Antibody subunit analysis workflow on a quadrupole-Orbitrap mass spectrometer: from optimized sample preparation to data analysis

Importance of the Topic

Subunit analysis of monoclonal antibodies plays a critical role in confirming molecular weight, verifying primary structure, and supporting biopharmaceutical development and quality control. A robust workflow covering sample preparation, chromatographic separation, mass spectrometric detection, and data analysis ensures accurate characterization with minimal sample and instrument time.

Objectives and Overview

This study aims to optimize sample preparation and LC MS/MS conditions for antibody subunit analysis using a quadrupole Orbitrap instrument. It provides a detailed protocol for generation of light and heavy chain fragments via IdeS digestion and reduction, high resolution analysis for mass confirmation, and middle down fragmentation for sequence verification.

Instrumental Setup

• Sample preparation tools including denaturation with guanidine hydrochloride and reduction with DTT or TCEP
• Liquid chromatography using Thermo Vanquish UHPLC with MAbPac RP column at 80 °C
• Mass spectrometry on Thermo Q Exactive BioPharma HF or HF-X with Intact Protein Mode and High Mass Range Mode
• Data analysis via Thermo BioPharma Finder for spectral deconvolution and fragment assignment

Methodology

• IdeS protease digestion to yield F(ab′)2 and scFc fragments
• Reduction with 50 mM DTT or TCEP at 57 °C in presence of 4 M guanidine hydrochloride for full disulfide cleavage
• Reversed-phase gradient separation on a 2.1×50 mm MAbPac RP column with 0.1% formic acid mobile phase
• MS acquisition in Intact Protein Mode at resolutions up to 240 000, variable isolation windows, and stepped collision energies
• Deconvolution for monoisotopic or average mass determination and targeted PRM fragmentation for middle down sequence coverage

Main Results and Discussion

• Complete reduction is only achieved with denaturant plus elevated temperature; incomplete protocols leave intra-chain disulfides intact
• Optimized LC gradients effectively separate light chain, heavy chain, scFc, LC, and Fd′ subunits
• Intact Protein Mode improves signal intensity and charge distribution of high-mass subunits
• High resolution and dynamic resolution switching enable accurate monoisotopic mass for light chains and average mass for heavy chains
• Wide isolation windows and stepped collision energies yield up to 33% sequence coverage in a single LC run
• Partially reduced species are identified by retention time shifts and fragmentation gaps corresponding to intact bonds

Benefits and Practical Applications

• Rapid subunit mass confirmation supports identity testing and lot release in QC
• Middle down sequence verification detects variants, glycoforms, and terminal clipping
• Minimal preparation time and single-platform workflow increase laboratory throughput
• Single instrument covers intact, subunit, and middle down analyses

Future Trends and Possibilities

• Integration of native and denaturing analyses for proteins above 100 kDa
• Improved algorithms for standardizing average mass calculations
• Automation of sample preparation to boost throughput
• Combining ion mobility and top down fragmentation for deeper structural insight

Conclusion

An optimized workflow combining IdeS digestion, complete denaturation and reduction, high-resolution LC MS in Intact Protein Mode, and targeted middle down fragmentation provides reliable subunit characterization for monoclonal antibodies. This approach delivers accurate mass confirmation and robust sequence verification in a streamlined protocol suitable for biopharmaceutical research and quality control.

References

• Scheffler K and Damoc E Protocols for antibody subunit generation via IdeS and reduction
• Scheffler K Viner R and Damoc E High resolution top down strategies on an Orbitrap platform J Proteomics 175 42–55 2018
• Bobaly B et al Protocols for mAb sample preparation J Chromatogr B 1060 325–335 2017
• Farrell A et al Monoclonal antibody sequence assessment using hybrid quadrupole Orbitrap Anal Methods 10 3100–3109 2018