfide linkages in the fusion protein Etanercept by leveraging multiple fragmentation techniques on an Orbitrap Fusion Tribrid mass spectrometer. Both reduced and non-reduced tryptic digests were analyzed by LC-MS/MS in data-dependent acquisition (DDA) mode, with targeted MS2 electron transfer dissociation (ETD) and MS3 higher-energy collisional dissociation (HCD) to validate complex bond arrangements.

Used instrumentation

• Liquid Chromatography: Thermo Fisher Vanquish UHPLC with Accucore C18 column (100 × 2.1 mm, 1.7 µm)
• Mass Spectrometry: Orbitrap Fusion Tribrid
• Software: BioPharma Finder for peptide identification and disulfide mapping

Methodology

Etanercept was partially deglycosylated (PNGase F and neuraminidase) and denatured at pH 5 with guanidine and N-ethylmaleimide (NEM) to block free thiols. After buffer exchange, the sample was trypsinized overnight at 37 °C. An aliquot of the digest underwent reduction with DTT as a control. The non-reduced digest was analyzed by LC-MS with DDA-HCD, followed by targeted MS2 ETD and MS3 HCD for selected disulfide bond peptides.

Main results and discussion

Key findings:

• Confirmed canonical CDR3 domain disulfide linkages: C104-C112, C112-C115, and C121-C139.
• Detected a free thiol at C115 via NEM labeling, indicating partial heterogeneity.
• Mapped an N-terminal truncated peptide with disulfide bonds C18-C31 and C32-C45, revealing sample processing variability.
• ETD preferentially cleaved S-S bonds, producing distinctive c- and z-type fragment ions for individual peptides.
• MS3 HCD significantly enhanced sequence coverage and confidence for low-abundance or complex linkage patterns.
• Identified a non-tryptic peptide (PGTETSDVVC139KPC142APGTFSNTTSSTDIC157RPHQI) by combined MS2 ETD–MS3 HCD, illustrating discovery potential for unknown linkages.

Benefits and practical applications

This integrated strategy enables:

• Accurate mapping of both expected and aberrant (scrambled) disulfide bonds.
• Detection of free cysteines and heterogeneity in therapeutic proteins.
• High confidence in structural characterization supporting quality control.
• Regulatory compliance by providing detailed product knowledge.

Future trends and possibilities

Emerging directions include:

• Integration of data-independent acquisition (DIA) with ETD/MS3 for increased throughput.
• Automation and machine-learning–based algorithms for de novo disulfide mapping.
• Application to a broader range of biotherapeutics, such as multispecific antibodies and fusion constructs.
• Top-down and hybrid fragmentation approaches to capture intact proteoforms and linkages directly.

Conclusion

Combining HCD, ETD, and MS3 HCD on an Orbitrap Fusion Tribrid platform delivers comprehensive and high-confidence disulfide bond characterization in biotherapeutic proteins, meeting stringent quality and regulatory requirements.

Reference

Houel S, Sutton J, Sharma S, Zhang T, Huguet R, Samonig M, Zabrouskov V, Josephs J. Disulfide Bond Identification of Biotherapeutic Proteins Using Various Fragmentation Techniques Available on an Orbitrap Fusion Tribrid Mass Spectrometer. Thermo Fisher Scientific Poster 64808-EN; 2016.