SEC Coupled to High-Resolution Mass Spectrometry for Detailed Characterization of mAbs and ADCs

Significance of the Topic

Monoclonal antibodies and antibody drug conjugates represent a rapidly growing class of biopharmaceuticals with critical applications in therapy and diagnostics. Detailed structural characterization of these complex proteins is essential for ensuring product quality safety and efficacy. Coupling size exclusion chromatography with high resolution mass spectrometry provides a powerful approach to monitor aggregation fragmentation glycosylation and drug to antibody ratio in a single workflow.

Objectives and Study Overview

The primary aim of this study was to demonstrate the application of MS compatible SEC conditions coupled to a high resolution Q TOF mass spectrometer for comprehensive analysis of intact mAbs subunit fragments and ADCs. Key goals included evaluation of volatile mobile phases for MS compatibility comparison with traditional buffers detailed profiling of glycoforms determination of drug distribution and assessment of purification strategies.

Methodology and Instrumentation

Sample preparation employed intact reduction and enzymatic digestions using IdeZ and papain under controlled pH and temperature conditions. Volatile mobile phase containing acetonitrile formic acid and trifluoroacetic acid enabled direct coupling of SEC to MS. SEC separations were carried out on an Agilent Bio SEC-3 column at 1 mL/min and 24 °C. MS data were acquired in positive ion mode over m/z 1000–3200 in high resolution profile mode.

• Intact mAb and ADC analysis at 2 mg/mL in Tris buffer
• IdeZ digestion at 0.5 mg/mL in phosphate buffer
• Papain digestion at 2 mg/mL with cysteine activation
• Reduction of subunits using DTT at 60 °C

• Liquid chromatography system Agilent 1290 Infinity series
• Column Agilent Bio SEC-3 7.8 × 300 mm 3 μm
• Mass spectrometer Agilent 6540 Q-TOF with Jet Stream ESI source
• Software Agilent MassHunter Acquisition Qualitative Analysis BioConfirm

Key Results and Discussion

• Volatile SEC mobile phase delivered sharp chromatographic peaks and a clean MS source compared to phosphate buffer which fouled the interface and suppressed signal
• Intact mAb analysis resolved major glycoforms with mass differences below 0.005 of theoretical values along with minor fragments
• IdeZ and papain digestions yielded well separated subunits including F(ab')2 Fab Fc and light and heavy chains with accurate mass confirmation
• ADC analysis allowed determination of drug to antibody ratio species distribution from DAR 0 to DAR 4 by deconvoluted MS spectra
• Protein A purification of culture supernatant enriched mAb enabling profiling of glycosylation C terminal truncation and light chain glycation

Benefits and Practical Applications

• Provides simultaneous size based separation and high accuracy mass measurement in a single run
• Enables rapid assessment of aggregation fragmentation and subunit profiles for quality control
• Details glycoform distribution critical for pharmacokinetics and immunogenicity studies
• Measures drug loading distribution in ADCs facilitating formulation optimization
• Supports clone screening and process monitoring via direct analysis of culture supernatants

Future Trends and Potential Applications

Advancements may include miniaturized SEC columns operated at lower flow rates to enhance sensitivity and reduce solvent consumption. Integration with native mass spectrometry could preserve noncovalent interactions enabling structural studies of higher order assemblies. Automated high throughput SEC MS workflows and online data processing will support real time process analytical technology and accelerate biopharmaceutical development.

Conclusion

The combined SEC HRMS approach delivers a versatile analytical platform for comprehensive characterization of mAbs and ADCs. MS compatible volatile mobile phases preserve chromatographic resolution while providing high quality mass spectra. This strategy supports detailed profiling of intact proteins subunits glycosylation and conjugation patterns critical for research and quality control of next generation biotherapeutics.

References

1. Sandra K Vandenheede I Sandra P Modern chromatographic and mass spectrometric techniques for protein biopharmaceutical characterization J Chromatogr A 2014 1335 81 103
2. Fekete S et al Chromatographic Electrophoretic and Mass Spectrometric Methods for the Analytical Characterization of Protein Biopharmaceuticals Anal Chem 2016 88 480 507