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.
Highlights
IgG Fc N-glycans consist of heterogeneous glycoforms that make the Drug-antibody ratio (DAR) analysis more complicated since the lysine-linked antibody-drug conjugates (ADCs) bear heterogeneous numbers of small molecules as well
For MMAE-SMCC, the protected 3-mercaptopropionyl group was introduced onto the N-terminal secondary amine of MMAE by coupling reaction, the thiol group was deprotected and conjugated with the maleimide moiety of SMCC to give the target intermediate (Scheme S2)
We have developed an efficient approach[8, 36] for IgG glyco-remodeling and the rapid deglycosylation activity of an Endo-N-acetylglucosaminidase from Streptococcus pyogenes (Endo-S)[37] inspired us to employ this enzyme for ADC deglycosylation and real-time DAR detection
Summary
IgG Fc N-glycans consist of heterogeneous glycoforms that make the DAR analysis more complicated since the lysine-linked ADCs bear heterogeneous numbers of small molecules as well. There are about 20–30 m/z deconvoluted peaks for a typical lysine-linked ADC and the deglycosylation could dramatically simplify the DAR measurement by reducing the m/z to about 5–8 deconvoluted peaks. Efficient deglycosylation of ADC is a crucial step for real-time DAR analysis. We report a rapid process of deglycosylation and LC-MS determination within 15 minutes for real-time DAR analysis. With this approach, we monitored the ADC conjugation reaction, optimized the conjugation conditions, and controlled the DAR as expected. Random lysine-linked ADCs, glycosite-specific ADCs (gsADCs)[8], and complicated dual-payload ADCs (dpADCs) were prepared with this real-time analytical method
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