Lanshoeft, Christian, Wolf, Thierry, Heudi, Olivier, Cianferani, Sarah, Barteau, Samuel, Walles, Markus, Picard, Franck and Kretz, Olivier (2016) The use of generic surrogate peptides for the quantitative analysis of immunoglobulin G1 in pre-clinical species with high-resolution mass spectrometry. Analytical and bioanalytical Chemistry.

Abstract

In the present study, the application of a liquid chromatography high-resolution mass spectrometry (LC-HRMS) analytical assay for the quantitative analysis of a recombinant humanized immunoglobulin G1 (IgG1) in rat serum is reported using three generic peptides GPSVFPLAPSSK (GPS), TTPPVLDSDGSFFLYSK (TTP) and VVSVLTVLHQDWLNGK (VVS). Moreover, the deamidation site of a fourth peptide FNWYVDGVEVHNAK (FNW) was identified and further excluded from the assay evaluation due to the inaccuracy of the quantitative results. The rat serum samples were spiked with a fully labeled IgG1 as internal standard (ISTD). The digestion with trypsin was performed onto the pellet prior to peptide analysis by LC-HRMS using a quadrupole time of flight (QTOF) mass analyzer operating in selected reaction monitoring (SRM) mode with enhanced duty cycles (EDC). The assay linearity for the three investigated peptides was established for a humanized IgG1 (IgG1A) from 1.00 to 1000 µg mL-1 with a mean coefficient of determination (R2) higher than 0.9868. The inter-day accuracy and precision obtained in rat serum over three days was ≤11.4 % and ≤10.5 %, respectively. Short-term stability on the auto-sampler at 6 °C for 30 h, at RT for 48 h and a 100-fold dilution factor were demonstrated. In addition, QC samples prepared in cynomolgus monkey serum and measured with the present method met the acceptance criteria of ±20.0 % and ≤20.0 % for all three peptides regarding accuracy and precision, respectively. The LC-HRMS method was applied to the analysis of samples from five individual cynomolgus monkeys dosed with a second IgG1 (IgG1B) and consistent data were obtained compared to the LC-MS/MS method (conventional triple quadrupole (QqQ) mass analyzer operating in SRM). The present data demonstrate that LC-HRMS can be used for the quantitative analysis of IgG1 in both species and that quantification is not only limited to classical QqQ instruments.

Item Type:	Article
Keywords:	high-resolution mass spectrometry; generic peptide; immunoglobulin G; pellet digestion, pre-clinical species, deamidation
Date Deposited:	09 May 2016 23:45
Last Modified:	09 May 2016 23:45
URI:	https://oak.novartis.com/id/eprint/27276