KR830001822B1 - Production method of euro kinase - Google Patents

Abstract

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Description

Production method of euro kinase

The present invention relates to a method for purifying and concentrating urokinase.

It is already known that urokinase is an enzyme present in small amounts in urine of mammals and humans. Eurokinase is an activator and is used to convert plasminogen to prasmin. This enzyme can also dissolve cellulose fusions. Thus, in pharmaceuticals, urokinase preparations are useful products for the treatment of thromboembolism.

A number of methods are known for separating urokinase in high yield from human urine and its tablets and concentrates. These methods are to contact urine with an adsorbent and then elute the adsorbate. The crude urokinase is then concentrated in high concentration in the extract. The extract is treated with a number of adsorption-eluting and other separation methods to obtain a highly concentrated and fixative urokinase solution.

As the known adsorbent, for example, aluminum hydride silicates, ion exchange silicates, molecular sieves and other organic and inorganic substances such as calcium carbonate, barium sulfate, aluminum oxide, calcium phosphate, zinc hydroxide, activated carbon, bentonite and kaolin are used.

The Swiss Federal Patent Application No. 10512/74 describes a novel adsorbent of this type, namely an acrylic resin cation exchanger. The present invention is inherently derived from German Patent No. 2,428,248.

On the other hand, a number of affinity-chromatography methods have been proposed for concentrating and purifying urokinase. These methods have been applied to affinity adsorption solid-phase matrices with the following compounds, mostly in surface-immobilized form: ε-amino caproic acid on Sepharose ^R and urokininase inhibitors of human placenta on p-amino benzamidine Sepharose and similar combinations. Use for

It is also known to fix aprotinin on the surface of a solid matrix and to separate and concentrate affinity chromatography other than non-urokinase by affinity chromatography using a resin prepared by this method. For example, affinity chromatography for concentrating trypsin on the resin was described in Biochemistry, Vol. 55, page 1323, published in 1973. A similar method was described in page 1976, page 15, 1976. In 1976, published in 1976, the Journal of Physiology and Chemistry, Vol. Clinical Chemistry and Clinical Biochemistry, Volume 15, page 479, published in 1977, describes a method for the isolation of kallikrein from humans.

On the one hand, the conventional methods describe only the preparation of urokinase with a number of surface binders except aprominin on the one hand and the immobilization of aprotinin on a solid matrix to concentrate and purify the majority of compounds except urokinase on the other hand. It has now been found that good and economically extremely suitable process results are obtained by isolating and concentrating urokinase in aprorinin immobilized in solids.

Prior to describing the present invention, it will be pointed out that in the known production process, urokinase generally results in two bond forms, a high molecular weight form having an average molecular weight and a low molecular weight form having an average molecular weight of 54,000. The molecular weight of the urokinase obtained by the method of the present invention described below was found to have a urokinase having only a slightly different molecular weight at an average value of 33,000. This improvement cannot be explained in the conventional product quality, and the present study is under consideration to determine whether the urokinase having a low molecular weight is a high molecular weight decomposition product.

6의 매질중에서 접촉 반융시켜 아프로티닌을 고착시키며, 담체매질을 분리시킨 다음 흡착된 유로키나제를 pH

4.5에서 용출제로 용출시킨 다음 상이한 의약품을 제조하기 의해 공지의 제조법을 사용한다.The present invention relates to a method for purifying lytokinase having high concentration and activity starting from a crude urokinase solution previously purified and concentrated by a known method, wherein the crude urokinase solution has a high specific surface area. Has a porous solid matrix and has a covalent pH on its surface

Contact affinity in the medium of 6 to fix the aprotinin, the carrier medium is separated and the adsorbed urokinase pH

The known recipe is used by eluting with eluent in 4.5 and then preparing different pharmaceuticals.

6의 매질에서 친화성 크로마토그라피로 분리시킨 다음에 이 방법으로 흡착시킨 유로키나제를 pH

4.5에서 용출제를 사용하여 용출시킨다.According to an unusual and industrially important method of this method, the crude eurokinase solution has a high specific surface area and pH in a porous solid matrix column which covalently bonds aprotinin on the surface.

The urokinase which was separated by affinity chromatography in the medium of 6 and then adsorbed by this method

Eluate with eluent in 4.5.

The action of apronin fixed for specific adsorption of urokinase is particularly surprising because conventional concentrated aprotinin does not inhibit urokinase action in solution.

Moreover, resins surface-activated with aprotinin have a longer and more consistent operating life than conventional chromatographic columns.

Crude eurokinase solution used is between 2,000 and 10,000 IU eurokinase / ml, suitably concentrations of 3,000 to 5,000 IU eurokinase / ml and 200 to 2,000 IU eurokinase / total protein mg, advantageously 300 to 1,000 IU eurokinase / total protein mg Should have a purity of.

The porous solid matrices used were in various resin forms, for example polysaccharides, cellulose based products, agarose such as Sepharose ^{R from} Pharmacia AB, Sweden, dextran such as Sephadex ^{R from} Pharmacia AB, Sweden, Or polyacrylamide based copolymer adducts having agarose, such as Sephacryl ^{R from} Swedish Pharmacia Avi, or Ultrogel ^{R from} LKB. Other solid matrices may also be used, the main feature being the ability to form aprotinin directly or through covalent bonds or via spacers. Examples of spacers include ε-aminocaproic acid (commonly known as EACA (6-aminohexanoic acid in the international literature). Bis-oxirane or other compounds of divinylsulfonda number can be used.

As is commonly done in affinity chromatography, solid phase matrices can be pre-activated using, for example, BrCN or other known active agents.

The method of the present method is not suitable for treating urine itself which is too low in urokinase concentration in urine. The total salt concentration of the crude urokinase solution may be 0.5 to 1 mol by adding various salts, for example, NaCl or KCl.

Before actually performing adsorption identification, if the adsorbent is present in the column, the adsorbent can be manipulated into the liquid medium at pH 6-9 and a total salt concentration of 0.5-1 mol. As the buffer, known phosphate buffers or Tris buffers and other buffers can be used. As modulating salt, NaCl or KCl or other suitable compounds can be used.

After adsorbing urokinase on a solid matrix with aprotinin, the solid matrix charged before actually eluting urokinase can be cleaned in an indirect manner. For this purpose, a liquid agent having a NaCl solution of 0.5 to 1 molar concentration can be used. This indirect cleaning removes unwanted proteins, especially from the solid matrix. The liquid may be cleaned until the leaking medium is optically inert at a wavelength of 280 nm, ie, until it substantially shows no further protein concentration.

4.5에서 행하므로 pH 2 ~ 4.5에서 상기염의 용액을 상기의 농도로 사용할 수 있다. 이 용액은 예를 들면 초산 또는 HCl 또는 기타 화합물을 가하여 조정할 수 있다.As the eluent, a solution of NaCl or KCl in a concentration of 0.5 to 1 mol is used. According to the present invention, the elution is pH

Since it is performed at 4.5, a solution of the salt can be used at the above concentration at pH 2 to 4.5. This solution can be adjusted, for example, by adding acetic acid or HCl or other compounds.

A well-known elution aid, such as an amino acid, for example, lysine and arginine, can be added to an eluent.

The urokinase solution having high concentration and high purity can be obtained by the method according to the present invention. For example, the method of the present invention can improve the concentration and / or purity of urokinase by 100 times over the raw material. According to the method of the present invention, a urokinase solution having a purity of 50,00 I.U eurokinase / mg of total protein or more can be obtained. This purity is considered to be high enough for products that will be used directly for clinical purposes. In addition to this advantage, the purified urokinase obtained by the present invention has a uniform molecular weight of about 50,000.

Yields and concentrations obtained from urokinase are listed in the table below.

[table]

The present invention will be described in detail based on the following examples.

Example 1

Separose-apro having a volume of 1 L of 12.10 ⁶ IU urokinase / 1 ml, i.e. 500 ml of crude urokinase solution containing urokinase having a concentration of 24,00 IU urokinase / ml in solution and a purity of 630 IU urokinase / total protein mg Filtered by kinin column. The tower was treated with 0.1 moles of Tris buffer at mill pH 8. The treatment liquid also contains 0.5 molar NaCl. After adsorption, the column was washed with the same control solution until it had an optical density of 280 nm or less. The tower was then washed with 1 L of 0.5 molar NaCl solution.

The adsorbed urokinase was finally eluted with 0.5 mol NaCl solution and then adjusted to pH 2.5 with HCl.

1.5 liters of eluate containing a total of 8,160,000 I.U (yield 68%) of urokinase were obtained. The purity of the solution obtained in this way was 53,000 I.U urokinase / total protein mg.

Example 2

A total of 22,260,000 IU of urokinase, ie, 5,200 IU urokinase / ml in solution and 4.2 l of crude urokinase solution containing urokinase having a purity of 370 I / U and urokinase / mg total protein, was filtered through a Sepharose-EACA-Aprotinin column. I was. The volume of the column was 1.5 liters. The column was previously equilibrated at pH 7.2 with 1.0, 0.02 molar phosphate buffer containing a concentration of NaCl. The filled resin was then rinsed with 3 liters of buffer and then washed with 2 liters of a 1.0 molar NaCl solution. Next, the adsorbed urokinase was eluted using a 1.0 mol NaCl solution adjusted to pH 3.9 with acetic acid.

2 L of urokinase solution containing a total of 15,915,000 I.U of urokinase and having a yield of 71.5% was obtained. The purity of the solution obtained was 61,000 I.U urokinase / mg total protein.

Claims (1)

A method for purifying high concentration and high activity of urokinase from crude urokinase solution previously purified and concentrated by a known method, using a porous solid matrix having a large specific surface area and immobilizing aprotinin on its surface by chemical covalent bonds. PH of the crude urokinase solution

6 contact with the medium, the carrier medium is separated and the adsorbed urokinase pH

After eluting with eluent in 4.5, the eluted urokinase is post-treated according to known different drug production methods.