Unconjugated Payload Quantification and Dar Characterization of Antibody–Drug Conjugates Using High-Resolution MS

Abstract

Aim: The application of high-resolution MS to antibody–drug conjugate (ADC) drug development may provide insight into their safety and efficacy. Quantification of unconjugated cytotoxic drug (payload) and characterization of drug-to-antibody ratio distribution were determined in plasma using orthogonal acceleration quadrupole-time-of-flight MS. Results: Unconjugated payload quantification determined by quadrupole-time-of-flight-based MRM^{highresolution} and triple quadrupole-based multiple reaction monitoring were comparable and achieved detection limits of 0.030 and 0.015 ng/ml, respectively. As determined by immunocapture and TOF-MS, drug-to-antibody ratio remained unchanged up to 3-weeks postdose for an ADC containing engineered glutamine linkers, but declined from four to three over 2 weeks in an ADC containing engineered cysteine linkers. Conclusion: The use of high-resolution MS in ADC drug discovery confirms its utility within the bioanalytical discipline.

Keywords::

• drug-to-antibody ratio
• high-resolution MS
• immunocapture
• mass resolving power
• MRM
• MRM^HR
• unconjugated payload quantification

Supplementary Data

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

Acknowledgements

The authors would like to thank Comparative Medicine and Drug Safety Research and Development (DSRD) colleagues for conducting the animal in-life portion of this study. We also thank the Pfizer Oncology ADC Bioconjugation group and the Pfizer Worldwide Medicinal Chemistry (WWMC) team for supplying the ADCs and payload standard.