RU2186581C2 - Method of highly purified monocomponent insulin preparing - Google Patents

Abstract

FIELD: medicinal industry, preparative biochemistry, hormones. SUBSTANCE: invention relates to preparing highly purified monocomponent insulin of different origin. Crude insulin is subjected for two procedures of column reverse-phase chromatography: at pH 1.5-4.5 and at pH 6.5-9.5. Purification is carried out on nonpolar sorbents exhibiting mean pores size 100-500

and particles size from 10 to 100 mcm. Aqueous-organic solvents containing ionic modifying agents are used as eluents. Process of chromatography treatment is carried out at temperature 5-30 C. Method provides preparing insulin of high quality containing 98.5% of basic substance, not less, 0.5% of desamido-A21-insulin, not above, 1.0% of unidentified impurities, not above, 1.0 mln^-1 of proinsulin, not above. EFFECT: improved method of purification, high quality and purity of insulin. 15 cl

Description

 The invention relates to the medical industry, and in particular to a method for producing highly purified monocomponent insulins: porcine, semi-synthetic human, human genetic engineering, used to treat diabetes mellitus.

 Modern requirements for the quality of highly purified monocomponent insulin are very high: the proinsulin content determined by the method of radioimmunological analysis (hereinafter referred to as RIA) should not exceed 1.0 p.p.m. (0.0001%), the content of unidentified impurities determined by high performance liquid chromatography (hereinafter referred to as HPLC) should not exceed 1.0%, the content of desamido-A21-insulin should not exceed 1.0% (HPLC).

 Known methods [1, 2, 3, 4, 5] to obtain highly purified insulin, which are based on the use of ion exchange and gel permeation chromatographic purification of insulin. However, the described methods do not allow to obtain insulin with a basic substance content of more than 97% (HPLC) and with a proinsulin content of less than 1 p.p.m. (RIA). Moreover, the yield does not exceed 60%.

 The known method [6] for the purification of insulin on weakly acidic ion exchangers with a hydrophobic shell in aqueous-organic solvents at pH values from 1.5 to 5.5. Due to the use of two adsorption mechanisms in this method, a deeper purification of insulin from impurities with an isoelectric point pI of more than 6.5 is achieved: arginininsulin, proinsulin, etc. However, the efficiency of cleaning impurities with a pI of less than 4.5: desamido-A21-insulin is reduced , desamido-B3-insulin. As a result, the purity of the resulting insulin does not exceed 96% (HPLC).

 Closest to the proposed is a method [7] for the production of highly purified insulin, which combines the use of liquid ion exchange chromatography, reverse phase (hereinafter referred to as RP) chromatography at a pH of about 3.0 and gel permeation chromatography. This method allows to obtain insulin with a basic substance content of more than 98% (HPLC) and with a proinsulin content of less than 4 p.p.m. (RIA) at an exit from 60 to 65%. However, due to the low selectivity for such impurities as monoarginininsulin, diargininsulin, ester derivatives of insulin, desamido-B3-insulins, des-B30-insulin, the level of impurities as a whole remains quite high. For the same reason, this method uses very low insulin loads on chromatographic columns during reverse phase chromatography: from 13 to 15 g / l of sorbent.

 The aim of the invention is to increase the yield and improve the quality of insulin during chromatographic purification.

и с размером частиц от 10 до 100 мкм. Максимальная эффективность очистки достигается при использовании сферических монодисперсных сорбентов со средним размером пор

с размером частиц от 10 до 15 мкм, упакованных в хроматографические колонки с динамическим аксиальным сжатием сорбента. Высота слоя сорбента в хроматографической колонке от 10 до 60 см, предпочтительно от 25 до 40 см. В качестве элюента применяют водно-органические растворители со значением рН от 1,5 до 4,5 (предпочтительно от 2,3 до 2,8) и от 6,5 до 9,5 (предпочтительно от 7,3 до 8,0) и содержащие ионные модификаторы: органические и неорганические кислоты, основания и их соли с молярной концентрацией от 0,001 до 0,3 моль/л. Предпочтительно использовать кислоты: уксусную, трифторуксусную, ортофосфорную, соляную; основания: трис(оксиметил)аминометан (далее по тексту ТРИС), аммиак, гидроокись натрия и их соли. Значение рН элюентов устанавливают добавлением необходимого количества кислот и оснований. В качестве органического растворителя применяют ацетонитрил, спирты; предпочтительно использовать ацетонитрил, метанол, этанол, пропанол-2 при первой и этанол при второй хроматографических очистках. Процесс хроматографической очистки и кристаллизации инсулина проводят при температуре от 5 до 30^oС, предпочтительно от 10 до 20^oС.To accomplish this task, the starting raw insulin, with a basic substance content of at least 85% (HPLC) and a proinsulin content of not more than 50 thousand ppm (RIA), is subjected to two chromatographic purifications in succession: RP column chromatographic purification at a pH value of 1.5 to 4.5 and RP column chromatographic purification at a pH value of from 6.5 to 9.5. The order of the first and second RP chromatographic purification can be arbitrary. Improving the quality is achieved due to the fact that during RP chromatographic purification at a pH from 6.5 to 9.5 compared with RP chromatographic purification at a pH from 1.5 to 4.5, the output order is reversed and the selectivity for a whole series is improved impurities (arginininsulin, desamido-B3-insulin, des-B30-insulin and a number of other impurities) and improved selectivity for desamido-A21-insulin. Thus, there is no need for low-performance ion-exchange chromatographic purification. Cleaning is carried out on non-polar macroporous sorbents: modified silica gels (C-8, C-18) or polymer sorbents with an average pore diameter

and with a particle size of from 10 to 100 microns. Maximum cleaning efficiency is achieved using spherical monodisperse sorbents with an average pore size

with a particle size of 10 to 15 μm, packed in chromatographic columns with dynamic axial compression of the sorbent. The height of the sorbent layer in the chromatographic column is from 10 to 60 cm, preferably from 25 to 40 cm. Water-organic solvents with a pH from 1.5 to 4.5 (preferably from 2.3 to 2.8) are used as eluent. from 6.5 to 9.5 (preferably from 7.3 to 8.0) and containing ionic modifiers: organic and inorganic acids, bases and their salts with a molar concentration of from 0.001 to 0.3 mol / L. It is preferable to use acids: acetic, trifluoroacetic, orthophosphoric, hydrochloric; bases: tris (oxymethyl) aminomethane (hereinafter referred to as TRIS), ammonia, sodium hydroxide and their salts. The pH of the eluents is established by adding the necessary amount of acids and bases. Acetonitrile, alcohols are used as an organic solvent; it is preferable to use acetonitrile, methanol, ethanol, propanol-2 in the first and ethanol in the second chromatographic purification. The process of chromatographic purification and crystallization of insulin is carried out at a temperature of from 5 to 30 ^o C, preferably from 10 to 20 ^o C.

 According to the proposed method receive highly purified monocomponent insulin with a basic substance content of more than 98.5%, with a content of desamido-A21-insulin of less than 0.5%, with an unidentified impurity content of less than 1.0% (HPLC) and with a proinsulin content of less than 1 p.p.m. (RIA), while the yield is at least 70% when using irregular sorbents with a particle size of from 50 to 100 μm and at least 75% when using spherical sorbents with a particle size of 10 to 15 μm in columns with dynamic axial compression of the sorbent.

 To carry out the first RP chromatographic purification at a pH of 1.5 to 4.5, the initial raw insulin is dissolved in the starting eluent A containing from 10 to 30 volume% of an organic solvent; the pH value is adjusted if necessary by adding the necessary amount of hydrochloric acid solution with mass shares from 5 to 10%. The concentration of insulin in solution is from 10 to 50 g / l. The load on the chromatographic column is from 15 to 50 g per liter of sorbent. Before entering the insulin solution into the chromatographic column, it is filtered through a hydrophilic membrane filter with an absolute retention capacity of 0.2 μm. The chromatographic column before insulin injection should be pre-balanced with starting eluent A in a volume equal to from three to five volumes of sorbent in the column. The insulin solution is introduced into the chromatographic column with a linear flow rate from 0.25 cm / min (for sorbents with a particle size of 50-100 μm) to 1.5 cm / min (for sorbents with a particle size of 10-15 μm). After entering the insulin solution, the chromatographic column is washed with the starting eluent A in an amount equal to the volume of the sorbent in the chromatographic column. The insulin is eluted using a continuous or stepwise gradient in the concentration of the organic solvent, the slope of the continuous gradient is from 0.5 to 3 (preferably from 0.8 to 1.5) vol% organic solvent per eluent volume equal to the volume of the sorbent in the chromatographic column. The linear flow rate during elution is from 0.5 cm / min (for sorbents with a particle size of 50-100 microns) to 3 cm / min (for sorbents with a particle size of 10-15 microns). Monitoring the elution of insulin is carried out by light adsorption at a wavelength of 276 nm. The eluate is divided into fractions with a volume equal to about 0.25 of the volume of the sorbent in the column. In each fraction, the content of impurities is determined by HPLC. According to the analysis results, three pools are formed: pool 1 - fractions with an impurity content of more than 40%, pool 2 - fractions with an impurity content of 3 to 40%, pool 3 - fractions with an impurity content of less than 3%. Pool 3 contains highly purified insulin with an impurity content of 2 to 3%, which is an intermediate for the second chromatographic purification. Pool 2 contains insulin with an impurity content of 7 to 12%, which is subjected to rechromatography under the same conditions as the original raw insulin; obtained after rechromatography, highly purified insulin with an impurity content of less than 3% is attached to pool 3. Pool 1, containing from 40 to 65% of the impurities, is sent to waste.

 After the end of the insulin purification process, the chromatographic column is regenerated with an eluent containing from 60 to 80% organic solvent in an amount equal to from three to five volumes of sorbent in the chromatographic column, and balance with the same volume of starting eluent A. Yield at the first chromatographic purification from 83 to 86 % The highly purified insulin contained in pool 3 is crystallized in a citrate buffer solution. To do this, the eluate is diluted with distilled water to reduce the concentration of organic solvent to 10-20 volume% (10% when using acetonitrile, propanol-2 and 20% when using ethanol, methanol), zinc chloride solution is added with a mass fraction of 10% from the calculation of 0, 75-0.80 ml per 1 g of insulin and a solution of citric acid with a molar concentration of 1.5 mol / l at the rate of 34-35 ml per 1 liter of diluted eluate. Then, in a crystallization solution, an ammonia solution with a mass fraction of 10-12.5% with constant stirring sets the pH value from 6.8 to 7.0, stirring is continued for 1-2 hours. Next, a solution of hydrochloric acid with a mass fraction of 5-10% in a crystallization solution, with constant stirring, a pH value of 5.9 to 6.1 is gradually established over 1-2 hours and stirring is continued for another 4-6 hours. The obtained crystals of highly purified insulin are separated from the mother liquor, washed with distilled water and transferred to second xp matograficheskuyu purification. Loss of insulin during crystallization from 1 to 3%.

 To carry out the second RP chromatographic purification at a pH from 6.5 to 9.5, a highly purified insulin intermediate obtained from pool 3 is dissolved in the starting eluent B containing from 10 to 30 volume% of an organic solvent. To bind zinc ions to a solution of insulin add a solution of Trilon B with a molar concentration of 0.1 mol / l at the rate of 0.5 ml per 1 g of insulin. The pH value, if necessary, is adjusted with an ammonia solution with a mass fraction of 10-12.5%. The concentration of insulin in solution is from 10 to 30 g / l. The load on the chromatographic column is from 15 to 50 g per liter of sorbent. Before entering the insulin solution into the chromatographic column, it is filtered through a hydrophilic membrane filter with an absolute retention capacity of 0.2 μm. The chromatographic column before entering the insulin solution must be pre-balanced with the starting eluent B in an amount equal to from three to five volumes of sorbent in the column. The insulin solution is introduced into the chromatographic column with a linear flow rate from 0.25 cm / min (for sorbents with a particle size of 50-100 μm) to 1.5 cm / min (for sorbents with a particle size of 10-15 μm). After entering the insulin solution, the chromatographic column is washed with the starting eluent B in an amount equal to the volume of the sorbent in the chromatographic column. The insulin is eluted using a continuous or stepwise gradient in the concentration of the organic solvent, the slope of the continuous gradient is from 0.5 to 3 (preferably from 0.8 to 1.5) vol% organic solvent per eluent volume equal to the volume of the sorbent in the chromatographic column. The linear flow rate during elution is from 0.5 cm / min (for sorbents with a particle size of 50-100 μm) to 3 cm / min (for sorbents with a particle size (10-15 μm). Control of elution of insulin is carried out by light adsorption at wavelength 276 nm. The eluate is divided into fractions with a volume equal to approximately 0.25 of the volume of the sorbent in the chromatographic column. The content of impurities is determined by HPLC in each fraction. Three pools are formed from the analysis: pool 4 - fractions with an impurity content of more than 20%, pool 5 - fractions with impurities from 1 to 20% and pool 6 - fractions with soda holding impurities of less than 1%. Pool 4 containing 30 to 45% of impurities is sent to waste. Pool 6 contains highly purified monocomponent insulin with an impurity content of less than 1%. Pool 5 contains insulin with an impurity content of 2 to 5%, which is subjected to rechromatography at the same regimen as insulin isolated from pool 3, with the exception of the load, which should be from 15 to 30 g per liter of sorbent in the column obtained after rechromatography of pool 5 highly purified monocomponent insulin with an impurity content of less than 1% is attached to pool 6 . Ambassador At the end of the insulin purification process, the chromatographic column is regenerated with an eluent containing from 60 to 80% organic solvent in an amount equal to from three to five volumes of sorbent in the chromatographic column and balanced with the same volume of the starting eluent B. The yield during the second chromatographic purification is from 91 to 94% . The highly purified monocomponent insulin contained in pool 6 is crystallized in a citrate buffer solution. For this, the eluate is diluted with distilled water to reduce the concentration of the organic solvent to 10-20 volume% (10% when using acetonitrile, propanol-2 and 20% when using ethanol, methanol), add a solution of ammonium citrate with a molar concentration of 1.5 mol / l and with a pH value of 7.5 to 8.5 based on 34-35 ml per liter of diluted eluate and a solution of zinc chloride with a mass fraction of 10% based on 0.75-0.80 ml per 1 g of insulin. The resulting crystallization solution is filtered through a sterilizing hydrophilic membrane filter with an absolute retention capacity of 0.2 μm. Then, in a crystallization solution of insulin with hydrochloric acid solution with a mass fraction of 5-10%, with constant stirring, the pH is first set to 6.8 to 7.0, and after 1-2 hours of exposure, the pH is gradually adjusted to 1-2 from 1-2 hours 5.9 to 6.1 and stirring is continued for another 4-6 hours. The obtained crystals of highly purified monocomponent insulin are separated from the mother liquor, washed with distilled water, ethanol and dried in a vacuum oven to constant weight. Loss of insulin during crystallization from 1 to 2%. Get highly purified monocomponent insulin containing not more than 1% unidentified impurities, not more than 0.5% desamido-A21-insulin (HPLC) and not more than 1 p.p.m. proinsulin (RIA). The yield during cleaning by the proposed method is from 70 to 80%.

Example 1. The process of chromatographic purification of insulin is carried out at a temperature of 11 ^o C. 110 g of technical weight (100 g in terms of dry matter) of raw porcine insulin containing 3.5% of desamido-A21-insulin and 9% of impurities are dissolved in 1 , 8 l of starting eluent A containing ammonium acetate with a molar concentration of 0.020 mol / l, propanol-2 with a concentration of 10 volume% and pH 2.5, the pH of the insulin and eluent solution is adjusted with hydrochloric acid with a mass fraction of 10%. The resulting insulin solution is filtered under an overpressure of 0.7 bar through a Bio-Inert NRLG membrane filter (Pall) with a diameter of 293 mm and with an absolute retention capacity of 0.2 μm, the filter is washed with 0.2 L of starting eluent A. Get about 2 L insulin solution.

). Колонку собирают и верхним подвижным адаптером сжимают слой сорбента. Затем через колонку в течение 15 мин пропускают пропанол-2 с линейной скоростью потока 5,1 см/мин (при этом давление на колонке не должно превышать 8 bar) и при необходимости опять верхним адаптером сжимают слой сорбента. Получают слой сорбента высотой 25,5 см и объемом 2 л. Для уравновешивания через колонку пропускают 8 л стартового элюента А.In a chromatographic column Superformance 500-100 (Merck) with an inner diameter of 10 cm and a height of 50 cm, 1 kg of irregular silica gel C18 from Amicon Matrex C18-100-50 (particle size 50 μm, pore size

) The column is collected and the sorbent layer is compressed by the upper movable adapter. Then, propanol-2 was passed through the column for 15 minutes with a linear flow rate of 5.1 cm / min (the pressure on the column should not exceed 8 bar) and, if necessary, the adsorbent layer was again compressed with the upper adapter. Get a layer of sorbent with a height of 25.5 cm and a volume of 2 L. To balance, 8 L of starting eluent A is passed through the column.

 The insulin purification process is carried out on a Gilson liquid chromatograph (Gilson company), equipped with model 305, 306 pumps with 200 Wti heads, model 807 pressure damper, model 112 photometric UV detector and FC-30 fraction collector (Fharmacia Biotech company).

The insulin solution is fed at a linear flow rate of 0.25 cm / min to the chromatographic column. Then the column is washed with 2 L of starting eluent A. The insulin is eluted with a linear flow rate of 0.5 cm / min in a linear gradient of the concentration of propanol-2 from 15 to 30 volume% for 8 hours. The gradient is formed automatically by mixing two eluents: the starting eluent A and an eluent containing ammonium acetate with a molar concentration of 0.020 mol / L, propanol-2 with a concentration of 30 volume% and with a pH value of 2.5. After the gradient is completed, the sorbent in the column is regenerated with an eluent containing ammonium acetate with a molar concentration of 0.020 mol / L, propanol-2 with a concentration of 60 volume% and a pH value of 2.5, which is supplied with a linear flow rate of 1.27 cm / min for 80 min The eluate containing the protein is crushed into fractions of 0.5 l each. Fractions are analyzed by HPLC for the content of impurities and three pools are formed from the analysis. Pool 1 contains 12 g of protein (7.5 l of eluate with a protein concentration of 1.6 g / l) of the following composition: insulin - 48.3%, desamido-A21-insulin - 6.7%, impurities - 45%. Pool 2 contains 13 g of protein (4.33 l of the eluate with a protein concentration of 3 g / l) of the following composition: insulin - 85.1%, desamido-A21-insulin - 4.0%, impurities - 10.9%. Pool 3 contains 75 g of protein (8.33 l of eluate with a protein concentration of 9.0 g / l) of the following composition: insulin - 94.2%, desamido-A21-insulin - 2.9%, impurities - 2.9%. Pool 1 is transferred to waste. Pool 2 is subjected to rechromatography under the same conditions, but on a smaller chromatographic column: Superformance 500-50 with an inner diameter of 5 cm, packed with 0.45 L of irregular silica gel C18 Matrex C18 - 100-20 with a particle size of 20 μm. After rechromatography of pool 2, an additional 9.4 g of insulin (1.15 l of eluate with a protein concentration of 8.17 g / l) of the following composition is obtained: insulin - 94.3%, desamido-A21-insulin - 2.7%, impurities - 3.0% After rechromatography of pool 2, the eluate is added to pool 3. 84.4 g of insulin are obtained in 9.48 l of eluate with a concentration of 8.9 g / l. The concentration of propanol-2 in the eluate of 17.5 volume%. 7.11 l of distilled water, 65 ml of zinc chloride solution with a mass fraction of 10%, 0.57 l of citric acid solution with a molar concentration of 1.5 mol / l and an ammonia solution with a mass fraction of 10% to pH are added to the eluate with stirring 6.8. The solution is stirred for 90 minutes, then the pH value is adjusted to 5.9 with hydrochloric acid solution with a mass fraction of 5% gradually over 120 minutes. Stirring is continued for another 4 hours, after which insulin crystals are separated from the mother liquor by suction filter filtration through an Ultipor N ₆₆ NH membrane (Pall) with a diameter of 293 mm and a pore size of 0.8 μm and washed with distilled water in an amount of 200 ml. Get 82.7 g of insulin in terms of dry matter of the following composition: porcine insulin - 94.2%, insamide-A21-insulin - 2.9%, unidentified impurities - 2.9% (HPLC), the content of proinsulin - 8.2 ppm (RIA).

 For the second chromatographic purification, the same packed chromatographic columns are used as for the first chromatographic purification: Super Form 500-100 and Superformance 500-50 and the same Gilson liquid chromatograph.

 After the first chromatographic purification (82.7 g, calculated on the dry matter), the obtained insulin crystals were dissolved in 2.5 L of starting eluent B containing TRIS with a molar concentration of 0.02 mol / L, ethanol with a concentration of 20 vol% and pH 7.6, the pH value is adjusted with hydrochloric acid with a mass fraction of 10%. When insulin is dissolved in eluent B, 41.4 ml of Trilon B solution with a molar concentration of 0.10 mol / L is added to adjust the pH. The resulting insulin solution is filtered under a pressure of 0.7 bar through a Bio-Inert NRLG membrane filter with a diameter of 293 mm and an absolute holding capacity of 0.2 μm, the filter is washed with 0.3 L of starting eluent B. About 2.9 L of insulin solution is obtained.

 Before entering the insulin solution, the Superformance 500-100 chromatography column is pre-equilibrated. To do this, 8 l of starting eluent B with a linear flow rate of 1.27 cm / min are passed through the column.

The insulin solution is fed at a linear flow rate of 0.25 cm / min to the chromatographic column. Then the column is washed with 2 L of starting eluent B. The insulin is eluted at a linear flow rate of 0.5 cm / min in a linear gradient of ethanol concentration from 25 to 37.5 volume% for 8 hours. The gradient is formed automatically by mixing two eluents: the starting eluent B and an eluent containing TRIS with a molar concentration of 0.02 mol / l, ethanol with a concentration of 50 volume% and with a pH value of 7.6. After the gradient is completed, the sorbent in the column is regenerated with an eluent containing a TRIS solution with a molar concentration of 0.020 mol / L and ethanol with a concentration of 70 volume%, which is supplied with a linear flow rate of 1.27 cm / min for 80 minutes. Fractions are analyzed by HPLC for the content of impurities and three pools are formed from the analysis. Pool 4 contains 4.1 g of protein (5 l of eluate with a protein concentration of 0.82 g / l) of the following composition: insulin - 46.7%, desamido-A21-insulin - 19.3%, impurities - 34%. Pool 5 contains 13.2 g of protein (3.8 l of eluate with a protein concentration of 3.47 g / l) of the following composition: insulin - 85.4%, desamido-A21-insulin - 11.1%, impurities - 3.5 % Pool 6 contains 65.3 g of protein (7.9 l of eluate with a protein concentration of 8.27 g / l) of the following composition: insulin - 98.8%, insamide-A21-insulin - 0.3%, impurities - 0.9 % Pool 4 is transferred to production waste. Pool 5 is subjected to rechromatography under the same conditions, but on a smaller chromatographic column: Superformance 500-50, packed with 0.45 L of irregular silica gel C18 Matrex C18 - 100-20 with a particle size of 20 microns. After rechromatography of pool 5, an additional 9.9 g of insulin (1.4 l of eluate with a protein concentration of 7.07 g / l) of the following composition is obtained: insulin - 98.1%, desamido-A21-insulin - 1.0%, impurities - 0.9%. After rechromatography of pool 5, the eluate is added to pool 6. 75.2 g of insulin in 9.3 L of eluate is obtained with a protein concentration of 8.09 g / L. The concentration of ethanol in the eluate of 33.5 volume%. 5.28 l of distilled water, 0.54 l of a solution of ammonium citrate with a molar concentration of 1.5 mol / l and 58 ml of a solution of zinc chloride with a mass fraction of 10% are added to the eluate with stirring. The resulting crystallization solution was filtered under a pressure of 1 bar through a Bio-Inert NRL (Pall) sterilizing membrane filter with a diameter of 293 mm and an absolute retention capacity of 0.2 μm, the filter was washed with 1.0 L of distilled water, and the washing water was added to the crystallization solution. In a crystallization solution with stirring with a solution of hydrochloric acid with a mass fraction of 5%, a pH value of 6.9 is established. The solution was stirred for 90 minutes. Then, the pH was adjusted to 6.0 with hydrochloric acid solution with a mass fraction of 5% gradually over 2 hours. Stirring was continued for another 4 hours, after which insulin crystals were separated from the mother liquor by filtration on a suction filter through an Ultipor N ₆₆ NH membrane (Pall ) with a diameter of 293 mm and a pore size of 0.8 μm. The insulin crystals are washed with 100 ml of distilled water, then three times with 100 ml of ethanol. The drying of insulin crystals is carried out in a vacuum oven to constant weight. Weight loss on drying 9.0%. Get 81.9 g by technical weight (74.5 g in terms of dry matter) of highly purified monocomponent porcine insulin of the following composition: porcine insulin - 98.7%, insamide-A21-insulin - 0.4%, unidentified impurities - 0, 9% (HPLC). The proinsulin content is 0.7 ppm (RIA). Yield 74.5%.

Example 2. The process of chromatographic purification of insulin is carried out at a temperature of 20 ^o C. 5.2 g of technical weight (4.8 g in terms of dry matter) of raw porcine insulin containing 4.3% of desamido-A21-insulin and 8, 7% of impurities are dissolved in 0.150 L of starting eluent A containing a solution of acetic acid with a molar concentration of 0.25 mol / L, acetonitrile with a concentration of 17.5 volume%, the pH in the insulin solution is adjusted to 2.7 with hydrochloric acid with a mass concentration 10%. The resulting insulin solution is filtered under an overpressure of 0.7 bar through a Bio-Inert NRLG membrane filter (Pall) with a diameter of 47 mm and an absolute retention capacity of 0.2 μm, the filter is washed with 0.010 L of starting eluent A. Obtain about 0.17 L insulin solution.

и размером частиц 10 мкм суспендируют в 0,16 л пропанола-2. В хроматографическую колонку Superformance 300-26 (фирма Merck) с внутренним диаметром 2,6 см и высотой 30 см загружают полученную суспензию сорбента. Перед загрузкой суспензии к верней части рабочей хроматографической колонки Superformance 300-26 присоединяют аналогичную упаковочную колонку. Колонки собирают и подают пропанол-2 с линейной скоростью потока 3,8 см/мин (при этом давление не должно превышать 50 bar). После формирования слоя сорбента упаковочную колонку снимают. Сорбент в рабочей колонке сжимают с усилием нижним и верхним подвижными адаптерами. Получают слой сорбента высотой 30 см и объемом 0,16 л. Для уравновешивания через колонку пропускают 0,64 л стартового элюента А.65 g of spherical silica gel C8 DuraSil S-100-C8 (APO company) with pore size

and a particle size of 10 μm suspended in 0.16 l of propanol-2. The chromatographic column Superformance 300-26 (Merck) with an internal diameter of 2.6 cm and a height of 30 cm is loaded with the resulting suspension of the sorbent. Before loading the suspension, a similar packaging column is attached to the top of the Superformance 300-26 working chromatographic column. The columns are collected and fed propanol-2 with a linear flow rate of 3.8 cm / min (the pressure should not exceed 50 bar). After the formation of the sorbent layer, the packing column is removed. The sorbent in the working column is compressed with force by the lower and upper movable adapters. Get a layer of sorbent with a height of 30 cm and a volume of 0.16 L. To balance, 0.64 L of starting eluent A is passed through the column.

 The insulin purification process is carried out on a Gilson liquid chromatograph (Gilson company), equipped with model 305, 306 pumps with 25Wti heads, model 805 pressure damper, model 811 dynamic mixer, model 112 photometric UV detector and FC-30 fraction collector (Fharmacia Biotech company) .

). После рехроматографии пула 2 получают дополнительно 0,432 г инсулина (0,055 л элюата с концентрацией белка 7,85 г/л) следующего состава: инсулина - 97,4%, дезамидо-А21-инсулина - 0,8%, примесей - 1,8%. Элюат, после рехроматографии пула 2 присоединяют к пулу 3. Получают 4,128 г инсулина в 0,511 мл элюата с концентрацией 8,08 г/л. Концентрация ацетонитрила в элюате 23 объемных %. В элюат при перемешивании добавляют 0,664 л дистиллированной воды, 3,2 мл раствора хлорида цинка с массовой долей 10%, 40,5 мл раствора лимонной кислоты с молярной концентрацией 1,5 моль/л и раствор аммиака с массовой долей 10% до значения рН 6,8. Раствор перемешивают в течение 90 мин, затем доводят значение рН до 5,9 раствором кислоты соляной с массовой долей 5% постепенно в течение 120 мин. Продолжают перемешивание еще 4 ч, после чего отделяют кристаллы инсулина от маточного раствора фильтрацией на нутч-фильтре через мембрану Ultipor N₆₆ NH (фирма Pall) с диаметром 47 мм и размером пор 0,8 мкм и промывают 15 мл дистиллированной воды. Получают 4,045 г инсулина в пересчете на сухое вещество следующего состава: инсулина свиного - 97,0%, дезамидо-А21-инсулина - 0,8%, неидентифицированных примесей - 2,2% (ВЭЖХ), содержание проинсулина 5,6 p.p.m. (РИА).The insulin solution is fed at a linear flow rate of 1.5 cm / min to the chromatographic column. Then the column is washed with 0.160 L of starting eluent A. The insulin is eluted at a linear flow rate of 3.0 cm / min in a linear gradient of the concentration of acetonitrile from 17.5 to 30 volume% for 125 minutes. The formation of the gradient is carried out automatically by mixing two eluents: starting eluent A and an eluent containing a solution of acetic acid with a molar concentration of 0.125 mol / l and acetonitrile with a concentration of 50 volume%. After the gradient is completed, the sorbent in the column is regenerated with an eluent containing a solution of acetic acid with a molar concentration of 0.125 mol / L and acetonitrile with a concentration of 50 volume% with a linear flow rate of 3.0 cm / min for 40 minutes. The eluate containing the protein is crushed into fractions of 0.040 L each. Fractions are analyzed by HPLC for the content of impurities and three pools are formed from the analysis. Pool 1 contains 0.528 g of protein (0.6 l of eluate with a protein concentration of 0.88 g / l) of the following composition: insulin - 15.1%, desamido-A21-insulin - 28.6%, impurities - 56.3%. Pool 2 contains 0.576 g of protein (0.24 L of eluate with a protein concentration of 2.4 g / L) of the following composition: insulin - 88.1%, desamido-A21-insulin - 5.1%, impurities - 6.8%. Pool 3 contains 3,696 g of protein (0.456 l of eluate with a protein concentration of 8.11 g / l) of the following composition: insulin - 97.1%, desamido-A21-insulin - 0.7%, impurities - 2.2%. Pool 1 is transferred to production waste. Pool 2 is subjected to rechromatography under the same conditions, but on a smaller chromatographic column: Superformance 300-10 with an inner diameter of 1 cm, packed with 0.023 L of spherical silica gel C8 DuraSil S-100-C8 (particle size 10 μm, pore size

) After rechromatography of pool 2, an additional 0.432 g of insulin (0.055 l of eluate with a protein concentration of 7.85 g / l) of the following composition is obtained: insulin - 97.4%, insamide-A21-insulin - 0.8%, impurities - 1.8% . The eluate, after rechromatography of pool 2, is attached to pool 3. 4.128 g of insulin in 0.511 ml of eluate is obtained with a concentration of 8.08 g / l. The concentration of acetonitrile in the eluate is 23 volume%. 0.664 L of distilled water, 3.2 ml of zinc chloride solution with a mass fraction of 10%, 40.5 ml of citric acid solution with a molar concentration of 1.5 mol / L and an ammonia solution with a mass fraction of 10% to pH are added to the eluate with stirring. 6.8. The solution is stirred for 90 minutes, then the pH value is adjusted to 5.9 with hydrochloric acid solution with a mass fraction of 5% gradually over 120 minutes. Stirring is continued for another 4 hours, after which insulin crystals are separated from the mother liquor by suction filter filtration through an Ultipor N ₆₆ NH membrane (Pall) with a diameter of 47 mm and a pore size of 0.8 μm and washed with 15 ml of distilled water. Obtain 4.045 g of insulin in terms of dry matter of the following composition: pork insulin - 97.0%, insamide-A21-insulin - 0.8%, unidentified impurities - 2.2% (HPLC), proinsulin content of 5.6 ppm (RIA )

 For the second chromatographic purification, the same packed chromatographic columns are used as for the first chromatographic purification: Superformance 300-26 and Superformance 300-10 and the same Gilson liquid chromatograph.

 The obtained insulin crystals after the first chromatographic purification (4.045 g in terms of dry matter) are dissolved in 130 ml of starting eluent B containing TRIS with a molar concentration of 0.02 mol / L, ethanol with a concentration of 20 volume% and with a pH value of 7.8, the pH value is adjusted with a solution of hydrochloric acid with a mass fraction of 10%. When insulin is dissolved in eluent B, 2.0 ml of Trilon B solution with a molar concentration of 0.10 mol / L is added to adjust the pH. The resulting insulin solution is filtered under a pressure of 0.7 bar through a Bio-Inert NRLG membrane filter with a diameter of 47 mm and an absolute holding capacity of 0.2 μm, the filter is washed with 5 ml of starting eluent B. Receive about 142 ml of insulin solution.

 Before entering the insulin solution, the Superformance 300-26 chromatographic column is pre-equilibrated. For this, 0.64 L of starting eluent B with a linear flow rate of 3.0 cm / min is passed through the column.

The insulin solution is fed at a linear flow rate of 1.5 cm / min to the chromatographic column. The column is then washed with 160 ml of starting eluent B. The insulin is eluted at a linear flow rate of 3.0 cm / min in a linear gradient of ethanol concentration from 25 to 37.5 volume% for 125 minutes. The formation of the gradient is carried out automatically by mixing two eluents: starting eluent B and an eluent containing TRIS with a molar concentration of 0.012 mol / l, ethanol with a concentration of 50 volume% and with a pH value of 7.8. After the gradient is completed, the sorbent in the column is regenerated with an eluent containing a TRIS solution with a molar concentration of 0.04 mol / L, ethanol with a concentration of 70 volume% and a pH value of 8.5, which is supplied with a linear flow rate of 3.0 cm / min for 40 min Fractions are analyzed by HPLC for the content of impurities and three pools are formed from the analysis. Pool 4 contains 0.121 g of protein (0.101 l of eluate with a protein concentration of 1.2 g / l) of the following composition: insulin - 50.5%, desamido-A21-insulin - 7.8%, impurities - 41.7%. Pool 5 contains 0.607 g of protein (0.148 L of eluate with a protein concentration of 4.1 g / L) of the following composition: insulin - 94.8%, desamido-A21-insulin - 2.7%, impurities - 2.5%. Pool 6 contains 3.317 g of protein (0.364 l of eluate with a protein concentration of 9.11 g / l) of the following composition: insulin - 99.1%, desamido-A21-insulin - 0.2%, impurities - 0.7%. Pool 4 is transferred to production waste. Pool 5 is subjected to rechromatography under the same conditions, but on a smaller chromatographic column: Superformance 300-10, packed with 23 ml of spherical silica gel C8 DuraSil S-100-C8. After rechromatography of pool 5, an additional 0.485 g of insulin (0.057 l of eluate with a protein concentration of 8.51 g / l) of the following composition is obtained: insulin - 98.9%, dzamido-A21-insulin - 0.3%, impurities - 0.8% . After rechromatography of pool 5, the eluate is attached to pool 6. 3.802 g of insulin in 0.421 l of eluate is obtained with a protein concentration of 9.03 g / l. The concentration of ethanol in the eluate of 32.5 volume%. 0.24 l of distilled water, 0.024 l of a solution of ammonium citrate with a molar concentration of 1.5 mol / l and 2.9 ml of a solution of zinc chloride with a mass fraction of 10% are added to the eluate with stirring. The resulting crystallization solution was filtered under a pressure of 0.7 bar through a Bio-Inert NRL (Pall) sterilizing membrane filter with a diameter of 47 mm and an absolute retention capacity of 0.2 μm, the filter was washed with 0.023 L of distilled water, and the washing water was added to the crystallization solution. In a crystallization solution with stirring with a solution of hydrochloric acid with a mass fraction of 5%, a pH value of 6.9 is established. The solution was stirred for 90 minutes. Then, the pH was adjusted to 6.0 with hydrochloric acid solution with a mass fraction of 5% gradually over 2 hours. Stirring was continued for another 4 hours, after which insulin crystals were separated from the mother liquor by filtration on a suction filter through an Ultipor N ₆₆ NH membrane (Pall ) with a diameter of 47 mm and a pore size of 0.8 μm. Insulin crystals are washed with 0.015 L of distilled water, then three times with ethanol in 0.010 L portions each. Drying of insulin crystals is carried out in a vacuum oven to constant weight. Weight loss on drying 8.0%. Obtain 4.091 g by technical weight (3.764 g in terms of dry matter) of highly purified monocomponent porcine insulin of the following composition: porcine insulin - 99.0%, insamide-A21-insulin - 0.3%, unidentified impurities - 0.7% (HPLC ) The proinsulin content of 0.6 ppm (RIA). The yield is 78.4%.

Example 3. The process of chromatographic purification of insulin is carried out at a temperature of 16 ^o C. 9.0 g of technical weight (8.19 g in terms of dry matter) of raw porcine insulin containing 5.1% desamido-A21-insulin and 9, 5% of impurities are dissolved in 0.300 l of starting eluent A containing a solution of trifluoroacetic acid with a mass fraction of 0.15% and acetonitrile with a concentration of 18.0 volume%, the pH in the insulin solution is adjusted to 2.4 with hydrochloric acid with a mass concentration of 10% . The resulting insulin solution is filtered under an overpressure of 0.7 bar through a Bio-Inert NRLG membrane filter (Pall) with a diameter of 47 mm and with an absolute retention capacity of 0.2 μm, the filter is washed with 0.030 L of starting eluent A. Obtain about 0.340 L of insulin solution .

Для уравновешивания через колонку пропускают 1,30 л стартового элюента А.For purification of insulin, a ready-made chromatographic column with a dynamic axial compression system Dynamax-300A C8 (Rainin), with an inner diameter of 4.14 cm and a length of 25 cm, is used. The column is packed with C8 spherical silica gel with a particle size of 15 μm and an average pore size

To balance, 1.30 L of starting eluent A is passed through the column.

 The insulin purification process is carried out on a Gilson liquid chromatograph (Gilson company) equipped with model 305, 306 pumps with 50SC heads, model 806 pressure damper, model 811 dynamic mixer, model 112 photometric UV detector and FC-30 fraction collector (Fharmacia Biotech company) .

The insulin solution is fed at a linear flow rate of 1.5 cm / min to the chromatographic column. Then the column is washed with 0.330 L of starting eluent A. The insulin is eluted with a linear flow rate of 3.0 cm / min in a linear gradient of the concentration of acetonitrile from 18.0 to 30.0 vol% for 100 min. The gradient is formed automatically by mixing two eluents: starting eluent A and an eluent containing a solution of trifluoroacetic acid with a mass fraction of 0.075% and acetonitrile with a concentration of 50 volume%. After the end of the gradient, the sorbent in the column is regenerated with an eluent containing a solution of trifluoroacetic acid with a mass fraction of 0.075% and acetonitrile with a concentration of 50 volume%, with a linear flow rate of 3.0 cm / min for 30 minutes. The eluate containing the protein is crushed into fractions of 0.080 L each. Fractions are analyzed by HPLC for the content of impurities and three pools are formed from the analysis. Pool 1 contains 0.983 g of protein (1.31 l of eluate with a protein concentration of 0.75 g / l) of the following composition: insulin - 6.9%, desamido-A21-insulin - 29.1%, impurities - 64.0%. Pool 2 contains 0.655 g of protein (0.35 l of eluate with a protein concentration of 1.87 g / l) of the following composition: insulin - 75.8%, desamido-A21-insulin - 14.1%, impurities - 10.1%. Pool 3 contains 6.552 g of protein (0.753 l of the eluate with a protein concentration of 8.70 g / l) of the following composition: insulin - 97.6%, desamido-A21-insulin - 0.6%, impurities - 1.8%. Pool 1 is transferred to production waste. Pool 2 is subjected to rechromatography under the same conditions, but on a smaller chromatographic column: Superformance 300-10 with an inner diameter of 1 cm, packed with 0.023 L of spherical silica gel C8 DuraSil S-100-C8 (particle size 10 μm). After rechromatography of pool 2, an additional 0.426 g of insulin (0.060 l of eluate with a protein concentration of 7.1 g / l) of the following composition is obtained: insulin - 97.2%, insamide-A21-insulin - 0.8%, impurities - 2.0% . The eluate, after rechromatography of pool 2, is attached to pool 3. 6.978 g of insulin in 0.813 l of eluate is obtained with a concentration of 8.58 g / l. The concentration of acetonitrile in the eluate of 24.4 volume%. 1.17 L of distilled water, 5.4 ml of a solution of zinc chloride with a mass fraction of 10%, 68 ml of a solution of citric acid with a molar concentration of 1.5 mol / L and an ammonia solution with a mass fraction of 10% to pH are added to the eluate with stirring. 6.8. The solution is stirred for 90 minutes, then the pH value is adjusted to 5.9 with hydrochloric acid solution with a mass fraction of 5% gradually for 2 hours. Stirring is continued for another 4 hours, after which insulin crystals are separated from the mother liquor by filtration on a suction filter through a membrane Ultipor N ₆₆ NH (Pall) with a diameter of 47 mm and a pore size of 0.8 μm and washed with 20 ml of distilled water. Get 6.84 g in terms of dry matter of insulin of the following composition: insulin - 97.5%, desamido-A21-insulin - 0.7%, impurities - 1.8% (HPLC), proinsulin content 4.7 ppm (RIA )

 For the second chromatographic purification, the same packed chromatographic column is used as for the first chromatographic purification: Dynamax-300 A C8 and the same Gilson liquid chromatograph.

 After the first chromatographic purification (6.84 g, calculated on the dry matter), the obtained insulin crystals were dissolved in 0.2 L of starting eluent B containing TRIS with a molar concentration of 0.01 mol / L, ethanol with a concentration of 20 vol% and pH 7.8, the pH value is adjusted if necessary with a solution of hydrochloric acid with a mass fraction of 10%. When insulin is dissolved in eluent B, 3.4 ml of Trilon B solution with a molar concentration of 0.10 mol / L is added to adjust the pH. The resulting insulin solution is filtered under a pressure of 0.7 bar through a Bio-Inert NRLG membrane filter with a diameter of 47 mm and an absolute retention capacity of 0.2 μm, the filter is washed with 28 ml of starting eluent B. Obtain about 0.25 L of insulin solution.

 Before entering the insulin solution, the Dynamax-300 A C8 chromatography column was pre-equilibrated. To do this, 1.3 L of starting eluent B with a linear flow rate of 3.0 cm / min is passed through the column.

 The insulin solution is fed at a linear flow rate of 1.5 cm / min to the chromatographic column. Then the column is washed with 0.33 L of starting eluent B. The insulin is eluted with a linear flow rate of 3.0 cm / min in a linear gradient of ethanol concentration from 25.0 to 37.5 volume% for 100 minutes. The formation of the gradient is carried out automatically by mixing two eluents: starting eluent B and an eluent containing TRIS with a molar concentration of 0.005 mol / l, ethanol with a concentration of 50 volume% and with a pH value of 7.8. After the gradient is completed, the sorbent in the column is regenerated with an eluent containing TRIS solution with a molar concentration of 0.01 mol / L, ethanol with a concentration of 70 volume% and a pH value of 8.0, is fed at a linear flow rate of 3.0 cm / min for 35 minutes . Fractions are analyzed by HPLC for the content of impurities and two pools are formed from the analysis. Pool 4 contains 0.27 g of protein (0.35 l of eluate with a protein concentration of 0.77 g / l) of the following composition: insulin - 48.7%, desamido-A21-insulin - 15.3%, impurities - 36.0 % Pool 5 contains 6.57 g of protein (0.792 L of eluate with a protein concentration of 8.3 g / L) of the following composition: insulin - 99.5%, desamido-A21-insulin - 0.1%, impurities - 0.4%. Pool 4 is transferred to waste. The insulin contained in pool 5 is crystallized.

The concentration of ethanol in the eluate 32.0% vol. 0.450 l of distilled water, 0.044 l of a solution of ammonium citrate with a molar concentration of 1.5 mol / l and 5.1 ml of a solution of zinc chloride with a mass fraction of 10% are added to the eluate with stirring. The resulting crystallization solution was filtered under a pressure of 0.7 bar through a Bio-Inert NRL (Pall) sterilizing membrane filter with a diameter of 47 mm and an absolute retention capacity of 0.2 μm, the filter was washed with 0.025 L of distilled water, and the washing water was added to the crystallization solution. In a crystallization solution with stirring with a solution of hydrochloric acid with a mass fraction of 5%, a pH value of 6.9 is established. The solution was stirred for 90 minutes. Then the pH was adjusted to 6.1 with hydrochloric acid solution with a mass fraction of 5% gradually over a period of 2 hours. Stirring was continued for another 5 hours, after which insulin crystals were separated from the mother liquor by filtration on a suction filter through an Ultipor N ₆₆ NH membrane (Pall ) with a diameter of 47 mm and a pore size of 0.8 μm. Insulin crystals are washed with 0.020 L of distilled water, then three times with ethanol in 0.015 L portions each. Drying of insulin crystals is carried out in a vacuum oven to constant weight. Weight loss on drying 9.1%. 7.15 g are obtained by technical weight (6.50 g in terms of dry matter) of highly purified monocomponent porcine insulin of the following composition: porcine insulin - 99.3%, insamide-A21-insulin - 0.2%, unidentified impurities - 0, 5% (HPLC). The content of proinsulin is 0.3 ppm (RIA). The yield is 79.4%.

Example 4. The chromatographic purification process is carried out at a temperature of 15 ^o C. Take semi-synthetic human raw insulin obtained by the method described in [8] from porcine desalanine-B30-insulin by interaction with a threonine derivative in the presence of trypsin. Due to the high content of impurities in semisynthetic raw human insulin (22.5%), it is first pre-purified by front-displacement column chromatography. To this end, 4.88 g by technical weight (4.44 g in terms of dry matter) of semisynthetic human raw insulin is dissolved in 40 ml of eluent A containing acetic acid with a molar concentration of 0.12 mol / L and acetonitrile with a concentration of 26 volume % In an insulin solution, a pH of 2.6 is established with a hydrochloric acid solution with a mass fraction of 10%. The solution is filtered through a Bio-Inert NRLG membrane filter (Pall) with an absolute retention capacity of 0.2 μm, the filter is washed with 5 ml of eluent A. The resulting insulin solution with a linear flow rate of 1.27 cm / min is passed through a Superformance 300-10 chromatographic column (Merck), filled with 55 ml of spherical silica gel C8 DuraSil S-100-C8 (particle size 10 μm). The column is then washed with 110 ml of eluent A. 140 ml of an eluate containing 3.99 g of insulin with a basic substance content of 84.9% are obtained. 68 ml of an aqueous solution of acetic acid with a molar concentration of 0.25 mol / L are added to the eluate, and further purification is carried out analogously to Example 2. 3.34 g of technical weight (3.04 g in terms of dry matter) of highly purified monocomponent semi-synthetic human insulin are obtained with a basic substance content of 98.9%. The content of desamido-A21-insulin is 0.3%, the content of unidentified impurities is 0.8%, including porcine insulin 0.2% (HPLC). The content of porcine proinsulin is 0.3 ppm (RIA). The yield is 68.4%, taking into account pre-treatment.

Example 5. The chromatographic purification process is carried out at a temperature of 15 ^o C. Take raw human insulin, which is obtained from genetically engineered (biosynthetic) human proinsulin under the action of trypsin and carboxypeptidase B according to the method described in [9]. The content of impurities in human raw insulin is 9.5%. Take 9.0 g by technical weight (8.1 g in terms of dry matter) of human raw insulin and further purification is carried out analogously to example 3. Receive 7.1 g by technical weight (6.4 g in terms of dry matter) highly purified monocomponent genetically engineered human insulin with a basic substance content of 99.0%, a content of desamido-A21-insulin - 0.2%, an impurity content of 0.7% (HPLC), a human proinsulin content of 0.3 ppm (RIA). The yield is 78.9%.

и размером частиц 10 мкм используют полимерный монодисперсный ОФ-сорбент Source 15RPC фирмы Fharmacia Biotech с размером частиц 15 мкм.Example 6. The chromatographic purification process is carried out at a temperature of 18 ^o C. 3.60 g are taken according to the technical weight (3.25 g in terms of dry matter) of raw porcine insulin with an impurity content of 8.7% and they are purified analogously to example 2 except that instead of spherical silica gel C8, DuraSil S-100-C8 (ARO company) with pore size

and a particle size of 10 μm using a polymer monodisperse OF-sorbent Source 15RPC sorbent from Fharmacia Biotech with a particle size of 15 μm.

 2.75 g are obtained by technical weight (2.50 g in terms of dry matter) of highly purified monocomponent porcine insulin of the following composition: porcine insulin - 98.8%, insamide-A21-insulin - 0.4%, unidentified impurities - 0, 8% (HPLC). The proinsulin content is 0.8 p.p.m. (RIA). The yield is 76.9%.

Example 7. The chromatographic purification process is carried out at a temperature of 17 ^o C. Take 5.20 g of technical weight (4.80 g in terms of dry matter) of raw porcine insulin with an impurity content of 8.7% and carry out cleaning similarly to example 2 for except that:
1. Change the order of the first and second chromatographic purifications: first, chromatographic purification is carried out at a pH of eluents of 7.6, and then chromatographic purification at a pH of eluents of 2.7.

 2. Use ethanol instead of acetonitrile in eluents.

 Receive 4.20 g of technical weight (3.79 g in terms of dry matter) of highly purified monocomponent porcine insulin of the following composition: porcine insulin - 98.6%, insamide-A21-insulin - 0.5%, unidentified impurities - 0, 9% (HPLC). The proinsulin content is 0.4 p.p.m. (RIA). The yield is 78.9%.

Example 8. The chromatographic purification process is carried out at a temperature of 16 ^o C. Take 5.20 g of technical weight (4.80 g in terms of dry matter) of raw porcine insulin with an impurity content of 8.7% and carry out purification analogously to example 2 for except that instead of a continuous linear gradient, a step gradient is used in the first chromatographic purification. Pool 3 is eluted with an eluent that contains acetic acid with a molar concentration of 0.20 mol / L and acetonitrile with a concentration of 26.5 volume%, which is supplied for 60 minutes with a linear flow rate of 1.5 cm / min. Pool 2 is eluted with an eluent containing acetic acid with a molar concentration of 0.20 mol / L and acetonitrile with a concentration of 28.0 vol%, which is supplied for 20 min with a linear flow rate of 3.0 cm / min. Further purification (rechromatography of pool 2, second chromatographic purification) and regeneration of the sorbent are carried out as described in example 2.

 Obtain 4.15 g by technical weight (3.81 g in terms of dry matter) of highly purified monocomponent porcine insulin of the following composition: porcine insulin - 98.9%, insamide-A21-insulin - 0.2%, unidentified impurities - 0, 9% (HPLC). The proinsulin content is 0.7 p.p.m. (RIA). The yield is 79.3%.

Example 9. The chromatographic purification process is carried out at a temperature of 14 ^o C. Take 5.20 g of technical weight (4.80 g in terms of dry matter) of raw porcine insulin with an impurity content of 8.7% and carry out the purification analogously to example 2 for except that to improve the degree of separation of insulin and argininsulin, instead of a linear gradient during the first chromatographic purification, an exponential gradient is used, which is described by the following expression:
C = 16 + 1.2e ^{N / 3} ,
where C is the concentration of acetonitrile in the mobile phase, expressed in volume%;
e is the base of the natural logarithm (2.71828 ...);
N is the ratio of the volume of eluent fed to the column to the volume of sorbent in the column (from 0 to 10).

 Get 4.21 g of technical weight (3.89 g in terms of dry matter) of highly purified monocomponent porcine insulin of the following composition: porcine insulin - 99.1%, insamide-A21-insulin - 0.3%, unidentified impurities - 0, 6% (HPLC). The proinsulin content is 0.7 p.p.m. (RIA). Yield 81.0%.

Bibliographic data
1. V.S. Belinsky, G.N. Khlyabich, I.A. Donetsk. Scaling and implementation of technology for obtaining highly purified insulin. Abstracts of reports. All-Russian Symposium "Bio-Separation-96". St. Petersburg, 1996

 2. Dillon, W. W., and Romans, R. G.: Heterogeneity of Insulin II. Chromatography of Insulin on Carboxymethyl Cellulose in Urea Buffers. Canad. J. Biochem. 1967.

 3. Smith, L. F .; Isolation of Insulin from Pancreatic Extracts Using Carboxymethyl and Diethylaminoethyl Cellulose. Biochem. Biophys. Acta. 1964.

 4. V.V. Kandaryuk, I.A. Donetsky, V.A. Gulyaev, and others. USSR Patent 2734204, 19.02.79.

 5. US patent 2105543, 03/15/78.

 6. French patent 2581647, 04.17.85,

 7. E. R. Kroeff, R. A. Owens, E. L. Campbell, R. D. Johnson and H. I. Marks. Production Scale Purification of Biosynthetic Human Insulin by Reversed-phase High-performance Liquid Chromatography., Journal of Chromatography, 461 (1989) 45-61.

 8. Morihara et al. Pat. 4511505 (US), 04.16.85 g.

 9. Ellie Lilly and Company. Bruce Hill Frank, Richard Eugene Heaney et al. U.S. Patent 1829939, 10.13.87.

Claims (14)

1. A method for producing highly purified monocomponent insulin contains not less than 98.5% of the main substance, not more than 0.5% desamido-insulin-A21, not more than 1.0% of unidentified impurities and not more than 1.0 million ^-1 proinsulin with using liquid column chromatographic purification of raw insulin at a temperature of 5-30 ^° C in aqueous-organic eluents with ionic modifiers on non-polar sorbents, characterized in that the raw insulin with a total amount of 15-50 g per 1 liter of sorbent in a chromatographic column is dissolved in eluent A with a pH value 1.5 to 4.5, containing ionic modifiers with a concentration of 0.001-0.3 mol / l and an organic solvent with a concentration of 10-30 vol.%, Is introduced at a linear flow rate of 0.25-1.5 cm / min into the chromatographic a column with a sorbent layer height of 10-60 cm previously balanced with eluent A and filled with a non-polar sorbent with an average pore size

particle size 10 - 100 μm, sorbed insulin is eluted from the chromatographic column with a linear flow rate of 0.5 - 3.0 cm / min using a stepwise or continuous gradient of the concentration of the organic solvent and a highly purified insulin intermediate is obtained, which is then isolated in crystalline form and dissolved in eluent B with a pH value of 6.5 - 9.5, containing ionic modifiers with a concentration of 0.001 - 0.3 mol / l and an organic solvent with a concentration of 10 - 30 vol.%, is introduced with a linear flow rate of 0.25 - 1 5 cm / min in chromatographic column with a sorbent layer height of 10-60 cm, previously balanced by eluent B and filled with a non-polar sorbent with an average pore size

particle size 10 - 100 μm, sorbed insulin is eluted from the chromatographic column with a linear flow rate of 0.5 - 3.0 cm / min using a stepwise or continuous gradient of the concentration of the organic solvent, and a solution of highly purified monocomponent insulin is obtained.  2. The method according to claim 1, characterized in that acetonitrile, ethanol, propanol-2, methanol are used as an organic solvent in eluent A.  3. The method according to claim 1, characterized in that ethanol is used as an organic solvent in eluent B.  4. The method according to claim 1, characterized in that C8 silica gel is used as a non-polar sorbent.  5. The method according to claim 1, characterized in that C18 silica gel is used as a non-polar sorbent.  6. The method according to claim 1, characterized in that as a non-polar sorbent use a polymer OF sorbent.  7. The method according to claim 1, characterized in that a non-polar sorbent with a particle size of 10-15 microns is used. 8. The method according to claim 1, characterized in that use non-polar sorbent with pore size

9. The method according to claim 1, characterized in that they use a spherical non-polar sorbent.  10. The method according to claim 1, characterized in that as the ionic modifier use organic and inorganic acids, bases and their salts.  11. The method according to claim 10, characterized in that acetic, trifluoroacetic, orthophosphoric, hydrochloric acids, TRIS, ammonia, sodium hydroxide and their salts are used as an ionic modifier.  12. The method according to claim 1, characterized in that the use of eluent A with a pH value of 2.3 - 2.8.  13. The method according to claim 1, characterized in that the use of eluent B with a pH value of 7.3 to 8.0.  14. The method according to claim 1, characterized in that chromatographic columns with a sorbent layer height of 25-40 cm are used. 15. The method according to claim 1, characterized in that the chromatographic purification process is carried out at a temperature of 10 - 20 ^o C.