DE3104949A1 - Process for the preparation of threonine<B30> esters of human insulin, and threonine<B30> ester of human insulin - Google Patents

Abstract

Insulin and insulin-like compounds of certain species can be transpeptidated in the presence of an L-threonine ester, water, a water-miscible organic solvent and trypsin to a threonine<B30> ester of human insulin, in which the protective group(s) can be removed with formation of human insulin.

Description

Process for the preparation of threonine esters human

Insulins as well as threonine B30 ester human insulin.

The invention relates to a method for producing B30 threonine -Ester human insulin or a salt or complex thereof, as well Threonine ester of human insulin.

In particular, the invention relates to the conversion of insulin and insulin-like compounds in human insulin.

The treatment of diabetes mellitus uses insulin compounds, made from pork or beef insulin generally related. Pig, bovine and human insulins show slight differences in their amino acid composition, being the difference between human and Porcine insulin is limited to a single amino acid in that the B30 The amino acid of human insulin is threonine, while that of porcine insulin Alanine is.

It can therefore at least be argued that the ideal insulin preparation for the treatment of humans an insulin with exactly the same chemical structure like that of human insulin.

The stands for the production of human, natural insulin Necessary amount of human pancreas glands not available.

Synthetic human insulin is very expensive on a small scale have been prepared, see Helv.Chim.Acta, 57, p. 2617, and 60, p. 27. Semi-synthetic human insulin is obtained from porcine insulin via time-consuming reaction pathways, as in Hoppe-Seyler's Zeitschrift für Physiologische Chemie, 356, 1631 and Nature 280, 412.

The US-PS 32 76 961 relates to a method of manufacture of semi-synthetic human insulin. Nonetheless, the yield is poor on human insulin because the procedure is carried out in water, and conditions in which trypsin causes cleavage of the Arg -Gly bond causes, see Journal of Biological Chemistry, 236, p.

743

A well-known semi-synthetic process for making human Insulin has the following three steps: B30 First, porcine insulin is converted into pigs-des- (Ala ) Insulin by treatment with carboxypeptidase A, see Hoppe-Seyler's journal from Physiological Chemistry, 359, page 799. In the second step, pigs-des- (AlaB3OY Trypsin-catalyzed insulin coupled with Thr-OBu, whereby the Th jU -tert-butyl ester of human insulin is formed. In the end this ester is treated with trifluoroacetic acid to deliver human insulin, see Nature 280, page 412. The first step provides A21 B30 with partial cleavage from Asz, thereby delivering des- (Ala AsnA2l) -insulin. This descendant leads after the two subsequent reactions to contamination of des- <Asn ) -Insulin in the manufactured semi-synthetic human insulin that is not can easily be removed with the known preparation method ent-A21. Des- (Asn ) -Insulin has low biological activity (about 5%) see American Journal of Medicine 40, p. 750.

The object of the invention is therefore to provide a method with which human insulin can be produced on an industrial scale, whereby non-human insulin is to be converted into human insulin.

Furthermore, a method is to be delivered, which pig insulin and certain impurities contained therein into human insulin converts a threonine ester of human insulin.

The task is through a process for the production of B30 threonine - Ester dissolved human insulin, which is characterized by transpeptidating an insulin compound or a salt or complex thereof, which are convertible into insulin threonine B30 esters, with an L-threonine ester or a salt of the same in a mixture of water, a water-miscible organic Solvent and trypsin, the water content in the reaction mixture being less than 50 vol .-% and the reaction temperature is below 500C, as well as through Threonine - Esters of human insulin in which the ester group is different from t-butyl and methyl differs.

The method according to the invention thus has the following advantages over the state of the art: a) The enzymatic hydrolysis for cleavage from AlaB3Oi, for example, with carboxypeptidase A, is omitted.

b) The isolation of an intermediate compound, such as pigs-des- (Ala ) -Insulins, is unnecessary.

A21 c) Contamination with the (Asn 1) insulin derivatives are avoided.

d) Proinsulin and other insulin-like impurities contained in the Crude insulin are present by the method according to the invention via the threonine - Esters of human insulin are converted into human insulin, thereby reducing the Yield is increased.

e) Antigen-like insulin-like compounds, see also GB-PS 12 85-023, are converted into human insulin.

Further features and advantages of the invention emerge from the claims and from the following description, in which exemplary embodiments are explained.

The invention is based on the discovery that those attached to the B29 carbonyl group Amino acid or peptide chain linked by Lys in the insulin compound with a Threonine ester can be exchanged. This exchange is referred to herein as transpeptidation designated.

The term (insulin compounds "used herein) includes insulins and insulin-like compounds which include the human des (ThrB30) insulin species The B30 amino acid of the insulin alanine (in insulin of for example Pig, dog, fin whale and sperm whale) or serine, as in rabbits. The term "Insulin-like compounds" as used herein includes proinsulin, derived from any of the above species and primates with intermediate products from the conversion of proinsulin to insulin. As an example of such intermediate products, split proinsulin, desdipeptide proinsulins, Desnonapeptide proinsulin and diarginine insulins are mentioned, see also R. Chance "In Proceedings of the Seventh Congress of IDF, Buenos Aires 1970, 292 to 305, Editor: .R.R. Rodriques & J.V.-Owen, Excerpta Medica, Amsterdam.

The method according to the invention involves the transpeptidation of an insulin compound or a salt or a complex thereof with an L-threonine ester or a salt of the same in a mixture of water, a water-miscible organic egg Solvent, and trypsin, the water content in the reaction mixture is less than about 50% by volume and the reaction temperature is below about 50 ° C lies.

Although trypsin is best known for its proteolytic properties those skilled in the art have recognized that trypsin B30 is capable of coupling catalyzing des- (AlaB30) -insulin and a threonine tert-butyl ester, vide Nature 280, p. 412. In the process according to the invention, the trypsin is used to catalyze the Transpeptidation reaction used.

The transpeptidation can be achieved by dissolving 1. the insulin compound, 2. an L-threonine ester, and 3. trypsin in a mixture of water and at least a water-miscible organic solvent, if desired in the presence of one Acid.

Preferred water-miscible organic solvents are polar solvents As specific examples of such solvents, methanol, ethanol, 2-propanol, 1,2-ethanediol, acetone, dioxane ,. Tetrahydrofuran, N, N-dimethylformamide, formamide, N, N-dimethylacetamide, N-methylpyrrolidone, hexamethylphosphoric triamide and acetonitrile to be named.

The choice of the water-miscible organic solvent used depends on the selected reaction temperature and on whether there is an acid in the reaction mixture is present, the '.Jassergehalt in the reaction mixture is less than about 50% by volume, preferably less than about 40% by volume and more than about 10 volume units should.

An advantage of reducing the amount of water in the reaction mixture is that due to this, the formation of by-products is reduced. By increasing the amount of acid in the reaction mixture, it is in a similar manner and Way possible to reduce the formation of by-products. Increasing the yield is positively correlated with a high percentage of organic solvents.

The type of trypsin is not essential to the practice of the invention. Trypsin is a well-characterized one that is commercially available in high purity Enzyme, namely from pigs and cattle. Furthermore, trypsin can be of microbial origin be used. The trypsin form, for example crystalline trypsin (soluble Form), immobilized trypsin or even trypsin derivatives (as long as the trypsin activity preserved); is not critical to the practice of the invention. The term Trypsin, as used herein, is said to be trypsin from all sources and all forms by trypsin, which have transpeptidating activity, included Proteases with trypsin-like specificity, for example Acromobacter lyticus protease, see Agric. Biol. Chem. 42, p. 1443.

As examples of active trypsin derivatives, acetylated trypsin, succinylated trypsin, glutaraldehyde treated trypsin, and immobilized Trypsin derivatives are called.

If an immobilized trypsin is used, it will be in the medium suspended.

Organic or inorganic acids such as hydrochloric acid, formic acid, acetic acid, Propionic acid and butyric acid or bases such as pyridine, TRIS (tris-hydroxymethylaminomethane) N-methylmorpholine and N-ethylmorpholine can be added to make an appropriate one Bringing buffer system into being. Organic acids are preferred. However, the reaction can also be carried out without such additives. The GE added acid is usually less than 10 equivalents per equivalent of L-threonine ester. The amount of acid is preferably between 0.5 and 5 equivxlante per equivalent of L-threonine ester. Ions that stabilize trypsin, such as calcium ions, can be added.

The process can be carried out in a temperature range of + 500C and the freezing point the reaction mixture can be carried out. Enzymatic reactions with trypsin will be usually carried out at about 37 ° C in order to achieve a sufficient reaction rate to ensure. Nonetheless, it is beneficial to inactivation of trypsin to avoid using the method according to the invention at a temperature below the Ambient temperature. In practice, reaction temperatures are above about 0 ° C preferred.

The reaction time is usually between a few hours and several days, depending on the reaction temperature, on the amount added Trypsins and other reaction conditions.

The weight ratio between trypsin and the insulin compound in the reaction mixture is usually above about 1: 200, preferred above about 1:50 and below about 1: 1.

The L-threonine esters contemplated for practicing the invention can by the general formula Thr (R5) oR4 II to be discribed, where R4 is a carboxyl protecting group and R5 is hydrogen or a hydroxyl protecting group or a salt thereof.

The threonine B30 ester of human insulin used in transpeptization can arise from the general formula (Thr (R5) -OR4) B30-h-In III where h-In represents the human des- (ThrB30) 7 insulin compound and R4 and R5 the same Residues as defined above are to be described.

L-threonine esters of the formula II which can be used are those in which R4 is a carbonyl protecting group derived from the threonine B30 ester of human insulin (Formula III) under conditions that do not have a substantial irreversible conversion cause in the insulin molecule, can be split off. As examples of such Carbxyl protecting groups can be lower alkyl groups, for example methyl, ethyl and tert-butyl, substituted benzyl such as p-methoxybenzyl, 2,4,6-trimethylbenzyl and Diphenylmethyl are mentioned, as well as groups of the general formula -CH2CH2SO2R , where R6 is a lower alkyl radical such as methyl, ethyl, propyl and n-butyl. Suitable R5 hydroxyl protecting groups are those of the threonine B30 ester of human insulin (formula III) can be split off under conditions which do not cause significant irreversible conversions in the insulin molecule. Tert-butyl can be mentioned as an example of such a group R5.

Lower alkyl groups contain fewer than 7 carbon atoms, preferred less than 5 carbon atoms.

Other protecting groups that are commonly used are from Wünch in: Methods of Organic Chemistry (Houben-Weyl), Volume XV / 1, editor Fugen Müller, Georg Thieme Verlag, Stuttgart 19/4, has been described. ~ Some L-threonine esters (formula II) are known compounds and the remaining L-threonine esters (Formula II) can be prepared analogously to the preparation of known compounds.

The L-threonine esters of the formula II can have free bases of salts suitable organic or inorganic acids thereof, preferably acetates, Propionates, butyrates and salts of hydrogen halides such as hydrochlorides.

It is desirable to use the reactants, namely the insulin compound and the L-threonine ester (formula II), to be used in high concentrations. Preferred becomes a molar ratio above about 5: 1 between the L-threonine ester and the Insulin compound, used.

It is desirable that the concentration of L-threonine ester (formula II) is above 0.1 molar in the reaction mixture.

B3O Human insulin can be made from threonine esters of human Insulin (formula III) by splitting off the protective group R4 and any protective group R5 can be obtained by known methods or methods known per se. In the event of, that R4 is methyl, ethyl or a group -CH2CH2SO2R6, where R6 is as described above is defined, the protective group can under slightly basic conditions in aqueous Medium are split off, preferably at a pH of about 8 to 12, for example at about 9.5. Ammonia, triethylamine or alkali metal hydroxides, such as sodium hydroxide.

If R4 is tert-butyl, substituted benzyl such as p-methoxybenzyl or 2,4,6-trimethylbenzyl or diphenylmethyl, said group can be replaced by acidic Cleavage to be removed, preferably with trifluoroacetic acid. the Trifluoroacetic acid can be anhydrous or contain some water or with a organic solvents such as dichloromethane. If R5 is tert-butyl, the group can be split off by acid cleavage as described above.

B30 Preferred threonine esters of human insulin of the formula III are compounds in which R5 is hydrogen = Rd these are derived from the L-threonine esters of formula II, in which R5 is hydrogen.

The transpeptidation of the insulin compounds in the threonine B3O ester of human insulin can be described in more detail on the basis of the following formula given to the insulin compounds: whereby human dthreonineB3O) -insulin compound, where Al 1 Gly is connected to the substituent denoted as R1 and LysB29 is connected to the substituent denoted as R2, where R2 denotes an amino acid or a peptide chain that contains no more than 36 amino acids and R1 represents hydrogen or a group of the general formula R3-X-, where X denotes arginine or lysine and R3 represents a peptide chain which does not contain more than 35 amino acids, or R2 together with R3 represents a peptide chain which does not contain more than Contains 35 amino acids with the proviso that the number of amino acids present in R¹ plus R² is less than 37.

The transpeptidation of this invention converts accordingly any of the above insulin compounds in threonineB O-esters human Insulins (Formula III), which then unblocks to form human insulin will.

Another advantage of the invention is that the in raw Insulin present insulin-like compounds that are available in some commercially available Insulin preparations are present and covered by Formula I by the inventive transpeptidation in threonine B30 ester human insulin which are then unblocked to form human insulin can be. Examples of insulin-like compounds of the formula I result from the following: porcine diarginine insulin (R1 is hydrogen and R is -Ala-3 2 -Arg-Arg), porcine proinsulin (R together with R is -Ala-Arg - Arg-Glu-Ala-Glu-Asn-Pro-Gln-Ala-Gly-Ala-Val-Glu-Leu-Gly - Gly -Gly-Leu-Gly-Gly-Leu-Gln-Ala-Leu-Ala-Leu-Glu-Gly-Pro-Pro-B29 -Gln-Lys-, with the terminal alanyl connected to Lys) dog proinsulin (R3, together with R2 is -Ala-Arg-Arg-Asp-Val - Glu-Leu-Ala-Gly-Ala-Pro-G ly-G lu-Gly-Gly-Leu-Gln-Pro-Leu-Ala - eu -Glu-Gly-Ala-Leu-Gln-Lys-, with the terminal alanyl B29 'connected to Lys), split porcine proinsulin (R3 'is Ala-Leu-Glu-Gly-Pro-Pro-Gln-Lys-, and R2 is -Ala-Arg-Arg-Glu-Ala-Glu-Asn-Pro-G ln-Ala-Gly-Ala-Val-Glu-Leu - Gly-Gly-Gly-Leu-Gly-Gly-Leu-Gln-Ala-Leu), desdipeptide-1 2 Porcine proinsulin (R is hydrogen and R is -Ala-Arg-Arg-Glu-Ala-Glu-Asn-Pro-Gln-Ala-Gly-Ala-Val-Glu - Leu-Gly-Gly-Gly-Leu -Gly-Gly-Leu-Gln-Ala-Leu-Alå-Leu-Glu-3 -Gly-Pro-Pro-Gln), human proinsulin (R together with R2ist -Thr-Arg-Arg-Glu-Ala-Glu-Asp-Leu-Gln-Val-Gly - Gln-Val-Glu-Leu-Gly -Gly-Gly-Pro-G ly-Ala-Gly-Ser-Leu-Gln - Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-Lys-, where the terminal Threonyl linked to Lysβ29 and monkey proinsulin (R¹ together with R² is -Thr-Arg-Arg-Glu-Ala-Glu-Asp-Pro-Gln-Val-Gly-Gln-Val-Glu-Leu-Gly-Gly-Gly -Pro - Gly-Ala-Gly-Ser-Leu-G ln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-1330 -Gln-Lys-, with the terminal threonyl connected to Lys).

With all of these impurities covered by formula I, the R1 substituent denoted by R3-X- is exchanged with hydrogen.

A preferred embodiment of the invention comprises reacting of raw porcine insulin, which contains insulin-like compounds or a salt or a complex thereof with an L-threonine ester (Formula II) or a salt thereof under the above conditions, after which the protecting group R4 and any protecting group R5 are split off. Through this procedure, the pig insulin is put together converted into human insulin with insulin-like compounds in the same.

As an example of a complex or a salt of an insulin compound (Formula I) can be called a zinc complex or a zinc salt.

When the reaction conditions are in accordance with the above explanations can be selected and the results obtained in the following examples be considered, it is possible to obtain a yield of greater than 60% ThreonineB3O Esters of human insulin, and even those higher than 80 %, and under particularly preferred conditions, one greater than 90%.

A preferred method of making human insulin is the following: 1. The starting material for transpeptidation is crude Pig insulin, for example crystalline insulin, which is made by using a citrate buffer, see US-PS 26 26 228 was made.

2. If some trypsin activity remains after transpeptization, this is preferred under conditions under which trypsin is not active, for example removed in acidic medium with a pH below 3.

Trypsin can be separated by molecular weight, for example by gel filtration on Sephadex G-50 or Bio-gel P-30 in 1 M acetic acid, see Nature 280, 412, can be removed.

3. Other contaminants, such as unreacted porcine insulin, can by anion or cation exchange chromatography, see Examples 1 and 2, to be removed.

4. Then the threonine B30 ester of human insulin unblocked and the human insulin in a manner known per se, for example by crystallization, isolated.

Through this procedure, insulin can be made an acceptable pharmaceutical Maintain purity and, if desired, subjected to further purification.

The invention also relates to novel threonineB3O esters human insulin, in which the ester compound is different from tert-butyl and is methyl.

The abbreviations used here correspond to those used by the IUPAC-IUB Commission in Biochemical Nomenclature, 1974, recommended rules, see Collected Tentative Rules & Recommendations of the Commission on Biochemical Nomenclature IUPAX-IUB, 2nd ed., Maryland, 1975.

Analytical studies: the conversion of porcine insulin and Porcine proinsulin in human insulin esters can be determined by DISC PAGE electrophoresis in 7.5% polyacrylamide gel in a buffer consisting of 0.375 M Tris, 0.06 Mt HCl and 8 M urea can be demonstrated. The pH of the buffer is 8.7. Ester of Formula III migrate at 75% the rate of porcine insulin. Porcine proinsulin migrates at 55% the speed of porcine insulin and is supported by the invention Process converted into the same product. Identification of the transformation products as compounds of the formula III takes place on the basis of the following criteria: a) The electrophoretic Migration of the human insulin esters of the formula III, based on porcine insulin, corresponds to the loss of a negative charge.

b) The amino acid composition of the stained protein bands in the Gel representing the compounds of formula III is identical to that human insulin, namely 3 moles of threonine and 1 mole of alanine per mole of insulin, and the composition of the porcine insulin is 2 moles of threonine and 2 moles of alanine per mole of insulin. The technique of amino acid analysis of protein bands in polyacrylamide gels is in Eur. J. Biochem. 25, 147.

c) Evidence that the built-in threonine is a C-terminal amino acid is bound in the B chain; becomeslX by oxidative sulfitolysis of the sulfur bridges of the insulin in 6 M guanidine chloride. followed by severing the basic B chains performed by ion exchange chromatography on SP Sephadex. Digestion of the B-chain S-sulfonate with carboxypeptidase A only releases the C-terminal amino acid.

This technique is on by Markussen in Proceedings of the Symposium Proinsulin, Insulin and C-peptide, Tokushima, July 12-14, 1978 (Editors: Baba, Kaneko & Yaniahara) Int. Congress Series No. 468, Excerpta Medica, Amsterdam-Oxford. The analysis is carried out after the Ester group has been split off from the compounds of the formula III.

These three analyzes clearly show that conversion into human Insulin has taken place.

The conversion of porcine insulin and porcine proinsulin to human Insulin esters can be quantitatively determined by HPCL (high pressure liquid chromatography) be followed in reverse phase. A 4 x 300 mm / u Bondapak C18 column (Waters Ass.) was used and the elution with a 0.2 M ammonium sulphate (with sulfuric acid 'auf adjusted to a pH of 3.5) and 26 to 50% by volume of acetonitrile Buffer carried out. The optimal acetonitrile concentration depends on which Ester of formula III one wishes to separate 4 from porcine insulin. In case that R4 is methyl and R5 is hydrogen, the separation is in 26% by volume acetonitrile achieved. Porcine insulin and (Thr-OMe) O-h-In (Me is methyl) are after 4.5 and 5.9 column volumes eluted as well separated, symmetrical peaks. Before applying on the HPLC column, the proteins in the reaction mixture were removed by adding 10 parts by volume of acetone precipitated. The precipitated was isolated by centrifugation, vacuum-dried and dissolved in 0.02 tt sulfuric acid.

The process for making human insulin esters and human Insulins is explained in more detail by the following examples, which are by no means considered Limitation should be viewed. The examples show some preferred embodiments of the method according to the invention.

Example 1 200 mg of crude porcine insulin, once crystallized, was added dissolved in 1.8 ml 3.33 po acetic acid. 2 ml of a 2 M solution of Thr-OMe (Me is methyl) in N, N-dimethylacetamide and 20 mg of trypsin dissolved in 0.2 ml of water were added. After standing for 18 hours at 37 ° C, the proteins were by adding 40 ml of acetone precipitated and the precipitated isolated by centrifugation. The supernatant was discarded. Analysis of the precipitate by HPLC using 26% acetonitrile (see "Analytical Investigations"), B30 shows a 60% conversion of the pig insulin in (Thr-OMe) h-In. The precipitate became 8 molar freshly deionized in 8 ml Dissolved urea solution, adjusted the pH to 8.0 with 1 molar ammonia solution, and the solution on a 2.4 x 25 cm column packed with QAE A-25 Sephadex with a 0.1 molar ammonium chloride buffer, which contained 60% by volume of ethanol and the pH of which had been adjusted to 8.0 with ammonia. the Elution was carried out with the same buffer and 1330 fractions of 15 ml collected. (Thr-OMe) -h-In was found in fractions 26 to 46, unreacted Porcine insulin in fractions 90 to 120. Fractions # 26 to 46 were combined, the ethanol evaporated in vacuo and (Thr-0Me) -h-In in a citrate buffer, as by Schlichtkrüll et al., Handbook of Internal Medicine 7 / 2A, 96, Berlin, Heidelberg, New York, described in 1975, crystallized. The yield was 95 mg of crystals with the same rhombic shape as crystallized in the same way Pig insulin.

The amino acid composition was found to be identical to that of human insulin. Further analytical studies to be carried out in the above Section "Analytical Investigations" are described, showed that the resulting 1330 was product (Thr-OMe) -h-ln.

Example 2 100 mg porcine insulin meeting the purity requirements of GB-PS 12 85 023 were sufficient, were dissolved in 0.9 ml of 3.33 molar acetic acid and then 1 ml of a 2 molar solution of threonine methyl ester in N, N-dimethylformamide and 12 mg treated with TPCK (tosylphenylalanine chloromethyl ketone).

Trypsin, dissolved in 0.1 ml of water, was added. After an incubation period of 24 hours at 37 ° C., the reaction was stopped by adding 4 ml of 1 molar phosphoric acid completed. The obtained hr-OMe) B30-h-In was from the unreacted porcine insulin by ion exchange chromatography on a 2.5 x 25 cm column with SP-Sephadex by means of an eluent which is 0.09 M. Sodium chloride and 0.02 ml sodium dihydrogen phosphate (pH of the buffer: 5.5) in 60% ethanol, separated off. Containing fractions (Thr-OMe) 1330-h-In were combined, the ethanol removed in vacuo and the product crystallized as in Example 1.

The yield was 50 mg (Thr-OMe) B30-h-In.

Example 3 100 mg of porcine proinsulin became 3.33 molar in 0.9 ml Acetic acid dissolved, converted into (Thr-QMe) 1330-h-In -and-as for porcine insulin described in Example 1, purified. The 1330 Conversion of Proinsulin to (Thr-OMe) -h-In was determined to be 73% by HPLC analysis of the acetone precipitate. the The yield of crystalline (Thr-OMe) B30-h-In was 54 mg.

Example 4 100 mg of porcine insulin are dissolved in 0.9 ml of 2.77 molar acetic acid dissolved in water and analogous to the method described in Example 2 for the reaction brought. After completion of the reaction, the proteins were removed by adding 10 Parts by volume of acetone precipitated. Analysis by DISC PAGE-1330 shows electrophoresis a conversion of 70% to (Thr-OMe) h-In.

Example 5 100 mg of porcine insulin was dissolved in 0.9 ml of 3.33 molar acetic acid dissolved and 1 ml of 2 r, Thr-OMe solution in N-rlethylpyrrolidone added. The reaction was carried out analogously to that described in Example 4 and the conversion to (Thr-OMe) -h-In was 20%.

Example 6 100 mg of porcine insulin was dissolved in 0.9 ml of 2.77 M acetic acid dissolved in water and 1 ml of a 2 M Thr-OMes solution in HMPA (hexamethylphosphoric triamide) admitted. The reaction was carried out analogously to that described in Example 4. The conversion to (Thr-OMe) B30-h-In was 80%.

Example 7 100 mg of porcine insulin was dissolved in 0.9 ml of 3.33 molar acetic acid dissolved and 1 -irl of a 2 M Thr-OMe solution in N, N-dimethylacetamide was added. the The reaction was carried out analogously to that described in Example 4. The transformation in 1330 (Thr-OMe) -h-In was 80%.

Example 8 100 mg of porcine insulin was dissolved in 0.9 ml of 3.33 molar acetic acid dissolved and 1 ml of a 2 M Thr-OMe solution in N, 0-dimethylacetamide was added. Afterward Trypsin with 2.00 U activity (measured against the substrate BAEE) was immobilized on 1 g glass beads, added. After an incubation time of 24 hours at 37 ° C became that bound to the glass Separated trypsin by filtration. After completion of the reaction, the proteins were by adding 10 parts by volume Acetone precipitated. Analysis by DISC PAGF electrophoresis showed conversion in 1330 in (Thr-OMe) -h-In of 40%.

Example 9 100 mg of porcine insulin was dissolved in 0.9 ml of 3.33 molar acetic acid dissolved and 1 ml of a 2M Thr-Oize solution in N, N-dimethylacetamide was added. Afterward was trypsin corresponding to an activity of 300 U (the activity was against the substrate BAEE measured) irmobilized -on 200 ug CNBr-activated "Sephadex G-'150 "was added. After an incubation of 24 hours at 37 ° C., the Separated trypsin bound to Sephadex by filtration. After the reaction has ended the proteins were precipitated by adding 10 parts by volume of acetone. analysis by DISC PAGE electrophoresis showed a conversion to (THr-OMe) B30-h-In of 70 %.

Example 10 The procedure described in Example 7 was repeated, with the prerequisite that the ester used is a 2 M solution of Thr-OBut (But is tert-butyl), in N, N-dimethylacetamide. The conversion to (Thr-OBut) ß30 -h-in was 80 ..

Example 11 The example described in Example 8 was repeated, the ester used being a 2 M solution of Thr-OBut in N, N-dimethylacetamide was. The conversion to (Thr-OBut) B30-h-In was 30%.

Example 12 The procedure described in Example 9 was repeated, the ester used being a 2 solution of Thr-OBut in N, N-dimethylacetamide. The reforestation in (Thr-OBu) B30-h-In was 70%.

Example 13 100 mg of porcine proinsulin became 3.33 molar in 0.9 ml Dissolved acetic acid and added 1 ml of 2 M Thr-OMe in N, N-dimethylacetamide. The reaction was carried out analogously to the manner described in Example 4. The Um-1330 Conversion in (Thr-OMe) -h-In was 80%.

Example 14 100 mg of porcine proinsulin became 3.33 molar in 0.9 ml Dissolved acetic acid and 1 ml, a 2 M solution of Thr-OMe in N, N-dimethylacetamide admitted. The mixture was immobilized with trypsin analogously to that in example 8 treated procedures. The conversion to (Thr-OMe) B30-h-In was 40 t: Example 15 100 mg of porcine proinsulin were dissolved in 0.9 ml of 3.33 molar acetic acid dissolved and added 1 ml of a 2 M solution from Doktor in N, N-dimethylacetamide. the Mixture was analogous to the method described in Example 9 with immobilized Trypsin treated. The conversion to (Thr-OMe) 30-h-In was 70%.

Example 16 100 mg of porcine prodinsulin was 3.33 molar in 0.9 ml Acetic acid and 1 ml of a 2 M ThrOBut solution in N, N-dimethylacetamide were added. The reaction was carried out analogously to that described in Example 4. The transformation in (Thr-OBut) B30 h-In was 80 t.

Example 17 100 mg of porcine proinsulin became 3.33 molar in 0.9 ml Dissolved acetic acid and added 1 ml of a 2 M Thr-OBut solution in N, N-bimethylacetamide. The mixture was made with trypsin immobilized on glass beads analogously to that in example 8 is carried out. The conversion to (Thr-OBut) B30-h-In was 40%.

Example 18 100 mg of porcine proinsulin became 3.33 molar in 0.9 ml Dissolved acetic acid and added 1 ml of a 2 M Thr-OBut solution in N, N-dimethylacetamide. The mixture was trypsin immobilized on CNBr-activated Sephadex G-150, treated analogously to the process described in Example 9.

The conversion to (Thr-OBut) B30-h-In was 70%.

Example 19 100 mg of porcine insulin was dissolved in 0.5 ml of 6 molar acetic acid dissolved and with 1 ml of 1 M solution of Thr-OTmb (Tmb is 2,4,6-trimethylbenzyl) in N, N-dimethylacetamide, added. Furthermore, 0.5 ml of N, N-dimethylacetamide and 5 mg of TPCK-treated trypsin in 0.1 ml of water were added. The reaction mixture was left to stand at 32 ° C for 44 hours. After completion of the reaction were the proteins precipitated by adding 10 parts by volume of acetone. Analysis through DISC PAGE electrophoresis showed a conversion to (Thr-OTmb) 1330-h-In of 50%.

Example 20 100 mg of porcine insulin was dissolved in 0.9 ml of 3 molar acetic acid dissolved and 1 ml of a 2 M solution of Thr «Ble in dioxane was added. the The reaction was carried out analogously to that described in Example 4; the conversion to (Thr-OMe) B30-h-In was 10%.

Example 21 100 mg of porcine insulin was dissolved in 0.9 ml of 3 molar acetic acid dissolved and 1 ml of a 2 × 4 solution of Thr-Otte in acetonitrile was added. The reaction was carried out analogously to the manner described in Example 4 and the Conversion 1330 to (Thr-0Me) -h-In was 10%.

Example 22 1330 250 mg of crystalline (Thr-OMe) h-In were in 25 ml Disperse water and add 1 N sodium hydroxide solution to pH of 10.0. The pH was kept constant at 10 for 24 hours at 250C.

The human insulin formed was by adding 2 g of sodium chloride, 350 mg sodium acetate trihydrate and 2.5 mg zinc acetate dihydrate followed by addition crystallized from 1N hydrochloric acid to obtain a pH of 5.52. After 24 hours The rhombohedral crystals were left to stand at 4 ° C. by centrifugation isolated, washed with 3 ml of water, isolated again by centrifugation and vacuum-dried. Yield: 220 mg of human insulin.

Example 23 1330 100 mg (Thr-OTmb) were dissolved in 1 ml of ice-cold trifluoroacetic acid dissolved and the solution left to stand at OOC for 2 hours.

The human insulin formed was by adding 10 ml of tetrahydrofuran and 0.97 ml of 1.03 molar hydrochloric acid precipitated in tetrahydrofuran. The educated Precipitate was isolated by centrifugation with 10 ml of tetrahydrofuran washed, separated by centrifugation and vacuum dried.

The precipitate was dissolved in 10 ml of water and the pH of the Solution adjusted to 2.5 using 1N sodium hydroxide solution. The human insulin was precipitated by adding 1.5 g of sodium chloride and isolated by centrifugation. The precipitate was dissolved in 10 ml of water and the human insulin passed through Addition of 0.8 g of sodium chloride, 3.7 mg of zinc acetate dihydrate and 0.14 g of sodium acetate trihydrate, followed by the addition of 1N sodium hydroxide solution to obtain pH of 5.52 precipitated. After standing at 4 ° C. for 24 hours, the precipitate became isolated by centrifugation, washed with 0.9 ml of water, again by centrifugation isolated and vacuum dried.

Yield: 90 mg human insulin.

Example 24 100 mg of porcine insulin was dissolved in 0.5 ml of 10 molar acetic acid dissolved and 1.3 ml of a 1.54 M solution of Thr9mS in N, N-dimethyl acetamide was added. The mixture was cooled to 12 ° C.

10 mg of trypsin dissolved in 0.2 ml of 0.05 M calcium acetate solution were added admitted. After standing for 48 hours at 120C, the proteins were by adding precipitated by 20 ml of acetone.

The conversion of the porcine insulin to (Thr-OMe) B30-h-In was 97 % as determined by HPLC.

Example 25 20 mg of porcine insulin was added in a mixture of 0.08 ml of 10 molar acetic acid and 0.14 ml of water were dissolved. 0.2 ml of a 2 fl solution of Thr-OMe in N, N-dimethylacetamide was added and the mixture was cooled to -100C. 2 mg of trypsin, dissolved in 0.025 ml of 0.5 M calcium acetate solution, were added. To The proteins were left to stand at -1OOC for 72 hours by adding 5 ml of acetone failed. The conversion of the porcine insulin to (Thr-OMe) B30-h-In was 64 % as evidenced by HPLC.

Example 26 20 mg of porcine insulin was placed in 0.1 ml of water.

Addition of 0.6 ml of a 2 M solution of Thr-OMe in N, N-dimethylacetamide caused the insulin to dissolve. The mixture was cooled to 70C. 2 mg of trypsin dissolved in 0.025 ml of 0.05 M calcium acetate was added. After 24 hours the proteins were precipitated by adding 5 ml of acetone. The transformation of the Porcine insulin in (Thr-OMe) 1330-h-In was 62% as demonstrated by HPLC could be.

Example 27 20 mg of porcine insulin was added to 0.135 ml of a 4.45 m Dissolved propionic acid. 0.24 ml of a 1.67 M solution of Thr-OMe in N, N-dimethylacetamide was admitted. 2 mg trypsin dissolved in 0.025 ml of a 0.05 M calcium acetate solution were added and the mixture held at 37 ° C for 24 hours. The proteins were precipitated by adding 10 parts by volume of 2-propanol. The conversion of pig insulin in (Thr-OMe) -h-In was 75% as verified by HPLC.

Example 28 20 mg of porcine insulin was dispersed in 0.1 ml of water.

0.4 ml of a 2 M solution of Thr-OMe in N, N-dimethylacetamide were added, followed by 0.04 ml of 10 N hydrochloric acid, which induced the insulin, to loosen up. 2 mg trypsin dissolved in 0.025 ml of a 0.05 M solution of calcium acetate was added and the mixture held at 37 ° C for 4 hours. Analysis by HPLC showed 46% conversion of porcine insulin to (Thr-OMe) 1330-h-In.

Example 29 20 mg of porcine insulin was dissolved in 0.175 ml of 0.57 M acetic acid solved. 0.2 ml of a 2 M solution of Thr-OMe in N, N-dimethylacetamide was added, followed by the addition of 0.02 ml of a 0.05 M solution of calcium acetate. 10 mg a crude preparation of Achromobacter lyticus protease was added and the Mixture held at 37 ° C. for 22 hours. The proteins were made by adding 10 parts by volume Acetone precipitated. The conversion of the porcine B30 insulin to (Thr-OMe) -h-In was 12%, as could be proven by HPLC.

Example 30 20 mg of porcine insulin was dissolved in 0.1 ml of 0.5 molar acetic acid solved. Addition of 0.2 ml of a 0.1 M solution of Thr-OMe in N, N-dimethylacetamide released the insulin. The mixture was cooled to 120C and 2 mg trypsin in 0.025 ml 0.05 M calcium acetate solution was added. After standing at 120C for 24 hours it showed HPLC analysis showed 42% conversion of porcine insulin to (Thr-OMe) h-In.

Example 31 2 mg rabbit insulin became 4.45 molar in 0.135 ml Acetic acid dissolved. 0.24 ml of a 1.67 M solution of Thr-OMes in N, N-dimethylacetamide was added followed by the addition of 1.25 mg of trypsin in 0.025 ml of 0.05 M Calcium acetate solution. The mixture was held at 37 ° C. for 4 hours. HPLC analysis showed 88% conversion of the rabbit insulin to (Thr-OMe) O-h-In. Rabbit insulin is eluted from the HPLC column before the porcine insulin, the ratio being between the eulution volumes of rabbit insulin to (Thr-OMe) B30-h-In is 0.72.

Example 32 1331 1332 2 mg of porcine diarginine insulin (Arg 1-ArgB3 Insulin) were dissolved in 0.135 ml of 4.45 molar acetic acid. 0.24 ml of a 1.67 M solution of Thr-OMes in N, N-dimethylacetamide was added, followed by the Add 1.25 mg of trypsin in 0.025 ml of a 0.05 M calcium acetate solution. The mixture was kept at 37 ° C. for 4 hours. Analysis by HPLC showed 91% conversion of diarginine insulin in (Thr-OMe) 1330-h-In. The arginine insulin comes before porcine insulin eluted from the HPLC column, the ratio between the elution volumes of Diarginine insulin to <Thr-0Me) 1330-h-In is 0.50.

Example 33 2 mg of pig intermediates (namely a mixture of desdipeptide (Lys62-Arg63) -proinsulin and desdipeptide 31 32 (Arg -Arg) -proinsulin) was allowed to react analogously to the reaction described in Example 32. analysis by HPLC showed an 88% yield of (Thr-OMe) B30-h-In.

Example 34 20 mg of porcine insulin was dissolved in 0.1 ml of 2 molar acetic acid dissolved 0.2 ml of a 2 M solution of Thr-OMe in N, N-dimethylacetamide was added and the mixture cooled to -180C. 2 mg trypsin dissolved in 0.025 ml 0.05 M calcium acetate solution were added and the mixture was kept at -180C for 120 hours. HPLC analysis showed that the conversion to (Thr-OMe) B30-h-In was 83%.

Example 35 The procedure described in Example 34 was followed under the Measure repeated that the reaction at 500C in 4 hours was carried out. IIPLC analysis showed that the conversion to (Thr-OMe) B30-h-In 23%.

Example 36 20 mg of porcine insulin was dissolved in 0.1 ml of 3 molar acetic acid solved. 0.3 ml of a 0.33 M solution of Thr-O (CH2) 2-S02-CH3, CH3COOH (Threonine-2- (methylsulfonyl) ethyl ester, hydroacetate) in N, N-dimethylacetamide was added. The mixture was cooled to 12 ° C and 2 mg of trypsin in 0.025 ml of a 0.05 M Calcium acetate solution added. HPLC showed a 77% strength after 24 hours at 120C Conversion to (Thr-0 (CH2) 2-SO2-CH3) 1330 - h-In. The product elutes approximately at the same place as 1330 (Thr-oMe) -h-In.

Example 37 20 mg of porcine insulin was dissolved in 0.1 ml of 10 molar acetic acid solved. 0.2 ml of a 2 M solution of Thr-OEt (Et is ethyl) in N, N-dimethlacetamide were added and the mixture cooled to 120C. 2 mg trypsin in 0.025 ml a 0.05 M solution of calcium acetate was added. HPLC showed after 24 hours 75% conversion to (Thr-OEt) B30-h-In at 120C.

The product eluted in one portion shortly after that of 1330 (Thr-OMe) -there.

Example 38 20 mg of porcine insulin was dissolved in 0.1 ml of 6 molar acetic acid solved. 0.3 ml of a 0.67 M solution of Thr (But) -OBut (But is tertiary butyl) in N, N-dimethylacetamide was added.

The mixture was cooled to 120 and 2 mg trypsin in 0.025 ml added to a 0.05 M calcium acetate solution. After 24 hours at 120 ° C., the Conversion to (Thr (But) -OBut) 1330-h-In 77%. The product was taken from the HPLC column by Applying an acetonitrile gradient from 27% to 40 % eluted.

Example 39 20 mg of porcine insulin were dissolved in 0.1 m 4 molar acetic acid solved. 0.2 ml of a 1.5 M solution of Thr-OMe, in tetrahydrofuran, was added and the mixture cooled to 120C. 2 mg trypsin dissolved in 0.025 ml 0.05 molar Calcium acetate was added. After 4 hours at 120 ° C. the analysis showed through HPLC found a 75% conversion to (Thr-OMe) h-In.

Example 40 20 mg of porcine insulin was dissolved in 0.1 ml of 4 molar acetic acid solved. 0.8 ml of a 2 M Thr-OMe solution in 1,2-ethanediol were added and the Mixture cooled to 120C. 2 ml of trypsin dissolved in 0.025 ml of a 0.05 M calcium acetate solution were admitted. After standing at 120 ° C. for 4 hours, the HPLC analysis showed a Conversion of 48% to (Thr-OMe) 1330-h-In.

Example 41 20 mg of porcine insulin was dissolved in 0.1 ml of 4 molar acetic acid solved. 0.2 ml of a 2 M Thr-OMe solution in ethanol was added and the mixture cooled to 12 ° C. 2 mg trypsin dissolved in 0.025 ml 0.05 M calcium acetate solution were added and the reaction was carried out at 12 ° C. for 4 hours.

Analysis by HPLC showed 46% conversion to (Thr-OMe) 1330-h-in.

Example 42 20 mg of porcine insulin was dissolved in 0.1 ml of 4 molar acetic acid solved. 0.2 ml of a 2 M solution of Thr-OMe in acetone were added admitted and the mixture cooled to 120C. 2 mg trypsin dissolved in 0.025 ml of a 0.05 M calcium acetate solution was added. After 4 hours at 120 ° C., the HPLC analysis showed a conversion of 48% to (Thr-OMe) -h-In.

Those disclosed in the above description as well as in the claims Features of the invention can be used individually or in any combination essential for the realization of the invention in its various embodiments be.

Claims (34)

Claims 1. A process for preparing human threonine B30 esters Insulin or a salt or complex thereof, characterized by transpeptidating an insulin compound or a salt or complex thereof, which are convertible into these, with an L-threonine ester or a salt thereof in a mixture of water, a water-miscible organic solvent and Trypsin, the water content in the reaction mixture being less than 50% by volume and the reaction temperature is below 50 ° C, optionally in the presence of a Acid. 2. The method according to claim 1, characterized in that the concentration of the L-threonine ester in the reaction mixture is above 0.1 molar that the reaction temperature is above the freezing point of the reaction mixture, and that the reaction mixture contains from 0 to 10 equivalents of an acid per equivalent of the L-threonine ester. 3. The method according to claim 1 or 2, characterized in that the insulin compound is a compound selected from the group consisting of porcine, Canine, fin whale, sperm whale and rabbit insulin, porcine diarginine insulin, split Pig proinsulin, pig desdipeptide proinsulin, pig, dog, monkey, and Human proinsulin and mixtures thereof. 4. The method according to any one of claims 1 to 3, characterized in that that the insulin compound is derived from pigs. 5. The method according to claim 4, characterized in that relative raw insulin is used as an insulin compound. 6. The method according to any one of claims 1 to 5, characterized in that that the reaction temperature is below 370C, preferably below ambient temperature, and is above OOC. 7. The method according to any one of the preceding claims, characterized in, that the water content in the reaction mixture is between 40 and 10 volume units. 8. The method according to any one of the preceding claims, characterized in, that the molar ratio between the L-threonine ester and the insulin compound is above 5: 1. 9. The method according to any one of the preceding claims, characterized in that that the organic solvent is a polar solvent. 10. The method according to claim 9, characterized in that the solvent Methanol, ethanol, 2-propanol, 1-2-ethanediol, acetone, dioxane, tetrahydrofuran, formamide, N, N-dimethylformamide, N, N-dimethylacetamide, N-methyl-pyrrolidone, hexamethylphosphoric triamide or Is acetonitrile. 11. The method according to any one of the preceding claims, characterized in, that the acid is an organic acid, preferably formic acid, acetic acid, propionic acid and butonic acid. 12. The method according to any one of the preceding claims, characterized in, that the amount of acid in the reaction mixture is between 0.5 and 5 equivalents per Equivalent of the L-threonine ester. 13. The method according to any one of the preceding claims, characterized in, that the weight ratio between trypsin and the insulin compound in the reaction mixture is above 1: 200. 14. The method according to any one of the preceding claims, characterized in, that the weight ratio between trypsin and the insulin compound in the reaction mixture above 1:50 and below 1: 1. 15. The method according to any one of the preceding claims, characterized in, that the transpeptidation is carried out in the presence of calcium ions. 16. The method according to any one of the preceding claims, characterized in that that the transpeptidation is carried out in a buffer system. 17. The method according to any one of the preceding claims, characterized in, that the L-threonine ester as acetate or propionate, butyrate or as hydrogen halide, such as hydrochloride, is added. 18. The method according to any one of the preceding claims, characterized marked, that the reaction temperature, the water content and the acid concentration in the Reaction mixture selected so that the yield of the threonine ester human insulin is higher than 60%. 19. The method according to claim 18, characterized in that the yield of the threonineB3O ester of human insulin is higher than 80%. 2o. Process according to Claim 19, characterized in that the yield of the threonine ester of human insulin is more than 90%. 21. The method according to any one of claims 1, 2 and 6 to 18, characterized in that the insulin compound is a compound of general formula I. is, where denotes the human des (ThrB30) -insulin unit in which GlyA1 is linked to the substituent R1 and Lys to the substituent R², where R² is an amino acid or a peptide chain which contains no more than 36 amino acids, and R1 is hydrogen or a group of the general forms R3-X-, where X represents arginine or lysine and R3 denotes a peptide chain which has no more than 35 amino acids, or R2 together with R3 denotes a peptide chain which contains no more than 35 amino acids, provided that that the number of amino acids present in R¹ plus R² is less than 37. 22. The method according to 21, characterized in that R1 is hydrogen is and R2 is -Ala, -Ser, -Ala-Arg-Arg, or -Ala-Arg-Arg-Glu-Ala-Glu-Asn-Pro-Gln-Ala-Gly-Ala-Val - Glu-Leu- G ly-Gly-Gly-Leu-Gly-Gly-Leu-Gln-Ala-Leu-Ala-3 -Leu-Glu-Gly-Pro-Pro-Gln, R Ala-Leu-Glu-Gly-Pro-Pro-Gln -Lys- is, and'R2 -Ala-Arg-Arg-Glu-Ala-Glu-Asn-Pro-Gln-Ala-Gly-Ala-Val-Glu-Leu-Gly-Gly-Gly-Leu-Gly-Gly-Leu -Gln - Ala-Leu, or R³ together with R² -Ala-Arg-Arg-Glu-Ala-Glu-Asn-P ro-Gln-Ala-Gly-Ala-Val-Glu-Leu-Gly-Gly-Gly-Leu-Gly-Gly -Leu-Gln-Ala-Leu-Ala-Leu-Glu-Gly-Pro-Pro-Gln-Lys-, where the terminal alanyl is linked to LysB29, -Ala-Arg-Arg-Asp-Val-Glu-Leu-Ala-Gly-Ala-Pro-Gly-Glu-Gly-Gly-Leu-Gln-Pro-Leu -Ala-Leu-Glu-Gly-Ala-Leu-Gln-Lys-, where the terminal alanyl is connected to LysB29, -Thr - Arg-Arg-Glu-Ala-Glu-Asp-Leu-Gln-Val-Gly-Gln-Val-Glu-Leu - Gly-Gly-Gly-Pro- Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu - Glu-Gly-Ser-Leu-Gln-Lys-, where the terminal threonyl is connected to LysB29, or -Thr-Arg-Arg-Glu-Ala-Glu - Asp-Pro-Gln-Val-Gly-Gln-Val-Glu-Leu-Gly-Gly-Gly-Pro- Gly - Ala-Gly-Ser-Leu-Gln-Pro-Leú-Ala-Leu-Glu-Gly-Ser-Leu-Gln-Lys-, the terminal threonyl being linked to Lys. 23. The method according to claim 22, characterized in that R1 is hydrogen and R2 represents alanine. 24. The method according to any one of the preceding claims, characterized in that dass.der L-threonine ester has the general formula II Thr (R5-OR4 (11), where R4 is a carbonyl protecting group and R5 is hydrogen or a hydroxyl protecting group means. 25, method according to claim 2, characterized in that R4 a lower alkyl, aryl (lower alkyl) such as a substituted benzyl or Diphenylmethyl or a group of the general formula -CH2CH2SO2R6, where R6 means a lower alkyl. 26. The method according to claim 25, characterized in that R4 is methyl Ethyl, tert-butyl, p-15ethoxybenzyl, 2,4,6-trimethylbenzyl or a group of general formula -CH2CH2 SO 2R6, where R6 is methyl, ethyl, propyl or n-butyl is. 27. The method according to any one of claims 1 to 6, characterized in, that the insulin compound is porcine insulin, the L-threonine ester Thr-OMe or Thr-OBut is that the water-miscible organic solvent is N, N-dimethylformamide or N, N-dimethylacetamide, that the reaction temperature is about 370 that the water content in the reaction mixture is between 41 and 43% that the Weight ratio between trypsin and the insulin compound is about 1: 8 that the molar ratio between the L-threonine ester and the insulin compound is about 1: 120 and that between 1.2 and 1.5 equivalents of acetic acid per equivalent L-threonine ester can be added. 28. The method according to claim 1 or 2, characterized in that it under the reaction conditions described in any one of Examples 1 to 21 or 24 to 42 are described, preferably among those in any of the examples 4, 7, 10, 24, 34, 36 and 39 are performed. 29. Threonine B30 ester of human insulin manufactured by a A method according to any one of claims 1 to 29. 3P process for the production of human insulin, characterized in that that a method according to any one of claims 1 to 24 is carried out first is unblocked and then the resulting threonine ester of human insulin will. 31. The method according to claim 30, characterized in that the deblocking in an aqueous medium in the presence of a base, preferably at a pH between 8 and 12. 32. The method according to claim 30, characterized in that the deblocking by acidolysis, preferably with trifluoroacetic acid. 33, method according to claim 30, characterized in that the deblocking carried out under the reaction conditions described in Examples 22 or 23 will. 34. ThreonineB30 ester of human insulin, being the ester group differs from tert-butyl and methyl.