Method for preparing medicine with proteinase inhibiting function and its detecting method

A protease inhibitor, protease technology, applied in biochemical equipment and methods, microbial determination/inspection, drug combination, etc., can solve the problems of low detection sensitivity, cumbersome detection technology operation, etc., and achieve high sensitivity, cost reduction, high active effect

Inactive Publication Date: 2005-05-04
爱华生物科技(香港)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] In order to achieve high yield, ensure the high purity and high activity of the product, simplify the process and reduce the cost at the same time, the present invention provides a preparation method of a drug capable o

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] After Cohn's Component IV was dissolved with 20 times the weight of purified water, 37% sucrose and 0.38M sodium citrate were added as stabilizers for virus inactivation treatment. Then tributyl phosphate with a final concentration of 0.3% and Triton X-100 with a final concentration of 1.0% were added and incubated at 30° C. for 4 hours. After virus inactivation, adjust the pH to 6.5, reduce the ionic strength to 2.5mS / cm with water, and add it to a DEAE Sepharose Fast Flow chromatography column pre-balanced at pH 6.5. The target protein α1-PI was selectively eluted with 20mM sodium dihydrogen phosphate and 100mM sodium chloride solution. Increasing the concentration of sodium chloride can elute strongly adsorbed foreign proteins, including ceruloplasmin, from the ion-exchange chromatography column. DEAE Sepharose Fast Flow resin can be reused after being sterilized with 0.5M sodium hydroxide.

[0030] The eluate containing α1-PI was passed through a 10 kD ultrafiltra...

Embodiment 2

[0038] The dissolved Cohn's fraction IV was adjusted to pH 5.4 with 1M hydrochloric acid and ionic strength to 1.5 mS / cm with water, and then added to a CM Sepharose Fast Flow column pre-equilibrated with 5 mM sodium citrate (pH 5.4). Collect the effluent from the ion exchange resin, adjust the pH to 6.5, adjust the ionic strength to 2.5mS / cm, and add it to a DEAE Sepharose Fast Flow chromatography column pre-balanced with 20mM sodium dihydrogen phosphate solution (pH6.5). Active α1-PI was selectively eluted using 20 mM sodium dihydrogen phosphate and 100 mM sodium chloride solution. In this example, 58.9 mg of α1-PI with a purity of 92.5% was purified from 5.34 g of fraction IV. The yield of active α1-PI was as high as 81.2%. Table 3 is the experimental data of this example.

[0039] Table 3. α1-PI activity

[0040] Total protein (mg) α1-PI activity (yield)

[0041] Component IV 1191.2 448.7U (100%)

[0042] CM chromatography 255...

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Abstract

The present invention relates to a method for preparing medicine with the action capable of inhibiting proteniase and its detection method. Said method includes making the aqueous solution containing alpha 1-proteinase inhibitor undergo the pocesses of virus inactivation treatment, ion exchange resin adsorption and elution; at the same time using the reaction of serine proteinase and alpha 1-proteinase to result in photoabsorption change of correspondent wavelength so as to quantitatively determine the activity of alpha 1-proteinase inhibitor. The invented preparation method can effectively implement high yield, can ensure high purity and high activity of product, at the same time its process is simple, cost is low, and its detection method is simple and easy to implement, and its sensitivity is high.

Description

【Technical field】 [0001] The invention belongs to the field of protein purification and detection, and in particular relates to a method for manufacturing and a detection method for a drug capable of inhibiting protease. 【Background technique】 [0002] α1-Protease Inhibitor (also known as α1-antitrypsin, alpha-l Antiryposin, α1-PI for short) is a glycoprotein with a molecular weight of about 53,000 Daltons, which contains an average of 1.5 grams per liter of plasma. The protein mainly inhibits the decomposition of elastase and other serine proteases in the body. When the concentration of active α1-PI in the body is significantly lower than the concentration of serine proteases, as occurs in people with a genetic defect of α1-PI, the lung tissue will be damaged by serine proteases and cause chronic diseases, such as pneumothorax swell. Patients with emphysema can use α1-PI as a long-term replacement therapy. However, the supply of α1-PI products on the market is in short su...

Claims

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Application Information

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IPC IPC(8): A61K38/55A61P11/00C12Q1/37
Inventor 黄炳镠谢亦武司徒子杰
Owner 爱华生物科技(香港)有限公司
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