RU2261107C2 - Method for preparing ready medicinal formulation of insulin with short effect - Google Patents

Abstract

FIELD: medicine, endocrinology, chemical-pharmaceutical industry, hormones.

SUBSTANCE: method involves preparing human insulin ester by transpeptidation reaction of porcine insulin in the molar excess of threonine di-tert.-butyl ester in an aqueous-organic medium in the presence of trypsin, inhibition of reaction by acidification, purification of preparing human insulin ester by chromatography, removal of protecting groups with trifluoroacetic acid and purification of prepared human crude insulin, its following dissolving in diluted acid and mixing with preserving agent solutions, isotonic agent and substances with buffer capacity. Preparing human insulin ester is carried out in the weight ratio trypsin to porcine insulin = 1:(300-1000) followed by additional inhibition of reaction by dilution of the reaction mixture with water by 2-3 times before acidifying. Purification of human insulin ester is carried out by HPLC method followed by precipitation of ester derivative fractions in the presence of zinc ions, removal of protecting groups and preparing human crude insulin crystals that is purified by repeated carrying out HPLC method. Both processes of purification are carried out by using sorbent DIASOGEL ODS (C18) as immobile phase with particles size from 15 mcm and pores size from 100

to 150

. At the first stage 0.06 M glycine - HCl buffer containing 0.015 M of ammonium sulfate and propanol-2 in the concentration from 20% to 35%, pH 2.5 is used as a mobile phase, and 0.05 M acetate buffer with the content of propanol-2 from 15% to 25%, pH 2.5 is used at the second stage. The purified human insulin is dissolved in diluted acid by stages wherein finely dispersed suspension of insulin crystals in water is prepared followed by addition a diluted acid to it. Invention provides the development of effective, economy method for preparing the ready medicinal formulation of insulin with short effect and with low immunological properties that provides reducing loss of insulin in the process of its preparing. Invention can be used in manufacturing ready insulin formulations of high quality and with short effect.

EFFECT: improved preparing method.

6 ex

Description

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

A known method of obtaining the finished dosage form (GLF) of porcine insulin by water-alcohol extraction of the pancreas of animals, purification of the extract from ballast proteins, dissolving the purified substance of insulin in portions in dilute acid, followed by mixing with solutions of methylparaben and sodium chloride (Schtichtkrull J. et al. , Insulin Rapitard and Insulin Actrapid. Acta Med Scand 177: 103-113).

This method allows to obtain a pharmaceutical preparation of insulin, which contains impurities of proinsulin, glucagon, self-statin, pancreatic polypeptide and other proteins (up to 45% -55%). But already 3 months after the start of insulin therapy in the blood serum of patients with the help of radio-immune research methods, antibodies to the above impurities are detected, which causes a number of undesirable allergic reactions, lipodystrophy, insulin resistance, the development of early complications. In addition, this method is characterized by significant loss of insulin in the preparation of its dosage form.

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, which is then dissolved in dilute acid and mixed with necessary additives to obtain short-acting GLF. But, as you know, such a two-stage preparation of an insulin ester derivative leads to an increase in the amount of impurities in the reaction mixture and to a decrease in the yield of human insulin ester in comparison with the transpeptidation reaction (US patent No. 4343898, p. 9, 1982), and therefore, does not allow get a good yield of pharmaceutical grade product with improved performance.

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, mixed with solutions of a preservative, isotonic agent and substances with a buffer capacity. As a preservative, m-cresol or phenol can be used, as isotonic and buffering agents, mono- or bisubstituted salts of phosphoric acid. 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%).

But the disadvantages of this method are still insufficient pharmaceutical purity of the drug and significant loss of insulin in the production process.

The objective of the invention is to provide an effective method for preparing a finished dosage form of short-acting insulin of pharmaceutical purity with improved characteristics, which guarantees a reduction in insulin losses during production.

, а 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 15% до 25% при рН 2,5, и растворение очищенного инсулина в разбавленной кислоте проводят поэтапно, причем сначала получают мелкодисперсную суспензию кристаллов инсулина в воде, а потом к ней добавляют разбавленную кислоту.The problem is solved in that in the method of preparing the finished dosage form of insulin of short action, including the production of human insulin ester by transpeptidation of porcine insulin with a molar 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 human insulin ester, deprotection with trifluoroacetic acid and purification of the obtained crude human insulin, its subsequent dissolution in a dilute acid and mixing with solutions of a preservative, isotonic agent and substances with a buffer capacity, 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, additional reaction inhibition is carried out by diluting the reaction mixture with water in 2-3 times before acidification, and the purification of human insulin ester is carried out by HPLC followed by precipitation of the fractions of the ether derivative in the presence of zinc ions, deprotection and crystals of crude human insulin, which is purified by repeated HPLC, both purification processes being carried out using a reversed-phase type sorbent as a stationary phase, for example DAISOGEL ODS (C18), with a particle size of 15 μm and a pore size of 100 to 150

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 pH 2.5, and in the second - 0.05 M acetate buffer with a propanol-2 content from 15% to 25% at pH 2.5, and the dissolution of purified insulin in diluted acid is carried out in stages, and a fine suspension of insulin crystals in water is first obtained, and then diluted acid is added to it.

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 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.

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 - satellites of the transpeptidation reaction.

The final cleaning at the first stage of obtaining human insulin crystals, 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.

, а в качестве подвижной фазы на первом этапе используют 0,06 М-глицин HCl буфер, содержащий 0,015 М аммония сульфат с концентрацией пропанола-2 от 20% до 35% при рН 2,5, а на втором - 0,05 М ацетатный буфер с концентрацией пропанола-2 от 15% до 25% при рН 2,5.As a stationary phase, the applicant uses a reverse phase sorbent, for example DAISOGEL ODS (C18), with a particle size of 15 microns and a pore size of 100 to 150

and as the mobile phase in the first stage use 0.06 M-glycine HCl buffer containing 0.015 M ammonium sulfate with a concentration of propanol-2 from 20% to 35% at pH 2.5, and in the second - 0.05 M acetate buffer with a concentration of propanol-2 from 15% to 25% at pH 2.5.

The conditions of the transpeptidation reaction, in particular the claimed enzyme-substrate ratio equal to 1: 300-1000, can increase the degree of conversion of porcine insulin or the yield 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 short-acting human insulin GLF, the crystals of human insulin are first suspended in water and mixed to obtain a finely divided suspension, and then added to the suspension with 1M HCl. 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, consequently, to an increase in the purity of the preparation.

As a preservative, m-cresol is used, as isotonic and buffering agents are glycerol and mono- or bisubstituted salts of phosphoric acid.

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. Фракцию 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 100 to 150 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 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. 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 15% to 25% propanol-2 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. An enzyme-linked immunosorbent assay revealed that the remainder of the 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 560 ml of water, and then mixed until a fine suspension was obtained, an amount of 1M HCl was added so that the pH of the solution was not lower than 3.1. Then, a buffer solution was prepared from 5.88 g of sodium phosphate monosubstituted two-water in 2.32 L of water, another 44.8 g of glycerol and 7.36 g of m-cresol were added, stirred for 30 minutes and the pH of the mixture was adjusted to 7.8. Then, the human insulin solution was mixed with a buffer solution and the pH was adjusted to 7.3. The volume of the solution was adjusted with water for injection to 2.8 L, it was sterilized by filtration, followed by aseptic filling into bottles and cartridges, capping and labeling. The study of the finished dosage form of short-acting insulin using an HPLC analytical system showed that the pharmaceutical preparation has an activity of 39.8 IU / ml, high molecular weight compounds of 0.25%, related related impurities of 0.75%, A21 insulin deamide of 0.3% .

Example 2

The conditions for obtaining human insulin crystals and other operations for the preparation of a short-acting insulin dosage form 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 in within 10 hours at a temperature of 23 ° C. Analysis by HPLC revealed that the number of related related impurities in the final product of the first stage of the process was 4.2%. Studies of the finished dosage form of short-acting insulin according to example 2 using HPLC showed that its activity was 38.2 IU / ml, the content of high molecular weight compounds 0.7%, related concomitant impurities 3.9%, A21-insulin desamide 1.5% .

Example 3

The conditions for the preparation of a short-acting 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 mixture was stirred for 20 hours at a temperature of 20 ° C. Analysis of the intermediate product by HPLC revealed that the number of related related impurities in it was 10%, which led to a decrease in the output of GLF compared with example 1. Studies of the finished dosage form of short-acting insulin in example 3 showed that its activity was 38.5 IU / ml, the content of high molecular weight compounds of 0.8%, related concomitant impurities of 1.5%, A21-insulin desamide 1.0%.

Example 4

. Исследования ГЛФ инсулина короткого действия показали, что его активность составляла 39,3 МЕ/мл, содержание высокомолекулярных соединений 0,5%, родственных сопутствующих примесей 1,8%, А21-дезамида инсулина 0,9%.The conditions for the production of short-acting insulin GLF, 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 to purify human insulin ester and human insulin crystals

. Studies of short-acting 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%, and insulin A21-deamide 0.9%.

Example 5

. Исследования ГЛФ инсулина короткого действия показали, что его активность составляла 39,0 МЕ/мл, содержание высокомолекулярных соединений 0,6%, родственных сопутствующих примесей 4,1%, А21-дезамида инсулина 1,6%.The conditions for the production of short-acting insulin GLF, as in Example 4, except that the DAISOGEL ODS sorbent (C 18) was used with a particle size of 40 to 60 μm and a pore size of 120

. Studies of short-acting insulin GLF showed that its activity was 39.0 IU / ml, high molecular weight compounds 0.6%, related related impurities 4.1%, insulin A21-desamide 1.6%.

Example 6

The conditions for obtaining short-acting insulin GLF, as in example 1, but as the mobile phase in the purification of human insulin ester, 0.1 M glycine-HCl buffer was used, which contained 0.1 M ammonium sulfate and propanol-2 with a concentration of 30% to 45% at pH 2.5. The characteristics of short-acting insulin GLF were as follows: activity was 39.5 IU / ml, high molecular weight compounds 1.2%, related co-impurities 2.9%, insulin A21-desamide 1.5%.

Example 7

The conditions for obtaining short-acting insulin GLF, as in Example 1, but 0.1 M acetate buffer, which contained propanol-2 with a concentration of 10% to 20% at pH 2.5, was used as the mobile phase in the purification of the obtained human insulin crystals. The characteristics of short-acting insulin GLF were as follows: activity was 38.0 IU / ml, high molecular weight compounds 0.7%, related co-impurities 3.1%, insulin A21-desamide 1.7%.

Example 8

The conditions for the operations of the first and second stages of the preparation of short-acting insulin HLP, as in Example 1, except that insulin crystals were directly dissolved in dilute acid. Studies of the finished dosage form of short-acting insulin in example 8 showed that its activity was 39.3 IU / ml, the content of high molecular weight compounds was 0.5%, related concomitant impurities were 0.9%, and A21-insulin insulin 2.1%.

Thus, the analysis of the performed examples shows that non-compliance with the ranges of the claimed enzyme-substrate ratio and deviation from the claimed parameters when purifying insulin ester and human insulin crystals by HPLC (examples 2, 3 and 4-7, respectively) lead to an increase in the content of high molecular weight compounds and related concomitant impurities in short-acting insulin GLF, that is, to a deterioration in the quality of the drug. Example 8 clearly reveals the dependence of the content of insulin A21-deamide, that is, one of the purity parameters of the preparation, on the method of obtaining a solution of insulin in dilute acid. At the same time, compliance with the limits of the claimed enzyme-substrate ratio, purification parameters using HPLC and the method of obtaining a solution of insulin in dilute acid (example 1) allows us to obtain short-acting high-purity insulin GLF with minimal loss of insulin during production.

Claims (1)

A method for preparing a short-acting 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 and mixing with solutions to onservant, isotonic agent and substances with a buffer capacity, characterized in that the production of human insulin ester is carried out at a weight ratio of trypsin and porcine insulin equal to 1: 300-1000, additional reaction inhibition is added by adding 2-3 times water to the reaction mixture before acidification, and the purification of human insulin ester is carried out by HPLC followed by precipitation of fractions of the ether derivative in the presence of zinc ions, deprotection and obtaining crystals of crude human insulin, which is purified by repeated HPLC, both cleaning processes using the DIASOGEL ODS sorbent (C 18) as a stationary phase with a particle size of 15 μm and a pore size of 100 to 150 Å, and 0 as the mobile phase at the first stage, 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 15 to 25% at pH 2.5, and the dissolution of purified human insulin in dilute acid is carried out in stages, and chalk is first obtained odispersnuyu insulin crystal suspension in water, and then thereto is added diluted acid.