RU2383624C2 - Method for extension of sorbent service life in industrial purification of genetically engineered insulin human - Google Patents

Abstract

FIELD: microbiology.

SUBSTANCE: method includes highly efficient liquid chromatography with application of movable phase, comprising mixture of organic modifier, organic acid and ester of organic acid, with further regeneration of sorbent with solution of 50% organic modifier in water for injections. As organic modifier, they use methyl, ethyl, isopropyl, propyl alcohol or acetonitrile. As organic acid, they use formic, acetic or propionic acid. As ester of organic acid, they use methyl acetate, ethyl acetate, methyl formate, ethyl formate or ethyl acetoacetate.

EFFECT: invention makes it possible to extend service life of sorbent operation in industrial purification of genetically engineered insulin human.

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Description

The invention relates to the field of biotechnology, in particular to the production of genetically engineered human insulin for the manufacture of drugs used in the treatment of diabetes mellitus.

Recombinant proinsulin obtained from genetically modified microorganisms, after the preliminary stages of the correct closure of disulfide bonds and purification, undergoes enzymatic cleavage to form insulin. However, in addition to the target protein, the hydrolyzate contains undesirable impurity products that have physicochemical properties similar to those of insulin, which complicates the task of purifying insulin. In this regard, the development of an effective method of purification of insulin is an urgent task.

A known method of purification of insulin using hydrophobic chromatography low pressure. The insulin obtained by enzymatic digestion of recombinant proinsulin with trypsin and carboxypeptidase B is purified by hydrophobic chromatography on a Butyl Toyoperl sorbent. Why insulin is precipitated with 2 M sodium chloride, the precipitate is separated and dissolved in 10 mm citric acid, sodium chloride is added to a concentration of 0.5 M and the pH is adjusted to 6.6. The chromatographic column is equilibrated with a solution of 0.5 M sodium chloride (pH 6.6). The insulin solution is applied to the column packed with the Butyl Toyopearl 650S sorbent. Insulin is desorbed with ethyl alcohol in citrate buffer (ethyl alcohol concentration from 10 to 23%). The main fraction of insulin eluted from the column was precipitated with 3 M sodium chloride. The precipitate was separated, dissolved in 1.0 M acetic acid and subjected to gel filtration on a Sephadex G50 sorbent, followed by crystallization and lyophilization of the product. The resulting insulin substance is used for the manufacture of finished dosage forms.

(Pat. RF №2322504, IPC С12Р 21/00, published on 04.20.2008.)

The disadvantages of the method include a low yield of the target product (not more than 40%) and a significant consumption of reagents for the preparation of mobile phases.

A known method of purification of insulin using high performance liquid chromatography (HPLC). Trifluoroacetic acid (to lower the pH to 3.0) and acetonitrile to a concentration of 20% vol. Are added to the insulin hydrolyzate solution obtained by enzymatic cleavage of the recombinant precursor. An HPLC column packed with the Cromasil C8 sorbent was equilibrated with an aqueous solution of acetonitrile (30% v / v) containing 0.25% pentafluoropropionic acid. The insulin solution is applied to the column at 30 ° C. The insulin is desorbed by the mobile phase containing 0.25% pentafluoropropionic acid in a gradient of acetonitrile concentration. The main fraction of the eluted insulin is collected and analyzed.

(Nilsson J., Jonasson P., Samuelsson E., Stahl S., Uhlen M. "Integrated production of human insulin and its C-peptide", Journal of biotechnology, 1996, v. 48, p.241-250.)

The disadvantages of the method include the relatively low yield of the target protein (54%), as well as the use of expensive and dangerous acetonitrile.

A known method of purification of insulin using high performance liquid chromatography (HPLC) on a polymer sorbent (for example, PLRP-S). The chromatographic column is equilibrated with a mobile phase containing 0.05 M ammonium acetate, 0.1 M glycine (pH 9.0). A sample of insulin is applied. The insulin is desorbed by the mobile phase containing 0.05 M ammonium acetate, 0.1 M glycine (pH 9.0), in a gradient of propanol-1 or propanol-2. The main insulin fraction is collected and analyzed.

(W. Sievers et al, Procedure of the Chromatographic purification of insulins, Patent USA No. 6710167, 2004.)

The disadvantages of the method include low productivity, the use of high pH values, as well as a low degree of purification of insulin from immunoreactive polypeptides.

A known method of purification of insulin using high performance liquid chromatography (HPLC) on a polymer sorbent (Source S 15). The chromatographic column is equilibrated with a mobile phase containing 30% propanol-1, 50 mM lactic acid, 0.01 M sodium chloride (pH 3.5). A sample of insulin is applied. Then again balance the column with the same mobile phase. The insulin is desorbed by the mobile phase containing 30% propanol-1, 50 mM lactic acid, in a concentration gradient of sodium chloride from 0.01 to 0.15 M. The main insulin fraction is collected and analyzed.

(R. Obermeier, Process for isolating insulin by high-pressure liquid chromatography, United States Patent No. 5977297, 1999.)

The disadvantages of the method include the use of an expensive sorbent, low productivity (no more than 6 grams of insulin is purified per liter of sorbent).

A known method of purification of insulin using high performance liquid chromatography on a sorbent Kromasil C8. The chromatographic column is equilibrated with a mobile phase containing ammonium acetate buffer (pH 4) and ethyl alcohol (10% vol.). A sample of insulin is applied. The insulin is desorbed by the mobile phase containing ammonium acetate buffer (pH 4), in a gradient of ethyl alcohol concentration from 27 to 30%.

(Degerman, N. Jakobsson, W. Nilsson, "Modeling and optimization of preparative reversed-phase liquid chromatography for insulin purification", Journal of Chromatography A (2007), V.1162, Issue 1, p.41-49.)

A disadvantage of the known method is the low concentration of insulin in the eluate, which is associated with the limited solubility of insulin in ammonium acetate buffer.

Known closest to the claimed method of purification of insulin, based on high performance liquid chromatography on a modified silica gel sorbent Cromasil C8. The chromatographic column is equilibrated with a mobile phase containing 10 mM citric acid (pH 2-3) and 20% v / v. ethyl alcohol. A sample of insulin is applied. The insulin is desorbed by the mobile phase containing 10 mM citric acid, 70 mM ammonium sulfate (pH 2-3), in a gradient of ethyl alcohol from 30 to 35%.

(D.A. Gusarov, V.V. Vostrikov, E.A. Ruchko, V.A. Lasman, A.V. Mikhalev, D.I. Bairamashvili, "Using RP HPLC for the purification of genetically engineered human insulin", Biotechnology, 2006, No. 2, pp. 44-49.)

The disadvantages of this method are the partial polymerization (fiber formation) of insulin and insulin-like peptides in the pores of the sorbent, which significantly reduces the life of the sorbent (not more than 30 cleaning cycles in the operating load on the sorbent, the authors of the known method), and therefore increases the cost of one stage of cleaning .

The invention solves the problem of extending the life of the sorbent by reducing the degree of polymerization (fiber formation) of insulin and insulin-like peptides inside the pores of the sorbent.

The problem is solved due to the fact that in the method of industrial purification of genetically engineered human insulin, which includes high-performance liquid chromatography using a mobile phase and sorbent regeneration, a mixture of an organic modifier, organic acid and an organic acid ester is used as a mobile phase, while methyl, ethyl, isopropyl, propyl alcohol or acetonitrile are used as an organic modifier; ants are used as an organic acid th, acetic or propionic acid, as a complex of an organic acid ester - methyl acetate, ethyl acetate, methyl formate, ethyl formate and ethyl acetoacetate, with the following ratio of the mixture components,% by vol .:

organic modifier 2.0-25.0 organic acid 0.2-5.0 organic acid ester 0.001-5.0 water for injections rest

The technical result of the invention is to increase the life of the sorbent (the number of insulin purification cycles of at least 50) due to the use of a mixture of an organic acid and an ester of an organic acid in the mobile phase, preventing the formation of insulin polymers or insulin-like peptides inside the pores of the sorbent.

The method is as follows.

A hybrid protein containing human proinsulin and obtained during the fermentation of the producer strain is isolated from inclusion bodies, dissolved with simultaneous restoration of disulfide bonds in a buffer with 5-10 mM dithiothreitol and 1 mM EDTA disodium salt, renatured and purified by ion exchange chromatography, then subjected to joint digestion with enzymes trypsin and carboxypeptidase B, during which human insulin and a number of impurities are formed. Purification of insulin is carried out using high performance liquid chromatography (HPLC) followed by gel filtration and crystallization in the presence of zinc salts.

The mobile phase used for chromatographic purification of insulin contains 2-25% vol. an organic modifier selected from the group consisting of methyl, ethyl, isopropyl, propyl alcohols and acetonitrile, 0.2-5.0% vol. organic acid in which the polymerization (fiber formation) of insulin or insulin-like peptides is minimized: formic, acetic or propionic, 0.001-5.0% of an ester of an organic acid selected from the group consisting of methyl acetate, ethyl acetate, methyl formate, ethyl formate or ethyl acetate (the rest is water for injections). The chromatographic column is packed with silica gel or polymer sorbent: YMC-Basic, Cromasil C8, Daiso C18, etc.

Balance the column with the mobile phase. The mobile phase contains 2-25% of an organic modifier, 0.2-5.0% of an organic acid, 0.001-5.0% of an organic acid ester and 50-98% of water for injection.

The insulin solution obtained by enzymatic digestion of the recombinant insulin precursor is precipitated with 0.1 M zinc acetate, the precipitate is separated and dissolved in the mobile phase to an insulin concentration of 5-25 mg / ml. The resulting solution was applied to a chromatographic column.

The insulin is desorbed by the mobile phase, increasing the concentration of the organic modifier in it by gradient elution until the start of the chromatographic peak corresponding to insulin or deamidated insulin (for example, A21-deamido insulin), after which the elution is carried out in an isocratic mode of concentration of the organic modifier in the mobile phase.

After the release of the main insulin fraction, the sorbent is regenerated with a solution of 50% vol. organic modifier in water for injection. Purified insulin is subjected to gel filtration, crystallization with zinc and subsequent lyophilization.

The invention is illustrated by examples.

Example 1

A Akta Design 100 chromatographic system (Amersham Biosciences, Sweden) was used, including a mobile phase feed pump, a gradient system, and a UV detector. The chromatographic column, size 20x100 mm, is packed with Daiso C18 silica gel sorbent (particle size 10 μm, porosity 20 nm). Prepare a mobile phase, which contains 2% vol. ethyl alcohol, 0.2% vol. acetic acid, 0.001% vol. methyl acetate, the rest is water for injection. Balance the chromatographic column with the mobile phase in a volume of 60 ml at a rate of 5 ml / min. Apply an insulin solution to the column. The insulin is desorbed by the mobile phase at a rate of 5 ml / min, increasing the concentration of ethyl alcohol using a system to create a gradient until the start of the chromatographic peak corresponding to insulin, after which the process is conducted in the isocratic mode of concentration of ethyl alcohol in the mobile phase until the end of the chromatographic peak corresponding to insulin. The main fraction containing insulin is collected and analyzed. The yield of the main fraction is 77%, the purity of insulin is 98.5%. Chromatographic column regenerate 50% vol. aqueous ethyl alcohol in a volume of 45 ml at a rate of 5 ml / min.

The operation of the sorbent in this way allowed for 56 cycles of insulin purification.

Example 2

A Akta Design 100 chromatographic system (Amersham Biosciences, Sweden) was used, including a mobile phase feed pump, a gradient system, and a UV detector. A chromatographic column, 20 × 100 mm in size, is packed with Cromasil C8 silica gel sorbent (particle size 10 μm, porosity 10 nm). Prepare a mobile phase, which contains 10% vol. ethyl alcohol, 2.5% vol. acetic acid, 2.5% vol. ethyl acetoacetate and 85% vol. water for injection. Balance the chromatographic column with the mobile phase in a volume of 60 ml at a rate of 5 ml / min. Apply an insulin solution to the column. The insulin is desorbed by the mobile phase at a rate of 5 ml / min, increasing the concentration of ethyl alcohol using a system to create a gradient until a chromatographic peak corresponding to insulin appears, after which the process is conducted in the isocratic mode of concentration of ethyl alcohol in the mobile phase until the end of the chromatographic peak corresponding to insulin . The main fraction containing insulin is collected and analyzed. The yield of the main fraction is 81%, the purity of insulin is 98.5%. Chromatographic column regenerate 50% vol. aqueous ethyl alcohol in a volume of 45 ml at a rate of 5 ml / min.

The operation of the sorbent in this way allowed for 62 cycles of insulin purification.

Example 3

A Akta Design 100 chromatographic system (Amersham Biosciences, Sweden) was used, including a mobile phase feed pump, a gradient system, and a UV detector. A chromatographic column, 20 × 100 mm in size, is packed with an HPR10 polymer sorbent (particle size 10 μm, porosity 10 nm). Prepare a mobile phase, which contains 2% vol. methyl alcohol, 5% vol. formic acid, 5% vol. ethyl acetate and 88% vol. water for injection. Balance the chromatographic column with the mobile phase in a volume of 60 ml at a rate of 5 ml / min. Apply an insulin solution to the column. The insulin is desorbed by the mobile phase at a rate of 5 ml / min, increasing the concentration of methyl alcohol using a gradient system until a chromatographic peak corresponding to insulin appears, after which the process is conducted in the isocratic mode of concentration of methyl alcohol in the mobile phase until the end of the chromatographic peak corresponding to insulin . The main fraction containing insulin is collected and analyzed. The main fraction yield 79%, insulin purity 98.4%. Chromatographic column regenerate 50% vol. aqueous methyl alcohol in a volume of 45 ml at a rate of 5 ml / min.

The operation of the sorbent in this way allowed for 68 cycles of insulin purification.

Example 4

An Agilent 1100 chromatographic system (Agilent Technologies, USA) was used, including a mobile phase feed pump and a UV detector. Use a chromatographic column measuring 20 × 250 mm, packed with YMC-Basic silica gel sorbent (particle size 10 μm, porosity 20 nm). Prepare a mobile phase, which contains 25% vol. acetonitrile, 1.0% vol. propionic acid, 0.05% vol. ethyl formate, the rest is water for injection. Balance the chromatographic column with the mobile phase in a volume of 80 ml at a rate of 5 ml / min. Apply an insulin solution to the column. The insulin is desorbed by the mobile phase at a rate of 5 ml / min in the isocratic mode of concentration of acetonitrile in the mobile phase until the end of the chromatographic peak corresponding to insulin. The main fraction containing insulin is collected and analyzed. The yield of the main fraction is 87%, the purity of insulin is 98.5%. Chromatographic column regenerate 50% vol. aqueous acetonitrile in a volume of 80 ml at a rate of 5 ml / min.

The operation of the sorbent in this way allowed for 69 cycles of insulin purification.

Example 5

An Agilent 1100 chromatographic system (Agilent Technologies, USA) is used, including a pump for supplying a mobile phase, a system for creating a gradient, and a UV detector. Use a chromatographic column measuring 20 × 250 mm, packed with YMC-Basic silica gel sorbent (particle size 10 μm, porosity 20 nm). Prepare a mobile phase, which contains 2% vol. isopropyl alcohol, 2.5% vol. acetic acid, 2.5% vol. methyl formate and 93% vol. water for injection. Balance the chromatographic column with the mobile phase in a volume of 80 ml at a rate of 3 ml / min. Apply an insulin solution to the column. The insulin is desorbed by the mobile phase at a rate of 3 ml / min, increasing the concentration of isopropyl alcohol using a gradient system until a chromatographic peak corresponding to insulin appears, after which the process is conducted in the isocratic mode of the concentration of isopropyl alcohol in the mobile phase until the end of the chromatographic peak corresponding to insulin . The main fraction containing insulin is collected and analyzed. The yield of the main fraction is 67%, the purity of insulin is 98.5%. Chromatographic column regenerate 50% vol. aqueous isopropyl alcohol in a volume of 80 ml at a rate of 3 ml / min.

The operation of the sorbent in this way allowed for 52 cycles of insulin purification.

Example 6

An Agilent 1100 chromatographic system (Agilent Technologies, USA) is used, including a pump for supplying a mobile phase, a system for creating a gradient, and a UV detector. Use a chromatographic column measuring 20 × 250 mm, packed with YMC-Basic silica gel sorbent (particle size 10 μm, porosity 20 nm). Prepare a mobile phase, which contains 2% vol. propyl alcohol, 2.5% vol. acetic acid, 2.5% vol. methyl acetate and 93% vol. water for injection. Balance the chromatographic column with the first mobile phase in a volume of 80 ml at a rate of 3 ml / min. Apply an insulin solution to the column. The insulin is desorbed by the mobile phase at a rate of 3 ml / min, increasing the concentration of propyl alcohol using a gradient system until a chromatographic peak corresponding to insulin appears, after which the process is conducted in the isocratic mode of the concentration of propyl alcohol in the mobile phase until the end of the chromatographic peak corresponding to insulin . The main fraction containing insulin is collected and analyzed. The yield of the main fraction is 65%, the purity of insulin is 98.3%. Chromatographic column regenerate 50% vol. aqueous propyl alcohol in a volume of 80 ml at a rate of 3 ml / min.

The operation of the sorbent in this way allowed for 52 cycles of insulin purification.

Example 7

A YMC chromatographic system (YMC Europe Co., Germany) is used, including a pump for supplying a mobile phase, a system for creating a gradient, and a UV detector. An axial compression chromatographic column measuring 150 × 600 mm (sorbent layer height about 200 mm) is packed with YMC-Basic silica gel sorbent (particle size 15 μm, porosity 20 nm). Prepare a mobile phase, which contains 10% vol. ethyl alcohol, 0.8% vol. acetic acid, 0.02% vol. ethyl acetate, the rest is water for injection. Balance the chromatographic column with the first mobile phase in a volume of 5000 ml at a rate of 200 ml / min. Apply an insulin solution to the column. The insulin is desorbed by the mobile phase at a rate of 200 ml / min, increasing the concentration of ethyl alcohol using a system to create a gradient until a chromatographic peak corresponding to insulin appears, after which the process is conducted in the isocratic mode of concentration of ethyl alcohol in the mobile phase until the end of the chromatographic peak corresponding to insulin . The main fraction containing insulin is collected and analyzed. The yield of the main fraction 82%, the purity of insulin 98.6%. Chromatographic column regenerate 50% vol. aqueous ethyl alcohol in a volume of 5000 ml at a rate of 200 ml / min.

The operation of the sorbent in this way allowed for 60 preparatory cycles of insulin purification.

Claims (1)

A method of increasing the life of the sorbent in the industrial purification of genetically engineered human insulin, including high-performance liquid chromatography using a mobile phase and regeneration of the sorbent with a solution of 50% organic modifier in water for injection, characterized in that a mixture of an organic modifier is used as the mobile phase, organic acid and an ester of an organic acid, while methyl, ethyl, isopropyl, prop octylphenyl alcohol or acetonitrile as the organic acid is formic, acetic or propionic acid, as a complex of an organic acid ester - methyl acetate, ethyl acetate, methyl formate, ethyl formate or ethyl acetoacetate in the following ratio of the mixture components,% vol:.
organic modifier 2.0-25.0 organic acid 0.2-5.0 organic acid ester 0.001-5.0 water for injections rest