RU2126690C1 - Method of purification of crude insulin obtained from pig pancreas - Google Patents

Abstract

FIELD: biotechnology, biochemistry. SUBSTANCE: method involves the successive combination of reversed-phase and anion-exchange methods on chromatography unit of low pressure. Method involves loading and elution processes of components to be separated by frontal-displacing regime. Highly purified insulin has, %: 7% basic substance, not less 7; proinsulin, not above 0.001; high-molecular proteins, not more, 1; insulin-like impurities, not more 1. The yield of end product is not less than 70%. EFFECT: improved method of purification, enhanced quality of product. 13 cl, 1 ex

Description

 The invention relates to medicine, specifically to methods for purifying insulin, a drug for the treatment of diabetes mellitus.

 Modern insulin preparations are made from highly purified substances. This is due to the fact that protein impurities that are usually present in insulins obtained from the pancreas of animals, especially proinsulins, insulin esters and polymer insulins, have high antigenic activity and can cause immunogenic damage to the vessels and internal organs of patients with diabetes who use drugs that are not sufficiently purified insulin. In the Pharmacopoeias of developed countries, in particular the British, European and USA, the proinsulin content in the substance of animal insulin is limited to 0.001%, high molecular weight proteins (dimeric and polymer insulin) - 1%, desamidoinsulin - 3% [1].

 For the production of highly purified insulin substance of various nature, various chromatographic methods are currently widely used [2]. The main criterion for the effectiveness of industrial chromatographic technology is the performance of the chromatographic column, expressed in the amount of purified product obtained per unit time per unit volume of the sorbent. Moreover, the performance of the column is proportional to the capacity, selectivity and efficiency of the sorbent [3]. For this reason, in modern industrial chromatographic processes, they strive to use highly efficient sorbents having, as a rule, a spherical or spheroid shape and a small particle size (5-10 microns), which, ceteris paribus, provides greater efficiency and, therefore, higher productivity these sorbents in comparison with less effective sorbents with larger particles of irregular shape.

 On the other hand, the use of highly efficient sorbents requires the use of high pressure (up to 200 atm) at the inlet to the column to realize optimal flow rates of the mobile phase. High-pressure industrial chromatographic systems are quite expensive, which to some extent impedes their widespread use in the production of pharmaceuticals.

 Most of the impurities that reduce the quality of insulin substance are its close analogues with minimal structural differences [4]. Therefore, for their separation from the main product on a preparative scale, one has to use either highly efficient sorbents and, accordingly, expensive high-pressure chromatographic systems, or sorbents and mobile phases that provide high selectivity for the separation of the main substance and impurities. Only in these cases it is possible to use high specific loads on the sorbent, which makes the chromatographic purification process economically viable. In this case, highly selective processes are economically more profitable than highly efficient, since in this case, relatively low-efficiency sorbents and, accordingly, cheaper low-pressure chromatographic systems can be used [5].

 In the general case, the preparative chromatographic separation process can be implemented in at least two different ways: elution or displacement. In the elution method, the separation of the components of the mixture is carried out due to the difference in their distribution coefficients between the mobile and stationary phases. Moreover, it is obvious that at high specific loads on the sorbent, there is a significant broadening and overlapping of the peaks of the components, which leads to a decrease in the yield of the target products [6].

 In the case of the displacement method, the ability of the components of the mobile phase or the components of the mixture to be separated to compete for places in the stationary phase is used in such a way that during the process of applying the mixture to the sorbent and subsequent elution, a so-called displacement front is formed, i.e. a sequence of zones of the separated components, in which each subsequent component displaces the previous one from the stationary phase [7].

 Theory of the process of displacement chromatography is just beginning to be developed, and in most cases the process parameters are selected empirically. Under well-chosen conditions, the degree of overlapping of the component zones in the displacement front is insignificant, which allows the use of much larger specific loads on the sorbent than in the case of the elution method [8].

As a rule, the effectiveness of the front-displacement preparative chromatographic process depends on the following parameters
- the composition of the initial separable mixture and the required quality of the target products, which determines the specific load on the sorbent, as well as the requirements for the sorbent and the initial composition of the mobile phase;
- characteristics of the sorbent used, which determine its capacity, selectivity, dynamics of mass transfer, compatibility with the components of the mixture being separated;
- the initial composition and speed of the mobile phase, which determine the composition of the dynamic stationary phase, as well as the conditions for the formation of the primary displacement front of the adsorbed components of the shared mixture due to the implementation of the self-displacement effect;
- elution conditions of the chromatographic column (gradient, flow rate, etc.), which determine the elution order and the degree of overlap of the chromatographic zones of the separated components, that is, the yield and quality of the products obtained.

 In the case of chromatographic purification of insulins, the problem of selecting process parameters is complicated by the high lability of insulin molecules, namely, their ability to rapidly and irreversibly denature under the influence of various factors, including upon contact with hydrophobic and some metal surfaces, in the presence of a number of inorganic and organic compounds, with an increase or a significant decrease in temperature, etc. [9].

 For this reason, only completely compatible materials and reagents must be used in the technological processes of insulin purification. The stability requirements of insulin impose certain restrictions on the temperature conditions of the process, on the materials of columns, filters and pipelines, as well as on sorbents and composition of mobile phases [10].

 The scientific, technical and patent literature provides examples of the use of various preparative chromatographic methods in the purification of insulin of various nature.

 It was proposed to use gel permeation chromatography for the purification of insulin from impurities of dimeric and polymer insulins [11]. However, this type of chromatography can be used only in the elution version, that is, at low specific loads and low elution rates, which makes these processes economically inefficient.

 It has been proposed to use preparative ion-exchange chromatography, in particular on carboxymethyl, diethylaminoethyl, and other ion-exchange sorbents, for the purification of insulins from insulin-like impurities, in particular, from desamidoinsulin [12]. However, in this case, the selected conditions allow using only low specific loads on the sorbent, which reduces the efficiency of the process.

 It is proposed to use reverse phase preparative chromatography for the purification of insulin from proinsulin and other insulin-like impurities.

 The data presented in the literature indicate the successful application in this case of the front-displacement method of chromatography. However, in the indicated studies, a rather high specific load on the sorbent (about 10% of the total sorption capacity of the sorbent) and the required yields of purified target products were achieved only using high-performance columns and high-pressure chromatographic systems [13].

 We found that a sufficiently effective purification of insulin from insulin-like impurities and proinsulin using the front-displacement chromatographic method can be achieved on low-efficiency sorbents using low pressure chromatographic systems by combining reverse-phase and anion-exchange preparative chromatography methods. In this case, the specific load on the sorbents is at least 30% of their total sorption capacity, which significantly exceeds the figures given in the literature.

 The closest analogue in technical essence and the achieved result to the proposed one is the method of purification of insulin by using chromatographic methods [14].

 The disadvantages [14] are the inability to obtain insulin of the required purity.

 The technical result of the invention is a high degree of purification of insulin with a content of not less than 97% of the main substance, not more than 0.001 proinsulin, not more than 1% high molecular weight proteins and not more than 1% insulin-like impurities with a yield of not less than 70%.

 The technical result is achieved by the fact that in the method of purification of natural raw insulin obtained from the pancreas of pigs, including various types of chromatography, according to the invention, chromatographic purification is carried out sequentially in two stages by reversed-phase and anion-exchange methods in frontal displacement mode using selective desorption target product from chromatographic columns.

The optimal conditions for achieving this result are as follows:
- raw insulin with a basic substance content of at least 80%, most preferably at least 90%, is used as a feedstock;
the content of proinsulin, desamidoinsulin, polymer insulin and other insulin-like impurities is not more than 5% each, most preferably the content is not more than 3% of each impurity;
- chromatographic purification is carried out after preliminary purification of the solution of the initial insulin-raw from insoluble and lipid-pigment impurities by sequential filtration through a membrane filter and a pre-column with a sorbent under conditions that ensure effective separation of lipid-pigment impurities to obtain a solution of technical insulin;
- stage reverse phase chromatography is carried out on a preparative chromatograph low or medium pressure, which allows you to implement a sequential and parallel operation of at least two chromatographic columns using direct and reverse flow of eluting solutions, as well as stepwise or gradient elution;
- stage reverse phase chromatography is carried out by applying the initial solution of technical insulin on serially connected columns in the mode of frontal displacement chromatography under conditions providing selective strong binding of contaminants-proinsulin and polymer insulin on the pre-column, followed by separation of the columns and gradient elution of the working column with a gradual increase in the content of the organic modifier in the eluting solution under specially selected conditions, about espechivayuschih selective desorption of insulin from the sorbent, and simultaneously effective displacement of the target insulin product impurities;
- the reverse phase chromatography step is carried out on hydrophobic sorbents based on silica gel, the surface of which is modified by monomeric or polymeric siloxanes containing C4-C20 hydrocarbon radicals, the most preferred are C12-C18 radicals; the pore diameter of these sorbents is 10-30 nm, the interval 15-20 nm is most preferred; the specific surface of the sorbents is 80-300 m / g, the interval 100-200 m / g is most preferred; the particle size is 30-70 microns, most preferably 30-50 microns; the particle shape of the sorbent is spherical, spheroidal or irregular, the most preferred spherical or spheroidal form; the total sorption capacity of the sorbents for insulin is at least 80 g / l of sorbent; the sorbent is fully compatible with insulin solutions, namely, under the conditions of the process, it does not cause the denaturation, aggregation and polymerization of insulin molecules;
- for application to the sorbent of the source of raw insulin, its solutions with a protein concentration of 50-200 mg / ml are used, the concentration of 80-150 mg / ml is most preferred in a mixture of aqueous solutions of ionic and organic modifiers, and sulfate, acetate are used as anions, chloride or phosphate, sulfate is most preferred; ammonium, sodium or potassium are used as cations; ammonium is most preferred; lower organic alcohols are used as an organic modifier: methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, tert-butanol, isobutanol, as well as acetonitrile, dimethylformamide or dimethylacetamide, ethanol is most preferred; the concentration of the ionic modifier is 0.01-0.1 mol / L, most preferably 0.03-0.07 mol / L; the concentration of the organic modifier is 10-20 vol.%, most preferably 12-15 vol.%; the pH of the solution of the source insulin is 2.0-3.5, most preferably 2.5-3.0; the linear velocity of the liquid phase inside the chromatographic column is 0.1-0.5 cm / min, the most preferred speed is 0.2-0.4 cm / min;
- the amount of partially purified raw insulin introduced into the columns is 10-40 weight. % of the total capacity of the used reversed-phase sorbent, most preferably an amount of 20-30 wt.%;
- the stage of anion exchange chromatography is carried out by introducing a solution of crude insulin partially purified by reverse phase chromatography into a column with an anion exchange sorbent in frontal chromatography under conditions providing selective strong binding of desamidoinsulin and other similar contaminants to the sorbent, followed by elution of the column with an increase the content of the ionic modifier in the eluting solution under conditions providing selective desorption of insulin from the anion-exchange rent;
-the stage of anion exchange chromatography is carried out on sorbents based on silica gel or a hydrophilic porous polymer, the surface of which is modified with chemical ionogenic groups having the properties of weak anion exchangers, diethylaminoethyl (DEAE) groups are most preferred; the pore diameter of these sorbents is 15-50 nm, most preferably a diameter of 20-30 nm; particle size is 20-150 microns, most preferred size is 20-50 microns; the particle shape of the sorbent is spherical, spheroidal or irregular, most preferred is a spherical or spheroidal form; the exchange capacity of the anion exchanger is at least 0.1 meq / ml of sorbent; the total sorption capacity for insulin is at least 30 mg / ml sorbent; the sorbent is fully compatible with insulin solutions, namely, under the conditions of the process, it should not cause the denaturation, aggregation and polymerization of insulin molecules;
- for application to an anion exchanger use an insulin solution with a protein concentration of 20-50 mg / ml, most preferably 25-30 mg / ml; in aqueous solutions of ionic and organic modifiers, with sulfate, acetate, chloride and phosphate being used as anions, sulfate and chloride are most preferred; sodium, potassium and ammonium are used as cations; potassium and ammonium are most preferred; lower alkyl alcohols are used as an organic modifier: methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, iso-butanol, tert-butanol, as well as acetonitrile, demethylformamide and dimethylacetamide, isopropanol is most preferred; the concentration of the ionic modifier is 0.01-0.05 mol / L; the concentration of 0.013-0.017 mol / L is most preferred; the volume concentration of the organic modifier in the solution is 25-35, the concentration of 28-30 vol is most preferred; the pH of the initial insulin solution is 7.5-8.5, most preferably 7.8-8.0; the linear velocity of the liquid phase inside the anion-exchange column is 0.1-0.5 cm / min, the most preferred speed is 0.2-0.3 cm / min;
- the amount of starting insulin introduced into the anion exchange column is 30-70 wt.% of the total sorption capacity of the used anion exchanger for insulin, most preferably the amount is 40-60.%;
- all technological operations for chromatographic purification of raw insulin are carried out at a temperature of 5 -20 ^o C, the most preferred temperature is 8-15 ^o C.

 The invention is illustrated by the following example.

Example
1.28 kg of raw insulin containing 90% of the main substance, 3% of proinsulin, 3% of desamidoinsulin, 3% of polymer insulins and 1% of other insulin-like impurities are dissolved in 10 liters of a 0.05 M aqueous-alcoholic solution of ammonium sulfate containing 10 vol.% ethanol and the amount of sulfuric acid necessary for dissolving insulin. The resulting solution with a protein concentration of 120 g / l, pH 2.5 is filtered through a 0.2 μm membrane filter and then passed through a forcolonna (10x30 cm) containing 1.5 kg of a hydrophobic sorbent (Octadecyl-Silica, for purification from lipid-pigment impurities) 15 nm, 35-75 microns) at a rate of 60 ml / min. For selective desorption of insulin, the forcolonna is washed with 5 liters of a 0.05 M solution of ammonium sulfate in a 10% aqueous solution of ethanol with a pH of 2.5.

 The resulting solution of partially purified insulin is applied to serially connected pre-column (20x30 cm, 1.5 L of Octadecyl-Silica sorbent, 15 nm, 20-30 μm) and a working column (20x50 cm, 7 L of Octadecyl-Silica sorbent, 15 nm, 35- 75 μm) at a rate of 50 ml / min. After applying insulin to the sorbent, the columns are washed with 6.5 liters of a 0.05 M solution of ammonium sulfate in a 10% aqueous solution of ethanol with a pH of 2.5 at a rate of 150 ml / min.

 After washing, the pre-column is disconnected from the working column and selective desorption of insulin from the pre-column is carried out with a 10 liter 0.04 M solution of ammonium sulfate in a 25% aqueous solution of ethanol with a pH of 2.5 at a rate of 150 ml / min. Selective desorption of insulin from the working column is carried out in a gradient mode at a rate of 200 ml / min for 300 minutes, gradually increasing the ethanol content in the eluting solution from 10% to 25% according to a given program.

In the process of desorption of insulin from the pre-column and the working column, fractions of 3-5 l are collected, the composition of which is determined using analytical quantitative HPLC under the conditions described in the literature. The fractions of purified insulin containing less than 0.001% proinsulin, less than 1% polymer insulin and less than 1% insulin-like impurities are combined. 30 l of a solution are obtained, to which 30 l of water is added to precipitate insulin, and the pH of the resulting solution is adjusted to 5.7 with a 7% aqueous ammonia solution. To complete coagulation of insulin, the solution is stirred for 10 hours at a temperature of 10-15 ^o C.

 After settling, the supernatant was decanted and the precipitate was centrifuged at 3000 x g for 30 minutes. After centrifugation, the supernatant is decanted, and the precipitate is washed twice with water at pH 5.8, followed by centrifugation under the same conditions.

 3.8 kg of a wet paste of partially purified insulin containing 28% protein are obtained in the reverse phase chromatography step. The yield is 92% of the loaded raw insulin.

 The above wet paste is dissolved in a calculated volume of 0.015 M aqueous-alcoholic solution of ammonium sulfate containing 30% isopropanol, with a pH of 9.5, so as to obtain a solution with a mass concentration of protein of 30 g / l. The prepared solution is filtered through a membrane filter and then applied to an anion exchange column (30x50 cm, Amicon DEAE-Silica sorbent, 20-50 μm, 8 L) at a rate of 200 ml / min. After applying the initial insulin solution, the column is washed with 10 liters of a 0.015 M aqueous-alcoholic solution of potassium chloride containing 30% isopropanol, with a pH of 7.8 at a rate of 200 ml / min.

 Selective desorption of insulin from the anion-exchange sorbent is carried out by washing with 20 liters of a 0.015 M aqueous-alcoholic solution of ammonium sulfate containing 30% isopropanol with a pH of 7.8 at a rate of 200 ml / min, followed by washing the column with 40 liters of the same eluting solution containing an additional 0.015 mol / l ammonium chloride.

 Fractions of 3-5 l are collected during the desorption process, the composition of which is determined by quantitative analytical HPLC under the conditions described in the literature. According to the results of the analysis, the fractions are combined in such a way as to obtain insulin containing less than 1% of desamidoinsulin.

 From the resulting solution, purified insulin is crystallized as a zinc complex under the conditions described in the literature. After filtration and drying in vacuo, a crystalline substance of purified porcine insulin is obtained that meets international and domestic pharmacopoeial requirements.

 The yield of the target product at the stage of anion exchange chromatography is 80% of the loaded insulin.

 The present invention allows the use of high specific loads on the sorbents, constituting at least 30% of their total sorption capacity, since the method involves loading and eluting the separated components in a front-displacement mode. The result is highly purified insulin containing not less than 97% of the basic substance, not more than 0.001% proinsulin, not more than 1% high molecular weight proteins and not more than 1% insulin-like impurities with a yield of not less than 70%.

Literature
1. British Pharmacopeia, 1993.

 2. US patent N 4129560 (12.12.78).

 3. Helenius et al., Proc. Nat. Acad Sci. USA, 1977, 74, N 2.

 4. UK patent N 881885.

 5. McLeod A et al. J. Chromatogr., 1984, 285, 319-331.

 6. Sokh G.B. J. Chromatogr., 1992, 599, 195-203.

 7. Kopaciewicz W. et al. - J. Chromatogr., 1987, 409, 111-124.

 8. Lee A.L. et al. - J. Chromatogr., 1988, 443, 31-43.

 9. Felinger A. et al. - J. Chromatogr., 1992, 609, 35-47.

 10. Snyder R. et al. - J. Chromatogr., 1991, 536, 57-73.

11. European patent N 0547544 A 2 (12/14/92)
12. Kroeff EP et al. - J. Chromatogr., 1989, 461, 45-61 /
13. European patent N 0474213 A 1.

 14. International application, WO 81 / 00674.0

Claims (13)

 1. The method of purification of natural insulin raw obtained from the pancreas of pigs, including various types of chromatography, characterized in that the chromatographic purification is carried out sequentially in two stages by reversed-phase and anion-exchange methods in frontal displacement mode using selective desorption of the target product from chromatographic columns.  2. The method according to claim 1, characterized in that the raw materials used are raw insulin with a basic substance content of at least 80%, most preferably at least 90%; the content of proinsulin, desamidoinsulin, polymer insulin and other insulin-like impurities is not more than 5% each, most preferably the content is not more than 3% of each impurity.  3. The method according to claim 1, characterized in that the chromatographic purification is carried out after preliminary purification of the solution of the source insulin-raw from insoluble and lipid-pigment impurities by sequential filtration through a membrane filter and a pre-column with a sorbent under conditions that ensure effective separation of lipid-pigment impurities to obtain a solution of technical insulin.  4. The method according to claim 1, characterized in that the stage of reverse phase chromatography is carried out on a preparative low or medium pressure chromatograph, which allows for the implementation of sequential and parallel operation of at least two chromatographic columns using direct and reverse flow of eluting solutions, and stepwise or gradient elution.  5. The method according to claim 1, characterized in that the reverse phase chromatography step is carried out by applying the initial technical insulin solution to the series-connected columns in the front-displacement chromatography mode under conditions providing selective strong binding of contaminants-proinsulin and polymer insulin to the pre-column , followed by separation of the columns and gradient elution of the working column with a gradual increase in the content of the organic modifier in the eluting solution in specially selected conditions providing selective desorption of insulin from the sorbent and at the same time effective displacement of insulin-like impurities by the target product.  6. The method according to claim 3, characterized in that the reverse phase chromatography step is carried out on hydrophobic sorbents based on silica gel, the surface of which is modified by monomeric or polymeric siloxanes containing C4-C20 hydrocarbon radicals, the most preferred are C12-C18 radicals; the pore diameter of these sorbents is 10-30 nm, the most preferred range is 15-20 nm; the specific surface area of the sorbents is 80-300 m / g, the most preferred range is 100-200 m / g; the particle size is 30 to 70 microns, most preferably 30 to 50 microns; the particle shape of the sorbent is spherical, spheroidal or irregular, the most preferred spherical or spheroidal form; the total sorption capacity of the sorbents for insulin is at least 80 g / l of sorbent; the sorbent is fully compatible with insulin solutions, namely, under the conditions of the process, it does not cause the denaturation, aggregation and polymerization of insulin molecules.  7. The method according to p. 4, characterized in that for applying to the sorbent the source of insulin-raw use its solutions with a protein concentration of 50 to 200 mg / ml, the most preferred concentration of 80 to 150 mg / ml; in a mixture of aqueous solutions of ionic and organic modifiers, with sulfate, acetate, chloride or phosphate being used as anions, sulfate is most preferred; ammonium, sodium or potassium are used as cations; ammonium is most preferred; lower organic alcohols are used as an organic modifier: methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, tert-butanol, isobutanol, as well as acetonitrile, dimethylformamide or dimethylacetamide, ethanol is most preferred; the concentration of the ionic modifier is 0.01 to 0.1 mol / L, most preferably 0.03, - 0.07 mol / L; the concentration of the organic modifier is 10-20% by volume, most preferably 12-15% by volume; the pH of the solution of the source insulin raw 2.0 to 3.5, most preferably a value of 2.5 to 3.0; the linear velocity of the liquid phase inside the chromatographic column is 0.1-0.5 cm / min, the most preferred is 0.2-0.4 cm / min.  8. The method according to claim 7, characterized in that the amount of partially purified crude insulin introduced into the columns is 10-40% by weight of the total capacity of the reversed-phase sorbent used, most preferably 20-30% by weight.  9. The method according to p. 1, characterized in that the stage of anion exchange chromatography is carried out by introducing a solution of crude insulin partially purified by reverse phase chromatography into a column with an anion exchange sorbent in frontal chromatography under conditions providing selective strong binding to desamidoinsulin sorbent and other such contaminants, followed by elution of the column with an increase in the content of the ionic modifier in the elution solution under conditions providing selective des orbit of insulin from the anion-exchange sorbent.  10. The method according to claim 9, characterized in that the anion exchange chromatography stage is carried out on sorbents based on silica gel or a hydrophilic porous polymer, the surface of which is modified by chemical ionogenic groups having the properties of weak anion exchangers, diethylaminoethyl (DEAE) groups are most preferred; the pore diameter of said sorbents is 15-50 nm, the diameter of 20-30 nm is most preferred; a particle size of 20 to 150 microns, most preferably a size of 20 to 50 microns; the sorbent particle shape is spherical, spheroidal or irregular, the most preferred spherical or spheroidal form; the exchange capacity of the anion exchanger is not less than 0.1 meq / ml of sorbent; the total sorption capacity for insulin is at least 30 mg / ml sorbent; the sorbent is fully compatible with insulin solutions, namely, under the conditions of the process, it should not cause the denaturation, aggregation and polymerization of insulin molecules.  11. The method according to claim 9, characterized in that for applying to the anion exchanger use an insulin solution with a protein concentration of 20 to 50 mg / ml, most preferably 25 to 30 mg / ml; in aqueous solutions of ionic and organic modifiers, with sulfate, acetate, chloride and phosphate being used as anions, sulfate and chloride are most preferred; sodium, potassium and ammonium are used as cations; potassium and ammonium are most preferred; lower alkyl alcohols are used as an organic modifier: methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, iso-butanol, tert-butanol, as well as acetonitrile, demethylformamide and dimethylacetamide, isopropanol is most preferred; the concentration of the ionic modifier is 0.01-0.05 mol / L; the concentration of 0.013-0.017 mol / L is most preferred; the volume concentration of the organic modifier in the solution is 25 to 35%, the concentration of 28 to 30 vol.% is most preferred; the pH of the initial insulin solution is 7.5-8.5, most preferably 7.8-8.0; the linear velocity of the liquid phase inside the anion exchange column is 0.1-0.5 cm / min; the most preferred is 0.2-0.3 cm / min.  12. The method according to claim 9, characterized in that the amount of source insulin introduced into the anion exchange column is 30 to 70% by weight of the total sorption capacity of the used anion exchanger for insulin, most preferably the amount is 40-60%. 13. The method according to claim 1, characterized in that all technological operations for chromatographic purification of raw insulin are carried out at 5 - 20 ^o C, the most preferred temperature is 8 - 15 ^o C.