Pharmacokinetic mass spectrometry method of monoclonal antibody medicine

A monoclonal antibody, pharmacokinetic technology, applied in the field of proteomics, can solve the problems of inability to distinguish endogenous protein interference, long development cycle, narrow quantitative range, etc., to achieve simple and easy sample processing and quantitative methods. , Conducive to the promotion of the method, the effect of the simple preparation method

Inactive Publication Date: 2016-05-11
JILIN UNIV
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Problems solved by technology

However, in monoclonal antibody pharmacokinetic experiments, 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 endogeno

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  • Pharmacokinetic mass spectrometry method of monoclonal antibody medicine
  • Pharmacokinetic mass spectrometry method of monoclonal antibody medicine
  • Pharmacokinetic mass spectrometry method of monoclonal antibody medicine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Analysis of monoclonal antibody drugs in rat plasma samples

[0041] Part I: Intravenous Administration in Rats

[0042] A. Rat intravenous administration process

[0043] (1) choose a male rat with a body weight of 200g ± 10g, and eat and drink freely;

[0044] (2) Purchase commercially available monoclonal antibody drug Bevacizumab injection with a concentration of 25 mg / mL;

[0045] (3) According to the conversion of the clinical dosage, administer the monoclonal antibody drug to rats by intravenous injection at a dosage of 50 mg / kg;

[0046] B. Preparation of Rat Plasma Samples

[0047] Different time points (0, 30min, 1h, 2h, 4h, 8h, 12h, 24h, 32h) were selected, and rat whole blood was collected from the vein, and the serum was centrifuged at 13000g for 5min and stored in a -80°C refrigerator.

[0048] Part II: Determination of the monoclonal antibody drug Bevacizumab in rat plasma samples

[0049] A. Plasma sample pretreatment

[0050] (1) Reduct...

Embodiment 2

[0089] Example 2 Rat plasma standard curve sample preparation

[0090] First prepare the monoclonal antibody standard series solution: dilute the monoclonal antibody standard solution stock solution (25 μg / μL) to 5, 10, 40, 100, 500, 1000, 1200 ng / μL with deionized water, respectively.

[0091] Then prepare the rat plasma standard curve sample: proceed in the following 7 steps,

[0092] (1) Reduction of rat plasma standard curve samples,

[0093] Add 2 μL monoclonal antibody standard series solution into the polyethylene tube respectively,

[0094] Add 2 μL rat blank plasma, vortex to mix,

[0095] Add 94 μL of pH buffer containing denaturant, vortex to mix,

[0096] Add 2 μL reducing agent, vortex to mix,

[0097] Incubate at 60°C for 1 hour to obtain a reaction solution;

[0098] (2) Blocking of cysteine,

[0099] Add 0.75 mg of solid cysteine ​​blocking agent to the reaction solution, and vortex to mix;

[0100] Incubate at 25°C in the dark for 40 minutes;

[0101] ...

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Abstract

The invention provides a pharmacokinetic mass spectrometry method of a monoclonal antibody medicine and belongs to the technical field of proteomics. With the adoption of the technical scheme, the pharmacokinetic mass spectrometry method comprises the following steps: selecting a specific peptide fragment obtained by hydrolyzing Bevacizumab with a pancreatic enzyme, wherein the specificity means that the peptide fragment is generated by only hydrolyzing the Bevacizumab with the pancreatic enzyme, and the peptide fragment cannot be generated by hydrolyzing other proteins and protein type medicines through the pancreatic enzyme; based on a liquid chromatograph-tandem mass spectrometry technology, scanning and analyzing the specific peptide fragment by using a parallel reaction monitoring mode, and establishing a stable and reliable LC/MS/MS quantifying method of the specific peptide fragment. The pharmacokinetic mass spectrometry method of the monoclonal antibody medicine has the characteristics of high flux, high sensitivity, good repeatability and the like; used reagents and medicines are low in price and easy to obtain and a preparation method is simple, so that the popularization of the method is facilitated; a sample treatment process and a quantifying method are simple and feasible, and the method has guide and reference meanings on qualitative and quantitative researches on the other protein type medicines and polypeptide medicines.

Description

technical field [0001] The invention belongs to the technical field of proteomics, and relates to a pharmacokinetic mass spectrometry analysis method for a therapeutic monoclonal antibody drug Bevacizumab. Background technique [0002] Monoclonal antibody drugs are a class of protein drugs with special therapeutic effects based on the structure of γ-type immunoglobulin (ImmunoglobulinG), and have been successfully used in the clinical treatment of various diseases such as cancer, autoimmune diseases, viral infections, and central nervous disorders. treat. Compared with traditional small molecule drugs or traditional Chinese medicine preparations, monoclonal antibody drugs have clear pharmacological activity and excellent pharmacokinetic properties; Elimination half-lives range from weeks to months on average. Therefore, it is of great significance to establish standardized and reliable analytical methods in time and to conduct pharmacokinetic studies of monoclonal antibody...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06G01N33/6848G01N2030/067
Inventor 胡良海丛宇婷杨波张章顾景凯
Owner JILIN UNIV
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