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Method for measuring free polyethyleneglycol content in sample or products

A polyethylene glycol and detection method technology, applied in the direction of measuring devices, material separation, instruments, etc., can solve the problems of unsuitable complex sample analysis, inapplicability, etc., achieve high sensitivity, avoid interference, and expand the application range

Active Publication Date: 2009-07-29
HAINAN SIMCERE PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method is susceptible to interference from many factors and is not suitable for the analysis of some complex samples.
And when the sample or product to be analyzed is a product containing modified polyethylene glycol, this method is not applicable

Method used

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  • Method for measuring free polyethyleneglycol content in sample or products
  • Method for measuring free polyethyleneglycol content in sample or products
  • Method for measuring free polyethyleneglycol content in sample or products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The detection sample is a recombinant human arginase (EC 3.5.3.1) modified by monomethoxypolyethylene glycol activated by succinimidyl propionic acid with 40 times the molar amount, the reaction is carried out at room temperature, and then 0.2 mol / The pH of the reaction mixture was adjusted to 4.0 with 1 liter of citric acid. The main components of the sample mixture are unmodified recombinant human arginase, modified recombinant human arginase and hydrolyzed monomethoxypolyethylene glycol propionic acid. The reaction mixture was diluted with ultrapure water to a concentration of the monomethoxypolyethylene glycol derivative of about 10-15 mg / ml.

[0037] The instrument is Agilent 1200 chromatographic system: Vydac 214TP54 (250×4.6mm, 5μm, ) chromatographic column, DAD diode array detector with SEDEX75 evaporative light detector, automatic sampler, quaternary pump, column thermostat, and workstation.

[0038] The mobile phase was chromatographic grade acetonitrile, ...

Embodiment 2

[0049] The detection sample is a polyethylene glycol-modified recombinant human arginase sample purified by gel exclusion chromatography well known in the art. The instrument is Agilent 1200 chromatographic system: Vydac 214TP54 (250×4.6mm, 5μm, ) chromatographic column, DAD diode array detector with SEDEX75 evaporative light detector, automatic sampler, quaternary pump, column thermostat, and workstation.

[0050] The mobile phase was chromatographic grade acetonitrile, trifluoroacetic acid and ultrapure water.

[0051] Monomethoxypolyethylene glycol succinimidyl propionic acid and monomethoxypolyethylene glycol (both molecular weight 20,000 Daltons) were purchased from NEKTAR Company.

[0052] Chromatographic conditions:

[0053] Running buffer: ultrapure water containing 0.1% trifluoroacetic acid by volume, which becomes buffer A in the present invention; acetonitrile containing 0.1% trifluoroacetic acid by volume, which becomes buffer B in the present invention;

[005...

Embodiment 3

[0061] The invention is used to detect the content of free polyethylene glycol in the reaction mixture.

[0062] The standard sample is monomethoxypolyethylene glycol purchased from NEKTAR Company, with a molecular weight of 20,000 Daltons. The sample to be analyzed was the same as the reaction mixture prepared in Example 1, except that the molar ratio of polyethylene glycol succinimide propionic acid / protein was 10:1, and the concentration of polyethylene glycol was 12 mg / ml. All samples are filtered through a 0.45 μm filter membrane, and the concentration of free polyethylene glycol in the sample to be analyzed is calculated (the molar amount of polyethylene glycol added is divided by the volume of the reaction solution), and the concentration of polyethylene glycol diluted with ultrapure water is lower than 5 mg / ml. Similarly, prepare monomethoxypolyethylene glycol standard solution with serial dilutions of 5236, analyze according to the following chromatographic condition...

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Abstract

The invention discloses an RP-HPLC detection method for the content of free polyethylene glycol in samples or products, which is characterized by making use of the non-polar difference of polyethylene glycol from other substances on the reversed-phase chromatography column and using UV detector combining evaporative light detector or diode array detector combining evaporative light detector signal contrast to screen signal peaks of free polyethylene glycol and to determine the content thereof by means of the external standard method. Based on the method, the invention also can be used for detecting the purity of polyethylene glycol modified products and the modification degree of modified products. The analysis method is highly sensitive, good in repetitiveness and selectivity, fast, efficient and simple, and is suitable for being used in the samples which have more complex components and are interfered in the polyethylene glycol-barium compound colorimetric determination provided by pharmacopoeia, especially suitable for being used in the polyethylene glycol modified samples or products.

Description

field of invention [0001] The invention belongs to a new method for detecting polyethylene glycol residues in samples or products by means of reverse-phase high-performance liquid chromatography, and is also applicable to the detection of the degree of modification of samples or products modified by polyethylene glycol. The method can be applied to the content analysis and detection of biological products, medicines, foods, cosmetics, health products, etc. added or modified with polyethylene glycol. Background technique [0002] Polyethylene glycol is composed of many repeating oxyethylene groups (-CH 2 -CH 2 -O-) is a linear or branched neutral inert macromolecular polymer composed of structural units, and its CAS registration number is 25322-68-3. Polyethylene glycol molecules are non-toxic, non-immunogenic and antigenic. Its application in the pharmaceutical industry continues to expand. It has been approved by the US Food and Drug Administration for use in many fields ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/74G01N30/89
Inventor 于鹏展覃丹秦国宏
Owner HAINAN SIMCERE PHARMA CO LTD
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