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Analytical and pharmacological consequences of the in vivo deamidation of trastuzumab and pertuzumab

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Abstract

A liquid chromatography-tandem mass spectrometry method is presented for the quantitative determination of the in vivo deamidation of the biopharmaceutical proteins trastuzumab and pertuzumab at an asparagine in their complementarity determining regions (CDRs). For each analyte, two surrogate peptides are quantified after tryptic digestion of the entire plasma protein content: one from a stable part of the molecule, representing the total concentration, and one containing the deamidation-sensitive asparagine, corresponding to the remaining non-deamidated concentration. Using a plasma volume of 10 µL and a 2-h digestion at pH 7, concentrations between 2 and 1000 µg/mL can be determined for the various protein forms with values for bias and CV below 15% and without unacceptable in vitro deamidation taking place. A considerable difference between the total and non-deamidated concentrations, and thus a substantial degree of deamidation, was observed in plasma for both trastuzumab and pertuzumab. After a 56-day forced deamidation test 40% of trastuzumab and 68% of pertuzumab was deamidated, while trastuzumab and pertuzumab showed up to 47% and 35% of deamidation, respectively, in samples collected from breast cancer patients during treatment with a combination of both drugs. A good correlation between the non-deamidated concentration results and those of a receptor binding assay indicate a loss of receptor binding for both trastuzumab and pertuzumab along with the deamidation in their CDRs. Deamidated trastuzumab also lost its capability to inhibit the growth of breast cancer cells in a cell-based viability assay, suggesting a relation between the degree of deamidation and pharmacological activity.

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Acknowledgements

We thank all patients for their participation, the staff of the NKI trial laboratory, in particular dr. T. Korse, who assisted in collecting the samples, and the Dutch Breast Cancer Research Group (BOOG) as the sponsor of the clinical trial.

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Authors and Affiliations

  1. Analytical Biochemistry, Department of Pharmacy, University of Groningen, A. Deusinglaan 1, 9700 AV, Groningen, The Netherlands

    Peter Bults, Rainer Bischoff & Nico C. van de Merbel

  2. Bioanalytical Laboratory, Early Development Services, ICON, Amerikaweg 18, 9407 TK, Assen, The Netherlands

    Peter Bults & Nico C. van de Merbel

  3. Division of Medical Oncology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, the Netherlands

    Anna van der Voort & Gabe S. Sonke

  4. Faculty of Medical Sciences, Department of Medical Oncology, University Medical Centre Groningen, Hanzeplein 1, 9713 GZ, Groningen, The Netherlands

    Coby Meijer

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  1. Peter Bults
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  2. Anna van der Voort
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  3. Coby Meijer
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  4. Gabe S. Sonke
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  6. Nico C. van de Merbel
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Correspondence to Nico C. van de Merbel.

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Ethical approval

Samples were collected after ethical approval by the medical ethics committee of the Netherlands Cancer Institute and all patients provided written informed consent prior to study inclusion. All procedures performed in this study involving human participants were in accordance with the ICH Harmonized Tripartite Guideline for Good Clinical Practice and with the 1964 Helsinki Declaration and its later amendments.

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Bults, P., van der Voort, A., Meijer, C. et al. Analytical and pharmacological consequences of the in vivo deamidation of trastuzumab and pertuzumab. Anal Bioanal Chem 414, 1513–1524 (2022). https://doi.org/10.1007/s00216-021-03756-z

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  • DOI: https://doi.org/10.1007/s00216-021-03756-z

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