Metabolism Analysis

Mimotope-based sample purification for mass spectrometric quantification of co-administered trastuzumab and pertuzumab in human serum*

  • WANG Jia-jun ,
  • WEI Qian-qian ,
  • HUANG Shuo ,
  • LIU Kai-di ,
  • SONG Yu-bo ,
  • LIU Jiu-yang ,
  • WU Jian-hua ,
  • LIU Liang ,
  • WU Dong-fang
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  • 1. Department of Pharmacy, Zhongnan Hospital of Wuhan University, Wuhan 430071, China;
    2. Wuhan University, Wuhan 430072;
    3. Department of Thyroid and Breast Surgery, Zhongnan Hospital of Wuhan University, Wuhan 430071, China

Online published: 2025-10-13

Abstract

Objective: To develop a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method that can simultaneously determine co-administered trastuzumab and pertuzumab in human serum based on mimotope purification technique. Methods: Capture probes based on mimotope were used to capture trastuzumab and pertuzumab from serum samples, followed by reduction, alkylation, trypsin digestion, and quantification using LC-MS/MS. The sample was detected in positive mode. The UPLC separation was conducted using an Agilent SB C18 column (30 mm×2.1 mm, 2.7 μm) at 40 ℃. Mobile phase A was water (0.1% formic acid), and mobile phase B was acetonitrile (0.1% formic acid). The flow rate was set at 0.3 mL · min-1 alongside an injection volume of 10 μL.Gradient elution was used. The monitoring ion pairs of trastuzumab were m/z 485.5/234.0, m/z 485.5/335.5, m/z 485.5/608.3, and the monitoring ion pairs of pertuzumab were m/z 419.4/249.4, m/z 419.4/476.2, m/z 419.4/589.6. In this process, we examined the adsorption isotherms and adsorption kinetics of mimotope capture probe, the digestion efficiency of the surrogate peptide, and validated the developed method. Results: Capture probes based on mimotope had a high-affinity enrichment ability to trastuzumab and pertuzumab, and the incubation process could be finished in 1 h. The linearity ranges for trastuzumab and pertuzumab were 0.200-200 μg · mL-1, and the limits of quantitation were both 0.2 μg · mL-1. The intra-day and inter-day precisions (RSD) for trastuzumab and pertuzumab were within 11.3% and 12.3%, and the accuracies (bias) were within 12.1% and 11.5%. This new method was successfully used for detecting the concentration of 12 patients with co-administration mAbs, the detected serum concentration range of trastuzumab was 13.75-59.12 μg · mL-1, 37.55-127.20 μg · mL-1 for pertuzumab. Conclusion: The developed method based on mimotope purification technique overcomes the inherent drawbacks of protein A/G-based affinity separation purification, such as high cost, low stability, and possible activity loss. It is a promising technical approach for achieving personalized and precise clinical treatment, especially for mAbs administered in combination.

Cite this article

WANG Jia-jun , WEI Qian-qian , HUANG Shuo , LIU Kai-di , SONG Yu-bo , LIU Jiu-yang , WU Jian-hua , LIU Liang , WU Dong-fang . Mimotope-based sample purification for mass spectrometric quantification of co-administered trastuzumab and pertuzumab in human serum*[J]. Chinese Journal of Pharmaceutical Analysis, 2025 , 45(4) : 634 -643 . DOI: 10.16155/j.0254-1793.2024-1322

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