Bioanalysis of the Bicycle ® Toxin Conjugate BT5528 and Released Monomethyl Auristatin E via Liquid Chromatography–Tandem Mass Spectrometry

Background: The Bicycle® toxin conjugate BT5528 is a novel peptide therapeutic conjugated to the cytotoxic agent monomethyl auristatin E (MMAE). A bioanalytical assay was developed to quantify BT5528 and unconjugated MMAE in human plasma. Methodology: BT5528 quantitation used a protein precipitation procedure followed by LC–MS/MS detection. Quantitation of MMAE required a selective offline and online solid-phase extraction with detection via LC–MS/MS. Results: BT5528 was quantified over 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 released MMAE, in a phase I/IIa clinical trial; to date, over 2000 human patient samples have been analyzed.