A PROCESS FOR THE PREPARATION OF INSULIN DERIVATIVES

Abstract: Insulin derivatives are prepared in one step by exchanging the C-terminal amino acid of the B-chain in an insulin by treatment with a proteolytic enzyme, such as trypsin, in the presence of an excess of an amino acid, e.g. L-threonine, with protected carboxyl group and in the presence of an organic solvent, whereafter the carboxyl protecting group is optionally split off.

Description: A PROCESS FOR THE PREPARATION OF INSULIN DERIVATIVES. TECHNICAL FIELD The present invention relates to a novel process for the preparation of insulin derivatives. BACKGROUND ART For many years Diabetes has been treated with insulin. It would be natural to treat human beings with human insulin; however, this is impossible in view of the existing great demand. Therefore, for practical reasons bovine and porcine insulin is used. However, to a larger or smaller extent, these insulins give rise to the formation of antibodies in the human body which i.a. involves a reduced effect of the further insulin treatment. This disadvantage is supposed to be caused partly by so- -called "impurities" in the bovine/porcine insulin, partly by the alien nature. The latter manifests itself in the hu¬ man insulin molecule differing from. other animal insulin molecules in certain minor dissimilarities in the amino acid sequence. Great improvements have been obtained as regards the insu¬ lin preparations after the introduction of the latest puri¬ fication methods, but formation of antibodies in the human body may still occur. It is believed that this can be reme¬ died by using human insulin instead of other animal insu- lin. OMPI US-PS No. 3,276,961, Bodanszky et al, 1963, discloses a pro¬ cess 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 an excess of threonine. However, by use of this prior process human insulin cannot be prepared to any appreciable extent. This is probably due to the fact that trypsin and carboxypeptidase A hydrolyze not only the lysyl-alanine peptide bond (B29-B30) but also the other positions in insulin under the working conditions. Trypsin preferably hydrolyzes the arginyl-glycine peptide bond (B22-B23) rather than the lysyl-alanine bond (B29-B30) , whereas carboxypeptidase A cannot split off the alanine at the C-teπninal of the B-chain alone without splitting off the asparagine at the C-terminal of the A-chain. It has la- ter been shown that a specific condition, i.e. reaction in an ammonium bicarbonate buffer solution is necessary in or¬ der to hinder the asparagine release, cfr. Hoppe-Seyler's 2. Physiol. Chem., 359, 799-802 (1978). Moreover, a consi¬ derable peptide formation scarcely occurs, since the velo- city of the hydrolysis reaction is higher than that of the peptide synthesis under the working conditions. That is prob¬ ably the reason why Bodanszky in the Examples of US-PS No. 3,276,961 describes the resulting product as transformed zinc insulin and not as human insulin. So far no process is known in which the undesired amino acid (B-30 Ala) is interchanged with B-30 Thr in one and the same step. However, methods for preparing human insulin comprising sev¬ eral separate steps are known, cfr. US-PS No. 3,903,068, Ruttenberg, 1975, and Hoppe-Seyler's Z. Physiol. Chem., 357, 759-767 (1976), Obermeier et al. Referring to the above, these methods prefer to work with the arginyl-glycine peptide bond and therefore comprise con¬ densation of a desoctapeptide-(B23-30) porcine insulin with -BURE OMPI a synthetic octapeptide corresponding to the positions B-23-30 in human insulin. However, 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 an demanding complicated purification procedures. Consequently these processes are not suitable for use on an industrial scale. Finally, Nature, Vol. 280, 2nd August 1979, 412-413, and Biochem. & Biophys. Res. Co mun., 29th January 1980, 92(2), 396-402 (both Morihara et al.) disclose a method for ex¬ changing the B-30 amino acid, in which treatment of porcine insulin with carboxypeptidase A at 25° C. for 8 hours in the presence of ammonium bicarbonate buffer results in a desala- nine insulin (DAI) , which is isolated. Then the coupling is carried out by adding trypsin to a solution of DAI and thre- onine butyl ester (Thr-OBu ) in an organic solvent compris¬ ing the organic solvent in an amount of about 60%, whereaf¬ ter the reaction proceeds at 37° C. for 20 hours to form (B30-Thr-OBu ) porcine insulin,which is isolated. Finally, the protecting group (butyl ester) is split off with tri- fluoro acetic acid in the presence of anisole. DISCLOSURE OF THE INVENTION It has now been found that it is possible, on an industrial base and using short reaction times, to exchange the B-30 amino acid for another amino acid in one step without pre¬ ceding isolation of a des-B30 insulin by treating an insu¬ lin with a proteolytic enzyme in the presence of an excess of an amino acid with protected carboxyl group and in the presence of an organic solvent and thereafter, optionally, splitting off the carboxyl protecting group. Accordingly, the process of the invention is characterized by treating an insulin with a proteolytic enzyme in the pre¬ sence of an excess of an amino acid with protected carboxyl - U E OMPI group and in the presence of an organic solvent to form a B-30-Thr-insulin derivative in an acceptable yield and of a high purity. The reaction may be completed within about 1 hour although longer reaction times may be used. As the proteolytic enzyme in the process of the invention trypsin or an enzyme related thereto can be used. It is surprising that the process of the invention can be carried out directly in one step, that the resultant insu¬ lin derivative can easily be separated from unreacted start- ing insulin, as well as that no considerable splitting of the insulin occurs at B22-arginine when using trypsin as the proteolytic enzyme, cfr. Hoppe-Seyler's Z. Physiol. Chem., 357, 759-767 (1976). The process of the invention is suitable for the preparation of human insulin from porcine insulin. As the starting mate¬ rial in the process of the invention even an unpurified in¬ sulin product can be used. Moreover, the unreacted insulin starting product is obtained in a pure form which can be used in insulin preparations for therapeutical use. Of course, the unreacted insulin starting product can also be reused as starting material. Furthermore, the human insulin prepared is obtained in a high purity and a reasonable yield in one step from an unpurified porcine insulin product. Con¬ sequently, the process of the invention is suitable for the preparation of insulin on an industrial scale. In the process of the invention transformed insulin with protected carboxyl group is formed, and this transformed in¬ sulin can be isolated from the reaction mixture and sepa¬ rated from unreacted insulin by well known chromatographical methods. By subsequent splitting off of the carboxyl pro¬ tecting group the transformed insulin is obtained in a pure form. The carboxyl group of the amino acid introduced in the B-30 position may be protected in the form of different esters and amides, however, when selecting these, regard should be paid to the stability of the insulin at the conditions whic are to be used in the removal of the protecting group. The t-butyl ester is particularly suitable, since the splitting off can be carried out by means of trifluoro acetic acid, but other ester or amide groups can also be removed by care ful, optionally enzymatically catalyzed hydrolysis. The enzyme used in the process of the invention must be ca- pable of splitting lysine carbony1 peptide bonds, and thus use can be made of 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__ (1978), pages 1443-1445. Optionally, the enzyme may have been treated with tosyl-L-phenyl alanine chloromethylketone (TPCK) to eliminate a possible contamination with chymotryp sin-like enzymes. The enzyme may be used in a dissolved for but may also be bound to an insoluble matrix, e.g. agarose or polyacrylamide or similar polymeric substances. The reaction is carried out under conditions where the en¬ zymatically catalyzed hydrolysis is sufficiently suppressed for the peptide forming reaction to proceed. The pH must be between 5 and 10, preferably between 6 and 9. The tempera¬ ture should be in the range of 4 to 50 C, preferably 20 to 40° C. The concentration of the reactants, i.e. starting insulin and amino acid ester, should be high, and moreover the ami- no acid ester used should be employed in a large excess, up to a molar ratio of 200:1, preferably in the range of 50:1 to 150:1." The reaction is carried out in the presence of a water-mis- cible organic solvent, whereby the hydrolysis reaction is hindered and the solubility of the reactants improved. The organic solvent may be dimethylformamide, acetamide, dimeth- UR " ylsulfoxide, ethanol, glycerin and similar solvents, option¬ ally in the form of their mixtures. The concentration of or¬ ganic solvent should be selected in the range of 10% to 80%, preferably 30% to 70%, calculated on the total volume of the reaction mixture. The reaction " time is chosen dependent on the remaining reac¬ tion conditions, but normally it does not exceed 24 hours, usually it is about 1 to about 4 hours. MODES FOR CARRYING OUT THE INVENTION The process of the invention is further illustrated by the following Examples. Example 1 200 mg of porcine insulin and 400 mg of L-threonine t-butyl ester acetate were dissolved in a mixture of 2.2 ml of eth- anol and 1.4 ml of 0.5 M tris-acetate buffer (pE = 7.5) . To this solution 20 mg of TPCK-treated trypsin were added, and the mixture was held at 37° C. for 2 hours. Then the reac¬ tion was stopped by adjustment of the pH to about 3 through the addition of 10 N acetic acid. High pressure liquid chromatographical analysis of the reac¬ tion mixture showed a conversion of 18%. The reaction mixture was subjected to gel filtration on a column of Sephadex ^G-50 Superfine (2.6 x 90 cm) in 1 M acetic acid. The fraction containing human insulin ester and unreacted porcine insulin was lyophilised. Yield: 180 mg of product mixture. Then the product mixture was ion-exchanged at 4 C. on a col¬ umn of DEAE cellulose (.-Thatmann DE-52) (5 x 2) equilibrated with 75 ml per hour of a buffer consisting of 0.02 M tris and 7 M urea, adjusted to a pH of 8.1 with hydrochloric acid. When the charging of the product was complete, the OMPI column was eluted for 2.5 hours using the above-mentioned buffer solution, thereafter for 2 hours using the above-men tioned buffer in admixture with 0.0045 moles of sodium chlo ide per liter and finally for 12 hours using the former buf fer in admixture with 0.011 moles of sodium chloride per liter. 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 unreacted porcine insu lin. The collected fractions were desalted on a column of Sephadex ^G-25 in 0.1 M acetic acid and lyophilised to yield 28 mg of human insulin t-butyl ester and 120 mg of un- reacted porcine insulin. The resultant insulin t-butyl ester (28 mg) was dissolved in 2 ml of trifluoro acetic acid in admixture with 0.2 ml of anisole, and the mixture was held at ambient temperature for 25 minutes. The trifluoro acetic acid was removed in va- cuo on a rotary evaporator, and the residue was extracted with 3 x 10 ml ether to remove the anisole. Upon drying in vacuo 25 mg of pure human insulin were obtained, identified by amino acid analysis and high pressure liquid chromato¬ graphy. Example 2 200 mg of porcine insulin and 400 mg of L-threonine t-butyl ester acetate were suspended in 2.2 ml of dimethyl forma- ide followed by the addition of 1.4 ml of 0.5 M tris-acet- ate buffer (pH 7.0) containing 20 mg of TPCK-treated tryp- sin. The reaction mixture was held at 37° C. for 3 hours, whereupon the reaction was stepped by adjustment of the pH to 3 by the addition of 10 :. acetic acid. High pressure liq¬ uid chromatographical analysis of the reaction mixture showed a conversion of 6%. The product mixture was isolated -BURE and fractionated using the procedure of Example 1 to yield 10 mg of human insulin t-butyl ester and 140 mg of unreacted porcine insulin. The isolated human insulin t-butyl ester (10 mg) was treated in the same way as stated in Example 1 with trifluoro acetic acid and anisole to yield 8.0 mg of pure human insulin. Example 3 To 100 mg of porcine insulin and 200 mg of L-threonine meth¬ yl ester 1.4 ml of 96% ethanol and 0.4 ml of 0.25 M tris- -acetate buffer (pH = 6.5) were added. Then 150 μliter of 5 N hydrochloric acid and 5 mg of porcine insulin dissolved in 200 uliter of 0.25 M tris-acetate buffer (pH = 6.5) were added. The mixture was held at 35 C. for 1 hour, whereafter the reaction was stopped by the addition of 10 N hydrochloric acid to obtain a pH of about 3. High pressure liquid chromatographical analysis of the reac¬ tion mixture showed a yield of human insulin methyl ester of 45%. The reaction mixture was purified in the same way as de¬ scribed in Example 1. Thereafter, the eluate from the ion- -exchange containing the resultant human insulin methyl es¬ ter, identified by high pressure liquid chromatography, was desalted on a column of Sephadex (R. G-25. The column was eluted using 0.05 M aqueous ammonium bicarbonate solution, the resultant eluate was adjusted to a pH of 9.5 using di¬ luted ammonia water, whereafter the mixture was held at 25 C. for 48 hours for the complete hydrolysis of the es¬ ter. Lyophilising of the hydrolysis mixture resulted in 37 mg of pure human insulin, determined by amino acid analysis and high pressure liquid chromatography. Example 4 To 100 mg of porcine insulin and 200 mg of L-threonine meth yl ester 1 ml of dioxane and 0.5 ml of 0.2 M tris-acetate buffer (pH = 7.5) were added. Then 50 jaliter of 5 N hydro- chloric acid as well as 10 mg of bovine trypsin dissolved i 0.5 ml of 0.2 M tris-acetate buffer (pH = 7.5) were added. The mixture was held at 35° C. for 45 minutes, whereafter the reaction was stopped by the addition of 10 N hydrochlor ic acid to obtain a pH of about 3. High pressure liquid chromatographical analysis of the reac tion mixture showed a yield of human insulin methyl ester o 40%. Purification of the reaction mixture and hydrolysis of the insulin methyl ester were carried out in the same manner as described in Example 1. Lyophilising of the hydrolysis mix¬ ture produced 30 mg of pure human insulin, determined by amino acid analysis and high pressure liquid chromatography Example 5 100 mg of porcine insulin and 200 mg of L-threonine amide were dissolved in 1.5 ml of 65% ethanol, whereafter 5 N hy¬ drochloric acid were added to adjust the pH to 7.5. There¬ after, a solution of 10 mg of trypsin in 0.5 ml of water was added, and the mixture was held at 35 C. for 1 hour. Then the reaction was stopped by the addition of 5 N hydro- chloric acid to a pH of 3. High pressure liquid chromato¬ graphical analysis of the reaction mixture showed a conver¬ sion of 37%. The product mixture was isolated and fractionated using the procedure described in Example 1. Yield: 30 mg of human in- sulin amide. Example 6 250 mg of L-threonine methyl ester and 100 μliter of gla¬ cial acetic acid were dissolved in a mixture of 500 μliter of dimethyl formamide and 400 uliter of water. 60 mg of por- cine insulin was dissolved in this mixture, whereafter a so¬ lution of 3 mg of trypsin in 700 μliter of water was added with stirring. The clear solution, the pH of which was 6.5, was then held at 20° C. for 3 hours, whereafter the reac¬ tion was stopped by adjusting the pH to about 3 using 10 N acetic acid. High pressure liquid chromatographical analysis of the reac tion mixture showed a conversion of 23%.

Claims: C L A I M S 1. A process for the preparation of insulin derivatives by exchanging the C-terminal amino acid of the B-chain under enzymatic action, c h a r a c t e r i z e d by treating an insulin with a proteolytic enzyme in the presence of an ex¬ cess of an amino acid with protected carboxyl group and in the presence of an organic solvent and subsequently, option¬ ally, splitting off the carboxyl protecting group. 2. The process of claim 1, c h a r a c t e r i z e d by using trypsin or an enzyme related thereto as the proteolyt¬ ic enzyme. 3. The process of claim 2, c h a r a c t e r i z e d by using trypsin as the proteolytic enzyme. 4. The process of claim 2, c h a r a c t e r i z e d by using achromobacterprotease as the proteolytic enzyme. 5. The process of claim 1, c h a r a c t e r i z e d by carrying out the reaction at a temperature of from about 20 to about 40 C. for a period of about 1 to about 4 hours. 6. The process of claim 1, c h a r a c t e r i z e d by carrying out the reaction at a pH in the range of about 5 to about 10. 7. The process of claim 1, c h a r a c t e r i z e d by using lower mono- or polyhydric alcohols, amides of lower carboxylic acids or dimethyl sulfoxide as the organic sol- vent. 8. The process of claims 1-7, c h a r a c t e r i z e d by using porcine insulin as the insulin. 9. The process of claims 1-8, c h a r a c t e r i z e d by using L-threonine as the amino acid with protected carb- URE~ oxyl group. 10. The process of claims 1-9, c h a r a c t e r i z e d by using the t-butyl ester group, other lower alkyl ester groups or amide groups as the carboxyl protecting group.

Inventors: ANDRESEN FINN HEDE, BALSCHMIDT PER
Applicants: ANDRESEN FINN HEDE, BALSCHMIDT PER, NORDISK INSULINLAB
Application year: 1982
Main classification: C12P21
IPC codes: C07K14, C12P21