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RP-HPLC detection method for recombinant human granulocyte colony stimulating factors

A technology of RP-HPLC and colony-stimulating factor, which is applied in the field of protein analysis, can solve the problems of insensitive and accurate detection of oxidation products, and achieve the effects of precise and rapid stability, high detection sensitivity and accuracy

Active Publication Date: 2011-07-20
HANGZHOU JIUYUAN GENE ENG
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Problems solved by technology

[0006] The liquid chromatographic method used in other public literatures about the stability test mainly includes molecular sieve HPLC [Xie Yihong, Zhong Ying, Huang Jianhong. China Pharmaceutical Industry Journal, 2000, 31 (03): 123-124; Zhao Xianliang, Huang Yulu, Wang Chunyu, etc. . Qilu Pharmaceutical Affairs, 2007, 26(06): 342-344], reversed-phase chromatography C4 column [Xie Yihong, Zhong Ying, Huang Jianhong. Chinese Journal of Pharmaceutical Industry, 2000, 31(03): 123-124; Zhong Ying. Chinese Modern Applied Pharmacy, 2000, 17(03): 220-222], cannot detect its oxidation products sensitively and accurately

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  • RP-HPLC detection method for recombinant human granulocyte colony stimulating factors
  • RP-HPLC detection method for recombinant human granulocyte colony stimulating factors
  • RP-HPLC detection method for recombinant human granulocyte colony stimulating factors

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example 1

[0045] Comparison of Example 1 Column Temperature

[0046] When the column temperature was increased from room temperature 25°C to 60±3°C, the resolution was greatly improved, the repeatability was better, and more oxidation products were detected. The results are shown in Table 4. The purity of the same sample detected by using a column temperature of 60 degrees is 1-3 percentage points lower than the purity detected by using a column temperature of 25 degrees.

[0047] Table 4. Effect of column temperature on purity determination

[0048]

[0049]

example 2

[0050] Influence of example 2 loading sample volume

[0051] After confirming that the temperature of 60 degrees is the optimal analysis temperature, the influence of different sample volumes on the analysis results was compared. As shown in Table 5, it was found that when the protein concentration was 0.3mg / mL, the sample loading of 10uL could only detect The main oxidation peak appears, while the loading volume of 20uL and above has basically no effect on the detection results. It can be seen that the detection method of the present invention has high sensitivity, and the lowest detection limit is 6 μg. However, according to the requirements of the Pharmacopoeia, the minimum sample amount required for general testing is 10 μg.

[0052] Table 5. Effect of sample volume on detection results

[0053]

example 3

[0054] Influence of example 3 column length on detection result

[0055] The influence of different column lengths of Vydac C4 columns on the detection results of accelerated oxidation samples was compared. The analysis results of column lengths of 15cm and 25cm are as follows: figure 2 As shown, although more oxidation products can be detected by using a column length of 25 cm, the separation of chromatographic peaks is not as good as that of a column length of 15 cm, and the purity test results are not much different.

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Abstract

The invention relates to a reversed-phase high-performance liquid chromatography (RP-HPLC) for recombinant human granulocyte colony stimulating factors. A Vydac C4 column is adopted in the method, acetonitrile gradient elution is performed, and the temperature of the column is 60+ / -3 DEG C. The C4 column used by the method is different from a C18 column used in the 2010 version of Chinese pharmacopoeia, and the detection sensitivity and the accuracy are higher. Although the C4 column is adopted in European pharmacopoeia, but the acetonitrile gradient range used by the C4 column is small, and the separation time is long, so the new method can measure the stability of the recombinant human granulocyte colony stimulating factors under different storage conditions more quickly and more accurately. The detection analysis method for the recombinant human granulocyte colony stimulating factors recorded by the conventional pharmacopoeia is improved, so that the method is more suitable for purity detection of an rhG-CSF product and has higher detection sensitivity and accuracy. The method also has important referential meaning for the development of stability testing methods for other recombinant proteins.

Description

technical field [0001] The invention relates to the field of protein analysis, in particular to an RP-HPLC method for accurately measuring the chemical stability of recombinant human granulocyte colony-stimulating factor. Background technique [0002] The emergence of recombinant DNA technology has enabled a large number of recombinant proteins to be successfully used in the treatment of various diseases, among which cytokines include erythropoietin (EPO), interferon (IFN-α, IFN-β, IFN-γ), granulocyte Colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (GM-CSF) and interleukin (IL), etc. Compared with small molecule chemical drugs, recombinant drug proteins have higher activity and specificity at lower concentrations. However, due to the physical and chemical instability of the recombinant protein itself, it is generally administered by injection. In order to ensure a certain validity period, pharmaceutical proteins generally need to be refrigerated or ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/89
Inventor 郭旺明方井晋陈海红沈玲李辉
Owner HANGZHOU JIUYUAN GENE ENG