![]() ![]() In addition, protein gel electrophoresis showed that there was only one enriched protein in the 144 h ADC-depleted and antipayload immunoprecipitated plasma sample, as compared to the 0 h plasma immunoprecipitated sample, and the mass of this enriched protein was slightly heavier than the mass of serum albumin. Intact protein mass analysis showed that at the 144 h time point, the mass of the major protein in ADC-depleted human plasma had an additional 1347 Da over the native albumin extracted from human plasma, exactly matching the mass of the linker-payload. We found that an enzymatic release of payload from ADC-depleted human plasma at 144 h was able to account for almost 100% of the DAR loss. In plasma stability assays, when the ADC (Trastuzumab-mcVC-PABC-Auristatin-0101) was incubated with plasma over a 144-h time-course, a discrepancy was observed between the measured released free payload concentration and the measured loss of drug-to-antibody ratio (DAR), as measured by liquid chromatography-mass spectrometry (LC-MS). The payload was conjugated to trastuzumab by a protease-cleavable linker, maleimido-caproyl-valine-citruline-p-amino-benzyloxy carbonyl (mcVC-PABC). An ADC conjugated via hinge-cysteines to an auristatin payload was used as a model in this study to understand the impact of the maleimide linkers on ADC stability. Currently, a numbers of ADCs in drug development are made by coupling a linker-payload to native or engineered cysteine residues on the antibody. The reactive thiol of cysteine is often used for coupling maleimide-containing linker-payloads to antibodies resulting in the generation of antibody drug conjugates (ADCs).
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