Analysis of Pembrolizumab in Human Plasma by LC-MS/HRMS. Method Validation and Comparison with Elisa

Pembrolizumab is a humanized immunoglobulin G4-kappa anti-PD1 antibody used in the treatment of different solid tumors or haematological malignancies. A liquid chromatography coupled with a high resolution mass spectrometry (orbitrap technology) method was fully developed, optimized, and validated for quantitative analysis of pembrolizumab in human plasma. A mass spectrometry assay was used for the first time a full-length stable isotope-labelled pembrolizumab-like (Arginine 13C6-15N4 and Lysine 13C6-15N2) as an internal standard; the sample preparation was based on albumin depletion and trypsin digestion and, finally, one surrogate peptide was quantified in positive mode. The assay showed good linearity over the range of 1–100 μg/mL, a limit of quantification at 1 μg/mL, excellent accuracy from 4.4% to 5.1%, and also a between-day precision below 20% at the limit of quantification. In parallel, an in-house ELISA was developed with a linearity range from 2.5 to 50 µg/mL. Then, results were obtained from 70 plasma samples of cancer patients that were treated with pembrolizumab and quantified with both methods were compared using the Passing-Bablok regression analysis and Bland-Altman plotting. The LC-MS/HRMS method is easy to implement in the laboratory for use in the context of PK/PD studies, clinical trials, or therapeutic drug monitoring.

Development of a chromatography-free method for high-throughput MS-based bioanalysis of therapeutic monoclonal antibodies

Aim: Our objective was to test the feasibility of developing an LC-free, MS-based approach for high-throughput bioanalysis of humanized therapeutic monoclonal antibodies. Methodology: A universal tryptic peptide from human IgG1, IgG3 and IgG4 was selected as the surrogate peptide for quantitation. After tryptic digestion, the surrogate peptide was fractionated via solid-phase extraction before being subjected to direct infusion-based MS/MS analysis. A high-resolution, multiplexed (MSX = 2) parallel reaction monitoring method was developed for data acquisition. Results & conclusion: This proof-of-concept study demonstrated the feasibility of achieving high-throughput MS-based bioanalysis of monoclonal antibodies using an LC-free workflow with sensitivity comparable to conventional LC–MS/MS-based methods.

P16 Towards determination of endogenous prorenin in paediatric samples using a hybrid approach – impact of antibody selection for immunocapture

BackgroundSince sample volume is limited in children, innovative bioanalytical methods and enrichment procedures are highly required. The analysis of endogenous substances by liquid chromatography coupled to mass spectrometry is a highly specific method because of its selectivity and accuracy. However reliable detection of endogenous substances can only be achieved by a hybrid assay approach combining immunocapture and mass spectrometry. Key element of the immunocapture procedure is the selection of the appropriate antibody for capturing the desired antigen. This study is meant to identify the most suitable antibody that facilitates the development of an hybrid assay approach concerning reliable detection of endogenous prorenin in paediatric samples.MethodsDynabeads magnetic beads were coupled to three different antibodies from three different vendors (GeneTex, Molecular Innovations, R&D systems). 500 µL human plasma which was spiked with 20 ng recombinant human prorenin (Cayman chemicals). The immunocapture step was followed by protease digestion and a custom-made µelution solid-phase extraction protocol. The digest was analyzed by Shimadzu Nexera LC-system coupled with Sciex TripleTOF 6600 mass spectrometer.ResultsThe analysis of the captured prorenin was performed by the surrogate peptide approach. In this case the surrogate peptide was identified as unique. The comparison of the three available antibodies showed that one antibody did not ensure reliable binding properties in human matrix. Among the two remaining antibodies only one showed sufficient binding capacities to be applied in small sample volumes commonly available in paediatric samples. Using this hybrid approach enabled the enrichment of the required volume by factor of 20.ConclusionThis study identified the most suitable antibody for the immunocapture procedure of the prorenin hybrid approach. This is now followed by further mass spectrometric method development and validation prior to its application in paediatric samples.Disclosure(s)Declaration of interest: none Ilja Burdman and Bjoern B. Burckhardt declare that there is no conflict of interest. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors

Development and Validation of Reagents and Assays for EZH2 Peptide and Nucleosome High-Throughput Screens

Histone methyltransferases (HMT) catalyze the methylation of histone tail lysines, resulting in changes in gene transcription. Misregulation of these enzymes has been associated with various forms of cancer, making this target class a potential new area for the development of novel chemotherapeutics. EZH2 is the catalytic component of the polycomb group repressive complex (PRC2), which selectively methylates histone H3 lysine 27 (H3K27). EZH2 is overexpressed in prostate, breast, bladder, brain, and other tumor types and is recognized as a molecular marker for cancer progression and aggressiveness. Several new reagents and assays were developed to aid in the identification of EZH2 inhibitors, and these were used to execute two high-throughput screening campaigns. Activity assays using either an H3K27 peptide or nucleosomes as substrates for methylation are described. The strategy to screen EZH2 with either a surrogate peptide or a natural substrate led to the identification of the same tractable series. Compounds from this series are reversible, are [3H]-S-adenosyl-L-methionine competitive, and display biochemical inhibition of H3K27 methylation.