CS237552B1 - A method for purifying crude pancreatic ethanol extract comprising insulin - Google Patents

Abstract

The invention relates to a method for purifying crude pancreatic ethanol extract containing insulin, obtained by extracting the gland with ethanol acidified to pH 3.0 to 4.0 with a mineral acid, preferably phosphoric acid.

Description

The invention relates to a process for purifying a crude insulin-containing pancreatic ethanol extract at a pH of 3.0 to 4.0 treated with a mineral acid, preferably phosphoric acid.

♦

As is known, insulin is obtained by extraction of bovine, porcine and pork. veal pancreas with ethanol, acidified with a mineral acid such as hydrochloric, sulfuric or phosphoric acid or an organic acid such as formic or acetic acid.

The acidic ethanolic extract is then adjusted to pH 7.2-8.0 to remove basic proteins and other ballasts, preferably ammonia, and the precipitated ballasts are separated by calibrating or centrifuging.

The clear filtrate obtained is again acidified to pH 3.2-3.5 (with sulfuric acid), then concentrated, freed from lipoids, and further processed to crystalline insulin by known methods.

It is further known that an extract of 1 kg of bovine or porcine pancreas contains about 3,500 to 4,500 IU and about 1 kg of veal pancreas about 15,000 to 20,000 IU. insulin. The amount of insulin depends on both the quality of the pancreas and the extraction method.

The final yield is directly influenced by the technological process used. During the processing of the crude extract, conventional insulin losses are still relatively high, so that the final yield is about 50% based on the original amount contained in the extract.

One of the main causes of these losses is the aforementioned treatment of the extract into a weakly alkaline region (pH 7.2 to 8.0), which was considered (in addition to the removal of basic proteins) necessary to facilitate further operations / lipoid separation, purification and crystallization /.

However, it is known that insulin is labile in an alkaline-alcoholic environment. In addition, the raw insulin solutions so treated are fairly dark and their discolouration is unpleasant, laborious and time-consuming, notwithstanding the losses associated with this purification.

In British Pat. No. 694227 discloses the use of salts with monovalent anions, with the exception of heavy metal salts, in the extraction of the gland with an acidified aqueous alcohol. Sodium chloride, ammonium chloride and calcium chloride are given.

This measure reportedly increases the extraction yield in the first feed, but the alkaline treatment (pH 7.6) and hence the possibility of destruction of the insulin contained in the raw extract again reduce the initial beneficial effect.

By examining the usual methods for increasing the overall yield, physicochemical methods have shown that the alkaline treatment of the extract results in the greatest loss of about 30% of the total insulin content of the extract.

Furthermore, it has been found that omitting the alkaline treatment makes it impossible to separate lipoids and makes it difficult to further purify the concentrate. Based on this knowledge and experience, a new improved method of isolating insulin without alkaline treatment of the crude extract has been developed.

The process comprises a process for purifying a crude insulin-containing pancreatic ethanol extract at a pH of 3.0 to 4.0 treated with a mineral acid, preferably phosphoric acid, and using the soluble alkaline earth metal salt, preferably calcium chloride, of the invention.

The essence of this process is to first prepare a crude, acidic ethanol extract and then clarify by adding a soluble alkaline earth metal salt, preferably calcium chloride, at a pH of 3.0 to 3.5, whereupon the precipitated ballast precipitate is separated, e.g. centrifuging and recovering insulin from the clear solution.

It is most preferred to add calcium chloride to the extract in an amount of 1.5 to 2.5% by weight, based on the volume of the crude, pancreatic ethanol extract. The method according to the invention has succeeded in significantly reducing the previous operating losses of insulin, simplifying the technology and making the entire production economical, without the need for increased consumption of auxiliary raw materials and special equipment. The total yield of crystalline insulin is thereby increased by about 20 to 30%.

He did

500 kg of ground pork and bovine pancreas are extracted at 15-20 ° C for 2 hours with an amount of ethanol such that the water content of the extract is not more than 30%. Ethanol acidified with phosphoric acid to pH 3.3-3.5 is used for extraction.

After separation of the first extract, the pancreatic pulp is extracted once more with 1000 liters of 70% ethanol, acidified with phosphoric acid to a pH of 3.3 to 3.5, for 2.5 hours. Centrifuge again and squeeze.

A solution of 20 kg of calcium chloride in 20 liters of water was added to the combined extracts with stirring. This reduces the pH of the extract to about 2.5, which is again adjusted to 3.2 by the addition of dilute ammonia (1: 1).

The extract thus treated is clarified on a centrifuge and the clear filtrate is concentrated under reduced pressure to 1/10 of the original volume at a temperature of not more than 35 ° C. Before discharge, the extract is heated to 45 ° C in a evaporator and, after cooling, discharged into a sedimentation vessel, where it settles and separates the lipoids within 2 hours.

Then the solution is clarified and salted by the addition of 25% w / w. volume / sodium chloride, maintaining the pH at 2.0-2.5 by the addition of HCl. The precipitate formed is filtered off, dissolved in 20 liters of water, the solution is clarified and salted again by the addition of 25% sodium chloride at pH 2.0 to 2.5.

The crude insulin thus obtained is further purified and processed by any of the known methods.

Using the procedure described, 2500 to 3000 IU are obtained from 1 kg of mixed / pork and beef / pancreas. crystalline insulin.

Example 2

The extract from the second extraction (see Example 1) and absolute ethanol are added per 500 kg of ground pancreas to a final concentration of 65%. The first extraction takes 2 hours at a temperature of about 15 ° C and at a pH of 3.3 to 3.5 treated with phosphoric acid.

After extraction, the pancreatic pulp is separated. Saturated calcium chloride solution was added to the extract and the procedure was as in Example 1.

The pancreatic pulp after the first extraction is extracted a second time by adding the extract from the third extraction. It is extracted for 2 hours at a temperature of 15 to 20 ° C, at a pH of 3.3 to 3.5 treated with phosphoric acid.

After the second extraction has been completed, the pancreatic pulp is separated by centrifugation and the resulting solution is used again for the first extraction of fresh pancreas. The remaining pulp is subjected to a third extraction. The extract obtained is used for the second extraction as described above.

The advantage of this process is that, with the same qualitative and quantitative results as in Example 1, 1/3 less solvent is consumed, thereby increasing by 1/3 the capacity of the production equipment.

Claims (2)

SUBJECT OF THE INVENTION A process for purifying a crude insulin-containing pancreatic ethanol extract at a pH of 3.0 to 4.0 treated with a mineral acid, preferably phosphoric acid, and using a soluble alkaline earth metal salt, preferably calcium chloride, characterized in that: First, a crude, acidic ethanol extract is prepared and then clarified by the addition of a soluble alkaline earth metal salt, preferably calcium chloride, at pH 3.0 to 3.5, whereupon the precipitated ballast precipitate is separated, eg by centrifugation, and isolated from the clear solution obtained. insulin. 2. The process according to claim 1, wherein the calcium chloride is added to the extract in an amount of 1.5 to 2.5% by weight, based on the volume of the crude pancreatic ethanol extract.