A method for industrial production of recombinant human insulin

 

(57) Abstract:

The invention relates to biotechnology and can be used to produce recombinant human insulin that is used for the preparation of drugs for the treatment of diabetes. The cultivation of the producer strain E. coli JM109/pPINS07 carried out in an industrial fermenter volume 200-1500 L. the Concentration of dissolved oxygen is maintained at 40±15%. After the destruction of the cells by the disintegration of Taurus include dissolved in a buffer containing 8 M urea, then add dithiothreitol. Renaturation hybrid protein insulin is carried out in one stage by incubation in 5-10-fold volume of buffer to the stage of purification by acid deposition. A hybrid protein chromatographic on KM-sepharose. Enzymatic cleavage is carried out sequentially at a ratio of trypsin : a hybrid protein and carboxypeptidase B : hybrid protein - 1:(500-1000). Between stages fission hybrid protein with trypsin and carboxypeptidase B spend chromatography on SP-sepharose. Insulin purified by the method of preparative reversed-phase high-performance liquid chromatography followed by gel-filtration and secretion in the presence and increase its output in an industrial scale. 4 C.p. f-crystals, 4 PL.

The invention relates to the field of biotechnology, namely the preparation of recombinant human insulin for the preparation of dosage forms for various duration in the treatment of diabetes.

Insulin is a pancreatic hormone that regulates carbohydrate metabolism and maintenance of normal blood sugar levels. The need for this drug may not be covered by insulin of animal origin due to the limited resource base. In addition, there is a need to use in the treatment of diabetes of different insulin preparations. Therefore, the development of ways to get insulin from recombinant strains of microorganisms is an actual medical problem.

A method of obtaining recombinant human insulin proposed by J. Nilsson and co-authors (1). The method consists in the cultivation of the producer strain E. coli producing proinsulin containing two synthetic IgG-binding domain of staphylococcal protein A. the Authors were able to achieve high productivity of the producer strain due to the use they have developed a rich nutrient medium. The yield of proinsulin was 3 g/l nutrient medium is Sulin, dissolution Taurus activation of oxidative sulfatase of proinsulin, its renaturation, purification denaturirovannogo protein by affinity chromatography on IgG-Sepharose, the cleavage of proinsulin proteolytic enzymes (trypsin and carboxypeptidase B) and final cleaning insulin high-performance reversed-phase liquid chromatography. The disadvantages of this method are:

using a rich nutrient medium for cultivation of the microorganism, long (about 34 hours) time of cultivation, the high cost of the target product caused by the use of purification affinity sorbent, expensive to manufacture and is short-lived when industrial use. The disadvantage can also be considered as the use of the technology for production of insulin detergent Tween 20. It is known that the use of detergents in the allocation of proteins leads to their adsorption on the protein and the presence of detergent in the final product.

A method of obtaining recombinant human insulin, namely, that cultured cells of the producer strain of Escherichia coli DN/V100 destroy bacterial cell disintegration, separate faction Taurus inclusion containing a hybrid protein, OK urea, 1 mm dithiothreitol, 0.1 M Tris Hcl, pH 8.0, for 12-16 hours any insoluble impurities are removed by centrifugation, and then increase the concentration of dithiothreitol to 10 mm and restore the disulfide bond at 4C for 1 h the Solution was diluted 5 times with cold water, the pH meter was adjusted pH to 4.5 and incubated for 2 h at 4C for forming sludge. The precipitate, containing a hybrid protein is separated by centrifugation and centurybut, which quickly dissolve in cold water at pH 10-12, and then diluted 10 mm glycine buffer pH to 10.8 and incubated at C for the night. After ultrafiltration, the solution is subjected to gel filtration on a column of Sephadex G-50 and elute 10 mm glycine buffer. Collect the fractions containing the hybrid protein, are ultrafiltration and freeze-dried. The resulting hybrid protein is dissolved in 0.08 M Tris Hcl buffer, pH 7.5 to a concentration of 10 mg/ml and digested simultaneously with trypsin and carboxypeptidase B in the ratio of 0.3:1:10 (protein carboxypeptidase B : protein tripsine:a hybrid protein) when C for 30 minutes Then add isopropanol to 40%. The mixture is immediately subjected to chromatographic purification on a column with DEAE-Sephadex A-25 and elute 0.05 M Tris Hcl buffer, pH 7.5 with 40% isopto sodium increased to 25% and shift the pH to 2.0, adding 4 N hydrochloric acid. Collect the precipitate of the target product - insulin (2).

However, the known method has a number of disadvantages.

The use of gel filtration in the initial stages causes significant difficulties when scaling the cleaning process, as it requires large amounts of sorbent. In the thus obtained material must contain impurities of non-protein nature, which are not reported. They can significantly impede regeneration of the sorbent. It is also known that the splitting of the hybrid protein by trypsin along with arginine and diagonalisation formed and destroysession due to the presence of lysine at position 29 of the b-chain of insulin. Division of destroysession and insulin presents a problem due to the proximity of the properties of these polymers. The authors do not report, we have analyzed whether the content of destroysession, although with such a high ratio of trypsin : a hybrid protein (1:10) the probability of accumulation of the drug is very high.

Known closest to the claimed method of production of recombinant human insulin, including cultivation of the producer strain E. coli JM109/pPINS07, destruction of bacterial cells devine and dithiotreitol, renaturation and cleaning denaturirovannogo hybrid protein by precipitation of impurity compounds in 40% isopropanol followed by chromatography on KM-sepharose, its sequential cleavage by trypsin and carboxypeptidase B, the products trypsinolysis chromatographic JV-sepharose, balanced of 0.03-0.1 M ammonium acetate buffer pH 5.0 to 6.0, containing 8 M urea, with elution fractions linear gradient of sodium chloride from 0 to 0.5 M in the starting buffer, and obtained after cleavage by carboxypeptidase B fraction of insulin purified by the method of reversed-phase high-performance liquid chromatography (RP HPLC) with subsequent gel filtration (3).

The disadvantages of the method include the use of a substantial amount of urea in phase chromatography on SP-sepharose and the use of organic solvents at the stage of selection of recombinant insulin.

The objective of the invention is the development of industrial technology for obtaining high-purity recombinant human insulin, making it easy to scale the process and to minimize the number of technological operations of production of the product.

The essence of the invention is producenta Escherichia coli, the destruction of the bacterial cell disintegration, separation Taurus inclusion containing a hybrid protein, dissolved in a buffer containing urea and dithiothreitol, renaturation and cleaning denaturirovannogo hybrid protein, its cleavage by trypsin and carboxypeptidase B, followed by purification and obtaining the target product, as a producer strain using strain Escherichia coli JM109/pPINS07, cleaning denaturirovannogo hybrid protein is carried out by deposition of doped compounds by acidification to pH 4.0-6.0, followed by chromatography of the supernatant liquid at KM-sepharose, splitting hybrid protein with trypsin and carboxypeptidase B exercise consistently, the products trypsinolysis chromatographic JV-sepharose, balanced of 0.03-0.1 M ammonium acetate buffer, pH 5.0 to 6.0, containing 3 M urea, with elution fractions linear gradient of KCl from 0 to 0.5 M in the starting buffer, and obtained after cleavage by carboxypeptidase B fraction of insulin purified by HPLC on reversed phases with subsequent gel-filtration and secrete insulin in the presence of zinc salts.

Developed by the authors defined placentas is tanzio recombinant human insulin with purity not less than 98% and the content of the bacterial peptide and proinsulin desired level (<10 ppm). This activity received insulin is not less than 27.5 u/mg In the proposed method, all the intermediate stages of the preparation of recombinant human insulin does not contain the crucial problems that prevent them from scaling. The used reagents are widely available, the sorbents can withstand repeated cycles. The obtained high-purity substance insulin meet the requirements of domestic and foreign pharmacopoeias, which allows the use of this technology in an industrial environment.

The method is as follows.

For the preparation of recombinant human insulin using recombinant strain of the bacterium Escherichia coli JM09/NS07 producing hybrid polypeptide containing human proinsulin. The strain is deposited in the Central collection of microorganisms of the Russian joint stock company “BIOLOGICAL product” CKMC “B” under No SMC IN-IN.

The growing seed and the main culture of the recombinant strain spend on a nutrient medium containing in g/l: casein hydrolysate hydrochloric acid - 20, Baker's yeast extract - 14, disubstituted phosphate potassium trihydrate - 6, one-deputizing phosphate potassium - 3, magnesium sulfate is 0.5, sodium chloride and 5 g of the fermenter with a total capacity of 200-1500 L. For the induction of the synthesis of a hybrid protein in the middle of the logarithmic phase of growth bring 1-isopropyl--D-1-thiogalactopyranoside. In the process of raising the concentration of dissolved oxygen is maintained at 40±15%, the amount of injected glucose regulate the level of dissolved oxygen and pH. After growing the culture concentrate on the cage and destroy the homogenizer Gaulin in 0.1 M Tris buffer containing 1.5 M urea and 1 mm EDTA. Taurus enable separated by centrifugation.

The selection of the hybrid protein of granul carried out according to the following scheme.

1. Taurus inclusion is dissolved in a buffer containing 0.1 M Tris, 8 M urea, and then add 5-10 mm dithiothreitol.

2. A hybrid protein insulin centurybut by incubation in 5-10-fold volume of 0.1 M glycine-NaOH buffer, pH 9-11 at t=10-14C.

3. Acid deposition is performed by bringing the pH to 4.0 to 6.0 with a solution of Hcl. The formed precipitate was separated by filtration.

4. Purification of the hybrid protein is carried out by chromatography on KM-sepharose, balanced of 0.05 Tris-HCl buffer, pH 7.0 and 7.5. Protein elute balanced buffer containing 0.25 M NaCl and 1.5 M urea.

5. Enzymatic cleavage gibr what Milenium hydrochloric acid to a pH of 3.8 to 4.0.

6. Products trypsinolysis chromatographic JV-sepharose, balanced 0.03 M ammonium acetate buffer, pH 5.0, containing 3 M urea. Adsorbed protein elute with a linear gradient of KCl from 0 to 0.5 M in equilibrating buffer. Combine the fractions containing not less than 95% of virginiensis.

7. Enzymatic cleavage of virginiensis carboxypeptidase B is carried out at the ratio of carboxypeptidase B : virginiensis - 1:(500-1000). The reaction stopped by the addition of acid (Hcl) to a pH of 3.8 to 4.0.

8. Clearance of insulin by the method of preparative reversed-phase high-performance liquid chromatography carried out on standard equipment.

9. The fractions containing not less than 98% of insulin, combine and add 10% zinc acetate, the pH of the solution was adjusted to 6.3-6.5 and the resulting precipitate is centrifuged. Final purification of insulin conduct of gel filtration on Sephadex G-50.

The invention is illustrated by the following example.

C. Example of preparation of a substance insulin on pilot equipment

C. 1. Growing biomass producer, a hybrid protein containing

The working culture of cells recombinantdna in water-glycerol solution in glass or plastic vials at a temperature of minus 40C.

At all stages of the multiplication of microbial cultures using a nutrient medium of the following composition (g/l) (see tab.1).

Pilot line for growing biomass producer consists of a rocking incubator for 12 katalozhnyh flasks and fermenters with a capacity of 15, 150 and 1500 liters of Seed culture prepared in the amount of 1/10 of the volume of the seeding medium. At the first stage of reproduction 2 flasks with culture medium seeded with the contents of one ampoule with the working culture and incubated in a rocking chair when S for 18 h Grown culture sow 10-12 flasks containing 100 ml of culture medium and grown until the optical density (OD) of 4-6 units (wavelength 540 nm, the optical path length 10 mm).

Chain fermentors, custom-designed, has agreed geometric, physical, and mass transfer characteristics. The management processes of preparation of nutrient media and carrying out the cultivation is carried out under the programs established in computers.

The results are given in table.2.

For all crops carry out structural-morphological analysis, which assesses not only the “purity” of cultures, but also morphological sesbania seed.

Growing main crop producer is carried out in a fermenter with a total capacity of 1500 liters of nutrient medium Composition and the basic conditions of cultivation are the same as for the crop, however, to induce the biosynthesis of hybrid protein at a certain stage of cultural development it is payable, the inductor 1-isopropyl--D-1-thiogalactopyranoside. The maximum accumulation of the hybrid protein occurs when the induction hybrid protein provoke in the middle of the logarithmic phase of growth. For the chosen growing conditions this corresponds to the accumulation of biomass 7-10% OP. After adding the inductor cultivation continued until the formation of intracellular inclusions hybrid protein “Taurus inclusion in 90-95% of the cells. The Express control of the process of accumulation Taurus enable (TV) is carried out using phase-contrast microscopy preparations “crushed drop”.

The main process parameters are given in table 3.

After the formation of the TV in 90-95% of the cells for further culturing may lead to partial lysis of the cells and the accumulated loss of the hybrid protein.

According to the results of the retrospective analysis in cultures up to enableval by a sharp reduction in the intensity of mixing, including aeration, and tahaliyani to 10-14C. Cooled culture concentrate on the separator 8-10 times. Get 100-125 l suspension in which the injected buffer concentrate (50 l of purified water to 15 kg of urea, 1.4 kg of sodium salt of ethylenediaminetetraacetic acid and 0.9 kg of TRIS buffer).

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

The cell homogenate is passed through flow-through centrifuge (g=18000). The bulk of Taurus on” (at least 90%) is deposited in the centrifuge rotor, and soluble proteins and the remains of cell walls is discharged from the centrifuge in the collection for inactivation. The wet sediment Taurus inclusion, 8-10 kg, unloaded from the centrifuge rotor, Packed in plastic containers, freeze and store in the freezer at minus 40C. Shelf life - up to 6 months. Loss of the hybrid protein in the allocation Taurus enable” do not exceed 15%.

C. 3. The selection of the hybrid protein

In a reactor with a volume of 250 l fill in 100 l of buffer solution containing 8M urea, download 10-12 kg of pasta Taurus on and include the item is Lieut stir for 10-12 hours

In jacketed reactors with a volume of 1200 l pour 900 l glycine-NaOH buffer, pH 9-11 and cool it to a temperature of 10-14C, feeding in the jacket water cooled. After the buffer has cooled down, start the pump feeding into the reactor a solution of a hybrid protein with the restored disulfide bonds. During 20-24 h incubated solution of the hybrid protein, stirring and maintaining the temperature in the reactor 10-14C.

After 75-80% of the hybrid protein (GB) sanatorium education properly closed S-S bonds (the control method OF HPLC), spend the acidification of the reaction medium in the reactor to a pH of 4.0 to 5.5 with hydrochloric acid. Stop stirring and after 4-5 h separating the formed precipitate on microfiltration installation. Correctly folded hybrid protein absorb from the filtrate on ionoobmennoe column volume of 8-10 l, filled KM-separate, pre-balanced 0.05 M Tris-Hcl buffer, pH 7,0-7,5.

Adsorbed protein elute from the column, passing through it equilibrating buffer with the addition of 0.25 M NaCl and 1.5 M urea.

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

C. 4. Trypticase Gee stainless steel equipped with a mixing device and a shirt.

The solution GB number 100-105 l (protein 1-1,2 kg) contribute to the reactor include a mixing device and fed to the jacket water cooled with a temperature of 10-12C. After 30-40 min after the solution GB cooled to a temperature of 10-12C, measure the pH GB and using a 1 M solution of Tris-(oxymethyl)-aminomethane bring its value up to pH 7.5 to 8.2. Then the reactor contribute trypsin solution from the calculation that the number inserted into the reactor trypsin is 1/500 part on the number of GB in the reactor. Through 100-120 min stop the cleavage reaction GB trypsin, podkisst the contents of the reactor hydrochloric acid to a pH of 3.8 to 4.0.

The resulting solution was applied to the chromatographic column volume of 20 l, filled JV-separate, pre-balanced 0.03 M ammonium acetate buffer, pH 5.0 by addition of 3 M urea.

After applying all of the solution from the reactor to the column it was washed with equilibrating buffer until reaching the baseline control flow densitometer. Sorbed material elute with a linear gradient of KCl from 0 to 0.5 M in equilibrating buffer for 8 h

what pcidata B. Processing carboxypeptidase B are in a reactor equipped with a stirrer, jacket, temperature and pH. The solution virginiensis fill the reactor include a mixing device and fed to the jacket water cooled with a temperature of 12-14C. After the solution virginiensis temperature 12-14C using a 1M solution of Tris-(oxymethyl)-aminomethane establish a pH of 7.8 to 8.0 and add a solution of carboxypeptidase B per gram of virginiensis 1 mg of carboxypeptidase B, i.e. in the ratio of 1000:1. The process of hydrolysis are 1.5 hours, then stopped by the addition of acid to the reactor until the pH of the solution of 3,8-4,0.

C. 5. Clearance of insulin by the method of preparative reversed-phase high-performance liquid chromatography (RP HPLC)

Clearance of insulin by the method OF HPLC performed on standard equipment Kiloprep 250 firms Waters, USA or similar hardware from other companies. Installation includes the following elements:

control unit,

- head diaphragm pump with high pressure,

the high - pressure pump for pumping sample separation,

- UV detector with variable wavelength

what eye on the exit,

a computer for control and data processing.

Preparation of chromatographic installation work carried out according to the instructions of the manufacturer for the individual blocks and the unit as a whole.

For carrying out work set a cartridge filled with silica gel grafted phase C18company "Videk, USA, in a column and make the shirt column pressure according to the attached cartridge passport. Connect column reinforced flexible hoses to the chromatograph. Wash the column sequentially with a solution of 0.1% TFU in the amount of 2-3 volume of the column, then 1-2 volumes of acetonitrile (A) and 1-2 volumes of water. The prepared column is used at the stage of purification.

In the prepared column, by means of a pump for supplying samples from the tank serves a solution of insulin from a previous stage in the amount of 35-40 grams of protein. Include a programming device and are split according to the following program (see tab.4).

Registration of the separation process is carried out at 220 nm at a flow spectrophotometer at the sensitivity of 2.0. The collected protein fractions are using collector chromatograph or manually. The collected fractions of the main peak of insulin analyze the P>The fraction of insulin, not containing impurities of desamethasone, transmit to the stage finish cleaning.

S. 6. Finishing purification and crystallization of insulin.

The fractions containing not less than 98% of insulin, combine in a glass reactor equipped with a jacket, temperature and pH.

Zakolerovat the contents of the reactor under stirring to 10-12C and add in the reactor 10% solution of zinc acetate at a rate of 5 ml 10% solution of zinc acetate per 1 g of insulin. Then bring the solution pH to a value of 6.3 to 6.5 with 10% ammonia solution. The stirrer is switched off and cooled suspension of leave for 10-12 hours for maturation of sediment. The precipitate was separated in a centrifuge at 5000-6000 rpm at a temperature of 10-12C.

Then spend clearance of insulin by the method of gel filtration on a column of size 250/60 filled with Sephadex G-50. Column balance 1M solution of acetic acid. The precipitate of insulin obtained in the preceding stage, dissolved in 1M acetic acid solution, and adjusted to an optical density of 80-100 optical units at a wavelength of equal to 277 nm. Before applying the solution of insulin on the column, it is filtered through a membrane with pore size of 0.22 μm. A solution of insulin injected into the upper part of the column with plait the introduction of a solution of insulin in column serves eluent, 1 M solution of acetic acid, with the same speed. A solution of 1 M acetic acid is passed through the column with the carrier to the lack of absorption in the ultraviolet part of the spectrum at 277 nm. Protein fractions are combined in accordance with the chromatographic zones on the output curve of distribution of the substance insulin, spectrophotometric determine the protein content and biological activity, which is at least of 27.5 IU/mg of insulin.

Thus, conducting chromatographic purification on neutral carriers allows to obtain human insulin with purity not less than 98% and not below of 27.5 IU/mg.

The authenticity of the obtained target product is confirmed by the following parameters:

- by matching the retention time of the principal peak of the target product and the international reference standard, USP (USP USP 23, USA);

- to assess the biological activity of the target product (VFS 42-3045-98), which is not less than 27 U/mg;

- coincidentally peptide maps of the target product and the reference standard, USP. The purity of the obtained target product was confirmed by Pharmacopoeia USP 23, United States and amounted to:

- the content of the target product, defined by the material of the peptides, less than 10 ppm;

- content desamethasone, less than 2%.

Sources of information

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. Chen J. - Q., Zhang H. - T. Or M. - N., Tang J.-G. Production of human insulin in an E. coli system with met-lys-human proinsulin expressed as the precursor. Applied Biochemistry and Biotechnology, 1995, v.55, p.5-15.

3. Kalinin, Y. T., Urakov N. N., Ivanov, C. T. and other Method of obtaining recombinant human insulin., The patent RU 2141531 from 26.05.99.

1. A method for industrial production of recombinant human insulin, including the cultivation of the producer strain E. coli JM 109/IS07 in uglevodosoderjati nutrient medium, the destruction of cell disintegration, separation Taurus inclusion, a hybrid protein containing insulin, their dissolution in buffer containing urea, renaturation, acid precipitation, and purification of the hybrid protein by chromatography on KM-sepharose, enzymatic cleavage with trypsin and carboxypeptidase B, followed by purification and isolation of insulin, wherein the cultivation is carried out in an industrial fermenter, while the concentration of dissolved oxygen support at (40±15)%, bullock include dissolved in b is in phase by incubation in glycine-NaOH buffer to the stage of acid deposition, the splitting of the hybrid protein is carried out sequentially at a ratio of trypsin : a hybrid protein and carboxypeptidase B : hybrid protein - 1:(500-1000), and selection - crystallization in the presence of zinc salts.

2. The method according to p. 1, wherein the cultivation is carried out in an industrial fermenter volume 200-1500 L.

3. The method according to p. 1, characterized in that in the process of cultivation, the quantity of injected glucose regulate the level of dissolved oxygen and pH.

4. The method according to p. 1, characterized in that the incubation of the hybrid protein is carried out in 5-10 times the volume of the glycine-NaOH buffer, pH 9-11.

5. The method according to p. 1, characterized in that dithiothreitol added to a final concentration of 5-10 mm.

 

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SUBSTANCE: recombinant plasmid DNA pHINS05 comprises gene encoding hybrid protein consisting of N-terminal fragment of human gamma-interferon, peptide linker His4GlySerArg and human proinsulin. E. coli cells are transformed with recombinant plasmid DNA pHINS05 and the strain E. coli JM109/pHINS05 is obtained that is a producer of hybrid protein with human proinsulin. Invention provides increasing yield of recombinant human proinsulin and to reduce cost of the technological process in preparing human insulin.

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4 cl, 2 dwg, 4 ex

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