Bioanalytical workflow for novel scaffold protein-drug conjugates: quantitation of total Centyrin protein, conjugated Centyrin and free payload for Centyrin-drug conjugate in plasma and tissue samples using liquid chromatography-tandem mass spectrometry

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. 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. 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.