Highly selective and sensitive LC-MS/MS quantification of a therapeutic protein in human serum using immunoaffinity...

We report the development, validation and application of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalytical method for the determination of a recombinant protein drug candidate, NVS001, in human serum. A unique surrogate peptide, IPAETTIYNR (IPA), was identified to distinguish NVS001 from its endogenous counterpart, i.e., the full length and the C-terminal of protein X in LC-MS/MS. The selection of IPA for the LC-MS/MS determination of NVS001 was supported by the absence of peak responses due to endogenous components in the LC-MS/MS chromatograms of the extracted blank human serum samples. The optimal chromatographic separation of IPA from the extracted matrix components was achieved on a Waters Cortecs C₁₈ (100 × 2.1 mm, 2.7 μm) column using gradient elution with a run cycle time of approximately 7.5 min. The mobile phases were water containing 0.1% formic acid (mobile phase A) and acetonitrile containing 0.1% formic acid (mobile phase B). The method was validated for specificity, sensitivity, matrix effect, recovery, linearity, accuracy and precision, dilution integrity, and stability. The validated assay dynamic range was 10.0 to 1000 ng/mL using a 50 μL sample volume. The accuracy and precision for the LLOQ (10.0 ng/mL) sample results were within ±9.2% bias and ≤6.0% CV, respectively. From the intra-day and inter-day assay performance evaluations, the precision of the QC sample (30, 500 and 750 ng/mL) results were ≤3.5% CV and the accuracy within ±3.3% bias, respectively. An additional assessment of incurred sample reanalysis (ISR) was conducted to demonstrate the ruggedness and robustness of the assay method. The validated method was successfully implemented in support of a first-in-human study.