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Capillary isoelectric focusing analysis method for polypeptide polymer

A technology of isoelectric focusing and analysis method, which is applied in the direction of material analysis, analysis material, and measurement device by electromagnetic means, and achieves the effect of high accuracy, low cost and fast speed.

Inactive Publication Date: 2017-07-07
HYBIO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Although capillary isoelectric focusing has been widely used in the quality analysis of monoclonal antibody drugs, its application to the analysis of consistency evaluation of peptide mixture drugs has not been reported

Method used

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  • Capillary isoelectric focusing analysis method for polypeptide polymer
  • Capillary isoelectric focusing analysis method for polypeptide polymer
  • Capillary isoelectric focusing analysis method for polypeptide polymer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Prepare capillary isoelectric focusing sample solution according to the composition in Table 1, place it in the autosampler of Agilent7100 capillary isoelectric focusing instrument, and use fluorine-coated capillary assembly (Agilent / 194-8111) to analyze the sample. The distances between the detector windows are 8.5cm and 24.5cm, respectively. The capillary electrophoresis system was equipped with an external water bath set at 15 °C. 2bar high pressure injection for 100s, 25kV focusing for 9min at 15°C, 200mM phosphoric acid as the anolyte, and 300mM sodium hydroxide as the catholyte. Migration conditions: 30kv migration 45min. The detection was performed at 280nm, and the isoelectric point of the measured sample was calculated using openLAB ControlPanel software. See Table 2 for the isoelectric points of each component of the trial-produced sample and the reference substance, and see the attached figure 1 , with figure 2 . Table 1 Proportion of loading solution f...

Embodiment 2

[0063] Prepare capillary isoelectric focusing sample solution according to the composition in Table 3, place it in the autosampler of Agilent7100 capillary isoelectric focusing instrument, and use fluorine-coated capillary assembly (Agilent / 194-8111) to analyze the sample. The distances between the detector windows are 8.5cm and 24.5cm, respectively. The capillary electrophoresis system was equipped with an external water bath set at 15 °C. 2.5bar high-pressure injection was used for 80s, at 15°C, 30kv focusing for 15min, the anolyte was 200mM phosphoric acid, and the catholyte was 300mM sodium hydroxide. Migration conditions are: 30kv migration for 40min. The detection was performed at 280nm, and the isoelectric point of the measured sample was calculated using openLAB ControlPanel software. The electrophoretic patterns of the trial samples and reference substances are attached image 3 , with Figure 4 .

[0064] Table 3 Proportion of sample solution for capillary isoel...

Embodiment 3

[0067] Prepare capillary isoelectric focusing sample solution according to the composition in Table 4, place it in the autosampler of Agilent7100 capillary isoelectric focusing instrument, and use fluorine-coated capillary assembly (Agilent / 194-8111) to analyze the sample. The distances between the detector windows are 8.5cm and 24.5cm, respectively. The capillary electrophoresis system was equipped with an external water bath set at 15 °C. 2.5bar high-pressure injection was used for 80s, at 15°C, 30kv focusing for 15min, the anolyte was 200mM phosphoric acid, and the catholyte was 300mM sodium hydroxide. Migration conditions are: 30kv migration for 40min. The detection was performed at 280nm, and the isoelectric point of the measured sample was calculated using openLAB ControlPanel software. The electrophoretic patterns of the trial samples and reference substances are attached Figure 5 , with Figure 6 .

[0068] Table 4 Capillary isoelectric focusing sample solution r...

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Abstract

The invention relates to a capillary isoelectric focusing analysis method for polypeptide polymer. The method includes the steps of firstly, preparing capillary isoelectric focusing sample-loading liquid, wherein the sample-loading liquid is mixed liquid comprising ampholyte, gel liquid, isoelectric point markers and to-be-analyzed polypeptide polymer; secondly, loading the sample-loading liquid to a capillary column, focusing under 15-30 DEG C and 10-40kV for 2-25 minutes, and migrating under 10-40kV for 20-50 minutes; using a capillary isoelectric focusing instrument or a capillary electrophoresis instrument for detection to obtain isoelectric focusing spectrogram; thirdly, using the isoelectric point markers to calculate the isoelectric point of each component of a polypeptide mixture, and inspecting through the isoelectric point value of each component and the peak pattern of each component. The method can fast and accurately judge whether the components of two polypeptide polymer are identical or not and is low in sample use amount, fast, low in cost and high in result accuracy.

Description

technical field [0001] The invention relates to the field of drug analysis, in particular to the field of separation and analysis of polypeptide drugs. Background technique [0002] Glatiramer acetate (abbreviated as GA) is a synthetic polypeptide mixture (relative molecular weight: 4700-11000 Daltons), composed of L-alanine, L-glutamic acid, L-tyrosine A random polymer composed of four amino acids of L-lysine and L-lysine, with a length of 45-100 amino acids. The molar ratio of each amino acid is about 0.392-0.462, 0.129-0.153, 0.086-0.100, 0.300-0.374. The drug was developed and manufactured by the Israeli pharmaceutical company Teva Pharmaceuticals (TEVA), and its trade name is It was approved by the US FDA in 1996 for the treatment of multiple sclerosis. There are two products, water for injection and lyophilized powder for injection, both of which are used for subcutaneous injection. [0003] Glatiramer acetate is a continuous copolymer with a structure as shown i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/447
CPCG01N27/44795
Inventor 何刚李勇唐洋明陶安进袁建成
Owner HYBIO PHARMA
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