Method for detecting purity of recombinant human prourokinase

A technology of pro-urokinase and detection method, applied in the field of detection and analysis, can solve the problems of poor separation effect of monomers and polymers, complex composition of finished preparations, and difficulty in detecting pro-urokinase polymers, etc.

Active Publication Date: 2021-11-19
TASLY BIOPHARMACEUTICALS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Due to the molecular characteristics of prourokinase, the separation effect of monomer and polymer is not good under routine experimental conditions, and the complexity of the components of the finished preparation poses a great challenge to the separation method
However, conventional methods, such as using a mobile phase of 10-250 mM phosphate buffered saline (PB), 100-400 mM sodium chloride (NaCl), pH 6.5-7.5, are difficult to detect the propolymer of urokinase

Method used

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  • Method for detecting purity of recombinant human prourokinase
  • Method for detecting purity of recombinant human prourokinase
  • Method for detecting purity of recombinant human prourokinase

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0065] This embodiment provides a method for detecting the purity of recombinant human pro-urokinase finished product, said detection method comprising the following steps:

[0066] (1) Preparation of sample solution: add the recombinant human prourokinase finished product into water to dissolve, prepare a sample solution with a prourokinase protein concentration of 1 mg / mL, centrifuge and take the supernatant, which is the sample solution;

[0067] (2) SEC-HPLC for detection: use Waters ACQUITYArc high performance liquid chromatography and ultraviolet detector for detection, the specific conditions are as follows:

[0068] Mobile phase A: 0.1vol% acetic acid aqueous solution containing 100mmol / L sodium chloride (pH is 3.1);

[0069] Mobile phase B: acetonitrile;

[0070] SEC-HPLC column: Waters XBridge TM BEH Protein SEC (7.8mm×300mm, 2.5μm, );

[0071] Test parameters: flow rate 0.5mL / min, detection wavelength 280nm, elution time 30min, injection volume 30μL per needle...

Embodiment 2

[0078] The present embodiment provides a method for detecting the purity of the original stock solution of urokinase, the detection method comprising the following steps:

[0079] (1) Preparation of sample solution: add the original urokinase solution into water to dissolve, prepare a sample solution with a concentration of 1 mg / mL of the original urokinase protein, centrifuge and take the supernatant, which is the sample solution;

[0080] (2) SEC-HPLC for detection: use Waters ACQUITYArc high performance liquid chromatography and ultraviolet detector for detection, the specific conditions are as follows:

[0081] Mobile phase A: 0.1vol% acetic acid aqueous solution containing 100mmol / L sodium chloride (pH is 3.1);

[0082] Mobile phase B: acetonitrile;

[0083] SEC-HPLC column: Waters XBridge TM BEH Protein SEC (7.8mm×300mm, 2.5μm, );

[0084] Test parameters: flow rate 0.5mL / min, detection wavelength 280nm, elution time 30min, injection volume 30μL per needle, set tem...

Embodiment 3

[0090]This embodiment provides a method for detecting the purity of recombinant human prourokinase finished product. The only difference from Example 1 is that mobile phase A is 0.1vol% TFA aqueous solution containing 100mmol / L sodium chloride, and NaOH is added to adjust the pH to 3.1, other test steps are the same as in Example 1;

[0091] The specific test results are shown in Table 3 below:

[0092] table 3

[0093] mobile phase A pH Aggregate and monomer resolution p / v 0.1% TFA, 100mM NaCl(NaOH) 3.1 3.6

[0094] From the comparison of Example 1 and Example 3, it can be seen that when the finished product of recombinant human urokinase is used as the test sample, if adding trifluoroacetic acid, it is necessary to add an appropriate amount of NaOH to adjust the pH to 3.1, and using acetic acid, not only the degree of separation can be further improved , and do not need to adjust the pH, easy to operate.

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Abstract

The invention provides a method for detecting the purity of recombinant human prourokinase. The method for detecting the purity of the recombinant human prourokinase comprises the following steps: diluting a recombinant human pro-urokinase sample into a sample solution, and detecting by adopting SEC-HPLC to obtain the purity of the sample, wherein a mobile phase A adopted by the SEC-HPLC is an aqueous solution of acid with the pH value of 2.5-3.5, and a mobile phase B adopted by the SEC-HPLC is acetonitrile. The detection method can be used for detecting the purity of a prourokinase finished product, is also suitable for analyzing the purity of a prourokinase stock solution, can accurately detect the prourokinase polymer in the finished product, and further improves the separation degree of the prourokinase polymer and the prourokinase monomer.

Description

technical field [0001] The invention belongs to the technical field of detection and analysis, and in particular relates to a detection method for the purity of recombinant human prourokinase. Background technique [0002] Size exclusion chromatography (SEC) can be used to analyze the purity of analytes based on molecular size. Macromolecules in the sample that exceed the exclusion limit cannot enter the gel pores and are completely excluded, and can only pass through the chromatographic column along the gaps between the porous gel particles, and are first eluted from the column by the mobile phase; The extreme molecules can enter the pores in the gel, and are eluted from the chromatographic column according to the size and time sequence; the solvent molecules that dissolve the sample have the smallest molecular weight, and finally flow out of the column, so as to achieve different molecular sizes. Separation of samples. The SEC method detects and calculates the purity of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/34G01N30/36G01N30/74
CPCG01N30/02G01N30/06G01N30/34G01N30/36G01N30/74Y02A50/30
Inventor 张文明王倩李春澍董艳叶韩进杨普
Owner TASLY BIOPHARMACEUTICALS CO LTD
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