Bioanalysis of the Bicycle R ? toxin conjugateBT5528 and released monomethyl auristatinE via liquid chromatography–tandem massspectrometry: supplementary materials

Background: The Bicycle R ? toxin conjugate BT5528 is a novel peptide therapeutic conjugated to thecytotoxic agent monomethyl auristatin E (MMAE). A bioanalytical assay was developed to quantifyBT5528 and unconjugated MMAE in human plasma. Methodology: BT5528 quantitation used a proteinprecipitation procedure followed by LC–MS/MS detection. Quantitation of MMAE required a selectiveoffline and online solid-phase extraction with detection via LC–MS/MS. Results: BT5528 was quantifiedover the assay range of 5–2500 ng/ml and free MMAE was quantified over the assay range of 0.05–50 ng/ml. Conclusion: Bioanalytical methods were used in the bioanalysis of intact BT5528 and releasedMMAE, in a phase I/IIa clinical trial; to date, over 2000 human patient samples have been analyzed.