CS225134B2 - A method for commercial or crude insulin storage - Google Patents

Abstract

When an aqueous solution of commercial or crude insulin at pH 6 to 10 is applied to an anion exchange column and the column is then eluted in fractions with a water-miscible aliphatic C1-C2 alkanol and the fractions showing essentially a single component (when analyzed by D1SC PAGE) are collected, the insulin obtained is free of pancreatic proteins of molecular weight higher than that of insulin and free of antigenic impurities similar to insulin of approximately the same molecular weight as insulin. The water-miscible alkanol contains water in an amount of 20 to 70% by volume and has a pH of 6 to 10.

Description

BACKGROUND OF THE INVENTION The present invention relates to a method of fusing commercial or crude insulin to deprive pancreatic proteins of molecular weight greater than that of insulin, namely over 6,000, and non-insulin-like antigenic insulins of approximately the same molecular weight as insulin. mania, anions.

British Patent Specification No. 1,285,023 discloses an injectable insulin preparation comprising insulin obtained from the pancreas, dissolved or suspended in an aqueous injection medium, wherein the insulin is free of proteins of pancreatic origin having a molecular weight of more than 6,000 and exhibits essentially a single component when analyzing its solution by polyacrylamide gel electrophoresis (the so-called Disc Pege method).

Commercial insulin, i.e., amorphous insulin or crystalline insulin, which can be recrystallized several times, is generally used as the starting material for insulin purification. It is also possible to use crude insulin, such as cakes, insulin-containing salts, which results from the isolation of insulin from the pancreatic glands and usually contains 10-31% by weight of insulin, but due to its high impurity content this material is less suitable for the inventive process. A better starting material can be obtained by treating the salt cake solution by isoelectric precipitation by adjusting the pH of the solution to 5.5 and isolating the precipitate by centrifugation.

Initially, it was generally believed that appropriately recrystallized, sharp-edged romboeders, coupled with flat crystal surfaces, is a pure protein, an insulin whose configuration has been determined by Sanger et al., See, for example, Biochemical Journal 60 (1955), 556. Later it was found not to be so. Scientific analytical investigations have shown that said crystalline insulin contains proteins of pancreatic origin with a molecular weight higher than that of pure insulin and, moreover, an insulin-like substance of approximately the same molecular weight as pure insulin (about 6000).

It has been shown that crystalline insulin dissolved in 1 M acetic acid can be fractionated in components (a), (b) and optionally (c) by gel filtration on a Sephadex 0-50 column (Sephadex is a trademark) which is dextrene cross-linked with epichlorohydrin a penetration limit of about 10,000 (minimum molecular weight of the excreted substance), which is in the form of grains (20 to 80 micron diameter). The three components can be isolated by conventional means from their solutions, for example by salting, zinc salt precipitation in a neutral reaction, lyophilization, etc. Both components (a) and (b) contain proteins of pancreatic origin and a molecular weight greater than 6000, while component (c) it contains pure insulin contaminated with insulin-like proteins of approximately the same molecular weight as pure insulin.

The amount of component (a) may comprise 2 to 5% by weight of the crystallized insulin and less than 1% by weight of the recrystallized insulin. The amount of component (b) may in both cases be 4 to 8% by weight and the amount of insulin-like protein in component (o) may be 5 to 13% by weight in component (c).

The animal immunological experiments underlying the present invention have shown that components (a) and (b) above and to some extent the insulin-like protein present in component (c) are essentially responsible for the anti -genic properties of the prior art insulin preparations.

The purpose of the invention is to eliminate the impurities contained not only in the raw insulin but especially in the commercial insulin in a simple and efficient manner such that the purified insulin shows no or substantially reduced antigenicity, since it contains neither high-molecular proteins nor insulin-like antigenic substances.

SUMMARY OF THE INVENTION A process for purifying commercial or crude insulin to deprive pancreatic proteins of molecular weight above 6,000 and similar impurities by column chromatography is to use, as an aqueous solution of the insulin to be purified,

225.34, a solution having a pH in the range of 6 to 10 is used, from which the fractions are eluted with a water-miscible aliphatic alkanol (1 to 3 carbon atoms per molecule) containing water in an amount of 20 to 70% by volume, preferably 30 to 50%, by volume, and having a pH in the range of 6 to 10, preferably 6.5 to 8.5, collects or collects the eluate fractions having essentially a single component when analyzed by polyacrylamide gel electrophoresis (DISC PAGE), and insulin thus purified is recovered from the collected fraction or collected fractions.

It is advantageous to use strongly basic anion exchangers as anion exchangers

For analytical purposes, polyacrylamide gel discontinuous alecophoresis has already been used, also in connection with crystalline insulin. This specific gel "slekirophoresis was developed by B, J, Davia and L. Oraateiite" is described in Asm. M. X., Aead. Sci. W, pp. 321-497 and 474-427 (1964). In this case, 7.58 polyacrylamide, which contains 8 U urea and a pH of 3 and a pH of 3, is used in the bottom gel, while the top gel and the applied insulin solution containing aul 0.1 mg of tasulin also contain 8 U urea. .

It is not only surprising that _, according to the present invention, high molecular weight proteins can be eliminated. Non-darkness criteria for these proteins, not known. Using this analytical method in connection with commercial itutilins provides an insulin pda containing a high molecular weight protein, a dimer, see. for example, an article by Steiner et al. in Diabetes, 17th century »» 728, right column.

It could no longer be expected that the eluent used could be capable of producing a monomerizing effect on the proteins present to allow separation of insulin-like substances.

Anion exchangers which should preferably be of the strongly basic type used in the process of the invention are known. Examples of weakly basic anion exchangers are Bio-Oel UM, manufactured on the basis of polyacrylamides, DEAE-Sephadex, made from sulfated dextrans, and DBAE-cellulose, made from cellulose. Examples of strongly anionic anion exchangers are Dowers (Dovex is a trademark), made of polystyrene and QAE-Sephadex, x.r.buttoned with crosslinked dextrans.

Examples of water-containing organic water-miscible solvents are tetramethylurea, dimethylformamide, dloxane, water-miscible ketones, for example acetone, and alkanols, for example methanol, ethanol and propanols. The alkanols are preferred organic elution solvents for use in the process of the invention and are preferably used in a form containing from 30 to 50% by volume of water.

The eluent always contains a buffering agent to control the pH of the eluent. It is preferred to carry out the process of the invention at a stable pH. The pH of the eluent should be maintained in the range of from 6.5 to 8.5, as measured by glass electrode, adjusted in the usual manner to a standard buffer.

Suitable buffers can be found in the available literature.

Constant temperature during ion exchange is essential. >ορ.>. Οΐ, υ. it can be cast in the range of -10 ° C to 40 ° C, preferably 0 ° C to 30 ° C. The reshaped insulin from the fractions may be isolated in the usual manner, for example by evaporation, salting out or precipitation in an arafic or crystalline form in the presence of zinc lions.

In the isolation of Insulin, it is known to subject acidic or neutralized alcoholic extracts from the pancreas to column chromatography on a cation exchanger, e.g. ) or on an anion exchanger, for example aa diethylaminoethylcellulose (British Patent No. 2,931,954). However, the insulin obtained by these methods contains impurities as well as the crystalline insulin obtained by conventional isolation methods including repeated precipitation of crude insulin solutions by salting out with sodium chloride.

British Patent Specification No. 881,855 also discloses column chromatography of unspecified zinc-insulin on a Dowex 50-XI cation exchanger (eulfonated styrene _T divinyl copolymer in granular form) using aqueous ethanol as the eluent, although using as starting material of recrystallized insulin, isolated insulin usually contains the same impurities as the starting material, since all the fractions containing insulin and not specific fractions containing insulin are collected which have a single component by the CISC PAGE method.

It is further known from British Patent No. 913,042 to purify crystalline insulin in admixture with amorphous or foreign microcrystalline materials by washing the impure crystallizate with an aqueous solution whose pH is maintained from 3.8 to 4.3 with a weak acid or a mixture of buffers. However, this purification step does not remove impurities such as proinsulin, dimer or an intermediate thereof.

Finally, British Patent Specification No. 871,541 discloses column chromatography of six times crystallized insulin and insulin purified as described by Lens (Biochem. Et Biophys. Acta (1948) 2, 76) on a styrene-divinylbenzene copolymer cation exchanger on a large surface inert diluent. however, insulin still contains impurities such as proinsulin, dimer and intermediate along with arginine-insulin and monodesaminoinsulin after this chromatography, because the presence of these impurities does not prevent the chromatogram from giving the impression that the material eluted from the column is substantially homogeneous.

The invention is illustrated by the following examples »

Examples of examples 1 and d 1

Prepare a buffer of the following composition:

0.1 M ammonium chloride solution

0.02 14 ammonia solution

60% ethanol (v / v)

The pH is 8.3 at 25 ° C.

g of the Dowex 1 x 2 anion exchanger (mesh: 50/100) is swollen in buffer and the finest particles are removed by decantation. The material was used to fill a column with a diameter of 1.6 cm and a height of 25 µm. Equilibrate the column with buffer. Dowex 1 x 2 is a copolymer of styrene with 2% divinylbenzene, which contains benzyltrimethylammonium as functional groups and is in granular form.

500 mg of insulin, once crystallized from citrate buffer, are dissolved in a mixture of 20 mg of ethylenediaminetetraacetic acid disodium salt, 5 ml of buffer, 5 ml of 60% (v / v) ethanol and 0.04 ml of 15.3 M ammonia solution (final pH 8.5). The insoluble material was removed by centrifugation and the clear solution was applied to the column. Elution is performed with buffer at 25 ° C and 7.5 ml / h. Fractions of 5 ml are collected.

Extinction values at 276 nm are measured and plotted against fraction numbers. The fraction corresponding to the middle main part of the insulin maximum (the highest maximum), which showed essentially a single component by polyacrylamide gel electrophoresis (DISC PAGE), is mixed and the insulin is precipitated by adding 100 ml of water, 1.3 ml of 1 N hydrochloric acid and 2 ml

225Ί34

N of zinc acetate solution per 100 ml of combined fractions. The precipitate is isolated by centrifugation and crystallized from acetone-citrate buffer in a known manner. The yield of purified insulin is 200 mg.

Example 2

Add the following buffer:

25.0 g of tris (hydroxymethyl) aminomethane 29.0 ml of a 6 N hydrochloric acid solution

1.2 liters of methanol

Water to make up the total volume to 2 liters. The pH is 7.3‘ at 25 ° C.

The QAE-Sephadex A-25 anion exchanger was swelled in buffer and the finest particles were removed by decantation. The material was used to fill a column with a diameter of 2.5 cm and a height of 25 cm. Equilibrate the column with buffer. QAE-Sephadex A-25 is epichlorohydrin cross-linked dextran with a penetration limit of about 5,000 (minimum molecular weight of excreted substances), which has diethyl- (2-hydroxypropyl) aminoethyl functional groups as granules (average 40 to 120 microns) .

- 500 mg of insulin crystallized with one of the citrate buffer are dissolved in a mixture of 25 mg of ethylenediaminetetraacetic acid dibasic salt, 94 mg of tris (hydroxymethyl) aminomethane, ml of 60% methanol (v / v) and 0.073 ml of 6 N hydrochloric acid (final value) pH 7.3). The insoluble material was removed by centrifugation and the clear solution was applied to the column. Elution is performed with buffer at 25 ° C and 30 ml / h. Fractions of 5 ml are collected. Extinction is measured at 276 nm and plotted against fraction numbers. The fraction corresponding to the middle main part of the insulin maximum (maximum maximum), which showed a single component by polyacrylamide gel electrophoresis (essentially a single component), is mixed and the insulin is precipitated by adding 100 ml of water and 2 ml of 1N zinc acetate solution ml of combined fractions. The precipitate is isolated by known centrifugation and crystallized from acetone-citrate buffer. The yield of purified insulin is 250 mg.

Example 3

Prepare a buffer of the following composition:

0.06 M tris (hydroxymethyl) aminomethane

0.02 M hydrochloric acid solution

0,075 M sodium chloride solution% ethanol (v / v)

The pH is 8.3 at 25 ° C.

g of the anion exchanger of Example 2 is swelled in buffer and the finest particles are removed by decantation. The material was used to pack a column with a diameter of 2.5 cm and a height of 50 cm. Equilibrate the column with buffer,

2.5 g of insulin crystallized with one of the citrate buffer were dissolved at 0 ° C in a mixture of 450 mg of tris (hydroxymethyl) eniinomethane, 100 mg of ethylenediaminetetraacetic acid, disodium salt, 25 ml of buffer, 25 ml of 6056 ethanol (v / v) and 0.08 ml of a 4 N hydrochloric acid solution (final pH at 25 ° C is 8.4). The insoluble material was removed by centrifugation at 2-4 ° C and the clear filtrate was applied to the column. Elution is performed with buffer at 4 ° C and 26 ml / h. Fractions of 20 [mu] l are collected.

te

Extinction is measured at 276 nm and plotted against fraction numbers. The fraction corresponding to the middle main part of the insulin maximum (the highest maximum), which showed essentially a single component by polyacrylamide gel electrophoresis (so-called DISC FAGE), is mixed and the insulin is precipitated by adding an equal volume of 0.02 M zinc acetate, 0.03 N hydrochloric acid. The precipitate is isolated by centrifugation and crystallized from the acetonitrile buffer mixture in a known manner. .

The yield of purified insulin is 1.3 g.

Example 4

Prepare a buffer of the following composition:

14.9 g of histidine hydrochloride

38.7 ml of 1 N sodium hydroxide solution

16.1 g sodium chloride 3.12 liters 96% ethanol (v / v)

Water to make up a total volume of 5 liters. The pH at 25 ° C is 6.5.

g of the anion exchanger in Example 2 is swollen. in buffer and the finest particles are removed by decantation. The material was used to fill a column of 2.5 cm diameter and 25 cm height. Equilibrate the column with buffer.

500 mg of insulin once crystallized from nitrate buffer are dissolved in a mixture of 20 mg of ethylenediaminetetraacetic acid disodium salt, 6.5 ml of buffer and 6 ml of 60% ethanol (v / v), and the pH is adjusted to 6.8 by adding 1 N solution sodium hydroxide. The insoluble material was removed by centrifugation and the clear solution was applied to the column. Elution is performed with a buffer at a temperature of 27 ° C and a rate of 30 ml / h. Fractions of 5 ml are collected.

Extinction is measured at 276 nm and plotted against fraction numbers. The fraction corresponding to the central major part of the insulin maximum (the highest maximum), which showed an essentially single component by electrophoresis on 1-amide gel electrophoresis (so-called DISC PAGE), is mixed and the insulin

It is then added by adding an equal volume of 0.02 κ zinc zinc solution. The precipitate is isolated by centrifugation and crystallized from the acetone-acetate buffer mixture in a known manner. The yield of purified insulin is 210 mg.

Example. 5

Prepare a buffer of the following composition:

i, 25 kg of trihydroxymethyl ieainometeEfttt

725 ml of a 12.3 N hydrochloric acid solution

62.4 liters of 96% ethanol (v / v)

Water to make up the total volume to 100 liters. The pH at 25 ° C is 7.3.

1.3 kg of the anion exchanger shown in Example 2 were swollen in buffer and the finest particles were removed by decantation. The material was used to load a column of 15 cm diameter and 15 cm height. Equilibrate the column with buffer.

g of recrystallized pork or bovine insulin containing 0.4% zinc is dissolved in a mixture of 0.72 g of disodium ethylenedietaetraacetic acid disodium, 10.2 g of tris; hydroxymethyl) aminomethane and 720 ml of 60% ethanol (v / v). .). Insoluble material is removed by centrifugation. To the clear solution was added 10.7 mL of 6 N hydrochloric acid

51 225134 and the solution is applied to the column, eluting with buffer at 25 ° C at a rate of 1.2 L / h. 0.5 liter fractions were collected.

Extinctions are measured at 276 nm (EGg) ^and plotted against fraction numbers. The fraction corresponding to the central major part of the insulin maximum (the highest maximum), which showed an essentially single component by electrophoresis on a polyacrylamide gel (so-called LISC PAGE), was pooled. Insulin is precipitated from the pooled fractions by adding an equal volume of 0.02 M zinc acetate solution. The precipitate is isolated by centrifugation and crystallized from the acetone-citrate buffer mixture in a known manner.

8.2 g of purified insulin are obtained. Insulin is recrystallized from a buffer of the following composition:

2.0 insulin

0.8% Zn ⁺⁺ (as chloride) based on insulin weight

0.1 U sodium acetate solution

7.0% sodium chloride

Hydrochloric acid up to a pH of 5.45 to 5.55.

The insulin is dissolved in water containing hydrochloric acid and zinc chloride and a solution containing the appropriate amounts of sodium acetate and sodium chloride is added. Crystallization is carried out with mechanical stirring at room temperature. Crystallization is complete within one or two days. The insulin is isolated by filtration, washed with water and dried in vacuo. Yield 8.0 g.

Purified insulin can be used to prepare all kinds of pharmaceutical insulin preparations for clinical use.

Claims (4)

1. A method of purifying commercial or crude insulin to deprive pancreatic proteins of molecular weight greater than that of insulin, i.e., above 6,000, of antigenic insulin-like impurities of approximately the same molecular weight as insulin, by applying to the column the desired insulin. anion exchanger, characterized in that a solution having a pH in the range of 6 to 10 is used as the aqueous insulin solution to be clarified, and the fractions are eluted from the column with an aliphatic water-miscible C 1 -C 8 -alkanol containing water in an amount of 20 to 70% v / v and having a pH in the range of 6 to 10, the eluate fractions having essentially a single component when analyzed by polyacrylamide gel electrophoresis (DISC PAGE) are collected or collected, and the insulin thus purified is isolated from the collected fraction or of the collected fractions. 2. A process according to claim 1, wherein the water-miscible O, -Cyalkanol containing 30 to 50% by volume of water is used as the elunet. 3. Method according to claim 1 or 2, characterized in that an elunet having a pH of 6.5 to 8.5 is used. 4. The method of claim 1, wherein the anion exchanger is a strongly basic anion exchanger.