Systematic LC/MS/MS Investigations for the IND-Enabling Extended Characterization of Antibody-Drug Conjugate Modifications.

Thomas Linz,David Rabuka,Dominick Yeo,Wesley Zmolek,William Haskins,Qiuting Hong,Robyn Barfield,Jesse Mcfarland

doi:10.3390/antib7040040

Abstract

We hypothesized that systematic liquid chromatography-tandem mass spectrometry investigations of an antibody–drug conjugate (ADC), its small and large molecular components, and surrogate small-molecule conjugates might comprise a simple and efficient approach for the extended characterization of ADCs. Furthermore, we envisioned that results from this work might allow us to assign specific composition changes in the ADC based on monoisotopic mass shifts of conjugatable modifications as detected in the surrogate small-molecule conjugates. We tested our hypothesis with a case study using an aldehyde-tag-based ADC conjugated to a noncleavable linker bearing a maytansine payload. Nearly quantitative bioconversion from cysteine to formylglycine was observed in the monoclonal antibody, and bioorthogonal conjugation was detected only on the formylglycine residues in the ADC. Using our method, both conjugatable and nonconjugatable modifications were discovered in the linker/payload; however, only conjugatable modifications were observed on the ADC. Based on these results, we anticipate that our approach to systematic mass spectrometric investigations can be successfully applied to other ADCs and therapeutic bioconjugates for investigational new drug (IND)-enabling extended characterization.

Highlights

IntroductionTherapeutic bioconjugates, including antibody–drug conjugates (ADCs), are an important class of biologic drugs that are well-represented in clinical trials for oncology and other indications
Therapeutic bioconjugates, including antibody–drug conjugates (ADCs), are an important class of biologic drugs that are well-represented in clinical trials for oncology and other indications.These leading-edge medicines, which harbor both chemical and post-translational modifications (PTMs) that require characterization, provide strong challenges for platform analytical methods that were built to address more traditional, less-complicated molecular formats, such as monoclonal antibodies
Formylglycine generating enzyme (FGE) catalyzes the cotranslational bioconversion of the thiol group in the Cysteine or free thiol (Cys) residue within the tag sequence to an aldehyde group within a formylglycine residue

Summary

Introduction

Therapeutic bioconjugates, including antibody–drug conjugates (ADCs), are an important class of biologic drugs that are well-represented in clinical trials for oncology and other indications. These leading-edge medicines, which harbor both chemical and post-translational modifications (PTMs) that require characterization, provide strong challenges for platform analytical methods that were built to address more traditional, less-complicated molecular formats, such as monoclonal antibodies. Antibodies 2018, 7, 40 linker/payloads), as well as assign chemical and post-translational modifications to specific amino acid residue(s) of large molecules (e.g., oxidation of methionine residues in antibodies and ADCs). Features are quantified by integrating the chromatographic peaks in extracted ion chromatograms (XICs) with a narrow m/z tolerance (e.g., 5 ppm) for each ion to characterize the relative abundance of various modifications in reference, partially degraded, and enriched minor variant samples

Methods

Results

Conclusion