Analysis of ADCs by Native Mass Spectrometry

Area of Science:

• Biophysical Chemistry
• Analytical Chemistry
• Mass Spectrometry
• Bioconjugation Chemistry

Background:

• Native mass spectrometry (MS) is crucial for analyzing antibody-drug conjugates (ADCs), particularly those with noncovalent interactions like cysteine-conjugated ADCs.
• Ion mobility spectrometry coupled with MS (IM-MS) enhances ADC characterization by differentiating ions based on collision cross-section, adding conformational insights.
• Traditional methods for ADC analysis can be time-consuming and may not fully capture critical quality attributes (CQAs).

Purpose of the Study:

• To evaluate the utility of online size exclusion chromatography (SEC) coupled with native MS/IM-MS for comprehensive ADC analysis.
• To demonstrate the capability of this integrated technique for assessing key CQAs required for ADC market release.
• To provide an automated workflow for buffer exchange prior to native MS/IM-MS analysis of ADCs.

Main Methods:

• Utilized native MS for analyzing ADCs under nondenaturing conditions.
• Hyphenated native MS with ion mobility spectrometry (IM-MS) for enhanced gas-phase ion separation and conformational analysis.
• Integrated size exclusion chromatography (SEC) online with native MS/IM-MS for automated buffer exchange and sequential analysis.

Main Results:

• Online SEC-native MS/IM-MS enabled global structural characterization of ADCs.
• The integrated method successfully assessed critical quality attributes including drug load distribution (DLD) and drug-to-antibody ratio (DAR).
• The relative amount of unconjugated mAb and average DAR (DARav) were accurately determined, facilitating ADC release assessment.

Conclusions:

• Online SEC-native MS/IM-MS is a powerful, automated technique for comprehensive ADC characterization.
• This approach provides essential data for assessing critical quality attributes, supporting ADC development and quality control.
• The hyphenated technique offers an efficient workflow for the quantitative and qualitative analysis of complex bioconjugates like ADCs.