Project Goal

To address the need for precise bioanalytical methodologies for patient inclusion/exclusion criteria and to support pharmacokinetics/pharmacodynamics for gene therapy trials.

Biggest Challenges Right Now

• Development and validation of methods for monitoring endogenous small molecule biomarkers in plasma and urine
• For quantitative bioanalysis of oligonucleotides, sample preparation is usually achieved via ion-exchange solid phase extraction, chromatographic separation is by ion-pairing, producing multiply charged ions to be resolved in the mass spectrometer. This bioanalytical complexity requires:
  • High-resolution mass spectrometry to recognize the isotopic forms
  • A software solution to quantitate by summing multiple charge states and multiple isotopic forms
  • A robust ion-source to run thousands of samples from Investigational New Drug (IND)-enabling studies without need of major cleaning
  • Stable calibration for high sample throughput
  • Uniform resolution over a large mass range to accurately determine the mass of multiply charged metabolites

The Solution

• CLIA- and GLP-validated UPLC-MS/MS small molecule biomarker assays which are more robust and reliable than the current LBA assays used by physicians for patient inclusion/exclusion criteria
• Validated UPLC-HRMS plasma, urine, and tissue assays for siRNA molecules to selectively quantitate both the antisense and the sense strand and understand metabolic clearance
• Metabolite identification to compare in vitro and in vivo metabolism of oligonucleotides in toxicology species and humans

Outcomes of Research

• A UPLC-HRMS workflow for siRNA quantitation to support preclinical and clinical studies for the largest gene therapy trial to-date
• A CLIA workflow for inclusion/exclusion criteria using LC-MS/MS biomarker monitoring that is more accurate than the current industry standard