GB2173503A

GB2173503A - Purification of insulin

Info

Publication number: GB2173503A
Application number: GB08509442A
Authority: GB
Inventors: Peter Slonina; Ulrich Krabiell; Gudrun Derle
Original assignee: Berlin Chemie AG
Current assignee: Berlin Chemie AG
Priority date: 1985-04-12
Filing date: 1985-04-12
Publication date: 1986-10-15
Also published as: FR2581647A1; GB8509442D0; DE3511269A1; NL8501105A

Abstract

A process for the purification of insulin using a weakly acid cation exchanger preferably with a hydrophobic matrix is provided. Fractionation of the insulin is obtained by step-wise or continuous alteration of the concentration of the solvent in the acid pH range.

Description

SPECIFICATION Process for purification of insulin The process relates to a process for the purification of insulin utilizing an ion exchanger preferably with a hydrophobic matrix.

One process employed on a large scale in industry for the production of insulin from animal and human raw material involves the extraction of pancreatic tissue with mixtures containing solvents. Such extracts can be freed of a considerable portion of extraneous substances present by the addition of suitable salts, by changing the pH values, and by cooling the extracts to low temperatures. After the non-destructive separation of the extraction solvents, aqueous solutions are obtained which contain insulin and from which the said impure insulin can be separated e.g. by salting it out in a solid state.

Fractionated precipitation with common salt or ammonium sulphate and/or by precipitating the insulin in the vicinity of its iso-electric point, then provides precipitates from which insulin can be obtained in a relatively pure form by crystallization. If the resulting product is analysed by sensitive methods such as discelectrophoresis or HPLC it is found that a number of subsidiary components are present in addition to the insulin.

It has been found, for example, that biochemical precursors in the synthesis of insulin are always present in considerable quantities in insulin obtained by the above process.

Extensive biological, pharmacological and medical tests have shown that such subsidiary components, which have a higher molar mass than the insulin, are responsible for undesirable side effects of the eventual pharmaceutical products.

For the production of particularly high quality insulin methods have already been developed for the separation of such impurities.

GB-A-1 285 024 proposes a process for the chromatographic purification of insulin on anion exchangers in the presence of organic solvents miscible with water, and having a pH range of 6-10. This process suffers from the drawback that relatively high amounts of anion exchanger are used (at least 30:1 in relation to the insulin) and low yields of insulin are obtained (with a maximum of 60%).

As an alternative, DD-B-1 19 216 and 119 217, corresponding to DE-B-2 505 306 & BR< 307, propose the purification of ammoniuminsulin or alkali insulin by gel filtration. However in addition to the high consumption of gel (1000 ml per 4.5 g of insulin) and the mechanical and chemical instability of the gel bed, particularly at pH values of over 7.0, this method suffers from the drawback of a relatively high pro-insulin content. A further disadvantage is that chromatography only enables low-concentration insulin solutions to be obtained.

DE-A-2 701 092 proposes a process for the production of pure insulin, which throughout the production process maintains the insulin in a dissolved state without any alteration of phase. This method suffers from the drawback of requiring expensive techniques, such as reverse osmosis, and the use of very low tempertures. Further the aforementioned specification fails to provide any method for the purification of impure crystal insulin.

DE-A-2 757 377 is directed to a purification process utilizing cation and anion exchangers whereby pure insulins are obtained at the pancreatic extract stage. This method suffers inter alia from a high consumption of ion exchanger material and requires the use of cation and anion exchangers. This process has also to be followed by gel chromatography for high quality purification.

DE-A-2 629 568 proposes the purification of peptides, particularly insulin, by ion exchange chromatography in the presence of non-ionic detergents. This process suffers from the drawback that it involves the use of relatively expensive and less available auxiliary substances, from the low concentration of insulin in the fractions obtained by chromatography (e.g. 0.35%) and hence from low rates of insulin yield.

The purpose of the invention is to provide a process for the purification of insulin which will alleviate the foregoing drawbacks.

The aim of the invention is to provide a process enabling insulin to be purified without high losses; without the use of mechanically and/or chemically unstable systems, but utilizing readily accessible auxiliary substances.

According to the present invention therefore there is provided a process for the purification of insulin in a weakly acid cation exchanger in the presence of an organic solvent miscibie with water, characterized in that fractionation is effected by altering the solvent concentration in the acid pH range. Although the raw materials used in this process are inexpensive they can be recuperated with little expenditure and effort. The invention best achieves its object if the insulin and its impurities are fractionated on a weakly acid ion exchanger with a hydrophobic matrix in the acid pH range at different concentrations of solvent and possibly with the addition of aromatic derivative substances.

A surprising feature of this method is the fact that fractionation is possible not only according to charge but also according to molecular size.

Suitable ion exchangers are macroporous copolymers of the type obtained from acrylic acid derivatives with divinyl benzene (e.g.

commercial Wofatit'R' of CKB).

Suitable organic solvents include liquid aliphatic alcohols, such as methanol, ethanol,npropanol, iso-propanol; liquid aliphatic ketones with a low boiling point such as acetone, semi-acetals such as methylal, and esters at least partly miscible with water, such as acetic acid ethyl ester; and mixtures thereof.

Suitable acids for setting the pH values include organic acids such as formic acid, acetic acid propionic acid, tartaric acid and citric acid; inorganic acids such as hydrochloric acid, sulphuric acid and phosphoric acid; and acid salts of these acids.

Suitable salts of said pH value setting acids include alkali and ammonium salts of the aforementioned acids, such as sodium acetate and ammonium phosphate.

Suitable aromatic substances include benzene and its derivatives.

The separation of the insulin impurities can be effected either by a "batch" process or by the "column" method. The "batch" process is suitable for preliminary purification, while the "column" method is recommended for final purification.

While known processes for the purification of insulin are based on an alteration of the ion intensity and/or pH value, the proposed solution avoids the drawbacks inherent in these methods, such as the contamination of the eluates with salts and a re-charging of the ion exchanger, because fractionation is obtained by step-wise or continuous alteration of the solvent concentration during eiution.

The process is generally carried out such that insulin of sufficient purity, such as contaminated crystallized insulin from human or animal sources or from iso-electric precipitations are passed through an ion exchanger in a through-flow reactor. The actual purifying operation follows, in which elution is carried out with acid mixtures of water and solvent with pH values of 1.3-5.0 at temperatures of 27-32"C with increasing solvent concentration. This enables insulin to be eluted so that it is free of impurities of higher molecular weight. By varying the amount of the exchanger and the through-flow speed, the insulin can be fractionated into a practically uniform insulin component by disc electrophoresis.Further fractions which are enriched with acid insulin components (such as the de-amidized forms), and fractions which are enriched in basic insulin components such as the arginine derivatives or the insulin esters may be selectively removed.

In addition to this form of fractionation a desorption process may be used to achieve a purification effect. In this adsorption of impurities on the exchanger in the "batch" may be achieved when solvent concentrations are just sufficient to prevent insulin adsorption.

Example 1 A column 300 mm high and 80 mm wide and having a volume of 1.5 litres and contain ing 900 ml of Wofatit was charged with 90 g of pig insulin dissolved in 1800 ml of 1 N acetic acid, at a rate of 600 ml/h. The column was then eluted with a solvent comprising 1 N acetic acid and a graduated amount of 0-60% isopropanol. The isolated insulin is free of pro-insulin (PAAGE,pH 8.4; Coomassi Blau).

The yield was 72 g.

Example 2 A column 300 mm high and 80 mm wide was packed with 900 ml of Wofatit CA20, and charged with 90 g of insulin dissolved in 1800 ml of 0.02 N hydrochloric acid, at a rate of 600 ml/h. Insulin so obtained was treated with 9 litres of 0.02 N hydrochloric acid and eluted with a solvent comprising graduated relative amounts of acetic acid/benzene.

Example 3 A column 150 mm high and 9 mm wide (volume 9.5 ml) was charged with 500 mg of insulin dissolved in 10 ml of acetic acid, at a rate of 10 ml/h. The column was then eluted with a solvent comprising 0.02 N sodium chloride, up to 25% methyal and 1 N acetic acid. The elution was conducted fractionally, the extinction points measured and the individual components separated.

Example 4 A column 150 mm long and 9 mm wide (V=9.5 ml) was charged with 500 mg of insulin dissolved in 10 ml 1 N acetic acid, at a rate of 10 ml/h, and eluted with 250 ml of a solvent comprising 1 N acetic acid, up to 25% ethanol and 0.05 N sodium chloride. After crystallization 400 mg of purified insulin was obtained.

Example 5 A column 300 mm high and 80 mm wide (V=1.5 I) was charged with 90 g of insulin, dissolved in 1800 ml of 0.03 N phosphoric acid. The column was then eluted with 12 litres of a solvent comprising 0.03 N phosphoric acid and up to 40% acetone.

The eluate containing insulin was adjusted by means of citric acid to a concentration of 0.5%, and directly crystallized in the presence of zinc salts.

The yield was 85%.

Example 6 A column 150 mm long and 9 mm wide (V=9.5 ml; Wofatit CP) was charged with 500 mg of human insulin, dissolved in 10 ml of 1 N acetic acid. After washing the column, the insulin was eluted with up to 60% etha nol, 1 N acetic acid and 0.1 N sodium chlo ride, at a rate of 5 ml/h.

Example 7 1 g of pig insulin was dissolved in 100 ml of 45% ethanol and 1 N acetic acid, and ad sorbed on 10 ml of up to 45% ethanol, 1 N acetic acid and equilibrated Amberlite IRC-50.

After stirring for 3 hours at 40"C a state of equilibrium was attained.

The ion exchanger is filtered off and washed. The filtrate contains the C component of the insulin, this component being free of constituents of higher molecular weight. The insulin is purified by distillation and subsequent crystallization.

Claims

1. A process for the purification of insulin in a weakly acid cation exchanger in the presence of an organic solvent miscible with water, characterized in that fractionation is effected by altering the solvent concentration in the acid pH range.

2. A process according to Claim 1, characerized in that a macroporous ion exchanger having a hydrophobic matrix with an acrylate base is used with divinyl benzene, its derivatives, or other cross-linking agents.

3. A process according to Claims 1 or 2, characterized in that the solvents used are selected from aliphatic alcohols, aliphatic ketones, semi-acetals and esters of aliphatic carboxylic acids and aliphatic alcohols, or mixtures thereof.

4. A process according to Claim 3 characterized in that aromatic derivatives are added to the solvents.

5. A process according to Claim 3 characterized in that the alcohol used is methanol.

6. A process according to Claim 3 characterized in that the alcohol used is ethanol.

7. A process according to Claim 3 characterized in that the alcohol used is n-propanol.

8. A process according to Claim 3 characterized in that the alcohol used is i-propanol.

9. A process according to Claim 3 characterized in that the ketone used is acetone.

10. A process according to Claim 3 characterized in that the ester used is acetic acid ethyl ester.

11. A process according to Claims 1 to 10 characterized in that the concentration of the solvent is between 3 and 70%.

12. A process according to Claims 1 to 11 characterized in that the solvent concentration is altered continuously.

13. A process according to Claims 1 to 11 characterized in that the solvent concentration is altered stepwise.

14. A process according to Claims 1 to 13 characterized in that the mixtures of solvent and water contain salts.

15. A process according to Claim 14 characterized in that the salts consist of buffer substances.

16. A process according to Claim 14 characterized in that the salts used are selected from alkali salts or ammonium salts of the acids used for setting the pH value.

17. A process according to Claims 1 to 16 charcterized in that impurities of high molecular weight are removed from the insulin by fractionation.

18. A process according to Claims 1 to 17 characterized in that the insulin obtained is free of subsidiary components.

19. A process according to Claims 1 to 18 characterized in that fractionation is effected by column chromatography.

20. A process according to Claims 1 to 18 characterized in that fractionation is carried out by a batch process.

21. A process according to Claims 1 to 20 characterized in that fractionation is carried out in a pH range of 1.5-5.5

22. A process according to Claims 1 to 21 characterized in that fractionation is effected at a pH range of 1.7-3.0

23. A process according to Claims 1 to 22 characterized in that an acid selected from formic acid, acetic acid, tartaric acid, citric acid, hydrochloric acid or phosphoric acid is used for setting the pH value.

24. A process according to Claims 1 to 23 characterized in that the insulin is directly crystallized from the eluate without further isolation.

25. A process substantially as hereinbefore set forth with reference to the foregoing Examples.