Figure 1 | Scientific Reports

Figure 1

From: Cysteine metabolic engineering and selective disulfide reduction produce superior antibody-drug-conjugates

Figure 1The alternative text for this image may have been generated using AI.

The TNB-capping strategy for chemoselective Cys-based conjugation for manufacturing high-quality clinical-grade site-specific ADCs. (A) TNB-capping and Cys-capping pathways to conjugatable antibody. (B) TSPP selective-reduction and subsequent direct-conjugation of mcvcPABC0101 linker payload reaction scheme and HIC analysis. TNB-capped Cys-mutant antibody trastuzumab HC-K290C-K334C produced ~ 90% DAR4 ADC. (C) Fully TNB-capped Cys-mutant antibody trastuzumab HC-K290C-LC-K183C was generated by stable CHO expression in proprietary basal medium with low fractional cysteine limitation ratios and a 1 mM DTNB bolus addition after the growth phase (Day 7). Conditioned media samples from culture conditions described in the table were purified and the antibody was digested with IdeS and subjected to LC/MS analysis to determine percentages of capping (Non-capped, glutathione (GSH)-capped, Cys-capped and TNB-capped). (D) DTNB feed concentrations ranging from 1 to 8 mM were evaluated as bolus additions on Days 7, 10, 11 or 12. The most optimal feed condition, 4 mM DTNB bolus on Day 10, yielded 70% of the desired TNB-capped DAR4 ADC species after TSPP reduction and conjugation.

Back to article page