Real-Time Analysis on Drug-Antibody Ratio of Antibody-Drug Conjugates for Synthesis, Process Optimization, and Quality Control

Abstract

Drug-antibody ratio (DAR) of antibody-drug conjugates (ADCs) is important for their therapeutic efficacy and pharmacokinetics, therefore control on DAR in synthesis process is a key for ADC quality control. Although various analytical methods were reported, the real-time monitoring on DAR is still a challenge because time-consuming sample preparation is usually needed during the analysis. Antibody deglycosylation of ADC simplifies DAR measurement, however long-time PNGaseF digestion for deglycosylation hampers the real-time detection. Here, we report a rapid DAR analysis within 15 min by robust deglycosylation treatment and LC-MS detection that enables real-time DAR monitoring for optimization on ADC synthetic process. With this approach, we were able to screen suitable conjugation conditions efficiently and afford the ADCs with expected DARs. To the best of our knowledge, this is the first report on real-time DAR analysis of ADCs for conjugation optimization and quality control, compatible with random lysine-linked ADCs, glycosite-specific ADCs, and the complicated dual-payload ADCs.

Challenges and new frontiers in analytical characterization of antibody-drug conjugates

“…In practice, deglycosylation by PNGase-F treatment prior to intact mass analysis can simplify the mass spectra significantly. 59 Another way of simplifying the mass spectra is to reduce the ADC molecules into heavy chain and light chain species, or cleave the ADC molecules into its Fc and Fd components by a combination of enzymatic treatment and reduction. 60 This approach allows for more detailed structural information and detection of low levels of variants such that the DAR can be determined with high accuracy and sensitivity.…”

Trastuzumab gold-conjugates: synthetic approach and in vitro evaluation of anticancer activities in breast cancer cell lines

“…Low DARs can result in reduced potency of the ADC while high DARs can result in increased pharmacokinetic of the ADC. 29 To mitigate such effects, we aimed for DAR comprised between 2 and 4 for our AGCs. Furthermore, Tras-1 and Tras-4 were obtained with purity > 95%.…”

Pharmacologic Considerations in the Disposition of Antibodies and Antibody-Drug Conjugates in Preclinical Models and in Patients

“…There have been extensive studies on the effects of DAR on ADC properties, which have also been reviewed [20,72,73,74].…”