RU2252782C2 - Method for production of insulin drug of durable action - Google Patents

Abstract

FIELD: pharmaceutical industry, in particular high quality insulin drug of durable action.

SUBSTANCE: claimed method includes providing of human insulin ester by transpeptization of porcine insulin at molar excess of threonine di-tert-butyl ester in aqueous/organic medium in presence of trypsin; acidifying for reaction inhibiting; chromatography purifying of obtained human insulin; deblocking of protective groups with trifluotoacetic acid and purifying of obtained crude human insulin; with subsequent dissolution in diluted acid, addition of acid solutions of zinc ions and protamine sulfate, mixing with buffered solution of m-cresol, phenol and glycerol; and solution conditioning to form crystals. Weight ratio of trypsin and porcine insulin is 1:300-1000. Before acidifying of reaction media reaction is additionally inhibited by mixture dilution with water by 2-3 times. Human insulin ester is purified using HPLC followed by deposition of ester derivatives fractions in presence of zinc ions and protective group deblocking to produce crystals of crude human insulin, which is purified again by HPLC, wherein both purifying processes are carried out using sorbent DIASOGEL ODS (C18) with particle size of 15 mum and pore size of 120 A as stationary phase, and as mobile phase in the first step 0.06 M-glycine HCl buffer, containing 0.015 M of ammonium sulfate and 20-30 % of propan-2-ol, having pH 2.5 is used, and in the second one 0.05 M acetate buffer containing 10-20 % of propan-2-ol with pH 2.5 is used. Then purified human insulin crystals are step-by-step dissolved in diluted acid to obtain fine dispersed suspension of insulin crystals in water; then diluted acid is added thereto, and mixture obtained after blending with buffered solution of cresol, phenol and glycerol, is held at 18-21°C for 20-22 h.

EFFECT: effective and economical method for insulin production with increased yield and purity; insulin of durable action and low immunological properties.

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Description

The invention relates to the pharmaceutical industry and can be used in the manufacture of high-quality finished dosage forms (GLF) of prolonged-acting insulins.

A known method of obtaining a pharmaceutical preparation of porcine insulin, which includes water-alcohol extraction of the pancreas of animals, purification of the obtained extract from ballast proteins, preparation of a concentrated solution of insulin in the presence of sodium chloride, sodium acetate, the corresponding amount of zinc ions and seeds, followed by dilution of this mixture in 10 times (Schlichtkrull J. (1956 b) Insulin Crystals IV. The preparation of nuclei, seeds and monodisperse insulin crystal suspensions. Fcta Chem Scand 11: 299-302).

Thus obtained finished dosage form of insulin contains a large number of impurities that cause unwanted allergic reactions and complications. In addition, this method is characterized by significant loss of insulin during the production process.

Also known is the “Method for the production of semi-synthetic human insulin” (US patent No. 4400465 from 1983), in which the human insulin ester is obtained in two stages: in the first stage, alanine is cleaved from porcine insulin with carboxypeptidase A at position B30, and sewn onto the second trypsin threonine ester. The reaction is carried out with a molar excess of the threonine derivative with respect to desalanininsulin from 5: 1 to 1000: 1, in the presence of trypsin or a trypsin-like enzyme. The resulting insulin ester is purified by low pressure chromatography, the protective groups are removed and human insulin is precipitated, which is then dissolved in dilute acid to obtain a sustained-release glyphosate, mixed with the necessary additives and the solution is kept until crystals form. But, as you know, such a two-stage preparation of an insulin ester derivative leads to an increase in the amount of impurities and to a decrease in the yield of human insulin ester in comparison with the transpeptidation reaction (US patent No. 4343898, p. 9, 1982), which means that it does not allow to obtain a good improved product yield.

Closest to the claimed method is the method described in US patent No. 4601852 A, published in 1986, which discusses a method of preparing insulin preparations, which receive human insulin ester by transpeptization of porcine insulin with a molar excess of threonine di-tert-butyl ether in water -organic medium in the presence of trypsin. The molar excess of threonine ester relative to insulin is claimed to be 10: 100 fold. The weight ratio of porcine insulin and trypsin is 1: 1-100. Then the reaction is inhibited by acidification of the reaction medium, human insulin ester is isolated, low-pressure chromatographic purification (gel filtration) is carried out, purified insulin ester is isolated by concentration and precipitation, the protective groups are removed in a known manner (for example, trifluoroacetic acid) and crude human insulin is isolated, which dissolved in dilute acid, add acidic solutions of zinc ions and protamine sulfate, mixed with buffered solutions of m-cresol, phenol and glycerol and incubated obtained mixture until a suspension of sustained-release insulin GLF crystals is formed. This method allows to increase the yield of human insulin ester, which determines the yield of human insulin during production, up to 60% (and under certain conditions even up to 90%) and significantly reduce the amount of impurities (up to 7%).

But the disadvantages of this method are still quite high immunological properties of the drug and significant loss of insulin in the production process.

The objective of the invention is the creation of an effective, economical method of preparing a finished dosage form of insulin prolonged action with low immunological properties, which guarantees an increase in the output of insulin in the production process.

The problem is solved in that in a method for preparing a sustained-release insulin dosage form, which comprises preparing human insulin ester by transpeptidating porcine insulin with a molar excess of threonine di-tert-butyl ether in an aqueous organic medium in the presence of trypsin, inhibiting the reaction by acidification, chromatographic purification of the obtained human insulin ester, deprotection with trifluoroacetic acid and purification of the obtained crude human insulin, its subsequent dissolution in dilute acid, adding acidic solutions of zinc ions and protamine sulfate, mixing with buffered solutions of m-cresol, phenol and glycerol, keeping the solutions until crystals form, according to the invention, the production of human insulin ester is carried out at a weight ratio of trypsin and porcine insulin equal to 1: 300- 1000, before acidification of the reaction medium, an additional inhibition of the reaction is carried out by diluting the reaction mixture with water by a factor of 2–3, and human insulin ether is purified by HPLC X, followed by precipitation of the fractions of the ether derivative in the presence of zinc ions, deprotection and obtaining crude human insulin crystals, which are purified by repeated HPLC, both purification processes being carried out using a reverse phase type sorbent as a stationary phase, for example, DAISOGEL ODS (C18 ), with a particle size of 15 μm and a pore size of 120 Å, and as the mobile phase in the first stage, 0.06 M-glycine Hcl buffer containing 0.015 M ammonium sulfate and propanol-2 with a concentration of from 20 to 35% at pH is used 2.5, and on the second is 0.05 M acetate buffer with a propanol-2 content of 10 to 20% at pH 2.5, then the purified crystals of human insulin are gradually dissolved in dilute acid, and a fine suspension of insulin crystals in water is first obtained, and then they are added to it dilute acid, and the mixture obtained after mixing with buffered solutions of m-cresol, phenol and glycerol is kept at a temperature of 18-21 ° C for 20-22 hours.

Associated related impurities of porcine insulin are pork proinsulin and products of modification and degradation of porcine insulin.

The aqueous-organic medium is water with a mixture of aprotic (e.g. dimethylacetamide) and protic (e.g. 1.2 ethanediol, 1.4 butanediol, 1.5 pentadiol) solvents.

Trypsin in the presented technical solution may be pork or bovine.

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 2-3 times before acidification allows you to directly apply the diluted mixture to the column of a high performance liquid chromatography (HPLC) system, which eliminates the need to perform a number of technological operations that are described in the prototype: that is, precipitation of the reaction mixture, separation sediment by centrifugation, dissolution of the precipitate. 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.

Final cleaning at the first stage of the preparation of HLF crystals of human insulin, which are obtained after removal of the protective groups in a known manner with trifluoroacetic acid and direct crystallization, is also carried out on a column of a high performance liquid chromatography system and allows to achieve even better results.

The applicant uses a reversed-phase sorbent, for example, DAISOGEL ODS (C18), with a particle size of 15 μm and a pore size of 120 Å as a stationary phase, and 0.06 M-glycine HCl buffer containing 0.015 M ammonium sulfate and propanol-2 with a concentration of from 20 to 35% at pH 2.5, and on the second - 0.05 M acetate buffer with a content of propanol-2 from 10 to 20% at pH 2.5.

The conditions of the transpeptidation reaction, in particular, the claimed enzyme-substrate ratio equal to 1: 300-1000, allows to increase the degree of conversion of porcine insulin or the output of human insulin ester, which characterizes the yield of the final product, up to 98% and reduce the number of difficult to remove impurities to 1.5 % An additional purification of the obtained crystals of human insulin is also aimed at increasing the degree of elimination of impurities - up to 0.9%.

In the second stage of the preparation of sustained-release human insulin GLF, the obtained crystals of human insulin are dissolved in diluted acid in stages: first, they are suspended in water and mixed well until a fine suspension is obtained, and then 1 M Hcl is added to it. The preparation of a finely dispersed suspension in the first stage, that is, ensuring that each individual small particle of insulin is surrounded on all sides by water, creates the conditions for their rapid dissolution in dilute acid in the second stage. Thus, the contact time of insulin crystals with acid decreases, which, in accordance with the studies of the authors, leads to a decrease in the amount of insulin A21-deamide formed and, therefore, to an increase in the purity of the drug. Then acid solutions of zinc ions and protamine sulfate are added, mixed with buffered solutions that contain m-cresol, phenol and glycerin. The resulting solution was maintained at a temperature of 18-21 ° C for 20-22 hours. Such a regime of crystal growth allows one to obtain a crystalline suspension with tetragonal crystals of a more uniform size from 1 to 25 μm in length, which provides a more uniform release of insulin in the human body (European Pharmacopoeia 2002, 4th edition, p. 1374, length crystals allowed from 1 to 60 microns).

The solution to this problem is illustrated by examples of specific performance.

Example 1

6 g of crude porcine insulin were suspended in 24 ml of water, 3.0 ml of acetic acid, 180 ml of 0.7 M threonine di-tert-butyl ether in a mixture of dimethylacetamide and 1.4 butanediol, 19 mg of trypsin in 12 ml of 0.05 were added M calcium acetate, the reaction mixture was stirred for 16 hours at a temperature of 23 ° C. The reaction was inhibited by 2 times dilution with water relative to the volume of the reaction mixture and the pH of the mixture was adjusted to 2.5 to 10% Hcl. The diluted reaction mixture was filtered through a 0.2 μm filter for application to an HPLC column. As a stationary phase, a DAISOGEL ODS (C 18) reverse phase sorbent with a particle size of 15 μm and a pore size of 120 Å was used. For the mobile phase, 0.06 M glycine-Hcl buffer was used, which contained 0.015 M ammonium sulfate with a concentration of propanol-2 from 20 to 35% at pH 2.5. The fractions of the ether derivative were 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. 5.0 g of the obtained human insulin ester was dissolved in 60 ml of trifluoroacetic acid, 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 carried out 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 with a propanol-2 content of 10 to 20% at pH 2.5 was used. The human insulin fraction was crystallized at pH 5.8, the crystal suspension was filtered off, washed with water and freeze-dried. The yield of human insulin was 4.5 g. Using an enzyme-linked immunosorbent assay, it was found that the content of porcine proinsulin in the resulting human insulin was less than 1 μg / g. As for the other related impurities, using the analytical system of HPLC, their content was established - 0.85%.

The resulting human insulin was placed in 320 ml of water, and then mixed until a fine suspension was obtained, an amount of 1 M Hcl was added so that the pH of the solution was not lower than 3.1. 0.355 g of protamine sulfate was dissolved in 58.4 ml of water, adding 1 M Hcl to the same pH. 0.0252 g of zinc chloride was added to the protamine sulfate solution. Then all the prepared acidic solutions were mixed. Then a buffer solution was prepared from 5.88 g of sodium phosphate monosubstituted two-water in 2.32 L of water, 44.8 g of glycerol, 5.25 g of m-cresol and 2.1 g of phenol were added, stirred for 30 minutes and the pH of the mixture was adjusted up to 7.8. Then, the buffer solution was subjected to sterilizing filtration and a mixture of acidic solutions, protamine sulfate, zinc chloride and insulin was filtered there. The resulting mixture was kept, stirring, at a temperature of 20 ° C for 21 hours to obtain a crystalline suspension of sustained-release insulin GLF. The shape and size of the obtained crystals were determined using a microscope at 400 × magnification. The crystals had a tetragonal shape and a length of 3 to 21 μm. Long-acting insulin GLF studies using an HPLC analytical system showed that the pharmaceutical preparation has an activity of 40 IU / ml, contains high molecular weight compounds of 0.4%, related cognitive impurities of 0.8%, insulin A21-desamide 0.4% and has supernatant activity 0.3 IU / ml.

Example 2

The conditions for obtaining human insulin crystals and other operations for the preparation of sustained-release insulin GLF, as in Example 1, except that at the first stage of the process 30 mg of trypsin in 12 ml of 0.05 M calcium acetate was added, and the reaction was carried out for 10 hours at a temperature of 23 ° C. The output of human insulin was 3.36 g, and according to HPLC, the number of related related impurities in it was 4.2%. Studies of the finished dosage form of prolonged-acting insulin according to Example 2 using HPLC showed that the activity of the pharmaceutical preparation was 39.2 IU / ml, the content of high molecular weight compounds was 0.7%, related concomitant impurities were 4.1%, A21-insulin desamide 2.0 %, supernatant activity of 0.4 IU / ml. The length of the crystals was 2-23 microns.

Example 3

The conditions for obtaining human insulin crystals and other operations for the preparation of a sustained-release insulin dosage form as in Example 1, except that 4.8 mg of trypsin in 12 ml of 0.05 M calcium acetate was added in the first stage of the process, and the reaction the mixture was stirred for 20 hours at a temperature of 20 ° C. The output of human insulin was 3.0 g. Studies of the pharmaceutical preparation according to Example 3 showed that its activity was 38.5 IU / ml, the content of high molecular weight compounds was 0.8%, related concomitant impurities were 1.8%, A21-insulin desamide 0, 8%, supernatant activity 0.5 IU / ml. The length of the crystals was 2-25 μm.

Example 4

The conditions for obtaining human insulin crystals and other operations for the preparation of a sustained-release insulin dosage form as in Example 1, except that in the first stage of the process 180 ml of 0.51 M threonine di-tert-butyl ether in dimethylacetamide mixture were added and 1.5 pentadiol, and for the purification of human insulin ester and human insulin crystals

DAISOGEL ODS sorbent (C 18) with a particle size of 15 μm and a pore size of 200 Å. An enzyme immunoassay of the obtained purified human insulin showed a pork proinsulin content of less than 1 μg / g, a content of related impurities determined by HPLC, 1.65%. Studies of sustained-release insulin GLF showed that its activity was 39.3 IU / ml, the content of high molecular weight compounds was 0.5%, related concomitant impurities were 1.8%, insulin A21-desamide 0.9%, and the supernatant activity was 0.35 IU / ml The length of the crystals was 1-26 μm.

Example 5

The conditions for obtaining human insulin crystals and other operations for the preparation of a sustained-release insulin dosage form as in Example 4, except that the DAISOGEL ODS sorbent (C 18) with a particle size of 40 to 60 μm and a pore size of 120 Å was used. An enzyme-linked immunosorbent assay of the obtained purified human insulin showed a pork proinsulin content of 2.1 μg / g and a content of related impurities of 3.75% (HPLC method). Studies of prolonged-acting insulin GLF showed that its activity was 39.0 IU / ml, the content of high-molecular compounds was 0.6%, related concomitant impurities were 4.1%, insulin A21-desamide was 1.9%, and the supernatant activity was 0.35 IU / ml The length of the crystals was 2-28 μm.

Example 6

The conditions for operations for the preparation of a sustained-release insulin dosage form as in Example 1, but 0.1 M glycine-Hcl, which contained 0.1 M ammonium sulfate and propanol-2 with a concentration of 0.15, were used as the mobile phase in the purification of human insulin ester from 20 to 35% at pH 2.5. The content of porcine proinsulin in the obtained human insulin was less than 1 μg / g (enzyme immunoassay), and the content of related impurities was 1.75% (HPLC method). The characteristics of prolonged-acting insulin GLF were as follows: activity was 39.2 IU / ml, high molecular weight compounds 0.5%, related co-impurities 2.1%, insulin A21-desamide 1.7%, and supernatant activity 0.39 IU / ml The length of the crystals was 2-36 μm.

Example 7

The conditions for operations for the preparation of a sustained-release insulin dosage form as in Example 1, but 0.1 M acetate buffer containing 10 to 20% concentration of propanol-2 at pH was used as the mobile phase in the purification of the obtained human insulin crystals 2.5.

The content of porcine proinsulin in the obtained human insulin was 2.2 μg / g (enzyme-linked immunosorbent assay), and the content of related impurities was 2.85% (HPLC method). The characteristics of prolonged-acting insulin GLF were as follows: the activity was 38.9 IU / ml, the content of high molecular weight compounds was 0.7%, related concomitant impurities were 3.1%, A21 insulin desamide was 1.5%, and the supernatant activity was 0.37 IU / ml The length of the crystals was 2-34 μm.

Example 8

The conditions for operations for the preparation of a sustained-release insulin dosage form as in Example 1, except that the solution obtained after mixing with buffered solutions of m-cresol, phenol and glycerol was kept at a temperature of 14 ° C. Studies of the pharmaceutical preparation of prolonged-acting insulin in Example 8 showed that its activity, the content of macromolecular compounds related to related impurities, A21-insulin desamide, and the activity of the supernatant were at the level described in Example 1, but within the limits of determination error. However, 95% of the crystals were 1 to 55 μm long.

Example 9

The conditions for operations for the preparation of a sustained-release insulin dosage form as in Example 1, except that the solution obtained after mixing with buffered solutions of m-cresol, phenol and glycerol was kept at a temperature of 25 ° C. Studies of the pharmaceutical preparation of prolonged-acting insulin in Example 9 showed that its activity, the content of high molecular weight compounds, related concomitant impurities, A21-insulin desamide were at the level described in Example 1, but within the limits of determination error. The crystals had a length of 1 to 55 μm and the activity of the supernatant was 1.2 IU / ml, indicating an incomplete crystallization process

Example 10

The conditions for conducting operations for the preparation of a sustained-release insulin dosage form as in Example 1, except that the solution obtained after mixing with buffered solutions of m-cresol, phenol and glycerol was kept for 17 hours. Studies of the pharmaceutical preparation of prolonged-acting insulin in Example 10 showed that its activity, the content of macromolecular compounds, related concomitant impurities, A21-insulin desamide and the activity of the supernatant were at the level described in Example 1, but within the limits of determination error. The number of crystals with a length of 1 to 25 μm was only 55%.

Example 11

The conditions for operations for the preparation of a sustained-release insulin dosage form as in Example 1, except that the solution obtained after mixing with buffered solutions of m-cresol, phenol and glycerol was kept for 25 hours. Studies of the pharmaceutical preparation of prolonged-acting insulin in Example 11 showed that its activity, the content of high molecular weight compounds, related concomitant impurities, A21-insulin desamide and the activity of the supernatant were at the level described in example 1, but within the limits of determination error. The number of crystals with a length of 1 to 25 μm was 95%. The same result is obtained by standing the solution for 22 hours, that is, here the process of preparing the finished dosage form of insulin is simply unreasonably delayed.

Thus, as the comparison of example 1 with other examples shows, non-compliance with the claimed limits of the enzyme-substrate ratio (examples 2 and 3) leads to an increase in the content of impurities in the preparations and to an increase in insulin losses during production, a change in the claimed parameters of the HPLC system (examples 4- 7) to an increase in the content of concomitant impurities, and hence to an increase in the immunogenicity of preparations, failure to comply with the declared limits of the crystal growth regime (examples 8-11) leads to either unreasonable delay of the process of the production process, that is, to a decrease in its efficiency, either does not allow to obtain crystals with a smaller spread in length, which reduces the quality of insulin GLF. At the same time, as evidenced by example 1, adherence to the claimed technology allows to obtain a cost-effective and effective method of a high-quality finished dosage form of insulin prolonged action with reduced immunological properties.

Claims (1)

A method of preparing a sustained-release insulin dosage form, including preparing human insulin ester by transpeptidating porcine insulin with a molar excess of threonine di-tert-butyl ether in an aqueous-organic medium in the presence of trypsin, inhibiting the reaction by acidification, chromatographic purification of the obtained human insulin ester, deprotection trifluoroacetic acid and purification of the obtained crude human insulin, its subsequent dissolution in dilute acid, the addition of acid solutions of zinc ions and protamine sulfate, mixing with buffered solutions of m-cresol, phenol and glycerol and maintaining the solutions until crystals are formed, characterized in that the preparation of human insulin ester is carried out at a weight ratio of trypsin and porcine insulin equal to 1: 300-1000, before acidification of the reaction medium provides additional inhibition of the reaction by diluting the reaction mixture with water by a factor of 2-3, and purification of human insulin ester is carried out by HPLC followed by precipitation of the ether fractions derivative in the presence of zinc ions, deprotection and obtaining crystals of crude human insulin, which is purified by repeated HPLC, both purification processes using DIASOGEL ODS sorbent (C 18) as a stationary phase with a particle size of 15 μm and pore size 120 Å, and the mobile phase in the first stage uses 0.06 M-glycine HCL buffer containing 0.015 M ammonium sulfate and propanol-2 with a concentration of 20-35% and a pH of 2.5, and in the second - 0.05 M acetate buffer with a propanol-2 content of 10-20% at pH 2.5, then purified f human insulin crystals are gradually dissolved in dilute acid, and a fine suspension of insulin crystals in water is first obtained, and then diluted acid is added to it and the mixture obtained after mixing with buffered solutions of m-cresol, phenol and glycerol is kept at a temperature of 18-21 ° C for 20-22 hours