(T1230-02-07) A Validated Immunocapture LC-MS/MS Hybrid Assay for the Quantitation of a Novel Multi-Domain Therapeutic Protein in Human Plasma with Clinical Sample Analysis

Purpose: BI X is a novel trivalent therapeutic protein with MW < 100KDa, developed for cancer treatment. BI X consists of three domains connected covalently by linkers and binds to two therapeutic targets as well as human albumin. It was critical to develop and validate a bioanalytical assay in understanding its clinical pharmacokinetics. In recent years, hybrid immunocapture liquid chromatography coupled with tandem mass spectrometry (IC-LC-MS/MS) has been increasingly used for the quantitation of large molecule drug candidates in the pharmaceutical industry. The purpose of this project was to develop and validate an IC-LC-MS/MS assay to support early phase clinical pharmacokinetic studies of BI X. In this presentation, we will report the experimental procedure and the assay elements evaluated to validate the IC-LC-MS/MS assay for the quantitation of BI X in human plasma. The validated assay was successfully applied to two clinical trials of BI X.

Methods: Reference standard BI X and the immunocapture reagent were prepared in house. The assay consisted of extraction of a 20 μL aliquot of human plasma containing BI X or blank control with pre-dilution/immunocapture/digestion combined with LC-MS/MS analysis of a unique surrogate peptide EGVSAIR of BI X. For the immunocapture, 0.1 mg/mL biotinylated monoclonal antibody to BI X and 5 mg/mL streptavidin magnetic beads were used. Subsequently, BI X was digested overnight with trypsin. The internal standard (IS) used was EGVSAI{ARG(13C6,15N4)}. Another unique surrogate peptide YDAVSLEGR of BI X and its IS YDAVSLEG{ARG(13C6,15N4)} were also measured for monitoring purpose only. The LC column was Waters Acquity peptide BEH C18 1.7μm, 2.1 x 50 mm with Waters Acquity protein BEH C18 1.7μm VanGuard pre-column. The gradient mobile phases, mobile phase A: water with 0.1% formic acid, mobile phase B: acetonitrile with 0.1% formic acid,were used. The injection volume was 10 μL. The surrogate peptides and IS’s were measured using an API 6500 mass spectrometer coupled with a UPLC system in the positive ion, electrospray, multiple reaction monitoring mode. The concentration of BI X was determined over the range of 0.500 to 100 μg/mL using linear calibration standard curve (eight concentration levels) with 1/x² weighting. A> benchtop automated Thermo KingFisher Flex bead handler was used to wash the beads and perform the elution in 96-well plates. During validation, calibration standards and curve range, accuracy and precision, specificity, recoveries and matrix effect, dilution linearity and analyte stabilities were evaluated.

Results: Validation criteria were met for both intra-run and inter-run accuracy and precision based on the analytical results for QC samples prepared at five concentration levels. Dilution accuracy and precision were evaluated by preparing over-the-curve QC samples and diluting 10-fold with blank plasma. The stabilities of the analyte in K2-EDTA human plasma were evaluated. These stability measurements included: freeze/thaw stability at -80ºC for four cycles, thawed matrix stability at room temperature for 42 hours, autosampler stability at 10 ºCfor 50.5 hours, processed sample stability at 10 ºC for 144.5 hours and long term freezer storage stability at -80 ºC for 938 days. Assay specificity was evaluated by 10 LLOQ samples from independent blank matrix lots, 10 LLOQ samples from independent cancer patient blank matrix lots and three zero samples (blank matrix with internal standard). Internal standard suitability, overall recovery, immuno-capture efficiency, peptide recovery, digestion efficiency, absolute and IS-normalized matrix effect including regular individual plasmas, hemolyzed plasma and lipemic plasma, carry-over and run size were all evaluated. All applicable validation acceptance criteria were met. The validated assay was used to support two clinical studies for BI X. For each accepted run, the acceptance criteria of calibration standards, QCs and/or Dilution QCs were met. Incurred sample re-analysis (ISR) were carried out for both studies and the results met the acceptance criteria for ISR.

Conclusion: A human plasma PK hybrid assay was developed and validated for the quantitation BI X using IC-LC-MS/MS. The assay was successfully used for clinical sample analysis in two BI X clinical studies and demonstrated adequate sensitivity, high specificity, robustness, and reliability in regulated bioanalysis for BI X.

Acknowledgements: The authors would like to thank David Dube and Jeffrey Duggan for their support of this project.