Monoclonal antibody mass confirmation using intact protein deconvolution for a compliance-ready manufacturing environment

Significance of the Topic

The confirmation of intact protein mass, especially for monoclonal antibodies (mAbs), is critical in biopharmaceutical development and quality control. Rapid, accurate mass analysis provides structural information on glycoforms and proteoforms without extensive sample preparation. A compliance-ready workflow that integrates data acquisition, deconvolution, and reporting under regulated conditions addresses the industry need for high throughput and reliable QC assays.

Study Objectives and Overview

This application note evaluates a fully integrated workflow for high-throughput mAb mass confirmation. The main goals are:

• To demonstrate the sensitivity, mass accuracy, and reproducibility of the Thermo Scientific™ Orbitrap Exploris™ 240 mass spectrometer for intact protein analysis under denaturing conditions.
• To showcase an end-to-end compliance-ready process using Thermo Scientific™ Chromeleon™ CDS with Intact Protein Deconvolution (IPD) and Thermo Scientific™ BioPharma Finder™ software.
• To validate targeted deconvolution of large sample batches (50 sequential injections) via an Intact Protein Workbook import and automated reporting.

Methodology and Instrumentation

Sample Preparation and Chromatography:

• MAb standard (NISTmAb) diluted to 1 µg/µL in 0.1% formic acid.
• Thermo Scientific™ Vanquish™ Horizon UHPLC with a MAbPac™ RP column (2.1×50 mm, 4 µm).
• 8-minute linear gradient from 25% to 80% acetonitrile (0.1% formic acid) at 0.25 mL/min.

Mass Spectrometry:

• Orbitrap Exploris 240 with BioPharma Option, Full MS only, m/z 2000–5000.
• Resolution set to 30,000 (FWHM at m/z 200), AGC target 3E6, max injection time 200 ms, source fragmentation 110 V.

Data Processing:

• BioPharma Finder 4.0 for sequence import and Intact Protein Workbook generation.
• Chromeleon 7.2.10 CDS with IPD option for targeted deconvolution via ReSpect algorithm, 10–100 charge states, 10 ppm mass tolerance.

Key Results and Discussion

Chromatographic and spectral reproducibility were demonstrated over 50 consecutive injections:

• Retention time RSD of 0.61% across runs.
• Consistent charge envelopes spanning m/z 2200–4000 and stable relative intensities for glycoforms at charge states +46 and +47.
• Automated targeted deconvolution matched five major glycoforms (A2G0F/A2G1F to A2G2F/A2G2F) within a 10 ppm window for every injection.

Report generation under Chromeleon provided pass/fail results, extracted ion chromatograms, and customizable views of raw and deconvoluted spectra without additional parameter editing.

Benefits and Practical Applications

This integrated platform offers:

• High-confidence mass confirmation for QC release testing and lead selection.
• Minimal manual intervention through workbook-driven deconvolution and automated reporting.
• Scalability for large sample sets with compliance-ready audit trails in Chromeleon CDS.

Future Trends and Opportunities

Emerging developments may include:

• Native MS workflows to preserve noncovalent interactions and detect antibody assemblies.
• Enhanced deconvolution algorithms leveraging machine learning for complex proteoform patterns.
• Integration of multi-attribute methods combining intact mass, peptide mapping, and post-translational modification profiling in one CDS.

Conclusion

The combination of the Orbitrap Exploris 240 and Chromeleon CDS with Intact Protein Deconvolution provides a robust, sensitive, and high-throughput solution for intact mAb mass confirmation. The targeted deconvolution workflow simplifies compliance requirements and supports large-scale QC operations with consistent accuracy and automated reporting.

Instrumentation

• Vanquish Horizon UHPLC (Thermo Scientific) with Binary Pump, Split Sampler HT, and Column Compartment.
• MAbPac RP column, 2.1×50 mm, 4 µm.
• Orbitrap Exploris 240 mass spectrometer with BioPharma Option.
• Chromeleon 7.2.10 CDS and BioPharma Finder 4.0 software.

References

1. Scheffler K, Damoc E. Full characterization of heterogeneous antibody samples under denaturing and native conditions on the Q Exactive BioPharma mass spectrometer. Thermo Scientific AN 72348 (2017).
2. Füssl F et al. Comprehensive characterisation of the heterogeneity of adalimumab via charge variant analysis hyphenated on-line to native high resolution Orbitrap mass spectrometry. MAbs (2018).
3. Füssl F et al. Cracking proteoform complexity of ovalbumin with anion-exchange chromatography–high-resolution mass spectrometry under native conditions. J Proteome Res (2019).
4. Kristensen DB et al. Increasing throughput of native SEC-MS in biopharmaceutical development using a tandem UHPLC setup. Thermo Scientific CAN 73423 (2020).
5. Carillo S, Bones J. Sensitive profiling of IgG1 monoclonal antibody variants under native conditions. Thermo Scientific AB 73554 (2020).