Comparison of Analytical Methods for Antibody-Drug Conjugates Produced by Chemical Site-Specific Conjugation: First-Generation AJICAP

Anal Chem. 2019 Oct 15;91(20):12724-12732. doi: 10.1021/acs.analchem.9b02192. Epub 2019 Sep 3.

Abstract

Antibody-drug conjugates (ADCs) have become a major class of oncology biopharmaceuticals. Traditional ADCs have a stochastic distribution of cytotoxic drugs attached at several different sites on the antibody. The heterogeneous nature of stochastic ADCs results in a complex compositional analysis. To improve on traditional ADC technology, we have developed a chemical conjugation platform termed "AJICAP" for the site-specific modification of native antibodies using a class of IgG Fc affinity reagents. Here we report further investigation focusing on several analyses of a first-generation AJICAP-ADC (Angew. Chem., Int. Ed. 2019, 58, 5592-5597). For drug-antibody ratio (DAR) determination, we examined and compared six different analytical methods. To the best of our knowledge, this is the first report of a comparison of analytical techniques to measure the DAR for ADCs produced by a site-specific technology such as AJICAP. Furthermore, a rapid analytical process for confirmation of the site selectivity of AJICAP conjugation was established by SEC-Q-TOF-MS. The analytical strategy reported here can be applied to the DAR determination of site-specific ADCs.

MeSH terms

  • Antineoplastic Agents / chemistry
  • Chromatography, High Pressure Liquid
  • Chromatography, Reverse-Phase
  • Immunoconjugates / analysis*
  • Immunoglobulin Fc Fragments / chemistry
  • Mass Spectrometry / methods*
  • Oligopeptides / chemistry
  • Trastuzumab / chemistry

Substances

  • Antineoplastic Agents
  • Immunoconjugates
  • Immunoglobulin Fc Fragments
  • Oligopeptides
  • Trastuzumab
  • monomethyl auristatin E