LC-MS/MS determination of a human mAb drug candidate in rat serum using an isotopically labeled universal mAb internal standard.

We report the application of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalytical method for the determination of a recombinant human immunoglobulin G1 (hIgG1), NVSMAb-1, in rat serum. A stable isotopically labeled universal monoclonal antibody (SILuMab), instead of stable isotopically labeled surrogate peptide, was employed as the internal standard. The internal standard was added to the sample matrix in the first step of the sample preparation process, which involved protein precipitation and pellet digestion. The digestion of the resulting pellet with trypsin was performed prior to analysis of surrogate peptides of both NVSMAb-1 and SILuMab using LC-MS/MS. Precipitation reagents (1% TCA in IPA, 75% MeOH and 14% PEG) and digestion conditions (50°C for 2h and 60°C for 0.5h) were evaluated by monitoring LC-MS/MS responses of GPS and VVS in the resulting sample extracts. Overall, the use of 1% TCA in IPA appeared to be more effective as compared to 75% methanol in protein precipitation and removal of unwanted matrix components, e.g., albumin, and more appealing than 14% PEG as it avoided additional steps that are necessary to remove PEG or reduce PEG to a negligible level. The yield (LC-MS/MS response) of GPS is less sensitive than VVS to the changes of digestion conditions (time and temperature). The results obtained using SILuMab over SIL surrogate peptide as the internal standard appeared unaffected by the suboptimal sample processing method. For the current assay, surrogate peptide GPSVFPLAPSSK (GPS) was selected as surrogate peptide over VVSVLTVLHQDWLNGK (VVS) for quantitative analysis of NVSMAb-1. The optimal chromatographic separation was achieved on a Waters Cortecs C18 (100×2.1mm, 2.7μm) column using gradient elution with a total cycle time of approximately 8min. The mobile phases were water containing 0.1% formic acid (mobile phase A) and acetonitrile containing 0.1% formic acid (mobile phase B). The current method was validated for specificity, sensitivity, matrix effect, recovery, linearity, accuracy and precision, dilution integrity, and stability. The validated assay dynamic range was 10-5000μg/mL using 20μL of rat serum. The accuracy and precision for the LLOQs (10μg/mL) were within ±6.0% bias and ≤6.5% CV, respectively. From the intra-day and inter-day assay performance evaluations, the precision of the other QC sample (30, 300, 2500 and 3750μg/mL) results were ≤6.8% CV and the accuracy within ±4.8% bias, respectively. 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 toxicity study in rats administered 30, 150 and 750mg/kg/week intravenous infusion and 150mg/kg/week subcutaneous injection of NVSMAb-1.