RU2146944C1 - Method of insulin purification - Google Patents

Abstract

FIELD: medicine, endocrinology, biochemistry. SUBSTANCE: invention relates to methods of purification of insulin isolated from animal raw or prepared by biotechnology approach (for example, human genetic engineering insulin). Insulin to be purified is subjected for gel-chromatography using 0.7-1.2 M acetic acid on column filled with hydrophilic gel obtained by polycondensation of polyglucin with epichlorohydrin and equilibrated with 0.7-1.2 M acetic acid solution. Insulin loading on column is 4-7 g/l of equilibrated gel at insulin concentration in applied solution 5-15%. Insulin is eluted from column at 8-15 C at the flow rate 2.0-5.0 ml/cm²/h and the end product is isolated by zinc precipitation and subjected for citrate crystallization. The yield of insulin is 75-85%, content of insulin monofraction is 98.0-98.5%, content of proinsulin is less 2 p.p.m., content of bacterial peptides (in human genetic engineering insulin) - is less 1 p.p.m. (i. e. less 0.0001%), amount of deamidoinsulin and nonidentified impurities (by methods of RIA and HPLC) - 1%, not above, in all cases. EFFECT: improved method of purification, increased yield, high purity of insulin. 5 cl, 3 ex

Description

 The invention relates to the medical industry, namely to the purification of insulin used in the treatment of diabetes mellitus from antigenic and other impurities that accompany natural insulin (from the pancreas of pigs and cattle) and biosynthetic human insulin produced by biotechnology.

 The main antigenic impurity in insulin preparations is proinsulin, which causes pronounced allergic reactions in the treatment of patients with diabetes mellitus. The content of proinsulin in insulin preparations according to modern pharmacopoeial requirements should not exceed 10 parts per million (10 ppm), i.e., should not be more than 0.001%. The same high demands are placed on the content of bacterial peptides in human insulin obtained by biotechnological methods.

 A known method of purification of insulin [1] by sorption of insulin-containing raw materials on macroporous sulfocationite mixed with an inert silicate filler in an equal volume ratio, followed by washing the mixture of microdispersion and filler 0.2 - 0.3 M acetic ammonium buffer at pH 5.2 - 6, 4 and insulin desorption of 0.02-0.03 M buffer solution (Tris: phosphate buffer: ammonium acetate) at a pH of 6.4-7.6. The disadvantages of this method include the low yield of insulin after purification (64-70%) and its high antigenicity (proinsulin content of 0.5% instead of 0.001%).

There is also known a method of producing highly purified insulin [2] by multistage purification, which includes first cation exchange chromatography of insulin in phosphate buffer on KU-23I sulfocationite (particle diameter 200-300 μm) using 0.018-0.022 M linear phosphate buffer in pH gradient from 6 to elute 7-6.8 to 7.9-8.0, followed by the release of insulin by isoelectric precipitation; the precipitated insulin is then subjected to gel permeation chromatography at 4-6 ^° C on Sephadex G-50 ultrafine (Pharmacia, Sweden) in 1 M acetic acid, and then chromatographed at 6-8 ^° C on a PAE-300 column with anion exchange silica gel (company Amicon, USA) in the eluent with a pH of 7.8 - 8.5, consisting of TRIS / HCl, Trilon B and ethanol (50-60 vol.%), with an exponential gradient of sodium chloride (0.0 - 1.0 M). After citrate crystallization, insulin is obtained that meets modern requirements for antigenicity, but this result is achieved by too much material and labor resources, while the insulin yield is low - 55 - 65%. The disadvantages of this method of purification of insulin should also include the use of imported media and equipment for column chromatography, which further limits the possibility of its practical use.

 Closest to the proposed method is the purification of insulin [3] by gel permeation chromatography on crosslinked dextran gels in 0.5 M acetic acid (prototype). After purification by this method of cattle insulin (cf.s.) with an initial content of insulin and proinsulin of 92.5 and 4.21%, respectively, insulin cf.s., still containing 0.44% of proinsulin, is obtained (instead the required content is not more than 0.001%). Using the same gel permeation chromatography of porcine insulin (the content of insulin and proinsulin in the starting material is 90.7 and 2.42%, respectively), insulin is obtained, which is also only partially purified from proinsulin: its amount in the target product is 0.57%, i.e. . exceeds the required 570 times.

 Thus, the known method does not allow to obtain insulin of the required degree of purity with respect to the main antigenic impurity - proinsulin.

 The aim of the invention is to develop a simple method for the purification of insulin, at the same time ensuring its high yield and quality that meets modern pharmacopoeial requirements for animal insulin and biosynthetic human insulin.

 To improve the quality and ensure a high yield of insulin according to the claimed method, pig insulin, insulin k.s. or biosynthetic, for example, genetically engineered human insulin, is subjected to gel permeation chromatography in aqueous acetic acid on a hydrophilic gel (Gefil-1) [4].

 The inventive method is as follows.

A solution of insulin (with a monofraction of insulin at least 80%) in 0.7-1.2 M acetic acid with a concentration of 0.7-1.2 M acetic acid is introduced into a column with a neutral Gefil-1 carrier balanced in 0.7-1.2 M acetic acid. insulin from 5 to 15% and with a total amount of insulin from 4.0 to 7.0 g per 1 liter of balanced gel. The insulin is eluted with a 0.7-1.2 M solution of acetic acid at a flow rate of 2.0 to 5.0 ml (cm ² • h) at 8-15 ^° C. From the bulk of the eluate in a known manner (by precipitation of amorphous insulin, followed by citrate crystallization) secrete highly purified monocomponent insulin. The purity of the released insulin is determined by high performance liquid chromatography (HPLC), and the content of proinsulin and bacterial peptides (in the case of biosynthetic, for example, genetically engineered human insulin) by radioimmunological analysis (RIA).

 The yield of the target product is 75-85% with the insulin monofraction content of 98.0-98.5%, while the proinsulin content is less than 2 ppm, and the content of bacterial peptides (in human biosynthetic insulin) is less than 1 p. lm (i.e., less than 0.0001%), the amounts of desamidoinsulin and unidentified impurities in all cases do not exceed 1% (HPLC).

 Thus, obtained by the present method, insulin (from animal raw materials or human biosynthetic) in its characteristics meets the requirements of world standards, which is not provided by the known method of purification of insulin.

Example 1. 2.0 g of porcine insulin with an activity of 25.0 U / mg (according to bioassay) and a proinsulin content of 2800 ppm (according to RIA) is dissolved in a 1 M solution of acetic acid to an insulin concentration of 10%. The insulin solution is injected into the column (inner diameter 5.0 cm, height 22 cm) filled with hydrophilic Gufil-1 gel balanced with 1M acetic acid (insulin load on the column is 4.6 mg / ml gel), and 1M insulin is eluted a solution of acetic acid at 10 ^o C with a speed of 2.4-2.6 ml / (cm ² • h). Highly purified monocomponent insulin is isolated from the main part of the eluate in a known manner. Obtain 1.6 g of porcine insulin with a content of prulinulin 1.0 ppm (according to RIA) and activity of 27.7 U / mg (according to bioassay). The mass yield is 80.0% of the initial insulin substance. The yield of insulin monofraction is 88.6%.

Example 2. 43.8 g of porcine insulin with an activity of 25.2 U / mg (according to bioassay) and a proinsulin content of 3500 ppm (according to RIA) is dissolved in 1 M acetic acid to an insulin concentration of 12%. The insulin solution is injected into the column (inner diameter 10.0 cm, height 100 cm) filled with hydrophilic gel gefil-1 balanced with 1M acetic acid (insulin loading on the column is 7.3 mg / ml gel) and 1M acetic insulin elute acid at 10 ^o C with a flow rate of 1.9-2.1 ml / (cm ² • h). Highly purified, multi-component insulin is isolated in a known manner from the portion of the eluate corresponding to the main peak. 32.9 g of porcine insulin with a proinsulin content of 1.7 ppm is obtained. (according to RIA) and activity of 27.5 U / mg (according to biotesting). The mass yield is 75.1% of the crude insulin substance. The yield of insulin monofraction is 82.0%.

 Example 3. Under the conditions described in example 1, purify 2.0 genetically engineered human insulin with an activity of 26 PIECES / mg (according to bioassay), with a proinsulin content of 2840 ppm and bacterial peptides 503.5 ppm (according to RIA). 1.55 g of human insulin is obtained with a proinsulin content of 0.6 ppm. and bacterial peptides 0.5 pfd (according to RIA), and activity of 28.5 U / mg (according to bioassay). The mass yield is 77.5% of the initial insulin substance. The yield of insulin monofraction is 84.9%.

Literature
1. USSR author's certificate N 1007675, A 61 K 37/26, 03.30.83. (Institute of Macromolecular Compounds of the Academy of Sciences of the USSR, Leningrad Plant of Medications "Lenmyasoprom").

 2. RF patent N 2120299, A 61 K 38/28, 10.20.98 (Joint-stock company Kurgan "Synthesis").

3. German patent N 2505307, C 07 C 103/52, 11.29.84 (Eli Lilly and Co., Indianapolis, Ind., US)
4. RF patent N 2076871, C 08 B 37/02, 04/10/97.

Claims (5)

 1. The method of purification of insulin by layering the initial preparation of insulin on a chromatographic column containing a hydrophilic gel, balanced in an aqueous solution of acetic acid, eluting the target product with a solution of acetic acid followed by zinc precipitation, characterized in that the chromatography uses a gel prepared by polycondensation of polyglucin with epichlorohydrin in an aqueous alkaline medium, the gel is equilibrated and eluted with a solution of acetic acid at a concentration of 0.7 - 1.2 M, and the obtained target the product is subjected to citrate crystallization.  2. The method according to claim 1, characterized in that the biosynthetic and semi-synthetic insulin preparation is purified.  3. The method according to claims 1 and 2, characterized in that the purification of human insulin obtained by genetic engineering method (recombinant).  4. The method according to PP.1 to 3, characterized in that the concentration of insulin when applied to the column is 5-15% by weight per volume of the applied solution.  5. The method according to PP. 1 to 4, characterized in that the amount of insulin when loaded onto the column is 4 to 7 g per liter of balanced gel.