Determination of drug-to-antibody ratio of antibody-drug conjugate in biological samples using microflow-liquid chromatography/high-resolution mass spectrometry

Bioanalysis. 2022 Dec;14(24):1533-1545. doi: 10.4155/bio-2022-0219. Epub 2023 Feb 24.


Background: Antibody-drug conjugates (ADCs) are a promising modality for cancer treatment; however, considering their complicated nature, analytical complexity in understanding their pharmacokinetics and pharmacodynamics in the body presents a significant challenge. Results: Vorsetuzumab maleimidocaproyl valine-citrulline p-aminobenzyloxycarbonyl monomethyl auristatin E was used to develop pretreatment and analytical workflows suitable for ADCs. Monomethyl auristatin E release and drug-to-antibody ratio retention were consistent in mouse plasma but inconsistent in monkey and human plasma. Further, metabolites were species-specific. Microflow-liquid chromatography/high-resolution mass spectrometry (LC-HRMS) resulted in a 4-7-fold improvement in detection sensitivity compared with conventional flow LC-HRMS. Conclusion: Microflow-LC-HRMS can be a useful tool in understanding the complex properties of ADCs in the body from a drug metabolism and pharmacokinetics point of view.

Keywords: antibody–drug conjugate; deconjugation; drug-to-antibody ratio; high-resolution mass spectrometry; metabolism of payload; microflow liquid chromatography; monomethyl auristatin E; species-specific; vorsetuzumab.

Plain language summary

Drug-to-antibody ratio (DAR), payload release and metabolite profile of deconjugated payload-linker of vorsetuzumab maleimidocaproyl valine-citrulline p-aminobenzyloxycarbonyl monomethyl auristatin E, an antibody–drug conjugate (ADC) with cleavable linker and monomethyl auristatin E as payload, are reported. Species-specific retention of DAR, payload release and metabolite patterns of deconjugated payload-linker of the ADC are summarized. Exploring the fate of payload-linker moieties deconjugated from ADCs in the body is also vital to understanding pharmacological activity and toxicity. Species-specific metabolite patterns of the ADC provided insight into the importance of optimization of the payload-linker moiety in biological samples, especially in humans. In terms of a more sensitive analytical platform for drug metabolism and pharmacokinetic evaluation, microflow-liquid chromatography/high-resolution mass spectrometry (LC–HRMS) in DAR analysis was found to take advantage of the improvement of detection sensitivity compared with conventional LC–HRMS. Because ADCs are a complex drug modality, these results indicated the importance of evaluation of ADCs from a drug metabolism and pharmacokinetics point of view to understand the pharmacology and toxicology of ADCs, more precisely.

MeSH terms

  • Animals
  • Antineoplastic Agents*
  • Chromatography, Liquid / methods
  • Haplorhini / metabolism
  • Humans
  • Immunoconjugates* / analysis
  • Mass Spectrometry / methods
  • Mice


  • Immunoconjugates
  • Antineoplastic Agents