The method of purification of insulin

 

(57) Abstract:

Usage: in the medical industry for more insulin. The inventive method of purification of insulin includes sorption insulinosoderzhaschego raw sulfonic cation exchanger with pre sulfonic cation exchanger with a solution of salt of acetic acid with subsequent zinc deposition and citrate crystallization. Use sulfonation with a grain size of 400-600 μm. Processing sulfonic cation exchanger conduct of 0.15-0.25 M solution of ammonium acetate or sodium acetate at pH 70-7,3. Sorption was carried out from 0,04-0,06 M solution of ammonium acetate or sodium acetate at pH 7.4 and 7.6. Elution conduct of 0.10-0.25 M solution of ammonium acetate or sodium acetate at pH of 6.9 to 7.1. table 1.

The invention relates to the medical industry and can be used to obtain preparations of insulin used in the treatment of diabetes mellitus.

A known method of purification of insulin by chromatography on anion exchanger Dowex 1X2 and QAE-Sephadex A-25 [1] the Disadvantage of this method is the low yield of purified insulin (40-60% ), as well as use in chromatography of complex water-organic mixtures containing 60% of combustible and toxic components, methanol or etano the percentage recrystallization.

In another method of purification of insulin [2] cation exchange chromatography on slightly acid cation exchanger (amberlite CG 50) is preceded by five additional stages: citrate crystallization, chromatography on Sephadex G-50, planting sodium chloride, isoelectric precipitation and the second citrate crystallization, and the allocation of purified insulin after cation-exchange chromatography involves three stages of planting sodium chloride, isoelectric precipitation and citrate crystallization. The disadvantage of this method, in addition to the complexity and labor intensity is low, the output of insulin (53%), as well as use with cation-exchange chromatography of complex buffer comprising of 0.13 M sodium phosphate, 7M urea and 1% n-butanol.

A known method of purification of insulin [3] (prototype) by sorption insulinosoderzhaschego materials on macroporous sulfonic cation exchanger with a particle size of 20-60 μm, mixed with inert silica filler (particle size of 40-120 μm) in equal volume ratio, followed by washing the mixture of microdisperse and filler 0,20-0,30 M oxymoronical buffer at pH 5,2-6,4 and desorption of insulin of 0.02-0.3 M buffer solution (Tris; phosphate buffer; ammonium acetate) at pH 6.4 and 7.6.

To insufficient is nalina after cleaning (64-70% ); high antigenicity of insulin (the content of proinsulin to 0.5%). In the prototype is not specified type of purified porcine insulin or bovine K. R. S.), although different types of insulin may differ materially as output and purification in the same conditions. In addition, the prototype did not specify methods of analysis of the fractional composition, neither the fractional composition of both the original and the purified insulin.

Process chromatography in the prototype possible because of the small grain size of the cation only in the presence of the filler. The presence of filler eliminates the possibility of regenerating the resin in the mode of fluidization, because the resin and the filler will delaminate. Thus, the use of filler in the prototype is a necessary measure, since in its absence chromatography will occur with very low velocities.

An object of the invention is to eliminate the use of filler in chromatography, the increased output of insulin and reduce its antigenicity.

The problem is solved in that the purification of insulin, including sorption insulinosoderzhaschego raw sulfonic cation exchanger with pre sulfonic cation exchanger R is m deposition and citrate crystallization, use sulfonation with a grain size of 400-600 μm, processing sulfonic cation exchanger conduct of 0.15-0.25 M solution of ammonium acetate or sodium acetate at pH 7.0-7,3, sorption was carried out from 0,04-0,06 M solution of ammonium acetate or sodium acetate at pH 7.4 and 7.6, and elution conduct of 0.10-0.25 M solution of ammonium acetate or sodium acetate at pH 6.9 and 7.1.

In the described conditions is provided, apparently, the differentiation of the interaction of negatively charged groups of a strong cation exchanger with oppositely charged groups (mainly ammonium and guanidinium arginine residues) shared substances at different pH values and concentrations of acetic acid sodium salt, or ammonium.

When using sodium acetate or ammonium acetate in the described conditions, the yield of purified porcine insulin is respectively 90% and 79% of These outputs is significantly more than the release of insulin when cleaning by known methods (50-70%).

Fractional composition of the purified insulin pigs and K. R. S. was estimated along with the disc-electrophoresis methods high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA). Received data (b) are presented in table 1.

As follows from the insulin from 88,7% to >96% in the case of insulin K. R. S. from 84.3 percent up to 95-96% of the content of the main antigenic impurities of proinsulin to insulin pigs is reduced from 4.5% to 0,04-0,05% i.e. reduced by approximately 100 times, and insulin K. R. C. number of proinsulin is reduced approximately 50 times from 4.9 to 0.1% at the same time in the purified insulin significantly reduced the content of desamethasone and other impurities.

The method is illustrated by the following examples.

Example 1. of 3.9 g of Sodium salt pork insulin, fractional composition of which is presented in the table.1, dissolved in distilled water upon acidification to a pH of 2.6-3,018% hydrochloric acid and the solution diluted to a value of optical density D2776,5-7,0 O. E. (optical unit). To the solution was added sodium acetate at the rate of 0.5 M, 5% sodium hydroxide solution (12,5% ammonium hydroxide solution for examples 2 and 4) establish a pH 7,500,05.

The column with sulfonic cation exchanger KU-23 (grain size 400-600 μm), balanced 0.2 M solution of sodium acetate with a pH of 7.2, serves freshly prepared solution of sodium salt pork insulin at a rate of 0.5 Vnumber/h (Vnumberthe column volume), and then with the same speed serves 0.2 M sodium acetate solution with a neutral pH value. Collection inset (after passing the maximum on the curve of elution) when values of D2770,5-0,6 O. E.

Fractions containing data disc electrophoresis in polyacrylamide gel of purified insulin, unite, dilute with water to D2773,0-3,5 O. E. and 18% hydrochloric acid to establish a pH of 3.0-3.5. Then add a 10% solution of zinc acetate and precipitated with zinc insulin at pH 6.0 to 6.1. Sediment zinc-insulin is filtered off and crystallized from a citrate buffer in a known manner.

Get 3.5 g of crystalline porcine zinc insulin (yield of 89.7%), fractional composition of which is presented in table.1.

Example 2. Cleaning 3.6 g of sodium salt pork insulin with the use of ammonium acetate is carried out in conditions analogous to example 1, but for equilibration of the column, dissolution of insulin and subsequent elution instead of sodium acetate using ammonium acetate.

After cleaning they receive 2.85 g of crystalline zinc insulin (output 79,2%), fractional composition of which is presented in table. 1.

Example 3. Cleaning charge of 40.0 g of sodium salt of insulin bovine K. p. C. ). Cleaning requirements are similar to the conditions described in example 1 (using sodium acetate).

After cleaning get to 33.7 g of crystalline zinc-inst of 3.85 g of sodium salt of insulin K. R. S. Cleaning requirements are similar to the conditions described in example 2 (using ammonium acetate).

After clearing the receive of 2.81 g of crystalline zinc insulin K. R. C. (yield 73%), fractional composition of which is presented in table. 1.

The method of purification of insulin, including sorption insulinosoderzhaschego raw sulfonic cation exchanger with pre sulfonic cation exchanger with a solution of salts of acetic acid and elution of insulin solution of salt of acetic acid with subsequent zinc deposition and citrate crystallization, characterized in that use sulfonation with a grain size of 400-600 μm, processing sulfonic cation exchanger conduct of 0.15-0.25 M solution of ammonium acetate or sodium acetate at pH 7.0-7,3, sorption was carried out from 0,04-0,06 M solution of ammonium acetate or sodium acetate at pH 7.4 and 7.6, and elution conduct of 0.10-0.25 M solution of ammonium acetate or sodium acetate at pH 6.3, and 7.1.

 

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