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Quantification of the anti-murine PD-1 monoclonal antibody RMP1-14 in BALB/c mouse plasma by liquid chromatography-tandem mass spectrometry and application to a pharmacokinetic study

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Abstract

RMP1-14 is a monoclonal antibody that targets the murine PD-1 protein, and has been used extensively to probe the effects of PD-1 inhibition in preclinical murine models. However, to date, no quantitative analytical methods have been published for RMP1-14. To evaluate its anti-tumor activity in BALB/c mice inoculated with CT26.WT murine colon cancer cells, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify RMP1-14 in BALB/c mouse K3EDTA plasma was developed and validated. The methodology used a signature peptide (GFYPPDIYTEWK) as a surrogate for RMP1-14 quantitation and an isotopically labeled analog of the signature peptide as the internal standard. Initial method development focused on a hybrid LC-MS/MS assay involving Protein G immunoprecipitation, but this strategy was abandoned due to lack of selectivity. The final validated method consisted of dilution with Tris-buffered saline, trypsin digestion, and desalting using micro solid-phase extraction. Analytical run time was 3.50 min, and the method demonstrated linearity between 0.500 and 50.0 μg/mL of intact RMP1-14. Accuracy, precision, and robustness were all acceptable, and the method was demonstrated to be comparable to a commercially available fit-for-purpose enzyme-linked immunosorbent assay (ELISA) capable of measuring RMP1-14. The validated method was used to generate pharmacokinetic parameters from tumor-bearing BALB/c mice dosed with RMP1-14 at either 2.50 or 7.50 mg/kg. Overall, the validated method represents a novel tool that can be used to evaluate RMP1-14 activity in future immuno-oncology studies.

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Acknowledgments

The authors gratefully acknowledge the assistance and advice provided by G.W. Sword in determining the signature peptides for RMP1-14; by T.S. Brus, R.D. Garton, Z. Gong, D.M. Good, K.A. Kowalski, X. Liu, B.L. Powers, J.M. Uhlenkamp, J.A. Usery, S. Voruganti, R.R. Voggu, and A. Zhou during method development and validation; by A.M. Radwan, C.A. Solenberg, and the rest of the Greenfield Oncology Team during the in-life phase of the study; and by J.M. Stangl in determining pharmacokinetic parameters for RMP1-14.

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Author notes

  1. Ryan C. Hill

    Present address: Eli Lilly and Company, 893 Delaware Street, Indianapolis, IN, 46225, USA

  2. Brandy L. Wilkinson

    Present address: Explora BioLabs, 11175 Flintkote Avenue, Suite B, San Diego, CA, 92121, USA

  3. Patrick B. Allison

    Present address: MI Bioresearch, 800 Technology Drive, Ann Arbor, MI, 48108, USA

Authors and Affiliations

  1. Covance Laboratories, Inc., 8211 SciCor Drive, Indianapolis, IN, 46214, USA

    Karan Agrawal, Ryan C. Hill & C. Eric Thomas

  2. Covance Laboratories, Inc., 671 South Meridian Road, Greenfield, IN, 46140, USA

    Brandy L. Wilkinson & Patrick B. Allison

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  1. Karan Agrawal
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Corresponding author

Correspondence to Karan Agrawal.

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In vivo studies were performed under protocols approved by the Covance Institutional Animal Care and Use Committee, and were in compliance with the U.S. Department of Agriculture’s Animal Welfare Act (9 CFR Parts 1, 2, and 3) and the U.S. National Research Council's Guide for the Care and Use of Laboratory Animals.

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Agrawal, K., Hill, R.C., Wilkinson, B.L. et al. Quantification of the anti-murine PD-1 monoclonal antibody RMP1-14 in BALB/c mouse plasma by liquid chromatography-tandem mass spectrometry and application to a pharmacokinetic study. Anal Bioanal Chem 412, 739–752 (2020). https://doi.org/10.1007/s00216-019-02292-1

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  • DOI: https://doi.org/10.1007/s00216-019-02292-1

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