ABSTRACT
A hybrid immunoaffinity LC-MS/MS assay was developed and validated for the quantitation of total antibody (TAb) from an antibody drug conjugate (ADC) PYX-201 in human plasma. PYX-201 was proteolyzed using trypsin, and a characteristic peptide fragment PYX-201 P1 with ten amino acids IPPTFGQGTK from the complementarity-determining regions (CDRs) was used as a surrogate for the quantitation of the TAb from PYX-201. Stable isotope labelled (SIL) peptide I(13C6, 15N)PPTFG(13C9, 15N)QGTK was used as the internal standard (IS). We performed chromatographic analysis using a Waters Acquity BEH Phenyl column (2.1 mm × 50 mm, 1.7 µm). Quantification of PYX-201 TAb was carried out on a Sciex triple quadrupole mass spectrometer API 6500 using multiple reaction monitoring (MRM) mode with positive electrospray ionization. To validate PYX-201 TAb, a concentration range of 0.0500 µg/mL to 20.0 µg/mL was used, yielding a correlation coefficient (r) of ≥ 0.9947. For intra-assay measurements, the percent relative error (%RE) ranged from -23.2% to 1.0%, with a coefficient of variation (%CV) of ≤ 14.2%. In terms of inter-assay measurements, the %RE was between -10.5% and -5.7%, with a %CV of ≤ 12.7%. The average recovery of the analyte was determined to be 81.4%, while the average recovery of the internal standard (IS) was 97.2%. Furthermore, PYX-201 TAb demonstrated stability in human plasma and human whole blood under various tested conditions. This assay has been successfully applied to human sample analysis to support a clinical study.
Subject(s)
Peptide Fragments , Tandem Mass Spectrometry , Humans , Chromatography, Liquid/methods , Tandem Mass Spectrometry/methods , Reproducibility of ResultsABSTRACT
PYX-201 is an investigational antibody drug conjugate (ADC) with an engineered, fully human IgG1 antibody, a cleavable chemical linker, and a toxin (Aur0101) with an average drug-antibody ratio (DAR) of â¼ 4. A sensitive and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and fully validated to determine the presence in human plasma, of free payload Aur0101 from PYX-201 to assess drug safety and efficacy. Aur0101 and its deuterated internal standard (IS), Aur0101_d8, were extracted from 25 µL of human plasma using a solid liquid extraction (SLE) method. Chromatographic analysis was carried out on a Waters Acquity UPLC BEH C18 (2.1 mm × 50 mm, 1.7 µm, 130 A) column. Quantitation of free Aur0101 was conducted on a Sciex triple quadrupole mass spectrometer API 6500 + using multiple reaction monitoring (MRM) mode via positive electrospray ionization. The calibration curve was linear over the concentration range of 25.0 to 12,500 pg/mL with correlation coefficient, r2 ≥ 0.9988. The intra-assay %RE was between -4.3% to 14.3% with % CV was ≤ 6.2%. The inter-assay %RE was between -0.2% to 9.5% with % CV was ≤ 6.1%. The average analyte recovery was 89.7% and the average IS recovery was 88.7%. Aur0101 was found to be stable in human plasma and human whole blood under various tested conditions with and without the presence of PYX-201. To our knowledge, this is the first published fully validated assay for free, unconjugated Aur0101 in any matrix, from any species. This assay has been successfully applied to clinical sample analysis to support clinical studies.
Subject(s)
Immunoconjugates , Tandem Mass Spectrometry , Humans , Chromatography, Liquid/methods , Tandem Mass Spectrometry/methods , Chromatography, High Pressure Liquid/methods , Reproducibility of Results , Spectrometry, Mass, Electrospray IonizationABSTRACT
PYX-201 is an anti-extra domain B splice variant of fibronectin (EDB + FN) antibody drug conjugate (ADC) composed of a fully human IgG1 antibody, a cleavable mcValCitPABC linker, and four Auristatin 0101 (Aur0101, PF-06380101) payload molecules. To better understand the pharmacokinetic (PK) profile of PYX-201 after it is administered to cancer patients, the development of a reliable bioanalytical assay to accurately and precisely quantitate PYX-201 in human plasma is required. In this manuscript, we present a hybrid immunoaffinity LC-MS/MS assay used to successfully analyze PYX-201 in human plasma. PYX-201 was enriched by MABSelect beads coated with protein A in human plasma samples. The bound proteins were subjected to "on-bead" proteolysis with papain to release the payload Aur0101. The stable isotope labelled internal standard (SIL-IS) Aur0101-d8 was added and the released Aur0101 was quantified as a surrogate for the total ADC concentration. The separation was performed on a UPLC C18 column coupled with tandem mass spectrometry. The LC-MS/MS assay was validated over the range 0.0250 to 25.0 µg/mL with excellent accuracy and precision. The overall accuracy (%RE) was between -3.8% and -0.1% and the inter-assay precision (%CV) was <5.8%. PYX-201 was found to be stable in human plasma for at least 24 h on ice, 15 days after being stored at -80 °C, as well as after five freeze/thaw cycles of being frozen at -25 °C or -80 °C and thawed on ice. The assay this paper reports on, has been successfully applied to human sample analysis to support clinical studies.
