Peptides linked with warheads are a novel class of drugs that can selectively target and destroy specific proteins in cells. These drugs consist of a peptide moiety that binds to the target protein and a warhead moiety that induces irreversible covalent modification of the protein, leading to its degradation or inactivation. However, quantifying these drugs in biological matrices, such as plasma, is challenging due to their complex structure, low stability, and high susceptibility to matrix interference. The complex structure of these drugs makes them difficult to separate and detect by conventional analytical methods. These drugs’ low stability means they can degrade or lose their activity during sample preparation, storage, or analysis. The high susceptibility to matrix interference means that the presence of exogenous or endogenous compounds in the biological samples can affect the accuracy and precision of the measurement. Therefore, there is a need for a sensitive, selective, and robust analytical method that can overcome these challenges and accurately quantify the peptides linked with warheads in plasma samples.
A liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed and validated to analyze a peptide linked with a warhead in rat, non-human primate (NHP), and human plasma. The method involved a simple protein precipitation step followed by chromatographic separation and detection using a triple-quadrupole mass spectrometer. A stable isotope-labeled peptide was used as an internal standard (IS) to correct for analyte losses and matrix effects. The protein precipitation step was performed by adding acetonitrile to the plasma samples. This resulted in the precipitation of proteins and other interfering substances while retaining the peptide linked with the warhead and the internal standard in the supernatant. The supernatant was then injected into the LC–MS/MS system, where the peptide linked with warhead and the internal standard(IS) were separated on a reversed-phase C18 column using a gradient elution of water and acetonitrile, both containing 0.1% formic acid. The detection was performed by the mass spectrometer in the positive(+ve) electrospray ionization mode (ESI), using multiple reaction monitoring (MRM) of the precursor and product ions of the peptide linked with the warhead and the internal standard. The MRM transitions were optimized to achieve the highest sensitivity and specificity for the analyte and the internal standard.
Outcome: The method was developed and validated according to the US FDA guidance for bioanalytical method validation. The method showed good linearity, accuracy, precision, selectivity, sensitivity, recovery, and stability for the peptide linked with warhead in all three species. The calibration curves were linear over the 0.5–500 ng/mL concentration range, with correlation coefficients greater than 0.99. The accuracy and precision of the method were within the acceptance criteria of ±15% for the quality control samples and ±20% for the lower limit of quantification. The method’s selectivity was demonstrated by the absence of any significant interference from the blank plasma samples or the plasma samples spiked with other drugs. The method’s sensitivity was sufficient to quantify the peptide linked with the warhead at the lower limit of quantification of 0.5 ng/mL. The method’s recovery was consistent and reproducible, with values ranging from 85% to 95% for the peptide linked with the warhead and 90% to 100% for the internal standard. The method’s stability was evaluated under various conditions, such as freeze-thaw cycles, short-term and long-term storage, and post-preparative storage. The results showed that the peptide linked with the warhead and the internal standard were stable under all the tested conditions, with no significant degradation or loss of activity. The method was applied to support pharmacokinetic drug studies in preclinical and clinical settings. The method demonstrated its utility and reliability for quantifying peptides linked with warheads in plasma samples, providing valuable information for assessing the drug’s safety, efficacy, and pharmacological properties.
The Peptide LC MS analysis method was validated according to the US FDA guidance for bioanalytical method validation. The LC-MS assay development for the quantification of metabolites, peptides, and proteins showed good linearity, accuracy, precision, selectivity, sensitivity, recovery, and stability for the peptide linked with warhead in all three species. The calibration curves were linear over the 0.5–500 ng/mL concentration range, with correlation coefficients greater than 0.99. The accuracy and precision of the method were within the acceptance criteria of ±15% for the quality control samples and ±20% for the lower limit of peptide quantification. The method’s selectivity was demonstrated by the absence of any significant interference from the blank plasma samples or the plasma samples spiked with other drugs, even in non-human primate testing scenarios. The Peptide LC MS method’s sensitivity was sufficient for peptide quantification linked with the warhead at the lower limit of peptide quantification of 0.5 ng/mL. The peptide quantification mass spectrometry’s recovery was consistent and reproducible, with values ranging from 85% to 95% for the peptide linked with the warhead and 90% to 100% for the internal standard. The method’s stability was evaluated under various conditions, such as freeze-thaw cycles, short-term and long-term storage, and post-preparative storage. The results showed that the peptide linked with the warhead and the internal standard were stable under all the tested conditions, including those relevant to non-human primate testing, with no significant degradation or loss of activity. The method was applied to support peptide pharmacokinetics in preclinical and clinical settings. The method demonstrated its utility and reliability for peptide quantification linked with warheads in plasma samples. Experimental strategies for protein and peptide analysis by LC MS played a crucial role in ensuring the accuracy and robustness of the results obtained. It provided valuable information for assessing the drug’s safety, efficacy, and pharmacological properties in human and non-human primate testing scenarios.