RU2201962C2 - Method of preparing human recombinant granulocytic colony-stimulating factor - Google Patents

Abstract

FIELD: biochemistry, biotechnology, medicine. SUBSTANCE: human recombinant granulocytic colony-stimulating factor is prepared from Escherichia coli transformed cells. Inclusion bodies comprising recombinant factor are dissolved in urea and reduction reaction is carried out with 10 mM of 2-mercaptoethanol. Renaturation is carried out by addition of EDTA-containing neutral buffer up to 0.8 M concentration of urea. Chromatography purification of recombinant factor is carried out on two successively linked columns filled with DEAE-cellulose and SP-Sephadex and the following elution of the end protein from SP-Sephadex column by linear gradient of sodium chloride in 0.02 M sodium acetate buffer, pH 4.4-4.5. Invention provides to increase the yield of product and retain its biological activity and purity. Invention can be used for isolation and purification of physiologically active human recombinant granulocytic colony-stimulating factor. EFFECT: improved method of preparing, increased yield and purity. 2 cl, 3 tbl, 3 dwg

Description

 The invention relates to the field of biochemistry and biotechnology and is a method for isolating and purifying a physiologically active recombinant human granulocyte colony stimulating factor (rhG-CSF), suitable for scientific research and as a medicine.

 G-CSF belongs to the group of cellular growth factors and plays an important role in stimulating the proliferation, differentiation and functional activity of granulocyte-containing white blood cells [1], rhG-CSF reduces the duration of neutropenia during chemotherapy of malignant tumors and bone marrow transplantation [2, 3], shortens the period granulocytopenia induced by radiation [4]. All this makes its use in medicine promising.

E. coli-produced recombinant human granulocyte colony stimulating factor is a 174-amino acid sequence containing additional Met at the N-terminus. The molecular weight of the protein determined by electrophoresis under denaturing conditions is 18800 D [5]. The molecule contains free cysteine at position 17 and two intramolecular disulfide bonds Cys ³⁶ -Sys ⁴² and Sys ⁶⁴ -Sys ⁷⁴ [5]. To restore the biological activity of rhG-CSF expressed in E. coli as insoluble inclusion bodies, renaturation is required [6].

 A known method of producing G-CSF from cells of a bladder carcinoma (line 5737) [7, 8]. The method includes concentration of the culture fluid with ammonium sulfate, dialysis, chromatography on DEAE cellulose, gel filtration on Ultragel AsA-54, and reverse phase high performance liquid chromatography (HPLC). The above method allowed to obtain a drug with a G-CSF content of more than 95% (according to the results of gel electrophoresis in polyacrylamide gel). There are no data on the yield and content of impurities.

 The disadvantages of the method: the use of somatic cells entails increased complexity in their conservation and cultivation, the process includes three chromatographic purifications, which also increases the complexity of the process and reduces the yield of the product. In addition, the use of reverse phase high performance liquid chromatography requires special equipment.

A method [7] for the preparation of rhG-CSF from E. coli transformed with p536hG-CSF2 plasmid is described. Cells are suspended in water, and the suspension is squeezed several times in a French press. The precipitate was separated by centrifugation and resuspended in water to a protein concentration of 5-6 mg / ml. The precipitate was separated by centrifugation and the precipitate material was dissolved in 1% sodium laurate containing 50 mM Tris, pH 8.5 and 5% ethanol. Insoluble material is separated by centrifugation, and the supernatant is applied to the column (C ₄ ) HPLC. The column was washed with 100 mM ammonium acetate, pH 6.0-7.0, and eluted with rhG-CSF using a gradient of isopropanol in ammonium acetate. The purity of the drug according to the results of gel electrophoresis in polyacrylamide gel is more than 95%. The disadvantage of this method is the use of reverse-phase high-performance liquid chromatography, which requires special expensive equipment. There is no data on the output of the rHG-CSF.

 Closest to the claimed method is a method for producing recombinant human G-CSF from Escherichia coli cells containing plasmid pCfBD28 [9] (prototype). Cells are destroyed by ultrasound in Tris-Hcl buffer. The inclusion bodies are separated by centrifugation and washed.

 The washed inclusion bodies are dissolved in 20 mM Tris-Hcl buffer, pH 8.0, containing 8 M urea.

Dithiothreitis is added to the resulting solution to a concentration of 0.1 mM and incubated for 5 hours at a temperature of 4 ^o C.

To the solution of the reconstituted protein for renaturation, oxidized glutathione is added to a concentration of 0.1 mM and incubated overnight at 4 ^° C.

 The renatured protein solution was applied to a DEAE-Toyoperl column, equilibrated with 10 mM Tris-HCl buffer, pH 8.0. rcG-CSF is eluted with a linear gradient of sodium chloride.

 Ammonium sulfate was added to an eluate containing rhG-CSF to a concentration of 0.25 M, and the solution was applied to a Butyl-Toyoperl column, equilibrated with 10 mM Tris-HCl buffer, pH 8.0, containing 0.25 M ammonium sulfate. rcG-CSF is eluted with a buffer solution with a decreasing concentration of ammonium sulfate.

 The fractions containing rcG-CSF were combined and desalted on a Sephadex G-25 column in 10 mM phosphate buffer, pH 7.2.

 The electrophoretic purity of the obtained rhG-CSF with a gel load of 8 μg of the drug was more than 99%. The yield of purified rhG-CSF was 33% relative to the amount of rhG-CSF in the inclusion bodies.

The disadvantages of the prototype method are:
- use for renaturation of a special oxidizing agent - oxidized glutathione;
- use to obtain purified rhG-CSF three stages of chromatography.

 The technical task of this invention is to simplify the method, reduce the loss of the target product and obtain the preparation of rhG-CSF with a low content of DNA impurities, lipopolysaccharides and proteins of the producer strain, suitable for use not only in scientific research, but also as a basis for creating a new medicinal product [10].

 The essence of the proposed method is as follows: the biomass of the recombinant strain of E. coli SG 20050 / pGGF8 [11], grown as described previously [12] and collected by centrifugation, suspended in 10 mm Tris-HCl buffer, pH 8.0. The cell suspension is sonicated for 5 minutes and centrifuged for 20 minutes.

 The obtained precipitate inclusion bodies washed in a known manner [9].

 The washed precipitate of inclusion bodies is dissolved in 10 mM Tris-HCl buffer, pH 8.0, containing 8 M urea.

 2-mercaptoethanol was added to the solution to a concentration of 10 mM and incubated overnight at room temperature.

rcG-CSF is renatured by diluting the resulting protein solution with 10 mM sodium phosphate buffer, pH 8.0, containing 10 mM EDTA and keeping the solution at 4-6 ^{° C.} for 20-24 hours.

 Acetic acid is added to the mixture of renatured proteins to a pH of 4.4-4.5, diluted twice with water.

 The resulting solution of the renatured protein is applied to two, connected in series and equilibrated with 20 mM sodium acetate buffer, pH 4.4-4.5 (buffer "A"), columns with DEAE-cellulose and SP-Sephadex (C-25). After applying the entire solution, the columns are washed with buffer "A". The DEAE cellulose column was disconnected and eluted with rhG-CSF with SP-Sephadex by a linear gradient of sodium chloride from 0 to 0.4 M in buffer “A”. The fractions containing rhG-CSF are combined.

 The resulting eluate is dialyzed against 0.05 M sodium acetate buffer, pH 4.4-4.5, containing 0.1 M sodium chloride.

The yield of electrophoretically homogeneous rhG-CSF is 24-30 mg from 1 l of a culture of E. coli SG 20050 / pGGF8 cells (57% of the amount of rhG-CSF in the inclusion bodies). The specific activity, determined by the formation of neutrophil colonies from mouse bone marrow precursor cells [13, 15], is 10 ⁷ -10 ⁸ IU / mg protein. The content of impurities of lipopolysaccharides, DNA and proteins of the producer strain meets the requirements of the Pharmaceutical Committee for the quality of genetically engineered medical products [10].

 The proposed scheme allows you to get drugs rhG-CSF with high yield and the necessary purity, which allows you to use them as a substance to create a new generation of the drug.

New in comparison with the prototype method is:
- renaturation of rhG-CSF by diluting the solution of inclusion bodies with a neutral buffer containing EDTA (instead of using expensive oxidized glutathione in the prototype method);
- one-step chromatographic purification of rhG-CSF on two series-connected columns with DEAE-cellulose and SP-Sephadex in a salt concentration gradient (instead of the three-stage in the prototype method).

 All this allows us to simplify the method of obtaining rhG-CSF and increase the yield of the final product from 33% in the prototype method to 57%.

 The proposed method was carried out using a producer strain obtained by transformation of E. coli SG20050 cells with plasmid pGGF8 [11]. Cell biomass was obtained as described previously [12].

 Cells are destroyed by ultrasound, centrifuged and the sediment of inclusion bodies is washed in a known manner. Dissolution of inclusion bodies occurs in a buffer solution containing 8 M urea.

 According to the results of electrophoresis of the obtained material in PAGE in the presence of SDS without treatment with 2-mercaptoethanol, the insoluble and inactive protein in the inclusion bodies is oligomers, where the polypeptides are connected by disulfide bonds. To break down disulfide bonds and produce monomeric protein, 2-mercaptoethanol reduction is used.

The resulting solution of the protein for renaturation is diluted with phosphate buffer to a concentration of urea of 0.8 M and the solution is kept at a temperature of 4-6 ^o C for 18-20 hours. As a result of lowering the concentration of urea, polypeptide molecules are folded (renaturation), and due to dissolved oxygen the formation of intramolecular disulfide bonds. To minimize the formation of intermolecular bonds, the process is carried out at a temperature of 4-6 ^o C.

 To stop the process of renaturation and reduce the number of oligomers using acidification of the mixture with acetic acid to a pH of 4.4-4.5.

 For protein purification using one-step chromatography on two columns: the first with DEAE-cellulose, the second with SP-Sephadex. The use of DEAE cellulose allows sorption of lipopolysaccharides, nucleic acids and proteins of the producer strain from the solution. In this case, the rhG-CSF on the first column is not adsorbed. On the second column, rhG-CSF is sorbed, and if some of the lipopolysaccharides and nucleic acids are not adsorbed on DEAE cellulose, they are not adsorbed on SP-Sephadex and elute when the column is washed. rcG-CSF is eluted with SP-Sephadex in a concentration gradient of sodium chloride (0-0.4 M) in 0.02 M sodium acetate buffer, pH 4.4-4.5. Using the above method allows to reduce losses during chromatographic purification of rhG-CSF by about 20% compared to the prototype method and to obtain rhG-CSF with a high yield of 24-30 mg / l of culture and a low content of lipopolysaccharides, producer strain proteins, DNA.

The present invention is illustrated by figures of a graphic image, which presents:
Figure 1. Electrophoretic analysis of proteins in a 12% polyacrylamide gel in the presence of SDS during the preparation of insoluble inclusion bodies:
lane 1 - destroyed cells of the producer strain;
lane 2 - supernatant after separation of inclusion bodies;
lane 3 - supernatant after washing with 1 M LiCl with urea;
lane 4 - supernatant after washing with buffer solution;
lanes 5, 6 - washed inclusion bodies in urea (5 and 10 μg of protein);
lane 7 — protein standards.

 Figure 2. An elution profile of rhG-CSF from an SP-Sephadex column.

Figure 3. Electrophoretic analysis of purified rhG-KSF:
lanes 1, 2 — rhG-CSF preparations (40 μg / gel each): sample preparation in the presence of 2-mercaptoethanol;
lane 3 — protein standards;
lanes 4, 5 - rhG-CSF preparations (40 μg / gel each): sample preparation in the absence of 2-mercaptoethanol.

 Examples of specific performance of the method are given below.

Example 1. Destruction of cells and obtaining inclusion bodies
10 g of the biomass of the producer strain E. coli SO 20050 / pGGF8 are suspended in 100 ml of 10 mM Tris-HCl, pH 8.0. Cells are destroyed by ultrasound and the suspension is centrifuged for 20 min at a temperature of 4 ^o C. the Precipitation of the inclusion bodies washed with salt solution in buffer. Wash solution contains a set of solubilized low molecular weight proteins, but does not contain rhG-CSF (figure 1, lanes 3, 4). The amount of protein in the resulting precipitate 100-120 mg (table 2) (figure 1, lanes 5, 6).

Example 2. Dissolution of inclusion bodies in urea and protein recovery
To the precipitate obtained in Example 1, 50 ml of 10 mM Tris-HCl, pH 8.0, containing 8 M urea was added and vigorously stirred until clear. The solution determines the concentration of protein. To the solution was added 10 mM Tris-HCl, pH 8.0, containing 8 M urea to a protein concentration of 1-2 mg / ml. To restore protein, 2-mercaptoethanol is added to a concentration of 10 mM and the mixture is left for 18-20 hours at room temperature (18-23 ^° C).

Example 3. Renaturation of rhG-CSF
The solution of the recovered protein obtained in the previous example is cooled to a temperature of 4-6 ^{° C.} and 9 volumes of 10 mM sodium phosphate buffer containing 10 mM EDTA cooled to the indicated temperature are added. The resulting mixture was kept at a temperature of 4-6 ^o C for 20-24 hours. Acetic acid was added to the solution of the renatured protein with stirring to a pH of 4.4-4.5.

Example 4. Purification of the renatured rcG-CSF
The solution of the renatured protein obtained in Example 3 is diluted twice with distilled water and applied to two columns connected in series with the first 100 ml of DEAE-cellulose, the second 300 ml of SP-Sephadex, balanced with 20 mM sodium acetate buffer, pH 4 , 4 (buffer "A"). After application, the columns are washed with buffer “A”, the column with DEAE-cellulose is disconnected and the proteins adsorbed on SP-Sephadex are eluted with a linear gradient of sodium chloride from 0 to 0.4 M in buffer “A”.

 Fractions were analyzed by electrophoresis under denaturing conditions in 12% PAAG. Fractions containing protein with a molecular weight of 18800 are combined.

 The resulting eluate of purified rhG-CSF was dialyzed against 50 mM sodium acetate buffer containing 0.1 M sodium chloride. The final volume of the obtained preparation was 157 ml, the protein concentration of 0.557 mg / ml.

 The total data on the purification of rhG-CSF from 10 g of biomass are shown in table 1.

 The electrophoretic purity of the obtained rhG-CSF protein at a well load of 40 μg is more than 95% (Figure 3).

Example 5. Determination of the biological activity of the drug rcG-CSF
A suspension of bone marrow cells is added to vials containing complete enriched medium, at the rate of 10 ⁵ cells per 1 ml, after which 20 μl of diluted rhG-CSF solution are added to the vials. A control sample was prepared by adding to the suspension of bone marrow cells in an enriched medium 20 μl of a 0.15 M sodium chloride solution. The resulting mixtures were poured into wells of a 24-well plate in a volume of 0.5 ml, using two wells for each dilution of rhG-β preparation. CSF (from 1 to 50 ng / ml of both the test sample and the standard preparation). The tablet with cells is incubated at 37 ^o C and 100% humidity for 7-8 days in an atmosphere with 5% carbon dioxide. At the end of the incubation, the colony-stimulating activity of the preparations is counted, counting under the microscope the number of colonies grown in the wells. Colonies mean the foci of hematopoiesis resulting from cultivation containing more than 50 cells.

 For 50 units of activity, the amount of rhG-CSF is taken, which stimulates the formation of 50% of the maximum number of colonies [13].

The biological activity of the obtained preparations of rhG-CSF is 10 ⁷ -10 ⁸ IU / mg protein.

Example 6. The selection of the concentration of the urea solution to dissolve the inclusion bodies of rhG-CSF
40 ml of a uniform suspension of inclusion bodies in Tris-Hcl pH 8.0 buffer is poured into 10 ml centrifuge tubes and the pellets are separated by centrifugation. To the resulting precipitates add 10 ml of Tris-Hcl buffer, pH 8.0, containing various concentrations of urea. The precipitates are dissolved by stirring for 20 minutes and centrifuged. In the supernatants, protein concentration, total protein, rhG-CSF content, total rhG-CSF amount and rhG-CSF yield relative to the amount of the target protein in the inclusion bodies are determined. The results obtained are summarized in table 2.

 Based on the data obtained, it is seen that the greatest extraction of the target protein occurs at a urea concentration of 8 M.

Example 7. Determination of the amount of impurities in the final product
Impurities of lipopolysaccharides were determined as described previously [14].

 The impurities of the proteins of the producer strain (E. coli SG20050 / pGGF8) were determined by enzyme immunoassay using polyclonal antibodies to E. coli proteins SG20050 [16].

 Impurities of DNA were determined by hybridization [10]. The results are presented in table 3.

 The data obtained indicate that the content of impurities in the rhG-CSF is insignificant and meets the requirements of the Ministry of Health for medical immunobiological preparations obtained by genetic engineering [17].

 Thus, the use of the proposed method in comparison with the prototype allows to simplify the method of purification of the target product (to perform renaturation without the use of a special oxidizing agent; to carry out chromatographic purification on two sorbents in one step (by connecting the columns in series); increase the relative yield of the target product by 20% (from 33% in the prototype up to 57% in the proposed method) and the purity of the product is not reduced.

LITERATURE
1. Metcalf D. Blood, 1986, v. 67, No. 2, p. 257-267.

 2. Morstin G., Souza L. M., Keech J., The Lancet, 1988, No. 26, p. 667-671.

 3. Bronchud, M. H., Scarffe, J. H., Thatcher, N. et al. Br. J. Cancer, 1987, v. 56, No. 6, p. 809-813.

 4. Groopman, J.E., Molina, J.-M., Scadden, D.T.N. Engl. J. Med., 1989, v. 321, No. 21, p. 1449-1459.

 5. Lu, H. S., Souza, L. M., Boone, T. S., Lai, P.-H. Arch. Biochem. Biophys., 1989, v. 268, No. 1, p. 81-92.

 6. Lu, H.S., Clogston, C. L., Narhi, L.O. et al. J. Biol. Chem., 1992, v. 267, No. 13, p. 8770-8777.

 7. Souza, L. M., Boone, T. C., Gabrilove, J., Lai, P. H. Science, 1987, v. 232, p. 61-65.

 8. Welte, K., Platzer, E., Lu, L. et al. Proc. Natl. Acad Sci. USA, 1985, v. 82, No. 5, p. 1526-1530.

 9. Motoo Yamasaki et al. Biosci. Biotechnol. Biochem., 1998, v. 62, No. 8, p. 1528-1534.

 10. Methods of control of medical immunobiological drugs administered to people. Guidelines, MUK 4.1 / 4.2 / 588-96, M., 1998.

 11. RF patent 2113483, cl. C 12 N 15/27, publ. BI 17, 1998

 12. RF patent 2158303, cl. C 12 N 1/21, publ. BI 30, 2000

 13. Nomura H., Imazeki I., Oheda M., Kubota M., Tamura M., Ono M., Ueyama Y., Asano S., EMBO J., 1986, v. 5, No. 5, p. 891-896.

 14. Denisova L.Ya., Baturina II, Zakabunin A.I., Afinogenova G.N., Pustoshilova N.M. ZhMEI, 1999, 5, pp. 109-112.

 15. Goldberg E. D., Dygay A.M., Zhdanov V.V. et al. Bulletin of Experimental Biology and Medicine, 1999, v. 128, 8, pp. 194-199.

 16. Afinogenova G.N., Gladchenko T.N., Verevkina K.N., Pustoshilova N.M. Proceedings of scientists of NIKTI BAS, Berdsk, 1999.

 17. General requirements for medical immunobiological preparations obtained by genetic engineering methods. RD 42-68-9-89, M., 1989.

Claims (2)

 1. A method of obtaining a recombinant granulocyte colony-stimulating human factor (rh G-CSF) from transformed Escherichia coli cells containing rh G-CSF in the form of insoluble inclusion bodies, comprising disrupting producer cells, separation, washing and dissolution of inclusion bodies using urea, restoration, renaturation and chromatographic purification of the target protein, characterized in that the restoration is carried out with 10 mm 2-mercaptoethanol, rn G-CSF is renaturated by diluting the solution of the recovered protein neutral buffer containing EDTA to a urea concentration of 0.8 M, and chromatographic purification of rh G-CSF is carried out by applying a renatured protein solution to two series-connected columns with DEAE-cellulose and SP-Sephadex, followed by elution of the target protein from a column with SP-Sephadex in a linear gradient of sodium chloride (from 0 to 0.4 M) in 0.02 M sodium acetate buffer at a pH of 4.4-4.5. 2. The method according to claim 1, characterized in that the renaturation of the RF G-CSF is carried out by diluting the solution of the recovered protein with phosphate buffer at a temperature of 4-6 ^o C for 18-20 hours

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