Fast Analysis of Therapeutic Monoclonal Antibodies, Fragments, and Oxidation Variants Using a Super-Macro Porous Reversed-Phase Column Coupled with an Orbitrap Mass Spectrometer

Importance of the Topic

Therapeutic monoclonal antibodies (mAbs) represent a rapidly expanding class of biopharmaceuticals used in oncology, autoimmune diseases, and other conditions. Structural heterogeneity arising from post-translational modifications such as oxidation can affect efficacy, safety, and stability. Rapid, high-resolution methods for fragment and variant analysis support quality control, accelerate development timelines, and ensure consistent product performance.

Study Objectives and Overview

This work describes a streamlined workflow to separate and characterize mAb fragments and detect oxidation variants within a 10-minute liquid chromatography (LC) run. Key goals include:

• Efficient separation of light chain (LC), heavy chain (HC), Fc, Fab, scFc, and F(ab′)₂ fragments generated by chemical reduction and enzyme digestion.
• Resolution of oxidized versus non-oxidized species without full peptide mapping.
• Demonstration of compatibility with high-resolution Orbitrap mass spectrometry for accurate mass measurement.

Methodology and Instrumentation

Sample Preparation and Fragment Generation:

• DTT reduction of inter-chain disulfides (4 mg/mL mAb, 20 mM DTT, 37 °C, 30 min).
• Papain digestion (2 mg/mL mAb, 0.04 mg/mL papain, Tris-HCl pH 7.6, 4 mM EDTA, 5 mM cysteine, 37 °C, 4 h).
• IdeS protease digestion (1 U enzyme per µg mAb, 50 mM phosphate pH 6.6, 37 °C, 30 min).

Chromatography:

• Column: Thermo Scientific™ MAbPac RP, 4 µm, 3 × 50 mm.
• Mobile phases: A) H₂O/FA/TFA (99.88:0.1:0.02 v/v/v); B) MeCN/H₂O/FA/TFA (90:9.88:0.1:0.02 v/v/v/v).
• Gradient: 0–10 min, linear elution; method duration 15 min; optimized with 0.1% TFA for improved separation of oxidized fragments.

Mass Spectrometry:

• Thermo Scientific™ Q Exactive™ Plus Orbitrap mass spectrometer.
• ESI in positive-ion mode; resolution set at 17,500 (FWHM at m/z 200).
• Mass range m/z 1,000–4,000; AGC target and in-source CID optimized for intact fragment analysis.

Results and Discussion

The 10-minute LC method achieved baseline separation of all mAb fragments including LC, HC, Fc, Fab, scFc, and F(ab′)₂. High-resolution MS spectra clearly resolved oxidized heavy chain (+16 Da) from non-oxidized HC. Similarly, oxidized and non-oxidized scFc fragments were distinguished at m/z 2525.60 and 2524.08, respectively. Increasing TFA to 0.1% in mobile phases further sharpened peak resolution for oxidation variant analysis.

Key observations:

• Fragment separation in under 15 minutes reduces analysis time by an order of magnitude compared to conventional peptide mapping.
• Orbitrap detection provided accurate mass confirmation of oxidation (+16 Da) on specific fragments without complete proteolytic digestion.
• Super-macro porous reversed-phase stationary phase supports fast, high-efficiency separations of large protein fragments.

Benefits and Practical Applications

This method offers a fast, robust approach for routine monitoring of mAb purity and oxidation status in QC and development laboratories. Benefits include:

• Reduced sample handling and digestion steps by analyzing large fragments directly.
• High throughput capability with 10-minute runs per sample.
• Compatibility with existing LC-MS platforms in regulated environments.

Future Trends and Potential Applications

Advancements may include integration of automated online digestion modules for seamless LC-MS workflows, development of even faster columns with sub-2 µm or core-shell technologies, and coupling with ion mobility spectrometry for enhanced variant resolution. Expansion to other antibody formats such as bispecifics or antibody–drug conjugates will extend the utility of rapid fragment profiling.

Conclusion

A super-macro porous reversed-phase LC coupled with high-resolution Orbitrap MS provides a powerful platform for the rapid separation and accurate mass analysis of therapeutic mAb fragments and oxidation variants. The 10-minute method delivers high throughput and reliable detection of critical quality attributes.

References

1. Liu H., Gaza-Bulseco G., Zhou L. Mass Spectrometry Analysis of Photo-Induced Methionine Oxidation of a Recombinant Human Monoclonal Antibody. J. Am. Soc. Mass Spectrom. 2009, 20, 525–528.