Method for measuring content of arginine in rhTNK-tPA (recombinant human TNK tissue-type plasminogen activator) with HPLC (high performance liquid chromatography)

A high-performance liquid chromatography and arginine technology, which is applied in the field of high-performance liquid chromatography to determine the content of arginine in Mingfule, can solve the problems of short peak time, high requirements for detection equipment, and cumbersome operation, and achieve quality improvement Effect

Inactive Publication Date: 2018-07-27
石药集团明复乐药业(广州)有限公司
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Problems solved by technology

There are also reports in the literature that ion chromatography and amino-bonded silica gel-packed columns are used to detect the content of amino acids in injection preparations, but there are problems such as narrow linear range, poor peak shape, and short peak time.
There are also some reports that use reversed-phase column (C18, C8 column) pre-column or post-column derivatization method, and detect the substance with ultraviolet absorption after the actual reaction with derivatization (determination of amino acid content in cerebroprotein hydrolyzate injection) Analytical method, Gong Jihua, Zhang Zexin, Qiu Deqing), but there are shortcomings such as insufficient repeatability and accuracy, cumbersome operation, and high requirements for testing equipment
More importantly, the above methods all involve the determination of the content of multiple amino acids, and the properties of each amino acid are quite different. The above methods are extremely likely not suitable for the detection of arginine content in Mefole

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  • Method for measuring content of arginine in rhTNK-tPA (recombinant human TNK tissue-type plasminogen activator) with HPLC (high performance liquid chromatography)
  • Method for measuring content of arginine in rhTNK-tPA (recombinant human TNK tissue-type plasminogen activator) with HPLC (high performance liquid chromatography)
  • Method for measuring content of arginine in rhTNK-tPA (recombinant human TNK tissue-type plasminogen activator) with HPLC (high performance liquid chromatography)

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1, the assay method of arginine content in Mingfule

[0049] Chromatographic conditions:

[0050] Chromatographic column: C8 acrylamide bonded silica gel column;

[0051] Mobile phase: 0.05mol / L potassium dihydrogen phosphate solution: acetonitrile=40:60, pH is 7.0, in parts by volume;

[0052] Detection wavelength: 208mm;

[0053] Column temperature: 30°C;

[0054] Flow rate: 1.0ml / min;

[0055] Injection volume: 10 μl.

[0056] Determination steps: take 1 bottle of the test sample and reconstitute it according to the marked amount, dilute it 10 times with the mobile phase and add the standard product (about 1.375g) into the mobile phase to dissolve, so as to obtain the test sample solution and the standard product solution, take 10 μl each, and inject them respectively High performance liquid chromatograph, record for test sample chromatogram ( figure 1 ) and standard chromatograms ( figure 2 ), obtained by calculating the peak area according to t...

Embodiment 2

[0057] Embodiment 2, the assay method of arginine content in Mingfule

[0058] The mobile phase described in Example 2 is 0.05 mol / L potassium dihydrogen phosphate solution: acetonitrile = 50: 50, and other parameters and operations are as shown in Example 1.

[0059] Determination steps are as shown in embodiment 1, measurement result image 3 shown. It can be seen from the figure that the ratio of potassium dihydrogen phosphate solution to acetonitrile is changed, and the peak eluting time is shortened, but there is a tailing phenomenon, and there are more impurity peaks.

Embodiment 3

[0060] Embodiment 3, the assay method of arginine content in Mingfule

[0061] The mobile phase described in Example 3 is 0.05 mol / L potassium dihydrogen phosphate solution:acetonitrile=60:80, and other parameters and operations are as shown in Example 1.

[0062] Determination steps are as shown in embodiment 1, and measurement result is as follows Figure 4 shown. It can be seen from the figure that changing the ratio of potassium dihydrogen phosphate solution to acetonitrile leads to poor peak shape, tailing phenomenon, poor separation effect and more impurity peaks.

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Abstract

The invention belongs to the field of biomedicine and particularly relates to a method for measuring content of arginine in an rhTNK-tPA (recombinant human TNK tissue-type plasminogen activator) withHPLC (high performance liquid chromatography). In terms of chromatographic conditions, chromatographic column adopts C8 column; for a mobile phase, the ratio of a 0.05 mol/L phosphate buffer solutionto acetonitrile is (20-60):(30-80); detection wavelength is 200-220 mm; column temperature is 30-35 DEG C; flow rate is 1.0 ml/min; sample size is 10 mu l. The method is particularly applicable to measurement of content of arginine in the rhTNK-tPA, pre-column/post-column derivatization is not needed, the method has the characteristics of high accuracy, high precision, good repeatability, wide linear range and the like, one scientific and strict inner quality standard is set for the rhTNK-tPA product, and quality of the rhTNK-tPA product is improved.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a method for determining the content of arginine in Mingfule by using high performance liquid chromatography. Background technique [0002] TNK-tPA is by far the safest, most effective and most convenient thrombolytic drug. The first generation of recombinant natural tPA products marketed in China is completely dependent on imports. Yuan. The second tPA product expressed in Escherichia coli, that is, Reteplase, "Ruitongli" produced by Shandong Ahua and "Paitoxin" developed by Beijing Aide Pharmaceutical Co., Ltd. were approved in 2007 and 2010, respectively. listed. Mingfule is the third-generation tPA product produced by Guangzhou Mingkang Bioengineering Co., Ltd. It is currently the only thrombolytic drug that can be injected intravenously in a single injection. , making it possible to carry out thrombolytic therapy outside the hospital, which has epoch-making signific...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02
CPCG01N30/02
Inventor 赵宇清何志辉霍碧桃张毛秀穆国会曹靖文
Owner 石药集团明复乐药业(广州)有限公司
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