JPH0150719B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing human insulin. The present inventors reacted des-B30-insulin with 1 to 100 times the molar amount of a threonine derivative by the action of trypsin or a trypsin-like enzyme that has specificity for basic amino acid residues on the carbonyl side, and then obtained a condensate. discovered a method to obtain insulin by removing the protecting group from.

Insulin is a valuable drug with no substitutes for the treatment of diabetes, and currently bovine insulin and porcine insulin are mainly used for treatment. However, some of the constituent amino acids of these insulins are different from human insulin, so
Antibodies may be produced in the body, and the production of antibodies causes problems such as a marked decrease in the therapeutic effect of insulin. Therefore,
There is a strong desire to establish a method for synthesizing human insulin that can be carried out on an industrial scale.

According to the method of the present inventors, human insulin can be easily synthesized using pig insulin, which differs from human insulin only in the amino acid at position 30 of the B chain, as a raw material. There is no doubt that the human insulin obtained in this way is an ideal therapeutic insulin.

Attempts to obtain human insulin from pig desoctapeptide insulin and human insulin octapeptide were conducted by M.A. Ruthtenberg (M.A.
Ruttenberg) Science vol. 177 623
(1972) and R. Obermeier et al.
Physiologische Chemie) Volume 357, Page 759 (1976)
Both methods are based on chemical means, and the former involves an alkali treatment at the end of the process, and accompanying side reactions are unavoidable. In the latter case, the reaction is nonspecific and many side reactions occur, making purification complicated and difficult and resulting in extremely low yields. Therefore, it cannot be carried out on an industrial scale.

On the other hand, the method of the present inventors obtains insulin by condensing des-B30-insulin and an L-threonine derivative through an enzymatic reaction. It has advantages such as no reaction, no racemization, and the ability to recover unreacted raw materials without damaging them.

Furthermore, in the same method, by using 1 to 100 times the molar amount of L-threonine derivative, the carbonyl side cleavage of arginine at position 22 of the B chain, which naturally occurs in normal enzymatic reactions, is prevented; Introduction of a protecting group to the arginine side chain, which is essential, is unnecessary.

This method uses trypsin or a trypsin-like enzyme that has specificity for basic amino acid residues on the carbonyl side.
1 for B30-insulin (hereinafter referred to as compound)
It consists of reacting an L-threonine derivative (hereinafter referred to as compound) in a molar amount of ~100 times, and then removing the protecting group from the resulting condensate. The compound is represented by the general formula below.

(In the formula, R ¹ is hydrogen or a hydroxy protecting group,
R ² each represents a carboxyl group protecting group. ) The above-mentioned compound is obtained by treating insulin of pig origin with carboxypeptidase A, and is obtained, for example, from E.
Hoppe-Seyler's Zeitschrift fu¨r
Physciologische Chemie) Volume 359, Page 799 (1978)
It can be manufactured by the method described in . Also, Achromobacter liteicus
lyticus), and can be easily produced using an enzyme that cleaves the carbonyl side of lysine. Regarding the isolation and properties of the enzyme, Masaki et al.
Agricultural and Biological Chemistry Volume 42
1443 (1978), in which the enzyme with the above properties is called protease.

A compound can be subjected to a reaction even if its side chain functional group, hydroxy group, is not protected, but if a protecting group is to be introduced, a protecting group commonly used in peptide synthesis reactions, such as t-butyl, benzyl , acetyl, etc. may be used. Furthermore, the carboxyl group of the compound needs to be protected, and a commonly used protecting group for carboxyl groups may be used. For example, modification in the form of alkyl and aralkyl esters such as t-butyl, benzyl, etc.

When selecting the above-mentioned protecting groups, consideration should be given to selecting those that do not denature or deactivate insulin during the introduction or removal treatment of the protecting groups. Regarding protecting groups used in peptide synthesis, M. Bodansudsky (M.
Peptide Synthesis, 2nd edition (1976) (John Wiley & Sons)
Sons)).

It goes without saying that when selecting a protecting group, it is preferable to select one that can be simultaneously removed by a single protecting group removal operation.

Enzymes used in the method include trypsin or trypsin-like enzymes that exhibit specificity for carboxyl-side basic amino acids derived from various animals, plants, and microorganisms. Examples of trypsin-like enzymes include enzymes extracted and separated from Streptomyces bacteria. Regarding the enzyme, Morihara et al. published Arch.Biochem.Biophys., Vol. 126, p. 971 (1968), and Yoshida et al. published FEBS Letters, Vol. 15. It is described on page 129 (1971). The enzyme used may be treated with tosyl-L-phenylalanine chloromethyl ketone (TPCK) or the like in order to remove contaminating chymotrypsin or chymotrypsin-like activity.

The condensation reaction between the compounds is carried out under conditions suitable for the peptide bond forming reaction of the enzyme described above. The pH is preferably around 5-8, especially around 6-7,
The reaction temperature is preferably 0 to 50°C, particularly 20 to 40°C. It is desirable that the compound and compound concentrations be as high as possible. Furthermore, the compound and the compound are 1:
The reaction is preferably carried out at a molar ratio of 1 to 100:1, particularly around 20:1 to 1 to 100:1. A suitable organic solvent miscible with water is added to the reaction solution. Addition of an organic solvent is effective not only in reducing the concentration of water in the reaction solution and suppressing the hydrolysis reaction, which is a reverse reaction, but also in significantly increasing the solubility of the compound and the compound. As the organic solvent, for example, methanol, ethanol, dimethylformamide, dimethyl sulfoxide, glycerin, etc. are used alone or in combination. Especially 0-65%, especially 40
It is recommended to use a concentration of ~60%. Generally, when using an organic solvent, the ratio to water is determined by considering the solubility of raw materials, denaturation of enzymes, hydrolysis reaction, etc. As a buffer for the reaction solution, trishydroxymethylaminomethane (Tris), carbonate, or the like is used.

The enzyme concentration in the reaction solution varies depending on the substrate concentration and enzyme activity, but when using commercially available crystalline trypsin, it is preferably around 1 mg/ml to 10 mg/ml. The enzyme may be used as it is, or may be used as an immobilized enzyme bound to or included in a suitable insoluble carrier.

The reaction time varies depending on the reaction conditions, but it is usually sufficient to take the time required for the enzymatic reaction to reach equilibrium, which is usually about 3 to 72 hours, and in most cases about 6 to 24 hours.

After the reaction, a combination of methods commonly used for separating peptides was used to obtain human insulin, in which the threonine at position 30 of the B chain has a protecting group on the carboxyl group and may have a protecting group on the hydroxyl group. , then the intended person gets insulin.

For example, after the reaction is completed, the reaction solution is subjected to gel filtration,
Unreacted compounds and enzymes are isolated and recovered. The recovered compounds and enzymes can be reused as they are. The remainder is subjected to appropriate chromatography, and the generated threonine at position 30 of the B chain has a protecting group on the carboxyl group and human insulin which may have a protecting group on the hydroxy group and unreacted des-
B30 - Separate insulin. The latter can be reused as a compound. The former is further subjected to a protecting group elimination reaction to produce human insulin.

The method for removing the protecting group differs depending on the protecting group used, but it can be carried out according to the usual method. It is eliminated upon treatment with trifluoroacetic acid in the presence of agents such as anisole. When a protecting group that can be removed by a single elimination process, such as the former, is used to protect the side chain functional group and carboxyl group of threonine, the elimination process is simple and the yield is improved. Even when the carboxyl terminal is converted to another ester, the protecting group can be removed by appropriate hydrolysis treatment.

The insulin obtained from human insulin, in which the threonine at position 30 of the B chain of the present invention has a protective group on the carboxyl group and may have a protective group on the hydroxyl group, has a hypoglycemic effect as described above for the treatment of diabetes. Useful as a medicine or reagent. Human insulin synthesized from pig insulin by the method of the present inventors showed an effect equivalent to that of bovine insulin in a hypoglycemic test on mice.

Human insulin synthesized from the compound of the present invention is
It is formulated and administered to humans in the same way as commercially available swine and bovine insulin. That is, adding zinc chloride etc. to form a zinc complex, adding buffering agents such as sodium hydrogen phosphate or sodium acetate, and adding sodium chloride to make it an isotonic agent.
Furthermore, an injection is manufactured using a preparation method used for commercially available insulin preparations, such as adding a preservative such as cresol, phenol, or alkyl ester of paraoxybenzoic acid (eg, methyl, ethyl, propyl, butyl ester, etc.). The dosage of such injections varies depending on the patient's symptoms, but
It may be administered in the same manner as commercially available insulin preparations, and it is recommended that adults administer about 1 to 100 units per day.

In the Examples below, production examples of the compounds of the present invention are shown, but the Examples are not intended to limit the present invention in any way.

In addition, the abbreviations used in the examples represent the following meanings.

Ala Alanine Ile Isoleucine Arg Arginine Leu Leucine Asn Asparagine Lys Lysine Asp Aspartic acid Phe Phenylalanine CySO ₃ H Cysteic acid Pro Proline Glu Glutamic acid Ser Serine Gln Glutamine Thr Threonine Gly Glycine Tyr Tyr Tyrosine His Histidine Val Valine OBu ^t t-Butyl ester residue Furthermore, des-B30-insulin refers to insulin in which the amino acid at position 30 of the insulin B chain is deleted, such as in porcine insulin, in which alanine is deleted.

Example 1 (1) Des-B30-insulin (pig type) 500 mg of porcine insulin was dissolved in 100 ml of 0.1 M ammonium bicarbonate (pH 8.3), and crystalline carboxypeptidase A (manufactured by Worthington Co., Ltd., treated with diisopropylfluorophosphate, 49u/
mg) and react at room temperature for 8 hours.
The reaction was stopped when the amount of alanine produced was 0.77M/1M insulin, and after freeze-drying the reaction product,
Ultra-fine particle Cephadex dissolved in 0.5M acetic acid
Adsorb onto G50 (trade name) column (3.5 x 95cm) and add 0.5M acetic acid to 11.5ml per fraction.
It elutes with Fractions No. 40 to 60 are collected and lyophilized to obtain 460 mg of the title compound. Yield 92%.

The amino acid analysis values obtained by subjecting the product to acid hydrolysis (6M hydrochloric acid, 110°C, 24 hours) are as follows. Values in parentheses indicate theoretical values. The same applies to the following.

Lys 1.00(1), His 1.91(2), Arg 0.95(1), Asp 3.21(3), Thr 2.09(2), Ser 2.97(3), Glu 7.35(7), Gly 4.29(4), Ala 1.26 (1), _CySO3H 5.94(6), Val 3.86(4), Ile 1.55(2), Leu 6.53(6), Tyr 4.08(4), Phe 3.22(3), Pro 1.2(1).

(2) [B30-Thr-OBU ^t ]-insulin (pig type) 100 mg (10 mM) of the product from (1) and 154 mg (500 mM) of L-threonine-t-butyl ester in an ethanol/dimethylformamide mixture (1:1)
0.5M containing 4 mg of crystalline trypsin (Worthington, recrystallized three times) dissolved in 1.05 ml.
Mix with 0.75 ml of borate buffer (pH 6.5). This mixed solution contains tosyl-L-phenylalanine chloromethyl ketone (hereinafter referred to as TPCK).
be contained at a final concentration of 0.01mM. The mixture was kept at 37°C overnight. The production of the target compound was confirmed by high performance liquid chromatography, and the yield was determined to be 75%. After making the mixture acidic with glacial acetic acid, ultrafine grains of Cephadex are added.
Gel filtration is performed using a G50 (trade name) column (4.2 x 130 cm), and the mixture is divided into a trypsin-containing fraction, a fraction containing the title insulin derivative, and a fraction containing L-threonine-t-butyl ester. Enzyme activity and ninhydrin reaction were measured, and it was confirmed that trypsin and threonine t-butyl ester were each recovered at 50%. The fraction containing the insulin derivative is lyophilized to obtain 89 mg of a crude powder of the title compound.

Then 0.01M Tris buffer (pH 7.6) and 7M
DEAE buffered with urea - Cephadex A25
(Product name) column (1.9 x 24.5 cm) at 4°C, and after flowing 800 ml of the above buffer, elution was performed with a linear concentration gradient up to a salt concentration of 0.3M, and 0.08 to 0.09M. Fraction A near the concentration and fraction B near the 0.13 to 0.14M concentration are sent sequentially. Each fraction was immediately dialyzed against 0.01M ammonium acetate solution in a cold place for 3 to 4 days, and then lyophilized. 35 mg of powder was obtained from fraction A and 27 mg from fraction B.
get. High performance liquid chromatography and polyacrylamide gel electrophoresis confirmed that the former was the title compound and the latter was a mixture of des-B30-insulin (pig type) and the title compound.

The values measured by high performance liquid chromatography and polyacrylamide gel electrophoresis of the title compound are shown below.

Polyacrylamide gel electrophoresis (pH 8; 10
% gel; 18.1 mA/cm ² ; electrophoresis time 100 minutes): Travel distance 4.3 cm.

High performance liquid chromatography [Nucleosil 7C ₁₈ (trade name) (Macherey-
Nagel); 4 mm x 50 mm (pre-column) and 4 mm x 250 mm (main column); 32% acetonitrile-buffer solution (5mM phosphoric acid,
5mM sodium n-butylsulfonate,
50 mM sodium sulfate, pH 3.0); flow rate: 2 ml per minute]: retention time 13.2 minutes.

(3) Human insulin trifluoroacetic acid 2 containing 0.2 ml of anisole
ml to 30 mg of the compound obtained in (2) above, and stirred at room temperature for 30 mg.
Keep it for a minute. After removing trifluoroacetic acid in a nitrogen stream and extracting anisole with 15 ml of ether in the presence of 2 ml of 1N acetic acid, the acetic acid portion was lyophilized to obtain 28 mg of the title compound. If the purity of the raw material is 77%, the yield is 43%.

This product was identified as the title compound based on amino acid analysis, slab gel electrophoresis, and high performance liquid chromatography.

The results of amino acid analysis conducted under the same conditions as (1) are as follows.

Lys 1.00(1), His 1.89(2), Arg 0.87(1), Asp 3.32(3), Thr 3.16(3), Ser 2.99(3), Glu 7.35(7), Pro 1.29(1), Gly 4.39 (4), Ala 1.26(1), CySO ₃ H 4.6(6), Val 3.93(4), Ile 1.61(2), Leu 6.51(6), Tyr 3.68(4), Phe 3.19(3).

Slab gel electrophoresis (pH 8; 10% gel;
18.1mA/cm ² ; migration time 100 minutes): moving distance 6.0cm.
High performance liquid chromatography [Nucleosil 7C ₁₈ (trade name) (Macherey-Nagel
); 4mm x 50mm (pre-column) and 4mm x
250mm (main column); 32% acetonitrile buffer solution (5mM phosphoric acid, 5mM sodium n-butylsulfonate, 50mM sodium sulfate,
pH 3.0); Flow rate: 2 ml per minute]: Retention time 4.97 minutes.

Claims (1)

[Claims] 1 Human insulin is obtained by subjecting human insulin, in which the threonine at position 30 of the B chain has a protecting group on the carboxyl group and may have a protecting group on the hydroxyl group, to a protective group removal treatment. Method for producing human insulin.