BG65045B1 - Method for the production of highly purified monocomponent insulin - Google Patents

Abstract

By the method monocomponent insulin of animal origin is produced. The processes comprise soft conditions of work, smooth temperatures and pH transitions ensuring the preservation of the insulin molecule and of the biological activity of the insulin produced, respectively.

Description

Technical field

The invention relates to a method for the production of highly purified monocomponent insulin, in particular monocomponent insulin of animal origin, used for the treatment of diabetes mellitus.

BACKGROUND OF THE INVENTION

Since its discovery in 1921, insulin has been a major agent worldwide for the treatment of diabetes. In the extraction processes used to extract insulin from the pancreatic tissue, significant amounts of non-insulin proteins are also recovered, which is why methods for purifying insulin for its separation from non-insulin proteins and other impurities are being developed.

From industrially relevant methods, one of the earliest used the general property of proteins to precipitate at their isoelectric point. Thus, US 1,469,994 refers to a purification method that involves the precipitation of non-insulin proteins contained in the aqueous pancreatic extract at their isoelectric points. Isoelectric precipitation of insulin from an aqueous pancreatic extract at pH 4 to 7 is described in US 1,520,673. According to another method described in US 1,547,515, precipitation of insulin is achieved by displacement, in particular with sodium chloride. A method of differential salting is described in US 2,449,076. A third industrially significant method for the purification of insulin uses the precipitation or crystallization of insulin from a solution as a zinc-insulin complex. US 3,878,186 discloses a method for purifying alkaline or ammonium insulin, comprising gel filtration of an aqueous alkaline or ammonium insulin solution having a purity of more than 80%, the concentration of insulin in the solution being from 1 to 8% by weight and volume, and pH the solution is 2.0 to 3.5. Elution of insulin from the gel filtration column is carried out with aqueous eluent with a pH within the same range as the insulin solution. For gel filtration, a swelling gel is used which, in the dry state, contains at least 4% water and has a particle diameter of less than 100 microns. With this method, a zinc-insulin yield of 91.9% by weight and 98.9% determined by biological activity is achieved. The biological activity of the zinc insulin obtained is 25.6 Units / mg. The content of proinsulin is 0.44%.

A method of purifying insulin of animal origin using low pressure preparative column chromatography is known from RU 2 057 542 as an aqueous solution of insulin crude pH 3.0 - 3.5 through ionite, which is a copolymerization product of methacrylic, acrylic acid and dimethacrylate of ethylene glycol with a swelling coefficient of Knab = 4,5 - 4,8 and a total exchange capacity of 9,2 - 9,8 mg.eq / g. Ionite is pre-equilibrated with 10 -16% aqueous isopropanol solution pH 3.0 - 3.5. The ionite is then washed with 10 -16% aqueous isopropanol solution pH 3.0 - 3.5 and the insulin is eluted with a 20-25% aqueous isopropanol solution at pH 3.4 - 3.9 containing 0.05 - 0.15 M ammonium acetate, from the resulting eluate the desired product is separated by precipitation. Insulin purification of up to 98.7% was achieved.

From RU 2 120 299, there is a method of producing highly purified monocomponent insulin of animal origin, according to which sodium insulin with a monofraction content of at least 85% is dissolved in 0.018 0.022 M phosphate buffer at pH = 6.7-6, 8 and fed into a chromatographic column filled with macroporous sulphocationite KU-23 equilibrated in the same buffer and having a particle diameter of 200 to 300 µm. Insulin was eluted at room temperature with 0.018 -0.022 M linear phosphate gradient phosphate buffer at pH: from pH = 6.7-6.8 to pH = 7.9-8.0. Insulin is separated from the resulting eluate by isoelectric precipitation. The insulin obtained after cation exchange chromatography was dissolved in 1M acetic acid and fed into a chromatographic column (Sephadex G-50 St from Pharmacia), eluting with 1M acetic acid. A portion of the insulin containing eluate eluting in the range of 0.6-0.7 to 0.9-1.0 column volume is returned to the inlet of the recirculation column and elution is continued with

1M acetic acid. Monopic insulin obtained after circulating gel permeation chromatography was fed into an anion-exchange silica gel PEA-300 column with a particle size of 35 to 70 micrometer. Elution was performed at a rate of 20 to 45 ml / cm ² . h ¹ , using an exponential gradient of sodium chloride concentration. The resulting insulin contains not less than 98.5% monofraction, not more than 0.5% desamidoinsulin, not more than 1.0% unidentified impurities and not more than 10 ppm proinsulin and its derivatives. In this case, the yield by mass is 55 to 65% relative to the starting material sodium insulin.

From RU 2 146 944, a method is known for purifying insulin derived from an animal feedstock or obtained by a biotechnological method. The starting insulin was gel chromatographed in 0.7-1.2 M acetic acid in a column filled with hydrophilic gel obtained by polycondensation of polyglucine with epichlorohydrin and equilibrated with a 0.7 - 1.2 M acetic acid solution. When loading the column, the amount of insulin is 4-7 g / Ι equilibrium gel, with the concentration of insulin in the solution being 5-15%. Elution of insulin was carried out at 8-15 ° C at a flow rate of 2.0 - 5.0 ml / (cm ^2. H), the desired product was separated by zinc precipitation and subjected to citrate crystallization to give insulin with yield 75 - 85% and insulin monofraction content 98.0 - 98.5%, wherein the proinsulin content is less than 2 ppm, bacterial peptide content (in genetically engineered human insulin) - less than 1 ppm, and in all cases the amount of desamidoinsulin and unidentified impurities does not exceed 1% (RIA and HPLC methods).

Products obtained by these methods cannot be classified as monocomponent insulin. The presence of impurities, especially of 1% unidentified impurities, makes the product insufficient quality, suggesting poor biological activity and risk of allergic and other adverse reactions.

UK 1 581 824 describes a method for producing monocomponent insulin with very little or no allergenic action. The method is as follows: frozen pancreatic glands are extracted with ethyl alcohol / 60-80% by volume / acidified with hydrochloric acid to a pH of about 3. After separation of the mass from the pancreatic cells, the pH of the extract is adjusted to 8, some proteins other than insulin are precipitated and removed. The pH is again adjusted to 3. Instead of so on. "PH-8-precipitation" for the separation of unwanted proteins, the acidic extract with pH = 3, can be subjected to ultrafiltration / hyperfiltration using appropriate membranes to separate said proteins, for example enzymes. The resulting clear extract was cooled in a special device to a temperature of -30 to -45 ° C, whereby the fat was crystallized in a compact mass over a small surface. The crystals are separated, for example by centrifugation, at the same low temperature. According to the invention, the already fat-free insulin extract is subjected to reverse osmosis with suitable membranes, while simultaneously reducing the volume of liquid and separating the insulin from impurities in the extract. Reverse osmosis is carried out at temperatures between + 10 ° C and -10 ° C and a pressure of 2-20 atm, the membrane type is selected to separate unwanted molecules larger than the insulin molecule. GRX-6 or GRX-7 (polysulfone membranes) membranes may be used. The permeate is subjected to reverse osmosis using membranes which release impurities with molecules smaller than that of insulin, for example membranes GRSP or 930 (cellulose acetate). Insulin remains in the concentrate. The extract may be concentrated to the desired extent, for example, from 1/5 to 1/40 of the starting volume. In doing so, the ratio of alcohol and water concentrations should remain the same as in the original extract. The resulting concentrate, containing virtually all of the insulin from the starting extract, is clear but colored. It is washed with 62% alcohol above a membrane, through which the washing solution with the coloring agents passes. The salts present in the extract are similarly washed. The small amounts of dissolved impurities remaining in the insulin-containing extract can be removed by ion exchange resin, cation exchange resin and anion exchange resin. Separation may also be carried out with a molecular sieve, for example Sephadex G-50 or by another suitable known separation method, but an ion exchange method is preferred. The ion exchange method may be column chromatography. Different types of ion exchange resins may be used. SP-Sephadex C-25 (cation exchange resin) and QAE-Sephadex A-25 (anion exchange) are preferred. Monocomponent insulin was obtained as demonstrated by polyacrylamide gel electrophoresis using a 15% polyacrylamide gel.

US 3,907,676 describes a method of purifying insulin to reduce the antigenic activity of insulin derived from animal pancreas. This method is most effective when the starting material is obtained by subjecting the solution of the salt precipitate to precipitation at the isoelectric point by adjusting the pH of the solution to 5.5, and isolating the precipitate by centrifugation. The method involves dissolving the insulin in aqueous medium, adjusting the pH in the range of 6 to 9, transferring the solution into an anion exchange resin chromatographic column, fractional elution from the eluent column consisting of water and water miscible monohydroxyl aliphatic alcohol with a concentration of alcohol 40 to 80% by volume, collection of insulin-containing fractions showing content of only one component, which is analyzed by polyacrylamide gel electrophoresis (DISC PAGE) and separation of the collected fractions.

The problem facing insulin manufacturers is the achievement of highly purified and ideally non-antigenic and allergenic monocomponent insulin while maximizing the recovery of insulin contained in the starting pancreatic material. Moreover, the methods and technical means by which the individual processes are carried out are efficient and technologically optimal for large-scale industrial production.

SUMMARY OF THE INVENTION

These problems are solved by a method that consists of the following steps:

I. Getting a salt cake:

- repeated extraction of the hormone from deep-frozen porcine pancreas with ethyl alcohol acidified with phosphoric acid at a temperature below 18 ° C;

- carrying out isoelectric precipitation in the alkaline and acidic regions at a temperature of 12 to 18 ° C;

- two-step concentration in vacuo;

- salting by adding pure sodium chloride to the salt (salt cake) at pH = 3.30 ± 0.02;

- staying at room temperature for 2 hours to 4 days;

I. Preparation of sodium - insulin:

isoelectric precipitation in the acidic range from pH = 2.8 to final pH = 5.3 ± 0.02, which is adjusted with sodium hydroxide at 4 ° C;

- addition of 96% alcohol and cooling to 4 ° C, pH adjustment = 5.30 ± 0.02;

- stay without stirring for 12 h;

- decanting the upper part of the liquid;

- centrifugation at 4 ° C;

- dissolving the isotopic acid in water;

- two-step pH adjustment with retention;

- adding to the insulin solution of disodium ethylenediamine tetraacetate dihydrate, adjusting the temperature to 18-25 ° C;

- addition of sodium chloride to a total salt content of 0.15 kg / Ι initial volume of solution and adjustment of pH = 8.40 ± 0.02;

- stirring for 8-12 h at room temperature;

- filtration and washing;

- storage of sodium insulin in a known manner, depending on the course of the process.

III. Purification of sodium - insulin by gel filtration:

Gel filtration is carried out in a known manner using chromatographic columns filled with, for example, dextran resin, type Sephadex G 50, elution is carried out with buffered acetic acid, whereby the separation of the components according to the size of their molecules is controlled by UV -absorption. Various known, appropriate modes may be used.

The monopic insulin fraction was collected and crystallized with sodium chloride to monopic sodium insulin, which was stored for further processing.

IV. Purification of monopic insulin by ion exchange chromatography to monocomponent insulin.

The method is based on the principle of absorption-desorption for the separation of insulin molecules that differ in their electrical charge.

- dissolution of monopic insulin in water.

- Insulin ion exchange of DEAE and QAE columns.

The ion exchange is performed on two parallel systems, each consisting of two KS-370/15 packet columns. 6 types of urea buffers are used.

The columns were filled with DEAE cellulose equilibrated with 0.025 M Tris / HCl buffer in 7 M urea buffer 1 and Pharmacia QAE Sephadex A 25 equilibrated in the same buffer. At 4 ± 1 ° C it is carried out

- submission of the sample to the DEAE columns at a specified flow rate;

- performing four elution with a certain type of buffer, controlling the operating parameters - pH, temperature, conductivity, UV-conductivity at 206 nm or 280 nm, flow rate;

- insulin was eluted from the QAE column with 0.025 MH ₃ PO ₄ containing 1% n-butanol (buffer 6). The parameters are monitored - pH, conductivity, UV signal at 280 nm, temperature and flow rate;

- regeneration of DEAE and QAE columns, combining the working mode of one pair of columns and regenerating the other;

- the monocomponent insulin fraction is filtered on filters 0.8 and 0.22 microns. The sterile solution thus obtained is fed to zinc crystallization.

V. Zinc crystallization: The zinc content should be 0.5% by weight of insulin, in the form of zinc acetate or zinc chloride (10 mg zinc per ml), pH = 6.5, temperature 15-20 ° C, stirring overnight. PH = 5.7 correction with 1M CH ₃ COOH. After 1 hour of stirring, the zincinsulin crystals were collected, stored at 4 ° C, and zinc insulin was graded by RIA and HPLC.

The mono-component zinc-insulin crystals thus obtained are formulated as fast acting zincinsulin preparations, intermediate and prolonged, with activity of 40 IU / ml, 80 IU / ml and 100 IU / ml.

All processes in the process according to the invention are characterized by mild operating conditions, smooth temperature and pH transitions, which ensure the preservation of the insulin molecule and, accordingly, the biological activity of the insulin produced. The advantages of the process according to the invention are: maximizing insulin recovery from the feedstock, minimizing purification steps, and simultaneously producing high-purity monocomponent insulin, proven by HPLC (FPLC, low antigenic and low immunogenic, proven by RIA) , with high biological activity, with purity proven by radioimmunoassay for contaminants. The method was developed as a waste-free technology, enabling the production of by-products and their utilization.

An exemplary embodiment of the invention

I. Getting a salt cake

500 kg of deep frozen at -25 ° C porcine pancreas is saturated to 0.2 mm (snow of meat) and extracted at 4 to 5 times with phosphoric acid acidified acid, the first extraction being carried out by 90% - ethanol, the second is carried out with the fourth extract of the previous charge, and the third and fourth extractions - with new / fresh / ethanol, the temperature is kept below 18 ° C. The separation of the meat and the extract is carried out rapidly on a decanting centrifuge, followed by purification with a clarifying separator (liquid-solid phase). Three consecutive extracts were collected, the pH was adjusted to 6, then slowly adjusted to 8.1 (with sodium hydroxide solution), with the temperature ranging from 12-17 ° C. The extract was purified through a decanter and clarified through a chamber filter press. The purified extract was acidified with 25% hydrochloric acid to pH = 4.65. The extract was then concentrated in vacuo. The temperature should not exceed 30 ° C. For the production of highly purified insulin, a second concentration to an ethanol content of less than 0.5% must be made.

After the second concentration, the precipitate is removed by decantation or filtration when the precipitate which is collected by precipitation is solid. The fat removed by curing is tetrachloromethane. The concentrated extract was purified by filtration through ancillary filter material. Salting is carried out to the salt cake and the concentrate is treated at pH = 3.30 by the addition of 18% v / v. pure sodium chloride and the reaction mixture was allowed to stand at room temperature for 2 hours. The salt cake is collected on a filter or in a single-chamber centrifuge, then dried and stored in a refrigerated warehouse for further purification;

II. Preparation of sodium - insulin

A salt pan obtained from 7.51 pancreas was dissolved in water and acidified with 2M phosphoric acid to pH = 2.8. The conductivity of the solution should not exceed 3 millisimens. Cool to 4 ° C, adjust pH to pH = 3.2 with sodium hydroxide at 4 ° C, add about 0.5 kg of acid-washed auxiliary filter material to the dissolved salt cake and filter the solution through a chamber filter. Isoedectric precipitation is followed as the solution is diluted, the pH is adjusted to pH = 5.30 ± 0.02 with IM NaOH, cooled to a temperature below 5 ° C, 96% ethanol is added in proportion to the 1: 4 solution, cooled again to 4 ° C, adjusted to pH = 5.30 ± 0.02 with 1M NaOH or IM HCl and remained without stirring for 12 h. The top of the liquid was then decanted to reach the surface of the precipitate, centrifuged at 4 ° C. The wash filtrates were collected for subsequent isoelectric precipitation. This is followed by dissolution of the precipitate in water. Add (NH ₄ ) ₂ HPO ₄ , adjust to pH = 7.2 ± 0.1 with IM NaOH, dilute the solution to a protein content of 20 g / Ι, filter through Rabbit EX blanks and then through a Millipore membrane filter, 0.45 microns. To the insulin solution was added 4 g of disodium ethylenediamine tetraacetate dihydrate (trilon B), adjusting the temperature to 18-25 ° C, adding sodium chloride 0.075 kg / твор solution, with vigorous stirring, followed by adjustment to pH = 8.40 ± 0.02 s IM NaOH, and stirring for 2 h, add the same amount of sodium chloride to a total salt content of 0.15 kg / Ι initial volume of solution, adjust the pH if necessary to pH = 8.40 +/- 0.02 with IM NaOH, followed by stirring 8-12 h at 1825 ° C. It was filtered through a calcined paper filter and washed with 15% sodium chloride solution. Sodium insulin is stored in three ways:

- first way - storage of wet crystals in cold storage - storage at 4 ° C for up to several days;

- the second way - drying the wet crystals under vacuum, storing them for up to several months at 4 ° C;

- third way - suspension of the crystals in ethanol 96%, filtration of the suspension and drying with clean compressed air - storage for up to several months at 4 ° C;

III. Purification of sodium - insulin by gel filtration

Sodium insulin was dissolved in 1M acetic acid and filtered. Insulin content check (should be 33 g / l);

Parmacia KS 370/15, a Sephadex G-50 Superfine gel of special quality, swollen in 1M CH ₃ COOH are used for gel filtration. Gel filtration can be carried out in automatic mode - each sample - 50 g insulin, flow rate 6-81 / h, eluent 1M CH ₃ COOH, temperature 4 ° C, pressure 0.3 bar.

IV. Purification of monopic insulin by ion exchange chromatography to monocomponent insulin.

The method is based on the principle of absorption-desorption for the separation of insulin molecules that differ in their electrical charge. The monopic crystalline insulin obtained in the previous step was dissolved in water, adjusting the pH to 8.10 ± 0.02. Work in a cold room. The solution was filtered through filters 0.8 and 0.22 microns. Insulin content was determined spectrophotometrically. The solution is adjusted to a specified concentration of insulin in the solution and to a specified conductivity (30 g / Ι and 2.0 millisimens);

The ion exchange is performed on two parallel systems, each consisting of two KS-370 / 15n bundled columns, which control the operating parameters of the technological mode. Urea buffers are used.

The columns were filled with DEAE cellulose equilibrated with 0.025 M Tris / HCl buffer in 7 M urea buffer 1 and Pharmacia QAE Sephadex A 25 equilibrated in the same buffer. Work at 4 ± 1 ° C. The sample was fed to the DEAE columns at a flow rate of 101 / h. Four elution with a certain type of buffer is performed, controlling the operating parameters - pH, temperature, conductivity, UV-conductivity at 206 nm or 280 nm, flow rate.

The insulin was eluted from the QAE - the column was 0.025 MH ₃ PO ₄ containing 1% n-butane (buffer 6). The parameters are monitored - pH, conductivity, UV signal at 280 nm, temperature and flow rate. This is followed by regeneration of the DEAE and QAE columns, combining the working mode of one pair of columns and regenerating the other. The MS-insulin fraction was filtered on filters 0.8 and 0.22 microns. The sterile solution thus obtained is crystallized by carrying out zinc crystallization. The zinc content should be 0.5% by weight of insulin, in the form of zinc acetate or zinc chloride (10 mg zinc per ml), temperature 15-20 ° C, pH = 6.5. Stir overnight. PH = 5.7 correction with 1M CH ₃ COOH. After 1 h stirring, the zinc-insulin crystals were collected. Store at 4 ° C. Zinc insulin is graded by RIA and HPLC. The mono-component zinc-insulin crystals thus obtained are formulated as fast acting zinc-insulin preparations, intermediate and prolonged, with activity of 40 IU / ml, 80 IU / ml and 100 IU / ml.

Claims (3)

Claims A method for the production of highly purified monocomponent insulin, including extraction of porcine pancreatic hormone with acidified ethanol, purification of the extract by isoelectric precipitation, subsequent purification steps by molecular filtration, and high purification by ion exchange chromatography, characterization chromatography, consists of the following steps: I. Getting a salt cake: - repeated extraction of the hormone from deep-frozen porcine pancreas with ethyl alcohol acidified with phosphoric acid at a temperature below 18 ° C; - carrying out isoelectric precipitation of impurities in the alkaline and acidic regions at a temperature of 12 to 18 ° C; - two-step concentration in vacuo; - salting by adding pure sodium chloride to the salt (salt cake) at pH = 3.30 ± 0.02; - staying at room temperature for 2 hours to 4 days; I. Preparation of sodium - insulin: isoelectric precipitation in the acidic range from pH = 2.8 to final pH = 5.3 ± 0.02, which is adjusted with sodium hydroxide at 4 ° C; - addition of 96% alcohol and cooling to 4 ° C, pH adjustment = 5.30 ± 0.02; - stay without stirring for 12 h; - decanting the upper part of the liquid; - centrifugation at 4 ° C; - dissolving the isotopic acid in water; - two-step pH adjustment with retention, - adding to the insulin solution of disodium ethylenediamine tetraacetate dihydrate, adjusting the temperature to 18-25 ° C; - addition of sodium chloride to a total salt content of 0.15 kg / Ι initial volume of solution and adjustment of pH = 8.40 ± 0.02; - stirring for 8-12 h at room temperature; - filtration and washing; III. Purification of sodium - insulin by gel filtration: - carrying out gel filtration, in particular by using a dextran resin-filled chromatographic column, type Sephadex G 50, eluting with buffered acetic acid, the separation of the components according to the size of their molecules being controlled by UV absorption; - crystallization of the monopic insulin fraction with sodium chloride to sodium monopic insulin; - Demonstration of monopic insulin with analytical gel filtration, Ornstein-Davis DISC-PAGE, subsequent densitometry, FPLC / HPLC. IV. Purification of monopic insulin by ion exchange chromatography to mono-component insulin: - dissolving monopic insulin in water and correcting pH = 8.1 ± 0.02 with 0.1 M hydrochloric acid or 0.1 M sodium hydroxide; - ion exchange in two parallel systems, each consisting of two KS-370/15 batch columns, the columns being filled with DEAE-cellulose, ex-10 vibrated with 0.025 M Tris / HCl buffer in 7 M urea-buffer and QAE Sephadex Pharmacy A 25 equilibrated in the same buffer at temperature 4 ± 1 ° C, carrying out four elution with a certain type of buffer, controlling the operating parameters - pH, temperature, conductivity, UV conductivity at 206 nm or 280 nm, flow rate at which insulin is eluted from the QAE column with 0.025 MH ₃ PO ₄ containing 1% n-butanol, following the operating parameters; - regeneration of DEAE and QAE columns, combining the working mode of one pair of columns and regenerating the other; - the monocomponent insulin fraction is filtered on filters 0.8 and 0.22 microns; V. Preparation of zinc insulin - crystallization of the sterile solution from the previous step as zinc-insulin with the addition of zinc acetate or zinc chloride, pH adjustment = 6.5, at a temperature of 15-20 ° C, stirring at slow rpm for at least 4 hours, preferably 1 night , pH adjustment = 5.7 with 1M CH ₃ COOH, zinc-insulin crystals are collected after a minimum of 1 hour of stirring; - qualification of zinc insulin by RIA and HPLC. The insulin produced by the method of claim 1, which is mono-component proven by RIA and HPLC. Use of insulin obtained according to claim 1 for the manufacture of insulin therapy preparations.