RU2261108C2 - Method for preparing human insulin combined preparation - Google Patents

Abstract

FIELD: chemical-pharmaceutical industry, pharmacy.

SUBSTANCE: method involves preparing human insulin ester by transpeptidation reaction of porcine insulin in the excess of threonine di-tert.-butyl ester in an aqueous-organic medium in the presence of trypsin, inhibition of reaction by acidification, purification of prepared insulin ester by chromatography method, removal of protecting groups with trifluoroacetic acid and purification of prepared human crude insulin. Preparing part of the combined preparation of short effect by dissolving the prepared insulin with concentrations 15%, 25% or 50% in diluted acid and mixing with preserving agent solutions, isotonic agent and substances with buffer capacity. Preparing part of the combined preparation of prolonged effect by dissolving remaining insulin in diluted acid also followed by addition of zinc ions acid solutions and protamine sulfate, mixing with buffered solutions of m-cresol, phenol and glycerol, keeping the solution up to formation of crystals and combination of prepared parts of the combined insulin preparation. The transpeptidation reaction is carried out in the weight ratio trypsin to porcine insulin = 1:(300-1000) and before acidification of reaction medium additional inhibition of reaction is carried out by dilution of the reaction mixture with water by 2-3 times. Insulin ester is purified by HPLC method followed by precipitation of fractions with ester derivative in the presence of zinc ions, removal of protecting groups and preparing crystals of crude insulin that are purified by repeated HPLC method. Both purifying processes are carried out by using sorbent DIASOGEL ODS (C18) as immobile phase with particles size from 15 mcm and pores size 120

, and at the first stage 0.06 M glycine-HCl buffer with 0.015 M of ammonium sulfate and propanol-2 with concentration from 20% to 35% at pH 2.5 is used as mobile phase, and at the second stage 0.05 M acetate buffer with the content of propanol-2 from 10% to 20% at pH 2.5 is used. In preparing both parts of the combined preparation crystals of insulin are dissolved in diluted acid by stage wherein firstly finely dispersed suspension of insulin crystals in water is prepared followed by addition of diluted acid to it. In preparing the combined preparation of prolonged effect a mixture obtained after mixing with buffered solutions of m-cresol, phenol and glycerol is kept at temperature 18-21°C for 20-22 h. Invention provides the development of effective, economy method for preparing the combined preparation of human insulin with low immunological properties that allows enhancing yield of insulin in the process of its manufacturing.

EFFECT: improved preparing method.

13 ex

Description

The invention relates to the pharmaceutical industry and can be used in the production of highly purified combined preparations of human insulin.

There is a method of obtaining a combined insulin preparation by obtaining insulin substance from the pancreas of animals, purifying the obtained extract from ballast proteins, preparing crystalline and amorphous parts of the combined preparation and mixing them (1951, Hallas-Moller 1956).

The combined preparation of insulin thus obtained contains a large amount of impurities harmful to human health, which cause undesirable allergic reactions and complications. This method is also characterized by significant losses of insulin during the preparation of its dosage form and the instability of the physical condition, because the amorphous part of the drug is conglomerated during storage, which significantly impairs the quality of the drug.

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: at 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, 15, 25 or 50% of which is then dissolved in dilute acid and mixed with solutions of preservative, isotonic agent and substances with buffer capacity to obtain part of a short-acting combined preparation, and to obtain part of a combined drug of prolonged action, the remaining human insulin is also dissolved in dilute acid, mixed with the necessary additives, solution until crystals and connect both parts combined insulin preparation. But, as you know, such a two-stage preparation of the insulin ether derivative leads to an increase in the amount of impurities in the reaction mixture and to a decrease in the insulin 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 to obtain part of a combined preparation of short action, and to obtain part of a combined preparation of prolonged action to acidic solutions of zinc ions and protamine sulfate are added to the creator of the obtained insulin, mixed with buffered solutions of m-cresol, phenol and glycerol and the resulting mixture is kept until a crystal suspension forms. 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 harmful impurities (up to 7%).

The objective of the invention is to provide an effective method for producing a combined preparation of insulin with a low content of impurities, which guarantees an increase in the output of insulin in the production process.

, и в качестве подвижной фазы на первом этапе используют 0,06 М-глицин HCl буфер, содержащий 0,015 М аммония сульфат и пропанол-2 с концентрацией от 20 до 35% при рН 2,5, а на втором - 0,05 М ацетатный буфер с содержанием пропанола-2 от 10 до 20% при рН 2,5, и при приготовлении обеих частей комбинированного препарата растворение кристаллов инсулина в разбавленной кислоте проводят поэтапно, причем сначала получают мелкодисперсную суспензию кристаллов инсулина в воде, а потом к ней добавляют разбавленную кислоту, причем при приготовлении части комбинированного препарата пролонгированного действия смесь, полученную после смешивания с забуференными растворами м-крезола, фенола и глицерина, выдерживают при температуре 18-21°С в течение 20-22 часов.The problem is solved in that in a method for producing a combined preparation of human insulin, including obtaining human insulin ester by transpeptization of porcine insulin with an excess of threonine di-tert-butyl ether in an aqueous organic medium in the presence of trypsin, acidification inhibition of the reaction, chromatographic purification of the obtained insulin ester, deprotection with trifluoroacetic acid and purification of the resulting crude human insulin, preparation of a portion of the short-acting combined preparation thereby dissolving 15, 25 or 50% of the obtained insulin in dilute acid and mixing with solutions of a preservative, isotonic agent and substances with a buffer capacity, preparing part of a prolonged combined preparation by dissolving the remaining insulin in dilute acid, followed by the addition of acidic solutions of zinc ions and protamine sulfate by mixing with buffered solutions of m-cresol, phenol and glycerol, keeping the solution until crystals form, and combining the resulting parts according to the invention, the transpeptidation reaction is carried out at a weight ratio of trypsin and porcine insulin equal to 1: 300-1000, before the acidification of the reaction medium, additional inhibition of the reaction is carried out by diluting the reaction mixture with water 2-3 times, and the human insulin ether is purified by HPLC followed by precipitation of the fractions of the ether derivative in the presence of zinc ions, deprotection and obtaining crude insulin crystals, which are purified by repeated HPLC Niemi, both the cleaning process is carried out using a sorbent as a stationary phase reversed-phase type, e.g. DAISOGEL ODS (C18), with a particle size of 15 microns and a pore size of 120

and as the mobile phase in the first stage use 0.06 M-glycine HCl buffer containing 0.015 M ammonium sulfate and propanol-2 with a concentration of from 20 to 35% at a pH of 2.5, and in the second - 0.05 M acetate a buffer with a propanol-2 content of 10 to 20% at pH 2.5, and in the preparation of both parts of the combined preparation, the dissolution of insulin crystals in diluted acid is carried out in stages, whereby a finely dispersed suspension of insulin crystals in water is obtained, and then diluted acid is added to it , moreover, when preparing a portion of the combined prolonged-release preparation, 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, and allows you to effectively clean the human insulin ester of porcine insulin, which is not reacted l, and from impurities-satellites of the transpeptidation reaction.

Re-use of HPLC for purification of obtained human insulin crystals is also carried out on a column of a high performance liquid chromatography system and allows achieving even better results.

, а в качестве подвижной фазы при очистке эфира инсулина использует 0,06 М-глицин НС1 буфер, содержащий 0,015 М аммония сульфат с концентрацией пропанола-2 от 20 до 35% при рН 2,5, а при очистке полученных кристаллов инсулина - 0,05 М ацетатный буфер с содержанием пропанола-2 от 10 до 20% при рН 2,5.As a stationary phase, the applicant uses a reverse phase sorbent, for example DAISOGEL ODS (C 18), with a particle size of 15 microns and a pore size of 120

and as the mobile phase in the purification of insulin ether uses 0.06 M-glycine HC1 buffer containing 0.015 M ammonium sulfate with a concentration of propanol-2 from 20 to 35% at pH 2.5, and when cleaning the obtained insulin crystals - 0, 05 M acetate buffer with a propanol-2 content of 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%.

For the preparation of parts of a combined preparation of both short and prolonged action, insulin crystals purified by HPLC are used. 15, 25 or 50% of all crystals are spent on the preparation of part of the short-acting combined preparation, and the remaining crystals are spent on the part of the prolonged-action combined preparation. In both cases, the crystals of human insulin are first suspended in water and mixed 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. So they get part of a short-acting combined insulin preparation. To obtain a part of a prolonged-acting insulin combined preparation, acidic solutions of zinc ions and protamine sulfate are added to a part of the crystals diluted in weak acid, 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).

Combined preparations in accordance with this method can be produced with different proportions of the soluble and crystalline parts of insulin, for example, 15/85, 25/75, 50/50. Such drugs retain their chemical and physical properties, regardless of the ratio of soluble and crystalline parts, and provide a simplification of the drug administration procedure and an increase in dosage accuracy.

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

Example 1

. Для подвижной фазы использовали 0,06 М глицин-HCl буфер, который содержал 0,015 М аммония сульфат и пропанол-2 с концентрацией от 20 до 35% при рН 2,5. Фракции эфирной производной осадили в присутствии ионов цинка при рН от 6,8 до 7,0, отцентрифугировали, полученный осадок промыли водой и высушили в вакуумном сушильном шкафу. 5,0 г полученного эфира инсулина человека растворили в 60 мл трифторуксусной кислоты, выдержали 40 минут при температуре 25°С, трифторуксусную кислоту отогнали на роторном испарителе при температуре не выше 30°С, полученный сырой инсулин человека растворили в воде и провели прямую цитратную кристаллизацию при рН 5,8.6 g of crude porcine insulin was 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 with 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 was used

. 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 pH 2.5. The ether derivative fractions 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 conducted 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, 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%.

Preparation of the crystalline part of a combined drug of prolonged action, which is 85% of the total amount of insulin.

2.295 g of human insulin crystals were placed in 170 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. 0.189 g of protamine sulfate was dissolved in 31 ml of water, adding 1 M HCl to the same pH. 0.0135 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 3.125 g of sodium phosphate monosubstituted two-water in 1.235 L of water, 23.8 g of glycerol, 2.23 m-cresol and 0.895 g of phenol were added, stirred for 30 minutes and the pH of the mixture was adjusted to 7.8. Then, the buffer solution was subjected to sterilizing filtration and a mixture of acid solutions of 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.

Preparation of the soluble portion of the combined short-acting preparation, which is 15%.

0.405 g of human insulin crystals were placed in 52.5 ml of water and then mixed well 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. Then, a buffer solution was prepared from 0.55 g of sodium phosphate monosubstituted two-water in 210 ml of water, 4.2 g of glycerol, 0.39 g of m-cresol and 0.157 g of phenol were added, stirred for 30 minutes and the pH of the mixture was adjusted to 7.4 . The insulin solution was mixed with a buffer solution and sterilized by filtration in a container with a crystalline suspension, stirred for an hour and poured into aseptic bottles. Studies of the combined insulin preparation using an HPLC analytical system showed that the preparation had a total activity of 39.9 IU / ml, the content of high molecular weight compounds was 0.3%, related related impurities 0.9%, A21 insulin desamide 0.4%, activity the supernatant was 6.5 IU / ml. The crystals examined under a microscope at 400x magnification had a tetragonal shape and a length of 3 to 21 microns.

Example 2

The conditions for obtaining the combined preparation of human insulin, as in example 1, except that the ratio of soluble and crystalline parts was 25/75. Studies of the combined insulin preparation of Example 2 using an HPLC analytical system showed that the activity of the drug, the content of high molecular weight compounds, related related impurities, insulin A21-deamide and the crystal sizes were at the level described in example 1, but within the limits of determination error. The activity of the supernatant was 10.3 IU / ml.

Example 3

The conditions for obtaining the combined preparation of insulin, as in example 1, except that the ratio of soluble and crystalline parts was 50/50. Studies of the combined insulin preparation according to Example 3 using an HPLC analytical system showed that the activity of the drug, the content of high molecular weight compounds, related related impurities, insulin A21-desamide and the crystal sizes were at the level described in example 1, but within the limits of determination error. The activity of the supernatant was 21 IU / ml.

Example 4

The conditions for obtaining the combined insulin preparation, 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 insulin yield was 3.76 g. Studies of the combined insulin preparation using an HPLC analytical system showed that the activity of the drug was 39.2 IU / ml, the content of high molecular weight compounds was 0.7%, related related impurities were 4.1%, and A21-insulin desamide 2.0%, and the activity of the supernatant of 6.4 IU / ml. The length of the crystals was 2-23 microns.

Example 5

The conditions for obtaining the combined insulin preparation, as in example 1, except that at the first stage of the process 4.8 mg of trypsin in 12 ml of 0.05 M calcium acetate was added, and the reaction was carried out for 20 hours at a temperature of 20 ° C. The insulin yield was 3.395 g. Studies of the combined insulin preparation according to Example 5 using an analytical HPLC system showed that the activity of the drug was 38.5 IU / ml, the content of high molecular weight compounds was 0.8%, related related impurities were 1.8%, A21-desamide insulin 0.8%, and the supernatant activity of 6.8 IU / ml. The length of the crystals was 2-25 μm.

Example 6

. Иммуноферментный анализ полученных кристаллов инсулина человека показал содержание свиного проинсулина менее 1 мкм/г. Исследования комбинированного препарата инсулина по примеру 6 с помощью аналитической системы ВЭЖХ показали, что активность препарата составляла 39,3 МЕ/мл, содержание высокомолекулярных соединений 0,7%, сопутствующих родственных примесей 1,8%, А21-дезамида инсулина 0,9%, а активность супернатанта 7,0 МЕ/мл. Длина кристаллов составляла 1-25 мкм.The conditions for the preparation of a combined insulin preparation as in Example 1, except that the DAISOGEL ODS sorbent (C 18) with a particle size of 15 μm and a pore size of 200 was used as the stationary phase

. An enzyme-linked immunosorbent assay of the obtained human insulin crystals showed a porcine proinsulin content of less than 1 μm / g. Studies of the combined insulin preparation of Example 6 using an HPLC analytical system showed that the activity of the drug was 39.3 IU / ml, the content of high molecular weight compounds was 0.7%, related cognitive impurities were 1.8%, A21 insulin desamide 0.9%, and the activity of the supernatant is 7.0 IU / ml. The length of the crystals was 1-25 μm.

Example 7

. Иммуноферментный анализ полученных кристаллов инсулина человека показал содержание свиного проинсулина 2,1 мкм/г. Исследования комбинированного препарата инсулина по примеру 7 с помощью аналитической системы ВЭЖХ показали, что активность препарата составляла 39,3 МЕ/мл, содержание высокомолекулярных соединений 0,6%, сопутствующих родственных примесей 4,1%, А21-дезамида инсулина 1,9%, а активность супернатанта 5,9 МЕ/мл. Длина кристаллов составляла 2-27 мкм.The conditions for obtaining a combined insulin preparation, as in example 1, except that the DAISOGEL ODS sorbent (C 18) with a particle size of 40 to 60 μm and a pore size of 120 was used as the stationary phase

. An enzyme-linked immunosorbent assay of the obtained human insulin crystals showed a porcine proinsulin content of 2.1 μm / g. Studies of the combined insulin preparation according to example 7 using an analytical HPLC system showed that the activity of the drug was 39.3 IU / ml, the content of high molecular weight compounds was 0.6%, related related impurities were 4.1%, insulin A21-insamide was 1.9%, and the activity of the supernatant is 5.9 IU / ml. The length of the crystals was 2-27 μm.

Example 8

The conditions for obtaining the combined insulin preparation as in Example 1, except that 0.1 M glycine-HCl buffer, which contained 0.1 M ammonium sulfate and propanol-2 with a concentration of 20, was used as the mobile phase in the purification of human insulin ester -35% at pH 2.5. An enzyme-linked immunosorbent assay of the obtained human insulin crystals showed a pork proinsulin content of less than 1 μm / g. Studies of the combined insulin preparation of Example 8 using an HPLC analytical system showed that the activity of the drug was 39.6 IU / ml, the content of high molecular weight compounds was 0.5%, related related impurities were 2.1%, A21 insulin desamide was 1.7%, and the activity of the supernatant is 5.9 IU / ml. The length of the crystals was 2-28 μm.

Example 9

The conditions for obtaining the combined insulin preparation as in Example 1, except that 0.1 M acetate buffer, which contained propanol-2 with a concentration of 20-35% at pH 2.5, was used as the mobile phase in the purification of the obtained human insulin crystals . An enzyme-linked immunosorbent assay of the obtained human insulin crystals showed a porcine proinsulin content of 2.2 μm / g. Studies of the combined insulin preparation of Example 9 using an HPLC analytical system showed that the activity of the drug was 39.4 IU / ml, the content of high molecular weight compounds was 0.7%, related cognitive impurities were 3.1%, A21-insulin desamide 0.9%, and the activity of the supernatant is 5.9 IU / ml. The length of the crystals was 2-26 μm.

Example 10

The conditions for obtaining the combined preparation of insulin, 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 combined insulin preparation of Example 10 using an HPLC analytical system showed that the activity of the drug, the content of high molecular weight compounds, related 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 11

The conditions for obtaining the combined preparation of insulin, 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 combined insulin preparation according to example 11 using the analytical HPLC system showed that the activity of the drug, the content of high molecular weight compounds, related related impurities, A21 -insulin insulin were at the level described in example 1, but within the limits of determination error. The length of the crystals ranged from 1 to 50 μm and the activity of the supernatant was 8.3 IU / ml, which indicated an incomplete crystallization process.

Example 12

The conditions for obtaining the combined preparation of insulin, 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 combined insulin preparation of Example 12 using an HPLC analytical system showed that the activity of the drug, the content of high molecular weight compounds, related related impurities, insulin A21-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 13

The conditions for obtaining the combined preparation of insulin, 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 combined insulin preparation according to example 13 using the HPLC analytical system showed that the activity of the drug, the content of high molecular weight compounds, related related impurities, insulin A21-deamide 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 when the solution is kept for 22 hours, that is, here the process is simply unreasonably delayed.

So, the analysis of examples of specific performance (examples 1-3) indicates that the claimed method can be obtained combined insulin preparations with different ratios of crystalline and soluble parts. Using this method allows to increase the output of human insulin, reduce the amount of impurities, that is, increase the purity of the drug. At the same time, the analysis of examples 4 and 5 shows that going beyond the claimed enzyme-substrate ratio leads to an increase in the amount of impurities in the preparation and to a decrease in insulin yield; a deviation from the claimed purification parameters of the human insulin ester and the obtained crystals of human insulin - examples 6-9 - also leads to a decrease in the purity of the drug, and failure to comply with the claimed crystal growth regimen (examples 10-13) does not allow to obtain more uniform crystals in length, and therefore ensure a more uniform release of insulin in the human body, that is, get better quality insulin preparations.

Claims (1)

A method of obtaining a combined preparation of human insulin, including the production of human insulin ester by transpeptidation of porcine insulin with an excess of threonine di-tert-butyl ether in an aqueous-organic medium in the presence of trypsin, inhibition of the reaction by acidification, chromatographic purification of the obtained insulin ester, deprotection of trifluoroacetic acid, and purification obtained crude human insulin, the preparation of part of a combined short-acting preparation by dissolving 15, 25 or 50% of the obtained and suline in dilute acid and mixing with solutions of a preservative, isotonic agent and substances with a buffer capacity, preparation of a part of a prolonged combined preparation by dissolving the remaining insulin also in dilute acid, followed by the addition of acidic solutions of zinc ions and protamine sulfate, mixing with buffered solutions of m-cresol, phenol and glycerol, keeping the solution until crystals form and combining the obtained parts of the combined insulin preparation, excellent In that the transpeptidation reaction is carried out at a weight ratio of trypsin and porcine insulin equal to 1: 300-1000, before the acidification of the reaction medium, additional inhibition of the reaction is carried out by diluting the reaction mixture with water by a factor of 2-3, and purification of human insulin ester is carried out by HPLC with subsequent precipitation of the fractions of the ester derivative in the presence of zinc ions, removal of the protective groups and obtaining crystals of crude insulin, which are purified by repeated HPLC, both cleaning processes they are bred using the DIASOGEL ODS sorbent (C 18) as a stationary phase with a particle size of 15 μm and a pore size of 120 Å, and 0.06 M-glycine HCL buffer containing 0.015 M ammonium sulfate and propanol-2 with a concentration of from 20 to 35% and a pH of 2.5, and on the second - 0.05 M acetate buffer with a content of propanol-2 from 10 to 20% at a pH of 2.5, and when preparing both parts of the combined preparation, dissolution crystals of insulin in dilute acid are carried out in stages, and a fine crystalline suspension is first obtained catching insulin in water, and then dilute acid is added to it, and when preparing part of a long-acting combined preparation, 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