science laboratory test tubes

Authors: Lanshoeft C*, Wolf T**, Walles M**, Barteau S**, Picard F**, Kretz O**, Cianférani S***, Heudi O****
Published in: J Pharm Biomed Anal. 2016 Aug 31;131:214-222. doi: 10.1016/j.jpba.2016.08.039.

Abstract
An increasing demand of new analytical methods is associated with the growing number of biotherapeutic programs being prosecuted in the pharmaceutical industry. Whilst immunoassay has been the standard method for decades, a great interest in assays based on liquid chromatography tandem mass spectrometry (LC-MS/MS) is evolving.

In this present work, the development of a generic method for the quantitative analysis of therapeutic proteins based on human immunoglobulin G (hIgG) in rat serum is reported. The method is based on four generic peptides GPSVFPLAPSSK (GPS), TTPPVLDSDGSFFLYSK (TTP), VVSVLTVLHQDWLNGK (VVS) and FNWYVDGVEVHNAK (FNW) originating from different parts of the fraction crystallizable (Fc) region of a reference hIgG1 (hIgG1A).

A tryptic pellet digestion of rat serum spiked with hIgG1A and a stable isotope labeled protein (hIgG1B) used as internal standard (ISTD) was applied prior LC-MS/MS analysis. The upper limit of quantification was at 1000μg/mL. The lower limit of quantitation was for GPS, TTP and VVS at 1.00μg/mL whereas for FNW at 5.00μg/mL.

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Accuracy and precision data met acceptance over three days. The presented method was further successfully applied to the quantitative analysis of other hIgG1s (hIgG1C and hIgG1D) and hIgG4-based therapeutic proteins on spiked quality control (QC) samples in monkey and rat serum using calibration standards (Cs) prepared with hIgG1A in rat serum. In order to extend the applicability of our generic approach, a bispecific-bivalent hIgG1 (bb-hIgG1) and two lysine conjugated antibody-drug conjugates (ADC1 and ADC2) were incorporated as well.

The observed values on spiked QC samples in monkey serum were satisfactory with GPS for the determination of bb-hIgG1 whereas the FNW and TTP peptides were suitable for the ADCs. Moreover, comparable mean concentration-time profiles were obtained from monkeys previously dosed intravenously with ADC2 measured against Cs samples prepared either with hIgG1A in rat serum (presented approach) or with the actual ADC2 in monkey serum (conventional approach).

The results of this study highlight the great flexibility of our newly developed generic approach and that the choice of the surrogate peptide still remains critical when dealing with different matrix types or modalities.


Author affiliation:* Novartis Institutes for Biomedical Research, Drug Metabolism and Pharmacokinetics, Fabrikstrasse 14 – Novartis Campus, 4056, Basel, Switzerland; Laboratoire de Spectrométrie de Masse BioOrganique, Institut Pluridisciplinaire Hubert Curien, CNRS-Université de Strasbourg, UMR 7178, 25 rue Becquerel, 67087, Strasbourg, France.
** Novartis Institutes for Biomedical Research, Drug Metabolism and Pharmacokinetics, Fabrikstrasse 14 – Novartis Campus, 4056, Basel, Switzerland.
*** Laboratoire de Spectrométrie de Masse BioOrganique, Institut Pluridisciplinaire Hubert Curien, CNRS-Université de Strasbourg, UMR 7178, 25 rue Becquerel, 67087, Strasbourg, France.
**** Novartis Institutes for Biomedical Research, Drug Metabolism and Pharmacokinetics, Fabrikstrasse 14 – Novartis Campus, 4056, Basel, Switzerland.

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