RU2451750C2 - Method of producing recombinant human proinsulin c-peptide - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology, particularly genetic and protein engineering, as well as a method of producing recombinant human C-peptide. The disclosed method involves culturing an Escherichia coli producing strain, breaking down bacterial cells by disintegration, separating "inclusion bodies" containing a hybrid protein, dissolution thereof in a buffer containing urea and dithiothreitol, renaturating and purifying the renatured hybrid protein, disintegration thereof with trypsin and carboxypeptidase B, separation via chromatography on SP-sepharose, followed by purification and obtaining the end product.

EFFECT: method enables to obtain C-peptide with high output and purity of not less than 95% from wastes formed when producing recombinant human insulin.

4 tbl, 6 ex

Description

FIELD OF THE INVENTION

The invention relates to biotechnology, in particular to genetic and protein engineering, and can be used to obtain a recombinant c-peptide (proinsulin connecting peptide) of a person.

State of the art

There are several forms of c-peptide with a length of 31 to 35 amino acid residues. The forms differ in the content of arginine and lysine residues at the C- and N-ends of the polypeptide. These amino acids are not part of the molecule of mature human insulin and, according to some researchers, should belong to the c-peptide. However, only the mature c-peptide with a length of 31 amino acid residues that do not contain basic amino acids is secreted into the bloodstream. The sequence of this form of c-peptide is given below.

Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly

Pro Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln

The c-peptide precursor is synthesized in the islets of Langerhans by specialized pancreatic cells as part of preprohormone (preproinsulin). The formation of the c-peptide occurs in the same place as a result of post-translational modifications. At the first stage, the signal sequence is cleaved from preproinsulin - an N-terminal fragment containing 23 amino acid residues. From the resulting proinsulin (86 amino acid residues) in the Golgi complex, with the participation of specific peptidases, the formation of a mature c-peptide (31 amino acid residues) occurs. Subsequently, it is secreted into the bloodstream in an equimolar ratio with insulin.

The human proinsulin c-peptide was described in the 60s of the XX century. Initial studies did not reveal the existence of a specific biological function. For a long time it was believed that it is necessary only for the formation of the correct conformation of the protein insulin molecule. In the early 90s of the XX century [Johansson et al., 1992] a lot of information appeared about the use of c-peptide for the prevention and treatment of complications associated with diabetes. c-Peptide has been successfully used to improve the condition of patients suffering from retinopathy, angiopathy and nephropathy - the main factors of disability and mortality in diabetes [Ido, Science, 1997, 277, 563-566; Forst, 1998, Exp. Clin. Endocrinol. Diabetes, 106, 270-276 et al.]. However, currently large-scale research and use in clinics are difficult due to the lack of c-peptide in the market, its high cost and low quality. So, for example, the well-known supplier of reagents - the Sigma-Aldrich corporation - offers a c-peptide having a purity of 85% at a price of 371.5 euros per 250 μg of the drug [Sigma Catalog 2006-2007, p.706]. Another corporation is Phoenix Pharmaceuticals, Inc. - Offers 200 mcg of the drug for $ 75.00.

However, the company does not indicate any data regarding the quality of the sold peptide.

In a healthy person, the c-peptide is secreted in an equimolar ratio with insulin. The exact amount needed by patients with diabetes is currently undetermined. If we assume that patients need equimolar administration of insulin and c-peptide preparations, then with a daily dose of 3 mg insulin, approximately 1.5 mg of c-peptide will be required. Thus, when using the c-peptide available on the market, the cost of treating one patient for a month will be more than $ 15,000. However, its quality does not meet the requirements for pharmacological preparations.

Unlike insulin, the amino acid sequence of the c-peptide is very different in different species of mammals, which makes it impossible to obtain it from animal raw materials. Existing methods for producing a c-peptide can be divided into three categories:

1) Obtaining c-peptide by chemical synthesis. This method is used to obtain the majority of the drug currently on the market [Sigma-Aldrich, Phoenix Pharmaceuticals, Inc. and etc.].

2) Obtaining a c-peptide by biosynthetic methods as part of fusion proteins [Huang, Acta Biochim. Biophys. Sin. 2006, 38: 586-592; Jonasson, Journal of Biotechnology 76 (2000) 215-226]. To obtain a c-peptide by this method, a chimeric protein is created in which the leader fragment is followed by several c-peptide sequences separated by amino acids that provide hydrolysis with specific proteases. At the first stage, microorganisms are cultured in fermenters, then synthesis of a recombinant polypeptide is induced in them; the cells are destroyed, and the recombinant protein is purified and processed with specific proteases, resulting in a c-peptide. At the final stage, the c-peptide is purified from impurities. This method can provide large volumes of production, but it requires the creation of producer strains, development of the conditions for the cultivation of microorganisms, methods for purification of recombinant protein, as well as the creation and validation of quality control methods.

3) Obtaining the c-peptide by biosynthetic methods together with insulin. This production method consists in introducing some modifications into the technology for producing recombinant insulin in order to optimize the production of c-peptide formed at certain stages of production, which is based on the production of non-modifying proinsulin. This method has several advantages. To obtain a c-peptide in this way, it is not necessary to create new producer strains, to develop protein purification and folding technology, or to create new instrumental methods for controlling the production process.

In addition, the raw material for the production of the c-peptide, in this case, is the waste generated by the production of recombinant insulin. Closest to the technical nature of the claimed invention is a method for producing a c-peptide proposed by J. Nilsson et al. (1). The method consists in cultivating a producer strain of E. coli , producing proinsulin, containing two synthetic IgG binding domains of staphylococcal protein A. The authors were able to achieve high productivity of the producer strain by using their developed rich nutrient medium. The proinsulin yield was 3 g / l of culture medium. The isolation scheme consisted of destruction of bacterial cells, production of inclusion bodies containing proinsulin, dissolution of inclusion bodies, oxidative sulfitolysis of proinsulin, its renaturation, purification of the renatured protein by affinity chromatography on IgG-sepharose, cleavage of proinsulin with proteolytic enzymes (trypsin b carboxypeptide) and final purification of insulin and c-peptide by reverse phase high performance liquid chromatography. The disadvantages of this method are:

A) the use of a rich nutrient medium for growing a microorganism, a long growing time (about 34 hours), the high cost of the target product due to the use of an affinity sorbent for cleaning, which is expensive to manufacture and short-lived in industrial use. The disadvantage can also be considered the use of Tween 20 detergent in the technology of insulin and c-peptide production. It is known that the use of detergents in the isolation of proteins leads to their sorption on the protein and the presence of detergent in the final product. Also, purification of the c-peptide only by reverse phase chromatography leads to a low yield at this stage, which is only 44%.

B) Isolation and purification of proinsulin as an intermediate product complicates the process and reduces the yield of target products.

The present invention eliminates the disadvantages of the methods for producing the c-peptide.

SUMMARY OF THE INVENTION

The present invention describes a method for producing a recombinant c-peptide, which is fully compatible with the existing technology for the production of recombinant human insulin, disclosed in the patent of the Russian Federation RU2144957, publication date January 27, 2000

The essence of the invention consists in a method for producing a recombinant human c-peptide, comprising cultivating a producer strain of Escherichia coli , destroying bacterial cells by disintegration, separating “inclusion bodies” containing the hybrid protein, dissolving them in a buffer containing urea and dithiothreitol, renaturation and purification of the renatured hybrid protein, its splitting with trypsin and carboxypeptidase B followed by purification and obtaining the target product. As the producer strain, the strain Escherichia coli JM109 / pPINS07 is used, the renatured hybrid protein is purified by precipitation of impurity compounds by acidification to pH 4.0-6.0, followed by chromatography of the supernatant on KM-sepharose, cleavage of the hybrid protein by trypsin and carboxypeptidase B is carried out at the same time, trypsinolysis products are separated by chromatography on SP-Sepharose, equilibrated with 0.03-0.1 M ammonium acetate buffer, pH 5.0-6.0, containing 3 M urea, eluting with a linear gradient of potassium chloride from 0 to 0.5 M in the start buffer. Protein material that is not sorbed under these conditions and contains a c-peptide is collected and then the c-peptide is purified on anion-exchange sorbents and by reverse phase high-performance chromatography.

The main difference of this method for producing the c-peptide is that its production does not require expenses for cultivating the microorganism, isolation and purification of the hybrid protein, as well as for enzyme treatment, since under the selected conditions for the hydrolysis of the hybrid protein, the c-peptide does not break down and retains its structure.

Among the direct costs for the production of c-peptide are those associated with two stages of chromatographic purification and lyophilization. Moreover, due to the use of low pressure anion exchange chromatography, it is possible to significantly extend the resource of sorbents used for high-performance chromatography and increase the yield of c-peptide at the purification stage.

BEST MODE FOR CARRYING OUT THE INVENTION

To obtain a recombinant human c-peptide, a recombinant strain of the bacterium Escherichia coli JM109 / pPINS07-producer of a hybrid polypeptide containing human proinsulin is used. The strain is deposited in the Central collection of microorganisms of the Russian joint-stock company "BIOPREPARAT" CCMK "B" under the number CCM V-66IN.

The cultivation of the seed and main culture of the recombinant strain is carried out on a nutrient medium containing in g / l: hydrochloric acid casein hydrolyzate - 20, baker's yeast extract - 14, disubstituted potassium phosphate three-water - 6, monosubstituted potassium phosphate - 3, magnesium sulfate - 0.5, sodium chloride - 5, glucose - 10, ampicillin sodium salt - 0.05, purified water - the rest. The seed is used for sowing a fermenter with a total capacity of 200-1500 liters. To induce the synthesis of a hybrid protein in the middle of the logarithmic growth phase, 1-isopropyl-P-B-1-thiogalactopyranoside is introduced. During the growing process, the concentration of dissolved oxygen is maintained at 40 ± 15%, the amount of glucose introduced is regulated by the level of dissolved oxygen and pH. After growing, the culture is concentrated on a separator and destroyed on a Gaulin homogenizer in 0.1 M Tris buffer containing 1.5 M urea and 1 mM EDTA. “Inclusion bodies” are separated by centrifugation.

The selection of the hybrid protein from the granules is carried out according to the following scheme.

“Inclusion bodies” are dissolved in a buffer containing 0.1 M Tris, 8 M urea, after which 5-10 mM dithiothreitol is added.

The hybrid protein is renatured by incubation in a 5-10-fold volume of 0.1 M glycine-NaOH buffer, pH 9-11 at t = 10-14 ° C.

Acid precipitation is carried out by adjusting the pH to 4.0-6.0 with a HCl solution. The precipitate formed is separated by microfiltration.

Purification of the hybrid protein is carried out by chromatography on KM-Sepharose, balanced with 0.05 Tris-HCl buffer, pH 7.0-7.5. The protein is eluted with a balancing buffer containing 0.25 M NaCl and 1.5 M urea.

Enzymatic cleavage of the fusion protein with trypsin and carboxypeptidase B is carried out at a ratio of carboxypeptidase: fusion protein of 0.3: 1000. The ratio of trypsin: carboxypeptidase B is 0.75: 1. The reaction is started at a temperature of 8.5-9.5 ° C, then the reaction mixture is gradually heated to 17-20 ° C. After completion of the process, the reaction is stopped by acidification with trifluoroacetic acid to a pH of 3.3.

Trypsinolysis products were chromatographed on SP-Sepharose, equilibrated with 0.03 M ammonium acetate buffer, pH 3.6, containing 3 M urea. At this stage, insulin is adsorbed onto the carrier, and the c-peptide elutes in the breakthrough fractions.

The breakthrough fractions are diluted in the ratio water: material - 3: 1 and the pH is adjusted to a value of 4.0-6.0. Then this material is subjected to chromatography on an anion-exchange sorbent. Elution is carried out in Tris-acetate buffer pH 5.0 by increasing the gradient of potassium chloride to 0.5 M.

Fractions containing pure material are purified using standard preparative reverse phase chromatography equipment.

Pure material is freeze dried and stored as such until used.

An example of obtaining c-peptide in experimental industrial equipment

1. Growing biomass producer containing a hybrid protein

The working cell culture of the recombinant strain of Escherichia coli JM109 / pINS07 producer of the hybrid polypeptide (protein) containing human proinsulin is stored in a water-glycerin solution in glass or polymer ampoules at a temperature of minus 40 ° C.

At all stages of reproduction of microbial cultures, a nutrient medium of the following composition is used (g / l):

Table 1 Casein hydrolyzate hydrochloride, type Difco twenty Baker's yeast extract fourteen Disubstituted Potassium Phosphate Three-Water 6 Monosubstituted Potassium Phosphate 3 Magnesium sulfate 0.5 Sodium Chloride 5 Glucose 10 Ampicillin sodium salt 0.05 Purified water Rest

A pilot industrial line for growing producer biomass consists of a rocking incubator for 12 rocking flasks and fermenters with a capacity of 15, 150 and 1500 liters. Sowing crops are prepared in the amount of 1/10 of the volume of sown nutrient medium. At the first stage of propagation, 2 flasks with a nutrient medium are inoculated with the contents of one ampoule with a working culture and incubated in a shaker at 37 ° C for 18 hours. A grown culture is seeded with 10-12 flasks containing 100 ml of culture medium and grown to an optical density (OD) of 4-6 units. (wavelength - 540 nm, optical path length - 10 mm).

A custom-made fermenter chain has a consistent geometric, physical and mass transfer characteristics. Management of the preparation of culture media and cultivation is carried out according to the programs embedded in computers.

The main conditions for growing crops

table 2 Fermenter working capacity 10 and 100 l The volume of the nutrient medium 9 and 90 liters Working temperature 36.5 ± 0.5 ° C pH 6.9 ± 0.2 PO ₂ 40 ± 15% Foaming (chemical and / or mechanical) Sensor Signal Aeration and stirrer operation The level of pO ₂ The amount of glucose and alkali administered According to the level of pO ₂ and pH Duration 4-6 hours Process end With OD 7-8

Structural morphological analysis is carried out for all inoculum crops, in which not only the “purity” of the crops is evaluated, but also the morphological structure of the microorganism cells. Based on the results of the analysis, a decision is made on the possibility of further use of seed.

The cultivation of the main culture of the producer is carried out in a fermenter with a total capacity of 1500 liters. The composition of the nutrient medium and the basic cultivation conditions are the same as for inoculum crops, however, to induce the biosynthesis of the hybrid protein at a certain stage of culture development, the inducer 1-isopropyl-p-0-1-thiogalactopyranoside is introduced into it. The maximum accumulation of the hybrid protein occurs when the induction of the hybrid protein is provoked in the middle of the logarithmic growth phase. For our growing conditions, this corresponds to an accumulation of biomass of 7-10 units. OP. After the addition of the inducer, growth is continued until the formation of intracellular inclusions of the fusion protein “inclusion bodies” in 90-95% of cells. Express control of the accumulation of "inclusion bodies" (TV) is carried out using phase-contrast microscopy of drugs "crushed drop".

The main process parameters are given in the table.

Table 3 No. Parameter Value Note one Full fermenter capacity 1,500 l 2 Fermenter working capacity 1000 l 3 The amount of culture medium 900 l four The number of crops 100 l 5 Working temperature 36.5 ± 0.5 ° C 6 pH 6.9 ± 0.2 7 PO ₂ 40 + 15% 8 Foaming (chemical and / or mechanical) Sensor Signal 9 Aeration and stirrer operation The level of pO ₂ 10 The amount of glucose and alkali administered According to the level of pO ₂ and pH Up to 15 kg eleven Inductor introduction After 4-6 hours of growth When OP 7-10 units. 12 Process end With the formation of TB in 90-95% of cells 7-9 hours

After the formation of TB in 90-95% of cells, further cultivation can lead to partial cell lysis and loss of accumulated hybrid protein.

According to the results of a retrospective analysis, up to 3 g / l of the hybrid protein is accumulated in the cultures.

2. Isolation of “inclusion bodies”

The growth of the culture in the fermenter is stopped by a sharp reduction in the intensity of mixing, including aeration, and cooling to 10-14 ° C. The cooled culture is concentrated on a separator 8-10 times. Get 100-125 l of suspension, into which a buffer concentrate is introduced (for 50 l of purified water - 15 kg of urea, 1.4 kg of sodium salt of ethylenediaminetetraacetic acid and 0.9 kg of Tris buffer).

The buffered suspension is passed twice through a Gaulin homogenizer at a pressure of 700-800 atm. The temperature of the suspension should not be higher than 15-20 ° C, which is achieved by cooling the feeder and receiver of the homogenizer.

Cell homogenate is passed through a flow centrifuge (g = 18000). The bulk of the “inclusion bodies” (at least 90%) is deposited in the centrifuge rotor, and soluble proteins and cell wall residues are discharged from the centrifuge into the collection tank for inactivation. Wet sediment “inclusion bodies”, 8-10 kg, is discharged from the centrifuge rotor, packed in a plastic container, frozen and stored in a freezer at a temperature of minus 40 ° С. Shelf life - up to 6 months. Losses of the hybrid protein during the isolation of “inclusion bodies” do not exceed 15%.

3. Isolation of the hybrid protein

100 l of a buffer solution containing 8 M urea is poured into a 250-liter reactor, 10-12 kg of inclusion Taurus paste is loaded and a mixing device is turned on. After dissolution of the inclusion body, 0.150 kg of dithiothreitol is added to the reactor and stirring is continued for 10-12 hours.

900 l of glycine-NaOH buffer, pH 9-11, is poured into a reactor with a 1200-liter jacket; it is cooled to a temperature of 10-14 ° C, feeding chilled water to the jacket. After the buffer has cooled, a hybrid protein solution with reduced disulfide bonds is pumped into the reactor. The hybrid protein solution is incubated for 20-24 hours, mixing and maintaining the temperature in the reactor 10-14 ° C.

After 75-80% of the hybrid protein (GB) is renatured with the formation of correctly closed S-S bonds (control by RP-HPLC), the reaction medium is acidified in the reactor to a pH of 4.0-5.5 with a hydrochloric acid solution. Stirring is stopped and after 4-5 hours the precipitate formed is separated on a microfiltration unit. A correctly folded hybrid protein is sorbed from the filtrate onto a 20 L ion-exchange column filled with KM-Sepharose, previously equilibrated with 0.05 M Tris-HCl buffer, pH 7.0-7.5.

Sorbed protein is eluted from the column by passing a equilibration buffer with 0.25 M NaCl and 1.5 M urea through it.

Fractions containing a hybrid protein with a purity of at least 95% (RP HPLC) are combined and used for further work.

4. Enzymatic hydrolysis of a hybrid protein

Cleavage of HB with trypsin and carboxypeptidase B is carried out in a stainless steel reactor equipped with a stirrer and a jacket.

The GB solution in an amount of 100-150 l (protein content 1-1.2 kg) is introduced into the reactor, a stirring device is turned on and chilled water is supplied to the jacket at a temperature of 8-9 ° C. 30-40 minutes after the GB solution is cooled to a temperature of 10-12 ° C, the pH of the GB solution is measured and, using a 1 M solution of tris- (oxymethyl) aminomethane, its value is adjusted to a pH of 7.0-7.5. Then, a solution of carboxypeptidase B and trypsin is introduced into the reactor on the basis that the mass ratio of carboxypeptidase B and fusion protein introduced into the reactor is 0.3: 1000, and the mass ratio of trypsin and carboxypeptidase B is 0.75: 1. After 1000-1200 min, the reaction of cleavage of GB with trypsin was stopped by acidifying the contents of the reactor with trifluoroacetic acid to a pH of 3.3.

The resulting solution was applied to a 20 L chromatographic column filled with SP-Sepharose, previously equilibrated with 0.03 M ammonium acetate buffer, pH 3.6 with 3 M urea, and fractions containing c-peptide were collected. At this stage, insulin is adsorbed on SP-Sepharose, and the c-peptide does not interact with the sorbent and leaves in the breakthrough fractions.

The fractions containing c-peptide are combined, diluted with water in a volume ratio of material: water of 3: 1, and after adjusting the pH to 4-6, apply to the column. The column is then washed with 20 mM Tris-Acetate buffer, pH 5.0, until the baseline of the control flow densitometer is reached. The sorbed c-peptide is eluted with a linear gradient of potassium chloride from 0 to 0.5 M in the same buffer.

The fractions containing the c-peptide with a purity of at least 90% are combined (control is carried out by analytical reverse phase high performance liquid chromatography — RP-HPLC) and transferred to the preparative RP-HPLC step. Then the pH of the solution is adjusted to a value of 6.3-6.5 with 10% ammonia solution. The average yield at this stage of purification exceeds 85%.

5. Purification of the c-peptide by preparative reverse phase high performance liquid chromatography (RP HPLC)

Purification of the c-peptide by RP HPLC was carried out using standard equipment Kiloprep 250 from Waters, USA, or similar equipment from other companies. The installation includes the following elements:

Control block,

double-head high pressure diaphragm pump,

high pressure pump for injection of separation sample,

variable wavelength ultraviolet detector,

preparative column with a device for radial compression,

pneumatically controlled outlet flow valves,

computer for managing and processing data.

The preparation of the chromatographic installation for work is carried out according to the manufacturer's instructions for individual units and the installation as a whole.

For work, install a cartridge filled with C ₁₈ grafted silica gel from Videk, USA, into the column and create pressure in the column jacket according to the passport attached to the cartridge. Connect the column with flexible reinforced hoses to the chromatograph. The column is washed sequentially with a solution of 0.1% TFA in an amount of 2-3 column volumes, then with 1-2 volumes of acetonitrile (B) and 1-2 volumes of water. The column thus prepared is used in the purification step.

A solution of c-peptide from the previous step in the amount of 35-40 g of protein is supplied to the prepared column using a pump for supplying a sample from the container. They turn on the programming device and carry out the division according to the following program:

Table 4 Time min The rate of elution, cm ³ % solution B 0 350 26 5 350 27.5 fifteen 350 28.5 twenty 350 thirty 25 350 70 thirty 350 26

The separation process is recorded at 220 nm on a flow spectrophotometer with a sensitivity of 2.0. Protein fractions are collected using a chromatograph collector or manually. The collected fractions of the main peak of the c-peptide are analyzed for impurities by analytical reverse phase high performance liquid chromatography. The yield of c-peptide at this stage exceeds 80%. Thus, the total yield of the two stages of chromatographic purification is more than 70%, which is more than 1.5 times higher than that obtained by Nilsson et al.

6. Finishing and lyophilization of c-peptide

Fractions containing at least 95% c-peptide are combined in a jacketed glass reactor with temperature and pH sensors. Then the pH of the solution is adjusted to a value of 6.3-6.5 with 10% ammonia solution. The material undergoes sterilizing filtration and is poured into vials under sterile conditions. Then freeze-drying of the preparation and capping of the bottles under sterile conditions are carried out. Thus, the use of this method allows to obtain in high yield a human pro-insulin c-peptide with a purity of at least 95% from waste generated in the process of producing recombinant human insulin.

Bibliography

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

2. Patent RU 2144957, 01.27.2000.

3. Johansson et al., 1992.

4. Ido, Science, 1997, 277, 563-566.

5. Forst, 1998, Exp. Clin. Endocrinol. Diabetes, 106, 270-276.

6. Huang, Acta Biochim. Biophys. Sin. 2006, 38: 586-592.

7. Jonasson, Journal of Biotechnology 76 (2000) 215-226.

Claims (2)

1. A method of producing a recombinant human c-peptide, including culturing a producer strain of Escherichia coli, disrupting bacterial cells by disintegration, separating “inclusion bodies” containing the hybrid protein, dissolving them in a buffer containing urea and dithiothreitol, renaturation and purification of the renatured hybrid protein, its cleavage with trypsin and carboxypeptidase B, which are processed simultaneously, while the trypsinolysis products are separated by chromatography on SP-Sepharose, balanced 0.03-0.1 M ammonium acetate tnym buffer, pH 3.6, containing 3M urea, eluting with a linear gradient of potassium chloride from 0 to 0.5M in the starting buffer, followed by purification and yield the desired product. 2. The method according to claim 1, characterized in that the producer strain is E. coli JM 109 / pPINS07, renaturation and purification of the renatured hybrid protein is carried out by precipitation of impurity compounds by acidification to pH 4.0-6.0, followed by chromatography on CM -sepharose.