WO1984003107A1

WO1984003107A1 - A process for the preparation of human insulin

Info

Publication number: WO1984003107A1
Application number: PCT/DK1984/000012
Authority: WO
Inventors: Finn Hede Andresen; Per Balschmidt; Kim Ry Hejnaes
Original assignee: Nordisk Insulinlab
Priority date: 1983-02-09
Filing date: 1984-02-08
Publication date: 1984-08-16
Also published as: DK55183D0; JPS60500401A

Abstract

Human insulin is prepared by treatment of a porcine insulin product, preferably raw porcine insulin, with carboxypeptidase A or a corresponding enzyme to form a desalanine-B30 insulin product, which is condensed, without the addition of a buffer, with a L-threonine alkyl ester in a mixture of a lower alkanol and water trypsin, a trypsin derivative or an enzyme related thereto at a pH value of from about 5.5 to about 7.5 and at a temperature between 0o and -30o C to form an insulin alkyl ester, which is hydrolyzed in an aqueous medium at a pH value of from about 8.5 to about 10.5.

Description

A process for the preparation of human insulin,

TECHNICAL FIELD

The present invention relates to a novel process for the preparation of a human insulin product being suitable for the preparation of therapeutical insulin preparations, in which process a porcine insulin produc •is treated with carboxypeptidase A or a corresponding enzyme to form a des- alanine-B30 insulin product, which is subsequently condensed with a L-threonine alkyl ester in the presence of trypsin or an enzyme related thereto and an organic solvent, where¬ after the insulin ester formed is hydrolyzed to human in¬ sulin.

BACKGROUND ART

Insulin is an indispensable medicine for the treatment of Diabetes. It would be natural to treat human beings with therapeutical preparations prepared from insulin of human origin. However, the number of diabetics and the individual need for insulin are disproportionate to the available amount of raw material (human Pancreas) .

It is known to prepare insulin chemically, vide US PS No.

3,903,068 and Hoppe-Seyler's Z. Physiol. Chem. , 357, 759-767 (1976) .

These processes comprise condensing a desoctapeptide- -(B23-30) porcine insulin with a synthetic octapeptide cor- responding to the positions B23-30 in human insulin. How¬ ever, in the first process an alkaline hydrolysis is carried out, which is accompanied by unfavourable side reactions. The second process comprises a non-specific reaction giving rise to many side reactions and demanding complicated puri-

' fication procedures. Consequently, these processes are not suitable for use on an industrial scale.

Moreover, US PS No. 3,276,961 discloses a process for the preparation of human insulin from other animal insulins by the action of an enzyme, e.g. carboxypeptidase A or trypsin, in the presence of threonine.

However, by this known process it is not possible to prepare human insulin to any appreciable extent. This is probably due to the fact that trypsin and carboxypeptidase A hydro- lyze not only the lysyl-alanine peptide bond (B29-B30) , but also other positions in insulin under the working condi¬ tions. Trypsin preferably hydrolyzes the arginyl-glycine peptide bond (B22-B23) rather than the lysyl-alanine bond (B29-B30) . However, carboxypeptidase A cannot exclusively split off the alanine at the C-terminal of the B-chain with¬ out also splitting off asparagine at the C-terminal of the A-chain. It has later been shown that a specific condition, i.e. reaction in an ammonium bicarbonate buffer solution, is necessary in order to hinder the asparagine release, cfr. Hoppe-Seyler's Z. Physiol. Chem. , 359, 799-802 (1978). More¬ over, a considerable peptide formation scarcely occurs, since the velocity of the hydrolysis reaction is higher than that of the peptide synthesis at the working conditions.

Finally, Nature Vol. 280, 2nd August, 1979, 412-413, Bio- chem. & Biophys. Res. Commun., 29th January, 1980, 92(2), 396-402, and European Patent Specification No. 0 017 938 (all Morihara et al.) disclose a method of exchanging the B-30 amino acid, in which treatment of porcine insulin with carboxypeptidase A, to which an inhibitor (diisopropyl flu- orophosphate) has been added, at 25o C. for 8 h<^■ours in the presence of "ammonium bicarbonate buffer results in a des- alanine insulin (DAI) , which is isolated. Thereafter the coupling reaction is carried out by adding a solution of trypsin in 0.5 M borate buffer solution and tosyl-L-phenyl alanine chloromethyl ketone (TPCKΪ to a solution of DAI and threonine butyl ester (Thr-OBu ) in a solvent containing an organic solvent mixture of a high concentration (about 60%) consisting of a mixture of DMF and ethanol, whereafter the reaction proceeds at 37 C. for 20 - 48 hours to form t (B30-Thr-OBu ) porcine insulin, which is isolated. Finally, the butyl ester protecting group is split off with trifluoro acetic acid in the presence of anisole.

This known process may be used on an industrial scale, but it involves considerable disadvantages as the threonine ter- tiary butyl ester, which must be present in a large excess, is difficult to prepare and consequently costly, and more¬ over, is difficult to recover. Besides, the use of trifluoro acetic acid in the great amounts necessary for the removal of the tertiary butyl ester group causes various difficul- ties when operating on a technical scale. Therefore, the method involving the tertiary butyl ester group in the B30

- position and thereby the intermediate (B30-Thr-OBu ) insul: is less appropriate for industrial purposes.

DISCLOSURE OF THE INVENTION It has now been found that it is possible to prepare a high¬ ly pure human insulin product free of the above-mentioned disadvantages of the human insulin products prepared by the known processes.

According to the invention, the coupling of desalanine-B30 insulin (DAI) with L-threonine alkyl ester is carried out at a temperature between 0 and -30 C. to form insulin ester in a high yield without any appreciable by-product formation. A mixture of a lower alkanol and water of a pH value in the range of from about 5.5 to about 7.5 is used as reaction medium. The insulin ester thus prepared is then converted into human insulin in a lenient manner, partly by an appropriate choice of the alkyl moiety of the ester, partly by carrying out the hydrolysis in an aqueous medium of a pH value in the range of from 8.5 to 10.5. Thus, the addition of enzyme, trifluoro acetic acid and anisole is avoided.

Accordingly, the process of the invention is characterized by suspending the desalanine-B30 insulin product in a lower al¬ kanol, mixing a solution of at least part of the L-threonine alkyl ester in water adjusted to a pH value of from about 5.5 to about 7.5 without addition of a buffer with the sus¬ pension, cooling the mixture to a temperature between 0 and -30 C, adding trypsin, a trypsin derivative or an enzyme related thereto, if desired dissolved in part of the solu- tion of the L-threonine alkyl ester in water, to the mix¬ ture, and leaving the resulting mixture for up to 5 days at. a temperature between 0° and -30° C.

When the process of the invention is carried out on a large scale such as industrial scale using e.g. from 50 g or more of the starting insulin product a preferred embodiment of the invention comprises dissolving separately trypsin or an enzyme related thereto in part of the solution of the L-threonine alkyl ester in water, adding the suspension of the desalanine-B30 insulin product to the remaining part of the solution of the L-threonine alkyl ester in water, adjust¬ ing the temperature and pH value of the resulting mixture and then adding the separately prepared solution of trypsin or an enzyme related thereto in part of the solution of the L-threonine alkyl ester in water. In this case it is pre- ferred that the temperature and the pH value of the result¬ ing mixture are adjusted to -2 to -15 C. and 5.8 to 6.2, respectively. Moreover, it is preferred to dissolve sepa¬ rately trypsin or an enzyme related thereto in 10 - 20% by weight of the solution of the L-threonine alkyl ester in water.

In the process of the invention the condensation reaction is carried out without the addition of a buffer, the buffering capacity of the reactants being sufficient - under the con¬ ditions given according to the invention - to maintain a pH in the range of 5.5 to 7.5. The above features are essential features of the process of the invention, and they are decisive of the obtaining of the end product in a high yield without any appreciable by-pro¬ duct formation, when the process is carried out on an indu- strial scale.

In the condensation reaction the reaction time is dependent on the remaining reaction conditions, especially the tempe¬ rature. Reaction times from about 30 minutes to about 120 hours are used.

As a consequence of the low temperature in the condensation reaction no appreciable side reactions occur, and, moreover, it is not necessary to use enzymes treated with tosyl-L- -phenyl alanine chloromethyl ketone (TPCK) .

In a particularly advantageous embodiment of the process of the invention raw porcine insulin, e.g. insulin salt cake, is used as starting material. It is worth noting that when using raw porcine insulin in the process of the invention it is possible to obtain a high yield of human insulin cor¬ responding to the yield which might be obtained when pre- paring chromatographically purified porcine insulin, when, in both cases, the total yields are calculated on the amount of Pancreas used.

It is surprising that the use of raw porcine insulin as starting material does not give rise to difficulties in the chromatographical separation of the obtained human insulin ester from the enzyme used and unreacted desalanine-B30 in¬ sulin.

The enzyme used in the condensation of the process of the invention must be capable of splitting lysine carbonyl pep- tide bonds, and thus use can be made of trypsin, trypsin de¬ rivatives (e.g. acetylated trypsin) or enzymes related thereto, e.g. achromobacterprotease I, the preparation and properties of which are described by Masaki et al., Agric. Biol. Chem., 4_2, 1443-1445 (1978). It is unnecessary to use enzymes treated with tosyl-L-phenyl alanine chloromethyl ketone (TPCK) to eliminate a possible contamination with chymotrypsin-like enzymes due to the low temperatures men- tioned above and the poor reactivity of such enzymes in the reaction mixture used.

The enzyme can be used in dissolved form, but can also be bound to an insoluble matrix, e.g. agarose or polyacrylamide or similar polymeric substances.

The condensation reaction is carried out under conditions where the enzymatically catalyzed hydrolysis is sufficiently suppressed for the peptide forming reaction to proceed. As mentioned above, the pH value must be between 5.5 and 7.5. The temperature is usually in the range of 0 to -30 C, preferably from 0° to -20° C.

In the condensation reaction the concentration of the reac- tants, i.e. des-B30 insulin and L-threonine alkyl ester, should be high, and moreover, the L-threonine alkyl ester used should be employed in a large excess, up to a molar ratio of 200:1, preferably in the range of 20:1 to 100:1.

The condensation reaction is carried out in the presence of water-miscible lower alkanols or mixtures thereof, whereby the hydrolysis reaction is hindered, and the solubility of the reactants is improved. The concentration of lower alka- nols should be selected in the range of 20 to 90%, prefer¬ ably 30 to 80%, calculated on the total volume of the reac¬ tion mixture.

When the condensation reaction is complete, the insulin-like proteins are separated from the remaining components by gel filtration, whereupon human insulin ester is separated from unreacted starting material by anion exchange chromatography. The unreacted starting material may possibly be reused in the process. The collected fractions from the anion exchange containing human insulin ester are desalted, whereafter the pH value of the collected eluate is adjusted to about 9.5 by means of NaOH. After 24 - 48 hours at ambient temperature pure human insulin is isolated by crystallization or other usual meth¬ ods.

The hydrolysis of the insulin alkyl ester proceeds smoothly at a pH value of from about 8.5 to about 10.5 in an aqueous solution. This has the effect that, after the. hydrolysis, the hydrolysis mixture can easily be worked up in a conven¬ tional manner and that the isolated human insulin is ob¬ tained in a high purity.

The human insulin prepared by the process of the invention is well-suited for the preparation of therapeutical insulin preparations, since it contains no proteolytic impurities, for which reason it can be used for the preparation of pre¬ parations having protracted activity, since it behaves in the same way as human insulin and porcine insulin prepared from Pancreas of human and porcine origin in the "Ames Test" mentioned above, and since it contains no impurities being difficult to remove by means of chromatographical methods generally used.

Moreover, in a preferred embodiment of the invention, where raw insulin is used as starting material, a yield of human insulin is obtained which in all essentials is equal to the yield of highly purified porcine insulin which can be ob¬ tained from the same amount of raw insulin. Thus, there is question of a method which is industrially as well as clin¬ ically satisfactory to a hitherto unknown degree.

MODES FOR CARRYING OUT THE INVENTION

The process of the invention is further illustrated by means of the following Examples. Example 1

Porcine insulin in the form of raw insulin corresponding to 1000 mg of porcine insulin was dissolved in 100 ml of 0.1 M aqueous NH₄HCO3 solution (pH value 8.4). To the solution 10 mg of carboxypeptidase A in the form of an aqueous solu¬ tion of a concentration of about 5 mg/ml were added. The mixture was left with gentle stirring for 3 hours at 20 C. Immediately thereafter the reaction mixture was freeze-dried.

Determination of released alanine showed a cleavage yield of 93% (amino acid analysis) .

The freeze-dried powder was suspended in 96% ethanol (12.7 ml) ,. 2000 mg of Thr-0-Me,HCl were dissolved in 5.00 ml of 1 mM Ca-acetate, and the pH was adjusted to pH 6.00 with 5 M NaOH.

10 mg of porcine trypsin were dissolved in 500 μlitres of the Thr-O-Me solution.

Then the ethanol suspension was added slowly to the Thr-O-Me solution with stirring. The mixture was cooled to -10 C, whereupon the trypsin solution was added with stirring. The reaction mixture was left at -10 C. for 22 hours. The reac¬ tion was stopped by adjusting first the pH value to 2.5 with 0.1 M HC1 and then the volume to 50 ml with distilled water.

High pressure liquid chromatographical analysis of the reac¬ tion mixture showed a production of human insulin ester of 76%.

The reaction mixture was gel filtered on a column of Sepha- dex® G-50 Superfine (8 x 80 cm) in 1 M acetic acid. The fraction containing human insulin ester and unreacted des- alanine-B30 insulin was freeze-dried.

Yield: 810 mg of product mixture. Thereafter the product mixture was ion-exchanged at 4 C. on a column of DEAE cellulose (Whatmann DE 52.5 x 23 cm), equi¬ librated with 140 ml/hour of a buffer consisting of 0.02 M tris and 7 M urea, adjusted to a pH value of 8.1 with hydro- chloric acid. When the charging of the product was complete, the column was eluted for 2.5 hours using the above-men¬ tioned buffer solution, then for 2 hours using the above- -mentioned buffer in admixture with 0.0045 moles of sodium chloride per litre and finally for 12 hours using the former buffer in admixture with 0.011 moles of sodium chloride per litre.

The eluate contained two proteinaceous main fractions. The fraction eluted- at first was identified by high pressure liquid chromatography as being human insulin ester and the fraction eluted thereafter as being desalanyl insulin.

The collected human insulin ester fraction was desalted on a column of Sephadex® G-25 in 0.1 M sodium acetate (pH value 8.0) at 4 C, whereafter the pH value was adjusted to 9.5 with 1 N NaOH solution. The fraction was left at 25 C. for 24 hours.

480 mg of pure human insulin were obtained, identified by a ino acid analysis and high pressure chromatography.

Example 2 100 mg of des-Ala B30 insulin (calculated on protein) ^•were slurried in 1880 μlitres of 96% ethanol. 200 mg of

Thr-O-Me,HC1 were dissolved in 500 μlitres of 1 M of Ca-

-acetate, and the pH value was adjusted to 6.0 with 5 M and

1 M NaOH.

1 mg of porcine trypsin was dissolved in 100 μlitres of the Thr-O-Me-ester solution.

B30 The des-Ala insulin suspension was added to the remaining part of the ester solution with stirring. The mixture was i cooled to -10 C. The trypsin solution was added, whereafter the mixture was left at -5 C. for 22 hours.

The yield of the condensation was determined by high pres¬ sure liquid chromatographical analysis using a mixture pre- pared by adding 75 μlitres of the above mixture to 2925 μlit¬ res of 0.5 M acetic acid by means of a pipette.

Yield of condensation: 83%.

ΪR1

Claims

1. A process for the preparation of human insulin in which process a porcine insulin product is treated with carboxy¬ peptidase A or a corresponding enzyme to form a desalanine- -B30 insulin product, which is subsequently condensed with a L-threonine alkyl ester in the presence of trypsin, a tryp¬ sin derivative or an enzyme related thereto and an organic cosolvent, whereafter the insulin ester thus formed is hy- drolyzed in an aqueous medium of a pH value in the range of from about 8.5 to about 10.5 to form human insulin, c h a r a c t e r i z e d by suspending the desalanine-B30 insulin product in a lower alkanol, mixing a solution of at least part of the L-threonine alkyl ester in water adjusted to a pH value of from about 5.5 to about 7.5 without addi- tion of a buffer with the suspension, cooling the mixture to a temperature between 0 and -30 C, adding trypsin, a trypsin derivative or an enzyme related thereto, if desired dissolved in part of the solution of the L-threonine alkyl ester in water, to the mixture, and leaving the resulting mixture for up to 5 days at a temperature between 0 and -30° C.

2. A process according to claim 1, c h a r a c t e r i z e d by the porcine insulin product used being raw porcine insu¬ lin.

3. A process according to claim 1, c h a r a c t e r i z e d in that the temperature and the pH value of the resulting mixture are adjusted to 0 - -20 C. and 5.8 - 6.2, respec¬ tively.

4. A process according to claim 1, c h a r a c t e r i z e d by the condensation reaction being carried out during a pe¬ riod of about 30 minutes to about 48 hours.

5. A process according to claim 1, c h a r a c t e r i z e d by the desalanine-B30 insulin product being isolated without separation of the employed carboxypeptidase A or correspond¬ ing enzyme.

6. A process according to claim 1, c h a r a c t e r i z e d by the L-threonine alkyl ester used being L-threonine methyl ester.

7. A process according to claim 1, c h a r a c t e r i z e d by the lower alkanol used being ethanol.

8. A process according to claim 1, c h a r a c t e r i z e d by dissolving separately trypsin, a trypsin derivative or an enzyme related thereto in 10 - 20% by weight of the solution of the L-threonine alkyl ester in water.