Michael Zorn (Presenter)
Immundiagnostik AG
Authorship: C. Christ1, M. Zorn1, K. Hartmann1, A. Dignass2,3, F. Hartmann2,3, J. Stein3,4, F. P. Armbruster1
(1) Immundiagnostik AG, Bensheim, Germany; (2) Agaplesion Markus Krankenhaus, Frankfurt/Main, Germany; (3) Interdisciplinary Crohn Colitis Centre Rhein-Main, Frankfurt/Main, Germany, (4) DGD Clinics Sachsenhausen, Frankfurt/Main, Germany
| Short Abstract Monoclonal antibodies have become commonplace biotherapeutics. Lack of response to monoclonal antibodies (mAbs) has been associated with inadequate mAb serum concentrations. Therapeutic drug monitoring (TDM) of mAbs has the potential to guide to more effective dosing in individual patients [1]. Here we show the development of an liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the absolute quantitation of Vedolizumab in serum. |
Long Abstract
Introduction:
Monoclonal antibodies have become commonplace biotherapeutics. Lack of response to monoclonal antibodies (mAbs) has been associated with inadequate mAb serum concentrations. Therapeutic drug monitoring (TDM) of mAbs has the potential to guide to more effective dosing in individual patients [1]. The therapeutic mAb Vedolizumab is an antagonist of α4β7 integrin. Blocking the α4β7- integrin by Vedolizumab results in gut-selective anti-inflammatory activity in the gastrointestinal tract of patients suffering from ulcerative colitis and Crohn`s disease. Vedolizumab has recently been approved for patients with moderately to severely active chronic inflammatory bowel disease [2]. Here we show the development of an liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the absolute quantitation of Vedolizumab in serum. The method was applied to evaluate the correlation between Vedolizumab trough levels and treatment response of patients.
Methods:
Over a time period of 6 months 155 Vedolizumab serum trough levels from 51 patients were assessed directly before the next scheduled application (standard scheme: week 0, week 2, week 6, every 4 or every 8 weeks). Quantitative method development and sample analysis was performed by LC-MS/MS on an Ultimate 3000 RSLC UPLC system (Dionex/Thermo Fisher Scientific) coupled to an TSQ Vantage triple quadrupole mass spectrometer (Thermo Fisher Scientific) equipped with H-ESI. The system was operated in positive-ion multiple reaction monitoring (MRM) mode. Calibrators (Cals) and Controls (QCs) were prepared by serial dilution of Vedolizumab stock solution (5.45 mg/ml) into human serum yielding in 3 Cal concentrations of 8.33, 25.0 and 75.0 µg/ml as well for QC levels at 16.6 and 50.0 µg/ml. Sample preparation started with the isolation of Vedolizumab by affinity purification using ImmuTube®-columns. The enriched mAb was subjected to proteolytic digestion for generation of surrogate peptides. The 13C and 15N heavy-labeled peptides were added prior to LC-MS/MS-analysis. Serum samples were additionally analyzed with an in-house validated ELISA for Vedolizumab quantification.
Results:
In silico analysis of Vedolizumab sequence allowed the identification of unique surrogate peptides that could be used for the quantification. Peptides containing KK or RR sequences or having residues prone to chemical modification, such as Met or Cys were excluded. Reproducibility of the surrogate peptides during proteolytic digestion, solubility, optimal ionization efficiency, susceptibilities to collision induced dissociation (CID) and optimal LC-MS/MS-settings were experimentally investigated in order to achieve the required sensitivity and specificity of the assay.
The calibration curves were linear over the concentration range from 8.33 to 75 µg/ml of Vedolizumab with correlation coefficient (r2) of duplicate calibration curves ˃0.99. Limit of detection and limit of quantification were determined with 1.6 and 8.33 µg/ml, respectively. The intraday (n=15) and interday (n=20) precision are within 20%.
Comparison with an in-house developed ELISA shows a correlation coefficient (r2) of > 0.91. LC-MS/MS-analysis of serum samples revealed considerably lower mAb trough levels in non-responders compared to immunotherapy responding patients.
Conclusion:
A simple and precise LC-MS/MS method was developed for the quantification of therapeutic mAb Vedolizumab in human serum. This study indicates a significant correlation between Vedolizumab trough levels and treatment response and can therefore be used as a tool for therapy monitoring.
References & Acknowledgements:
[1] Oude Munnink et al. Therapeutic drug monitoring of monoclonal antibodies in inflammatory and malignant disease: Translating TNF-α experience to oncology. Clin Pharmacol Ther (2016); 99 (4) : 419-31.
[2] Soler D. et al. The binding specificity and selective antagonism of vedolizumab, an anti-alpha4beta7 integrin therapeutic antibody in dvelopment for inflammatory bowel diseases. J Pharmacol Exp Ther (2009); 330: 864-75.
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