Capillary Chromatography for Therapeutic Proteins and Their Subunit Analysis Using MAbPac RP Column

Importance of the Topic

The accurate characterization of intact monoclonal antibodies (mAbs) and their subunits is critical in biopharmaceutical development, supporting process optimization, quality control and biosimilarity assessments. Capillary-flow chromatography coupled with high-resolution mass spectrometry enables analyses on minimal sample quantities, addressing challenges in early-stage process development where material is scarce.

Objectives and Overview of the Study

This work introduces a novel capillary Thermo Scientific™ MAbPac™ RP column (4 µm, 0.15 mm × 150 mm) designed for intact therapeutic protein and subunit analysis. The study aims to demonstrate:

• Enhanced sensitivity at low sample loads (10–20 ng).
• Compatibility with intact mass, glycoform profiling and top-down workflows.
• Application to innovator and biosimilar trastuzumab, Rituximab, NIST mAb and a glycosylated Fab.

Methodology and Used Instrumentation

Sample Preparation:

• Intact mAbs buffer-exchanged using 10 kDa cutoff filters.
• Endo S digestion for fucosylation profiling; IdeS digestion and reduction for subunit generation (scFc, LC, Fdʹ).

Chromatography and MS Conditions:

• Thermo Scientific™ UltiMate™ 3000 RSLCnano system.
• Thermo Scientific™ MAbPac™ RP capillary column (0.15 mm × 150 mm, 4 µm).
• Thermo Scientific™ Q Exactive™ Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer.
• Resolution: 17,500 for intact workflows, 140,000 for subunit mode and targeted MS2.
• Data acquisition with Thermo Scientific™ Chromeleon™ CDS and Thermo Scientific™ Xcalibur™; data analysis via Thermo Scientific™ BioPharma Finder™ 3.2.

Main Results and Discussion

Intact mAb Analysis:

• Trastuzumab innovator and biosimilar showed co-elution but distinct glycoform distributions.
• Capillary flow provided up to 140-fold sensitivity gain over high-flow systems, enabling detection of low-abundance glycoforms.

Subunit Profiling:

• Clear separation of scFc, LC and Fdʹ subunits at 140,000 resolution.
• scFc fragment glycoform mirror images highlighted differences between innovator and biosimilar trastuzumab.

Top-Down and Targeted MS2:

• High-accuracy fragment ions (<5 ppm) achieved in top-down experiments for sequence confirmation.
• Multiplexed targeted MS2 enabled confident assignment of glycoforms G0, G1, G2 and levels of glycation.

Benefits and Practical Applications

• Unprecedented sensitivity allows use of scarce or precious samples early in process development.
• Supports intact mass, glycoform profiling and subunit workflows on a single platform.
• Improves throughput and data quality for biopharma QC, biosimilarity testing and structural characterization.

Future Trends and Applications

Advances in capillary chromatography and high-resolution MS will drive:

• Deeper integration of native MS and top-down proteomics for comprehensive PTM mapping.
• Automation and miniaturization to further reduce sample requirements.
• Real-time monitoring of bioreactor processes using online capillary LC-MS.

Conclusion

The capillary MAbPac RP column combined with Orbitrap MS delivers exceptional sensitivity, resolution and workflow flexibility for intact and subunit analysis of therapeutic proteins. This platform meets the demands of early-stage development and QC, enabling detailed PTM profiling on minimal sample loads.

Reference

Sun X., Szabo Z., Robson B., Baynham M., Lin S. Capillary Chromatography for Therapeutic Proteins and Their Subunit Analysis Using MAbPac RP Column. Thermo Fisher Scientific Symposium, 2020.