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Preclinical pharmacokinetic study mass spectrometry analysis method of bevacizumab

A technology of pharmacokinetics and mass spectrometry, applied in the analysis of materials, scientific instruments, material separation, etc., can solve the problems of inability to distinguish endogenous protein interference, long development cycle, narrow quantitative range, etc., to achieve sample processing and The quantitative method is simple and easy to implement, which is conducive to the promotion of the method and the effect of the simple preparation method

Inactive Publication Date: 2019-07-23
南京智谱分子医学技术研究院有限公司
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the pharmacokinetic experiments of monoclonal antibodies, the ELISA method has a narrow quantitative range (usually one to two orders of magnitude), cannot simultaneously analyze different forms of monoclonal antibody (ie, monoclonal antibody) drugs, and there is a development cycle Long time (half a year to several years), high cost (hundreds of thousands to millions per target drug), inability to distinguish interference from endogenous proteins, etc., greatly affecting the reliability and practicability of ELISA quantification
For the RIA method, the target antibody of some radiolabeled molecules will show different absorption, distribution and metabolic characteristics from the prototype drug; in addition, the radiolabeled monoclonal antibody usually only exists stably for 2-3 weeks, and cannot adapt to medium and long-term Monoclonal Antibody Pharmacokinetic Study

Method used

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  • Preclinical pharmacokinetic study mass spectrometry analysis method of bevacizumab
  • Preclinical pharmacokinetic study mass spectrometry analysis method of bevacizumab
  • Preclinical pharmacokinetic study mass spectrometry analysis method of bevacizumab

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Analysis of monoclonal antibody drugs in rat plasma samples

[0042] Part 1: intravenous administration in rats

[0043] A. Rat intravenous administration process

[0044] (1) Choose a male rat with a body weight of 200 g±10 g and eat freely;

[0045] (2) Purchase the commercially available monoclonal antibody drug Bevacizumab injection with a concentration of 25 mg / mL;

[0046] (3) According to the clinical dosage conversion, the rat monoclonal antibody drug was given by intravenous injection at a dose of 50 mg / kg;

[0047] B. Preparation of rat plasma samples

[0048] Select different time points (0, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 32 h), collect rat whole blood from vein, centrifuge at 13000 g for 5 min , Store in a refrigerator at -80℃.

[0049] Part 2: Determination of the monoclonal antibody Bevacizumab in rat plasma samples

[0050] A. Plasma sample pretreatment

[0051] (1) Reduction of plasma sample, add 2 mL rat plasma sample into polyethylene tube, add 9...

Embodiment 2

[0073] Example 2 Preparation of rat plasma standard curve sample

[0074] First configure the monoclonal antibody standard series solution: Dilute the monoclonal antibody standard solution stock solution (25 mg / mL) to 5, 10, 40, 100, 500, 1000, 1200 ng / mL with deionized water.

[0075] Then prepare the rat plasma standard curve sample and proceed in the following 7 steps:

[0076] (1) Reduction of rat plasma standard curve samples. Add 2 mL of monoclonal antibody standard series solution to polyethylene tube, add 2 mL of rat blank plasma, vortex and mix, and add 94 mL of pH buffer containing denaturant , Vortex and mix, add 2 mL reducing agent, vortex and mix, and incubate at 60°C for 1 h to obtain a reaction solution;

[0077] (2) For cysteine ​​blocking, add 0.75 mg of solid cysteine ​​blocking agent to the reaction solution, vortex and mix; incubate at 25°C in the dark for 40 min;

[0078] (3) Trypsin digestion of rat plasma standard curve samples, add 700 mL digestion buffer to the...

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Abstract

The invention discloses a pharmacokinetic mass spectrometry analysis method of monoclonal antibody drugs, and belongs to the technical field of proteomics. According to the technical scheme, a specific peptide fragment obtained by pancreatin hydrolysis of Bevacizumab is selected, wherein specificity means that the peptide fragment is only generated by pancreatin hydrolysis of Bevacizumab, and other proteins and protein drugs cannot generate the peptide fragment by pancreatin hydrolysis; and based on liquid chromatography-tandem mass spectrometry technology, a parallel reaction monitoring modeis adopted to scan and analyze the specific peptide fragment to establish a stable and reliable LC / MS / MS quantitative method for the specific peptide fragment. The pharmacokinetic mass spectrometry analysis method of the monoclonal antibody drugs has the characteristics of high flux, high sensitivity, good reproducibility and the like. Reagents and drugs used are cheaper and easier to obtain, thepreparation method is simple, and the method is beneficial to popularization. A sample treatment process and quantitative method are simple and easy to implement, and guiding significance for qualitative and quantitative research of other protein drugs and polypeptide drugs is achieved.

Description

Technical field [0001] The invention belongs to the technical field of proteomics, and relates to a pharmacokinetic mass spectrometry analysis method of a therapeutic monoclonal antibody drug Bevacizumab (Bevacizumab). Background technique [0002] Monoclonal antibody drugs are a class of protein drugs with special therapeutic effects based on the structure of gamma immunoglobulin (Immunoglobulin G). They have been successfully used in cancer, autoimmune diseases, viral infections, and central nervous disorders. Clinical treatment. Compared with traditional small molecule drugs or traditional Chinese medicine preparations, monoclonal antibody drugs have clear pharmacological activity and excellent pharmacokinetic properties; monoclonal antibodies have a low degree of metabolism in the body and can maintain a high concentration in body fluids for a long time. The elimination half-life averages from several weeks to several months. Therefore, the timely establishment of standardi...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06G01N2030/045G01N2030/062
Inventor 胡良海李潇影张海洋陶纬国
Owner 南京智谱分子医学技术研究院有限公司
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