Development of a validated liquid chromatography tandem mass spectrometry assay for a PEGylated adnectin in cynomolgus monkey plasma using protein precipitation and trypsin digestion

摘要

A liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed and validated for a PEGylated adnectin therapeutic protein in cynomolgus monkey plasma. The validated method was performed using protein precipitation coupled with trypsin digestion, followed by LC-MS/MS detection of a surrogate peptide generated from the PEGylated adnectin protein. A tryptic peptide generated from a PEGylated adnectin protein analog was used as the internal standard to standardize the digestion, extraction, and quantitation processes. The protein precipitation extraction of the protein from cynomolgus plasma was performed using an acidic 2-propanol organic solution. Following the extraction, the supernatant was removed and a 45. min trypsin digestion was performed at 60. °C on the supernatant layer. The linear dynamic range of the assay was 50.0-25,000. ng/mL. Chromatographic separation was performed with an Acquity BEH C18 (1.7. μm particle size, 2.1. mm. ×. 50. mm) column using gradient elution. The assay proved to have robust accuracy, precision, and stability for the representative surrogate peptide of the PEGylated adnectin protein being evaluated. The validated method was implemented as a high throughput assay for a PEGylated adnectin protein using a similar PEGylated adnectin therapeutic protein as the internal standard that can be used for future monkey toxicokinetic (TK) studies.