https://orcid.org/0000-0002-2418-1112
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ABSTRACT
Production of site-specific cysteine-engineered antibody-drug conjugates (ADCs) in mammalian cells may produce developability challenges, fragments, and heterogenous molecules, leading to potential product critical quality attributes in later development stages. Liquid phase chromatography with mass spectrometry (LC-MS) is widely used to evaluate antibody impurities and drug-to-antibody ratio, but faces challenges in analysis of fragment product variants of cysteine-engineered ADCs and oligonucleotide-to-antibody ratio (OAR) species of antibody-oligonucleotide conjugates (AOCs). Here, for the first time, we report novel capillary zone electrophoresis (CZE)-MS approaches to address the challenges above. CZE analysis of six ADCs made with different parent monoclonal antibodies (mAbs) and small molecule drug-linker payloads revealed that various fragment impurities, such as half mAbs with one/two drugs, light chains with one/two drugs, light chains with C-terminal cysteine truncation, heavy chain clippings, were well resolved from the main species. However, most of these fragments were coeluted or had signal suppression during LC-MS analysis. Furthermore, the method was optimized on both ionization and separation aspects to enable the characterization of two AOCs. The method successfully achieved baseline separation and accurate quantification of their OAR species, which were also highly challenging using conventional LC-MS methods. Finally, we compared the migration time and CZE separation profiles among ADCs and their parent mAbs, and found that properties of mAbs and linker payloads significantly influenced the separation of product variants by altering their size or charge. Our study showcases the good performance and broad applicability of CZE-MS techniques for monitoring the heterogeneity of cysteine-engineered ADCs and AOCs.
Supplementary material
Supplemental data for this article can be accessed online at https://doi.org/10.1080/19420862.2023.2229102
Abbreviations
| AA | = | Acetic acid |
| ADC | = | Antibody-drug conjugate |
| AOC | = | Antibody-oligonucleotide conjugate |
| BGE | = | Background electrolyte |
| CE-SDS | = | Capillary electrophoresis-sodium dodecyl sulfate |
| CZE-MS | = | Capillary zone electrophoresis-mass spectrometry |
| DAR | = | Drug-to-antibody ratio |
| ESI | = | Electrospray ionization |
| HC | = | Heavy chain |
| HIC | = | Hydrophobic interaction chromatography |
| icIEF | = | Imaged capillary isoelectric focusing |
| Lc | = | Light chain |
| Lc_DAR1/DAR2 | = | Light chain conjugated with one/two drug |
| LC-MS | = | Liquid phase chromatography-mass spectrometry |
| mAb | = | Monoclonal antibody |
| OAR | = | Oligonucleotide-to-antibody ratio |
| ON | = | Oligonucleotide |
| pCQA | = | Product critical quality attribute |
| PTM | = | Post-translational modification |
| RPLC | = | Reverse-phase liquid chromatography |
| SEC | = | Size exclusion chromatography |
Acknowledgments
The authors would like to acknowledge the contributions of all the members of the Merck & Co., Inc., South San Francisco, CA, USA Protein Sciences Department within Discovery Biologics (protein expression, protein purification, and characterization groups) as well as the Discovery Chemistry group in South San Francisco for ADCs and AOCs conjugation. The authors also acknowledge Dr. Liangliang Sun at Michigan State University for support on method development.
Disclosure statement
No potential conflict of interest was reported by the author(s).