Final ProgramCPSA 2011
Science and Technology Coming Together to Make a Difference
October 3 - 6, 2011
Bucks County Sheraton Hotel
Langhorne, PA
Poster Abstract #15
Development and Validation of a Method for the Determination of a Genetic Fusion Protein in Human Plasma using UHPLC-MS/MS for First Time in Human (FTIH) Study Support
GlaxoSmithKline Pharmaceuticals, King of Prussia, PA USA
Currently, immunoassay is considered the ‘gold standard’ for detection and quantitation of biopharmaceuticals for the support of pharmacokinetic exposure studies as it provides an extremely sensitive platform to develop quantitative assays in biological matrices. The use of LC-MS/MS has gained considerable attention as a method to quantitate proteins from biological matrices as a result of its increased dynamic range and selectivity compared with immunoassay methods.
Data will be presented detailing development and validation of the GLP compliant method to support the FTIH study. The validated analytical method is based on protein digestion using the endoproteinase enzyme Lys-C. A specific peptide fragment is then extracted by solid phase extraction, followed by UHPLC/MS/MS analysis. The lower limit of quantification (LLQ) is 50 ng/mL, using a 50 µL aliquot of human plasma with a higher limit of quantification (HLQ) of 10,000 ng/mL. Additionally, the assay needed to take into account any interference caused by concomitant drug during study (acetaminophen).
The data will show selection of the precursor ion for monitoring based on the analysis of the peptide map generated using the endoproteinase enzyme Lys-C. Selectivity, sensitivity and linearity of the method in addition to stability in human plasma and blood at various temperature conditions will be presented.