FI58260B - FOER REFRIGERATION FOR RENTING INSULIN - Google Patents

Description

-ι ** ιτι— - ΓβΊ f11, ADVERTISEMENT rq «/ a lBJ (11) UTLÄGGNINGSSKRIFT 5 82 60 C Patent granted 12 01 1901 ^ Patent meddelat ^ (51) Kv.ik? /into.3 A 61 K 37/26 , C 07 C 103/52

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(22) Hmkainltplivt - Anaflknlnpdif 15.07 · 77 (23) AlloiplM — GIMghet * de | 15.07.77 (41) Interpreters for public * - Bllvlt offentHj 16.01.79

Patent and registration (44) Date of issue and date of issue. -

Patent and registration requirements AmMcm utitgd och utUkriften pubticend 30.09 · 80 '(32) (33) (31) Claim claimed — Be * lrd prlorltet (71) Laboratories Leo SA, 99 Avda.de Pio XII, Madrid, Spain-Spain (ES ) (72) Willy Henry Jensen, Madrid, Spain-Spain (ES) (7 * 0 Oy Kolster Ab (5 * +) Method for the production of pure insulin - Förfarande för fram-ställning av rent Insulin

The invention relates to a process for purifying a crude pancreatic extract containing insulin, water and ethanol, which process is characterized in that the crude extract, after cooling to remove fat by crystallization, followed by separation of fat crystals from the extract, and possibly further purification, the extract has an ethanol content of 60 to 80% by volume, is subjected to gel filtration with Sephadex® LH-60, an epi- “chlorohydrin-crosslinked dextran, with an exclusion limit of about 10,000 MP and a water-binding capacity of 5.3-6, 3 ml / g, and containing hydroxypropyl groups bonded to the glucose units of the dextran chains via ether bonds, and which gel is swollen in ethanol or an aqueous solution of ethanol, preferably having an ethanol content of at least 60% by volume, and preferably having the same ethanol content and pH than the extractant used, while ethanol or et an aqueous solution of an anole, preferably having an ethanol content of at least 60% by volume.

2,58260 The term "crude insulin extract" is used in its conventional sense, i.e. an extract obtained by treating the pancreas as an aqueous organic solvent, in particular aqueous ethanol, usually 60-80%, and also extracts which, in addition to being degreased, have undergone various treatments, for example by mixing different proteins from the insulin solution by changing the pH (so-called pH-8 precipitation), and / or treating the solution in a reverse osmosis device, however, so that the insulin remains in the liquid phase during these treatments.

Thus, the term does not include insulin-containing solutions formed by dissolving isolated, isolated, solid insulin from the original extract in a solvent. ~

It is now generally assumed that the antigen content in insulin preparations is primarily due to impurities in the preparations, whereas it has previously been thought that insulin antibodies evolved under the influence of insulin itself. These impurities may be foreign proteins of pancreatic origin, proinsulin, which is a precursor to insulin, intermediate insulin, dimer, insulin arginine, insulin ethyl ester, insulin desamido, which is deamidated to varying degrees, and other insulin variants, and.

Therefore, the aim is to prepare insulin preparations consisting of pure insulin, the so-called one-component insulin, free of all impurities and concomitant substances.

For this purpose, it has been proposed to subject amorphous or crystalline insulin prepared in the usual way to a thorough further purification, e.g. by ion exchange treatment, gel filtration using the so-called molecular sieve, or by fractional chromatography.

Purification using fractional chromatography is described, for example, in German Application No. 2,212,695. It is known to introduce a solution formed by dissolving solid, still impure insulin in an aqueous solvent system containing alcohols having 1 + ... 5 carbon atoms. , contains carboxylic acids having 1, 3 carbon atoms and / or ammonia or an organic base with pK values between about 5.1 and 11.11 for fractional chromatography, e G * using Sephadex as the gel @ LH-20. The aforementioned gel is dextran crosslinked with epichlorohydrin and containing hydroxypropyl groups linked by ether bonds to the glucose units of the dextran chains.

3,58260

Purification of insulin by gel filtration is described in U.S. Patent No. 3,876,623. Starting from alkali metal or ammonium insulin, a gel having well-defined properties, such as a specified water-binding capacity and a defined grain size, is used. The crude insulin to be purified must have an insulin content of more than 80 ts of insulin and the amount of blood sugar-lowering proteins resembling it must be at least 80% of the total amount of proteins present. It has been mentioned that the gel is e.g. crosslinked dextran, such as the Sephadex gel manufactured by Pharmacia Fine Chemicals, but it is not specified which Sephadex gel is relevant.

The highly purified insulins obtained by the above-mentioned known purification methods have a greatly reduced antigen content, but this has not been completely eliminated. The reason for this is undoubtedly that, despite the purification steps, the purified preparations contain substances other than insulin.

A method for preparing very pure one-component insulin has previously been developed (see Finnish application No. 770 112, which corresponds to Danish application No. 1U1 / 77) -

The method described therein may comprise, inter alia, the step of treating the defatted concentrated crude insulin extract (solvent mixture: water / organic solvent) with an ion exchanger, whereby the insulin binds to the ion exchanger from which it is eluted. Ko. the crude insulin extract may be pre-purified by precipitating the fat at a low temperature, e.g. at about -10 ° C, and removing some of the dye as well as a substantial portion of the molecules larger or smaller than the insulin molecule by ultrafiltration in a reverse osmosis device. In this case, it is difficult to separate the insulin both in very pure form and in good yield, because as the processing time increases, aggregates of insulin molecules are formed which - are difficult to elute later.

The inventors have therefore sought to find a purification method which can supplement or replace the treatment of the crude extract with ion exchangers, so as to achieve an efficient separation without insulin loss or with lower insulin losses than before.

Thus, it has now been discovered that a crude ethanol extract of insulin can be purified in about 100% insulin yield by subjecting the crude extract to gel filtration using a special gel and taking into account the special conditions.

Sephadex® LH-60 is a spherical gel with both hydrophilic and lipophilic properties. Sephadex®LH-60 is manufactured by Pharmacia Fine Chemicals, Uppsala, Sweden. Originally, its grain size was UO-120 Jim, but today there are other grain sizes. The grain size is not critical in the process of the invention.

In the gel filtration according to the present invention, insulin molecules penetrate the gel particles if their molecular weight is below the gel exclusion limit. Elution yields an insulin fraction which is completely free of medium and which does not contain, at least practically, more proteins with or without molecules. smaller than insulin molecules, but contain only insulin derivatives that have virtually the same molecular size as insulin. The insulin yield is practically 100%.

This method can be used particularly advantageously in connection with the method described in Finnish application No. 770 112 (corresponding to Danish application No. 1 ^ 1/77), since gel filtration complements or replaces the treatment with ion exchangers described in the description of this earlier application.

However, the gel filtration of the present invention is not limited to use with said prior art methods, but may be used in a selectively degreased aqueous / ethanol-containing crude extract and may be either a final purification step to isolate solid insulin or an intermediate purification step.

With respect to the aforementioned process, known from German Patent Application No. 2,212,695, comprising a solution formed by dissolving solid insulin in an aqueous solvent system containing, in addition to alcohols having U to 5 carbon atoms, a carboxylic acid and / or ammonia or subjecting the organic base to fractional chromatography using a Sephadex ®LH-20 gel, it is noted that this is a separation technique other than gel filtration. The method is not suitable for purifying the crude insulin extract, e.g. because it uses a solvent system which differs from the solvents normally used for the extraction of the pancreas. It should also be noted that Sephadex® LH-20 differs from Sephadex® LH-60 in that it has a lower exclusion limit than the latter, cf. page U, 3 of the German application publication complete piece so that the insulin molecules do not penetrate into the gel.

When purifying the crude insulin extract according to the invention, the procedure can be as follows:

The gel is swollen in ethanol or aqueous ethanol, preferably with an ethanol content of at least 60 volumes, for example in a mixture corresponding to the solution used for the extraction of the pancreas, or possibly in a mixture with a somewhat higher concentration of organic solvent.

5,58260

The column is used in the usual way; a crude extract having an ethanol content of between 60 and 80% by volume is introduced into the column, and an eluent, namely ethanol or aqueous ethanol, is made to flow through the column. The eluent may be the same as the swelling agent.

The pH value in the liquid phase of the Sephadex layer is preferably between 3 and 8. (Eluates with a pH below about 3 are too acidic to be taken directly to ion exchangers and at pH above 8 insulin may decompose).

The insulin is eluted and the fraction is collected. It is quite free of dyes. A practically 100 to # # insulin yield is achieved.

In order to obtain pure one-component insulin, the eluate is further purified, e.g. with ion exchangers, and finally the insulin is isolated, e.g. by mixing.

Example

Insulin-containing crude extract - obtained by treating frozen fine porcine pancreas with 85 ~ # ethanol acidified to a pH of about 3 with sulfuric acid (2 1 85 - # ethanol / 1 kg glands, which together with the water in the glands 65% aqueous extract of ethanol), degreasing the extract by cooling to -30 ° C, separating the formed fat crystals, and concentrating to 1/10 of the original volume by reverse osmosis - gel filtration as follows: 50 L of the above concentrate was applied to a column ( diameter U5 cm, (K) height 75 cm) with Sephadex® LH-60 swollen and equilibrated with 65% ethanol (acetate buffer, ionic strength 0.025, pH 7.3).

Elution was performed with an eluent identical to the swelling agent.

The fractions containing the insulin peak were pooled. Of these, the dyes had been completely removed and contained no or virtually no proteins with a molecular size larger or smaller than the insulin molecule.

Claims (1)

A method for purifying a limb pancreatic extract containing insulin, water and ethanol, characterized in that the crude extract, after cooling to remove fat by crystallization, followed by separation of fat crystals from the extract, and optionally after further pre-purification, The extract has an ethanol content of 60 to 80% by volume, gel-filtered with Sephadex® LH-60, an epichlorohydrin-crosslinked dextran, with an exclusion limit of about 10,000 MP and a water-binding capacity of 5.3 to 6.3. ml / g, and containing hydroxypropyl groups bonded to the glucose units of the dextran chains via ether bonds, and which gel is swollen in ethanol or an aqueous solution of ethanol, preferably having an ethanol content of at least 60% by volume and preferably having the same ethanol content and pH as the extractant used. , while ethanol or an aqueous solution of ethanol, e.g. the ethanol content is preferably at least 60% by volume.