RU2252225C2 - Method for production of semisynthetic human insulin - Google Patents

Abstract

FIELD: biotechnology, in particular for biochemical, immunological and biological investigation and for production of insulin pharmaceuticals.

SUBSTANCE: semisynthetic human insulin is prepared by transpeptidation of swine insulin in presence of trypsin in mass ratio trypsin/swine insulin of 1:300-1000 and molar excess threonyn di-tert-butyl ester in relation to swine insulin, in aqueous-organic medium. Reaction is inhibited by pH reducing and reaction mixture dilution with water by 2-3 times. Obtained human insulin ester is purified by HPLC followed by providing of human crude insulin crystals and repeated HPLC-purifying. Method of present invention makes it possible to increase human insulin ester yield up to 98 % and decrease by-product contamination in finished product up to 0.9 %.

EFFECT: method of improved yield; product of improved purity.

6 ex

Description

The invention relates to the field of biochemistry and pharmacy and can be used in biochemical, immunological and biological studies, as well as in the manufacture of insulin preparations for therapeutic purposes.

Known methods for producing a semisynthetic substance of human insulin from porcine insulin and from porcine insulin with related relatives by processing the feedstock with an excess of threonine ester in the presence of trypsin in an aqueous medium at a pH below the isoelectric point (Patent No. 4 601 852, USA, 1986) or the same feedstock using a combination of two types of enzymes: carboxypeptidase A and followed by addition of threonine di-tert-butyl ester, which is catalyzed by trypsin in free or immobilized gated state (Norre Seylers, Z. Physiol, Chem. 356, 1631).

The disadvantages of these methods is the formation of an intermediate product of the type Des- (Asp.sup.A21) -insulin, which has a low biological activity and a rather low yield of the final product due to cleavage of the Arg.sup.B22-Gly.syp.B23 bond with trypsin in an aqueous medium .

Closest to the present invention, the technical essence and the result that is achieved is a method for producing a semi-synthetic substance of human insulin, which involves the production of human insulin ester by transpeptidation of porcine insulin with concomitant related impurities in excess of threonine tert-butyl ether in an aqueous organic medium in the presence of trypsin and organic acid. This transpeptidation reaction is carried out with a molar excess of threonine tert-butyl ester with respect to porcine insulin of more than 5 and with a weight ratio of trypsin and porcine insulin from 1 to 1-200. Inhibition of the reaction is carried out by creating an acidic medium with pH 3, purification of the obtained human insulin ester is carried out by anionic and / or cationic chromatography, deprotection is carried out in a known manner by trifluoroacetic acid, after which crystallization of crude human insulin is carried out. Reaction conditions: the amount of water in the reaction mixture is more than 10% and less than 50% volume, the reaction temperature is less than 50 ° C (patent No. 4 343 898, USA, C 12 P 021/04).

This method allows to obtain the yield of human insulin ether, which determines the yield of the final product, more than 60% -90%, but it also has the same drawback - when using an enzyme-substrate ratio of 1: 1-200, under the conditions of the transpeptidation reaction, impurities (up to 7%) that cannot be removed in the subsequent stages of obtaining human insulin by known methods, resulting in a final product contaminated with these impurities. In addition, this method is time-consuming and multi-stage.

The objective of the invention is to provide a method for producing semisynthetic human insulin, which guarantees an increase in the yield of human insulin ester as a result of the transpeptidation reaction, a decrease in the amount of difficult to remove impurities in the final product, thus providing a biochemically active, immunologically safe human insulin applicable for therapeutic purposes and simplification is already existing method by reducing the number of operations.

The problem is solved in that in a method for producing semisynthetic human insulin, which includes producing human insulin ester with transpeptidium of porcine insulin with accompanying related impurities, which is carried out in the presence of trypsin with a molar excess of threonine di-tert-butyl ether in relation to porcine insulin in aqueous organic medium, inhibition of the reaction by lowering the pH, purification of the human insulin ester, deprotection and crystallization of human insulin, according to the invention, the transpeptidation of they are carried out at a weight ratio of trypsin and porcine insulin equal to 1: 300-1000, additional reaction inhibition is carried out by diluting the reaction mixture with water by a factor of 2-3, and human insulin ester is purified by HPLC followed by obtaining crystals of crude human insulin and repeated HPLC .

As the feedstock in the inventive method, pig insulin with concomitant related impurities, such as porcine proinsulin (up to 2%) and products of modification and degradation of porcine insulin, is used. Such insulin is also called crude insulin.

An aqueous-organic medium, which is water with a mixture of aprotic (e.g. dimethylacetamide, dimethyl sulfoxide) and protic (e.g. 1,2 ethanediol, 1,4 butanediol, 1,5 pentadiol) solvents.

Trypsin in the presented technical solution can be pork or bovine with an activity of 40-60 U / mg.

The threonine di-tert-butyl ester or its salt may be used in the transpeptidation reaction.

Additional inhibition of the transpeptidation reaction by diluting the reaction mixture with water by a factor of 2–3 allows directly applying the diluted mixture to a column of a high performance liquid chromatography (HPLC) system, which eliminates the need for a number of technological operations described in the prototype: that is, precipitation of the reaction mixture, separation of the precipitate by centrifugation dissolving the precipitate. The need to perform these operations after removal of the protective groups is also eliminated due to direct crystallization.

In addition, the use of the HPLC system makes it possible to efficiently purify human insulin ester from porcine insulin that has not reacted, and from impurities that accompany the transpeptidation reaction.

The final purification of the already final product - human insulin, which is obtained after removal of the protective groups in a known manner with trifluoroacetic acid and direct crystallization of crude human insulin, is also carried out on a column of high performance liquid chromatography and can achieve even better results.

The conditions of the transpeptidation reaction, in particular the claimed enzyme-substrate ratio, which distinguishes the claimed solution, can increase the degree of conversion of porcine insulin or the yield of human insulin ester up to 98% and reduce the number of difficult to remove impurities to 1.5%. An additional purification of the final product by the HPLC system is also aimed at increasing the degree of elimination of impurities - up to 0.9%.

Various methods of HPLC, peptide mapping and enzyme-linked immunosorbent assay were used for step-by-step control of the method for producing semisynthetic human insulin, evaluation of the final product and its identification.

The solution of the tasks is illustrated by examples of specific performance.

Example 1

100 mg of crude porcine insulin was suspended in 0.4 ml of water, 0.05 ml of acetic acid, 3 ml of 0.63 M threonine di-tert-butyl ether in a mixture of dimethylacetamide and 1.4 butanediol were added, 0.25 mg of trypsin with activity 50 U / mg in 0.2 ml of 0.05 M calcium acetate, the reaction mixture was stirred for 16 hours at a temperature of 23 ° C. The reaction was inhibited by diluting with water twice in relation to the volume of the reaction mixture, and the pH of the mixture was adjusted to 2.5 with 10% Hcl. Analysis of the reaction mixture showed that the yield of the insulin ester derivative was 98%. The diluted reaction mixture was filtered through a 0.2 μm filter for application to an HPLC column. The DAISOGEL ODS-AP (C-18) sorbent with a particle size of 15 μm and a pore size of 120 Å was used as the stationary phase. For the mobile phase, 0.06 M glycine-Hcl buffer was used, which contained 0.015 M ammonium sulfate and propanol-2 with a concentration of 20 to 35% at a pH of 2.5. The ether derivative fraction was precipitated in the presence of zinc ions at a pH of 6.8 to 7.0, centrifuged, the resulting precipitate was washed with water and dried in a vacuum oven. 82 mg of the obtained human insulin ester was dissolved in 1.0 ml of trifluoroacetic acid, the mixture was kept for 40 minutes at a temperature of 25 ° C, trifluoroacetic acid was distilled off on a rotary evaporator at a temperature not exceeding 30 ° C, the obtained crude human insulin was dissolved in water and spent direct citrate crystallization at pH 5.8.

The crystalline suspension was filtered, the crystals were washed with water, dissolved in 0.25 M acetic acid and applied to a column of an HPLC system. As the mobile phase, 0.05 M acetate buffer was used, which contained propanol-2 from 10 to 20% at pH 2.5. The human insulin fraction was crystallized at pH 5.8, the crystal suspension was filtered off, washed with water and freeze-dried. The output of human insulin was 75 mg. Using peptide mapping, it has been proven that this product is indeed human insulin. Then, by enzyme-linked immunosorbent assay, it was found that the remainder of the porcine proinsulin in the final product was less than 1 μg / g, which meets the pharmacopoeial requirements according to which the content of porcine proinsulin in human insulin should not exceed 10 μg / g. As for other related impurities, their content was established using an analytical system of HPLC - not more than 0.85%.

Example 2

50 mg of crude porcine insulin was suspended in 0.2 ml of water, 0.025 ml of acetic acid, 1.5 ml of 0.858 M threonine di-tert-butyl ether in a mixture of dimethylacetamide and 1.5 pentadiol, 0.165 mg of trypsin with an activity of 40 U / mg were added. in 0.1 ml of 0.05 M calcium acetate. The reaction mixture was stirred for 23 hours at a temperature of 18 ° C. Then, as in example 1, they did, but the reaction mixture was diluted with water three times in relation to the volume of the reaction mixture. The yield of human insulin ester was 40.5 mg, the content of related impurities was 0.8%.

Example 3

75 mg of crude porcine insulin was suspended in 0.3 ml of water, 0.04 ml of acetic acid, 2.25 ml of 1.14 M threonine di-tert-butyl ether in a mixture of dimethyl sulfoxide and 1.2 ethanediol, 0.08 mg of trypsin were added with an activity of 60 U / mg in 0.15 ml of 0.05 M calcium acetate. Then, as in example 1. The output of human insulin ester according to analytical HPLC was 97%. Analysis of the final product showed that the content of related related impurities in it is 0.84%.

Example 4

The reaction mixture and reaction conditions as in Example 2, except for the amount of trypsin-trypsin, was added 0.25 mg in 0.1 ml of 0.05 M calcium acetate. The reaction was carried out for 10 hours at a temperature of 23 ° C. According to HPLC, the yield of human insulin ester was 75%, the amount of difficult to remove impurities was 5%.

Example 5

The reaction mixture, as in example 4, except for the amount of trypsin. For the reaction, 0.04 mg of trypsin in 0.1 ml of 0.05 M calcium acetate was used. The reaction mixture was stirred for 20 hours at a temperature of 20 ° C. The yield of human insulin ester was 70%, and the amount of porcine insulin that did not react was 10%.

Example 6

50 mg of porcine proinsulin was suspended in 0.25 ml of water, 0.022 ml of acetic acid, 1.5 ml of 0.7 M threonine di-tert-butyl ether in a mixture of dimethylacetamide and 1.4 butanediol, 0.17 mg of trypsin at 0 were added. 1 ml of 0.05 M calcium acetate. The reaction conditions were, as in example 4. The reaction mixture was precipitated with 10 volumes of acetone and dried. Analysis of the precipitate by HPLC showed an 85% yield of the human derivative of insulin ether.

Thus, as examples of specific performance show, the use of the proposed method provides an increase in the yield of human insulin ester up to 98% and a decrease in the content of related related impurities in the final product to 0.9% (examples 1-3). At the same time, as shown by examples 4 and 5, in which the enzyme-substrate ratio of trypsin and porcine insulin are outside the claimed limits, the tasks in relation to the quality and quantity of the output product cannot be solved. Example 6 proves that under optimal conditions for the transpeptidation reaction, porcine proinsulin, which is an impurity in the feedstock, is 85% converted to human insulin ether, which, as is clear, also contributes to an increase in the yield of the final product.

Claims (1)

A method of producing semisynthetic human insulin, including the production of human insulin ester by transpeptidation of porcine insulin with concomitant related impurities, which is carried out in the presence of trypsin with a molar excess of threonine di-tert-butyl ether in relation to porcine insulin in an aqueous-organic medium, inhibition of the reaction by lowering pH, purification of the ether human insulin, deprotection and crystallization of human insulin, characterized in that the transpeptidization is carried out at a trypsin weight ratio a and porcine insulin equal to 1: 300-1000, additional reaction inhibition is carried out by diluting the reaction mixture with water 2-3 times, and human insulin ester is purified by HPLC followed by obtaining crystals of crude human insulin and re-performing HPLC.