Increased sensitivity and throughput for native intact mass analysis of mAb and ADCs using an online buffer exchange column

Importance of the Topic

Native intact mass analysis preserves all noncovalent interactions and provides critical insights into the heterogeneity of monoclonal antibodies (mAbs) and antibody–drug conjugates (ADCs), including drug-to-antibody ratios (DAR) and glycosylation patterns. Such detailed profiling is essential for quality control, regulatory compliance, and accelerating biopharmaceutical development.

Study Objectives and Overview

This study aimed to establish a rapid, high-sensitivity native intact mass spectrometry workflow for cysteine-linked ADCs—polatuzumab vedotin and trastuzumab deruxtecan—as well as a reference mAb (trastuzumab). Key goals included determining limits of detection (LOD), linear dynamic ranges (LDR), and evaluating throughput improvements using an online buffer exchange system.

Methodology

Samples were diluted in water and injected onto a NativePac OBE-1 SEC column with 100 mM ammonium acetate at 100 µL/min and 25 °C. Each run lasted 3 minutes. The eluent was directed into an Orbitrap Exploris 240 mass spectrometer operating in native mode, and data were processed using Thermo Scientific BioPharma Finder 5.2 software.

Instrumention

• UHPLC: Thermo Scientific Vanquish Flex
• Online SEC column: NativePac OBE-1 (P/N 43803-052130)
• Mass spectrometer: Orbitrap Exploris 240 with Biopharma option
• Data analysis: Thermo Scientific BioPharma Finder 5.2

Main Results and Discussion

The workflow achieved LODs of 2 ng for trastuzumab, 5 ng for polatuzumab vedotin, and 10 ng for trastuzumab deruxtecan on column (S/N > 10). Linear dynamic ranges covered three orders of magnitude with R2 > 0.99 for both raw MS signal and deconvoluted intensities. Compared to conventional SEC, the OBE-1 column required up to 20-fold less sample to deliver equivalent or superior sensitivity, while reducing analysis time to 3 minutes per injection.

Benefits and Practical Applications

• Enhanced sensitivity enables detection of low-abundance ADC species without sample enrichment.
• Fast 3 minute cycle time supports high-throughput screening in drug discovery and process development.
• Robust LDR and accurate DAR/glycoform profiling improve QC and release testing workflows.

Future Trends and Opportunities

Further integration with automated sample handling and AI-driven data interpretation could expand this approach to next-generation biotherapeutics, multi-attribute methods, and real-time process monitoring, pushing the limits of throughput and sensitivity even further.

Conclusion

The combination of an online buffer exchange SEC column and Orbitrap Exploris 240 mass spectrometry delivers a streamlined, ultra-sensitive native intact analysis platform for mAbs and ADCs. With LODs as low as 2 ng, broad dynamic ranges, and 3 minute runtimes, this workflow meets the growing demands for speed and sensitivity in biopharmaceutical characterization.

References

1. Pharmaceuticals 2020, 13, 245; doi:10.3390/ph13090245.