Aim: Alternative scaffold proteins have emerged as novel platforms for development of therapeutic applications. One such application is in protein-drug conjugates (PDCs), which are analogous to antibody-drug conjugates.
Methodology: Liquid chromatography-mass spectrometry methods for quantitation of total protein, conjugate and free payload for a PDC based on Centyrin scaffold were developed. Tryptic peptides generated from a region of the Centyrin that does not contain a conjugation site, and another that has the conjugation site with the linker-payload attached were used as surrogates of the total and conjugated Centyrin, respectively.
Conclusion: The methods were successfully applied to analysis of samples from mice to quantify the plasma and tissue concentrations. This same workflow can potentially be applied to other PDCs and site-specific antibody-drug conjugates.
Keywords: ADC; CDC; Centyrin; Centyrin–drug conjugate; LC–MS; PDC; alternative scaffold; antibody–drug conjugate; bioanalysis; conjugation; protein–drug conjugate; quantitation.