RU2487885C2 - Method for large-scale production, separation and purification of recombinant human granulocyte colony-stimulating factor - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: disclosed is a method of producing, separating and purifying the human granulocyte colony stimulating factor (hG-CSF). Cells of a recombinant producer strain are obtained and the culture fluid is then concentrated. Bacterial cells are broken down while separating inclusion bodies via double treatment with high pressure. Protein impurities are removed. The inclusion bodies are dissolved and protein is recovered. Protein renaturation is carried out for 70-90 hours while diluting the protein solution to 400 ml. The obtained solution is deposited on an anion-exchange chromatographic column with diameter of 300 mm with linear rate of deposition of 600-1000 ml/min. The hG-CSF is then concentrated successively on chromatographic columns with anion-exchange or cation-exchange sorbents. The hG-CSF is then purified on a cation exchanger in fraction recycling conditions and stabilised by dialysis.

EFFECT: method enables to obtain, in one cycle, a stable preparation of hG-CSF in amount of 15-20 g with high homogeneity on HPLC and high hemostimulating activity.

5 cl, 1 dwg, 2 ex

Description

The invention relates to biotechnology, specifically to the production of a recombinant granulocyte colony stimulating human factor (rhG-CSF) on an industrial scale and is a method for producing isolation and purification of recombinant human G-CSF as a drug, including for medical purposes, from biomass of bacterial cells containing recombinant plasmid DNA with the human G-CSF gene.

The human granulocyte colony-stimulating factor is the main regulator of neutrophil production. G-CSF is a glycoprotein with a molecular weight of 18-20 kDa, an isoelectric point of 5.5, which is active in monomeric form (Dr. Ken Alibek 27s lecture G-CSF). It is produced by many body cells: bone marrow stromal cells, endothelial cells, macrophages, granulocytes, fibroblasts, astrocytes (Dr. Ken Alibek).

The functions of G-CSF are: stimulation, differentiation and functional activation of neutrophil precursor cells; effect on the immune system by regulating the functions of T cells and dendritic cells (Dr. Ken Alibek); effects on the nervous system through anti-inflammatory effects, suppression of apoptosis and stimulation of neurogenesis (Solaroglu I, et al, A novel neuroprotectant granulocyte-colony stimulating factor, Stroke 2006).

In medicine, recombinant G-CSF is used, both glycosylated and non-glycosylated. Its clinical effect is independent of the glycosylation of the molecule (Bönig H, Silbermann S, et al. Glycosylated vs non-glycosylated granulocyte colony-stimulating factor (G-CSF) - results of a prospective randomized monocentre study, 2001; Hoglund M. Glycosylated and non -glycosylated recombinant human granulocyte colony-stimulating factor (rhG-CSF) - what is the difference? Med Oncol. 1998 Dec)

Recombinant human G-CSF is widely used in oncology and hematology in the treatment of neutropenia of various etiologies (David C. et al, “A Randomized Controlled Phase 3 Trial of Recombinant Human Granulocyte Colony-Stimulating Factor (Filgrastim) for Treatmentof Severe Chronic Neutropenia Blood”, 1993) human autoimmune diseases (Rutella S et al, Granulocyte colony-stimulating factor: a novel mediator of T-cell tolerance, 2005), myocardial infarction, stroke, HIV infection (Dr. Kuritzkes. Neutropenia, Neutrophil Dysfunction, and Bacterial Infection in Patients with Human Immunodeficiency Virus Disease: The Role of Granulocyte Colony-Stimulating Factor, 2000), Neutropenia in Newborns (A Randomized, Placebo-Controlled Trial of Recombinant Human Granulocyte Colony-Stimulating Factor Administration in Newborn Infants With Presumed Sepsis: Significant Induction of Peripheral and Bone Marrow Neutrophilia Blood, 1994).

The growing demand for G-CSF, the high efficiency in the treatment of a number of diseases of the immune and blood-forming systems, the high cost of foreign analogues make it an urgent task to develop a large-scale method for its production.

The use of microbiological synthesis based on the technology of recombinant DNA molecules provides the ability to scale the process of obtaining the target product with a high yield from a relatively inexpensive and safe raw material.

The literature describes several methods for producing rhG-CSF from E. coli cells (Welte K. et al // Proc. Natl. Acad. Sci. USA, 1985, V.82, No.5, p.1526-1530; Nomura H . et al. // EMBO J., 1985, V.5, No.5, p. 891-896; Morioka E. et al. // Res. Exp. Med. (Berl.), 1990, V.190 , No.4, p.229-238). However, some use organic solvents that lead to partial protein denaturation (Welte K. et al), while others use expensive reagents, such as oxidized glutathione (Morioka E et al). A common disadvantage is the low yield of the target protein, and the scaling of the described processes is complicated by the use of low-speed chromatography.

A known method of producing G-CSF from E. coli SG20050 cells transformed with pGGF8 plasmid (RU 2201962). Cells were destroyed by ultrasound, inclusion bodies were washed and dissolved using 8M urea, rhG-CSF renaturation was performed by dilution with neutral buffer. Chromatographic purification of rhG-CSF was carried out using single-stage ion-exchange chromatography on two series-connected columns with DEAE-cellulose and SP-sepharose at pH 4.4, followed by elution of the target protein from a column with SP-sepharose in a linear gradient of sodium chloride (0-0.4 M) in 20 mM sodium acetate buffer at pH 4.4-4.5. The yield of the target product was 24-30 mg from one liter of cell culture E.Coli SG20050 / pGGF8. The purified preparation contained 200 ng of bacterial proteins and 80 pkg of DNA per 1 mg of rhG-CSF. The disadvantages of this method include the large loss of the target protein in the preparatory stages when washing the NFC, as well as in the main stages due to the low efficiency of the renaturation process. The resulting preparations of rhG-CSF are characterized by a significant content of impurity proteins and DNA of the producer strain (about 200 ng and 80 pc per 1 mg of rhG-CSF, respectively).

Closest to the claimed method is a method for producing rhG-CSF from E. coli cells SG20050 / pGGF8 (RU 2278870), in which the transformed E. coli / pGGF8 cells grown as described previously (RU 2326169) are destroyed using a French press or ultrasound at a temperature of (6 ± 3) ° C. Inclusion bodies containing rcG-CSF are separated from the soluble cell protein fraction by laboratory centrifugation. To remove impurity bacterial proteins, the inclusion bodies are washed with a buffer solution with a pH of 7–9 containing detergents and 2 M urea. The precipitate of the inclusion bodies is again separated by centrifugation and dissolved in a buffer solution with a pH of 7–9 containing 8 M urea and 10–30 mM 2-mercaptoethanol or 2–5 mM dithiothreitol. Renaturation of reduced rhG-CSF is carried out by dilution with a buffer solution with pH 7-9 containing detergents, glycerin and EDTA to a total protein concentration of 0.1-0.3 mg / ml at a temperature of (4-20) ° C for 20-48 hours or by dialysis in dialysis tubes of reduced rcG-CSF against a buffer solution with a pH of 7–9 containing detergents, glycerin and EDTA at a temperature of (4–20) ° C for 20–48 hours.

After the renaturation process is completed, the obtained solution of the oxidized protein is passed through a chromatographic column with a diameter of 50-90 mm with an anion-exchange sorbent balanced with a buffer solution with a pH of 7-9, with a bulk velocity of 5-10 ml / min. Acetic acid was added to the combined fractions that did not bind to the sorbent to pH 4-5, and the resulting solution was applied to chromatographic columns with anion exchange and cation exchange sorbents with a diameter of 50 mm and 25 mm and a sorbent layer height of 50 and 70 mm, respectively. The rhG-CSF is eluted from the cation exchange sorbent in a linear gradient of sodium chloride with a bulk velocity of 1-2 ml / min. Fractions containing the target protein are combined and dialyzed in dialysis tubes against a buffer solution with a pH of 4-5. The resulting preparations of rhG-CSF contain 25-33 ng of impurity proteins and 10-13 pc of DNA of the producer strain per 1 mg of rhG-CSF.

The disadvantages of the prototype method are:

1) The described method is laboratory, carried out on laboratory equipment (laboratory centrifuges, chromatographic columns of small sizes, FPLC with a maximum flow rate of 10 ml / min, etc.) with appropriate methods (elution in a linear gradient, dialysis in dialysis tubes, etc. .d.) and approaches, which makes the described process open and discrete. However, the direct transfer of the results of obtaining the target protein from laboratory technology to the industrial method is not obvious, since the methods of the theory of similarity, allowing to obtain the criteria for scaling, are not suitable in biotechnology;

2) The laboratory number of cells involved in the technological process of 10 grams does not allow to obtain the target protein in a quantity sufficient for production.

The objective of the invention is to develop and search for optimal conditions for large-scale production (15-20 g per cycle), isolation and purification of human G-CSF with a high degree of homogeneity and purity, as well as with improved quality, suitable for creating a medicinal product, including medical purpose.

The essence of the proposed method is as follows:

1) The culture fluid obtained from the stage of culturing the producer strain E. coli in a fermenter with a working volume of 250 L is concentrated 10 times, the cells are destroyed using a French press, and insoluble fractions of cells (NFCs) are separated.

2) To remove impurity bacterial proteins, NFCs are washed with a pH 7-9 buffer solution containing detergents and urea. NFCs are again separated by separation.

3) The washed precipitate of the inclusion bodies is dissolved in a pH 8.0 ± 0.5 buffer containing Tris HCl, urea and 2-mercaptoethanol for 20 hours.

4) Renaturation of the recovered protein is carried out by 10-fold dilution, adding a solution of denatured protein in a cooled buffer. Renaturation ends after 70-90 hours. The percentage of homogeneity of the renatured protein is 60-80%.

5) The solution of the renatured protein is subjected to additional purification from impurity proteins, passing through a column with an anion-exchange sorbent at a speed of 500-700 ml / min, balanced with a buffer solution of pH 7-9. In the total volume of fractions not bound to the sorbent, the pH of the buffer is lowered to 4-5. The precipitate formed at a temperature of 10-12 ° C for 24 hours, is separated using membranes.

6) The concentration of the target protein is carried out on series-connected columns with anion-exchange and cation-exchange sorbents. Fractions containing rcG-CSF with HPLC homogeneity above 80% are combined. In this case, the working volume of the protein solution decreases by 40-50 times.

7) Purification is carried out using cation exchange chromatography in the mode of fraction recycling to increase the degree of extraction of the target protein and increase its purity and homogeneity by HPLC.

8) The combined target fractions of rhG-CSF are diafiltered on ultrafiltration membranes with a cutoff threshold of 10 kDa.

As detergents, non-ionic detergents Triton X-100, Tween-20, or a mixture thereof are used.

The RFH-KSF purification process is closed. All operations are carried out under sterile conditions using pyrogen-free sorbents and buffer solutions.

As a cation exchange sorbent, SP-sepharosa, KM-sepharosa, SO ₃ fractogel are used.

DEAE-sephacel, DEAE sepharosa are used as an anion-exchange sorbent.

The yield of electrophoretically homogeneous rhG-CSF with a purity of more than 98% is 60-80 mg from 1 l of a culture of E. coli strain SGK25 / pA3GF (Methods for the control of medical immunbiological drugs administered to humans. Guidelines, MUK 4.1 / 4.2 / 588-96, M., 1998). The specific (hemostimulating) activity of the obtained rhG-CSF preparation, determined in vivo in mice in which hematopoiesis hypoplasia was reproduced by cytostatic administration, is 1000-1400%. The specific activity determined in vitro by the stimulation of murine M-NFS-60 cells is more than 10 + 8 IU / mg protein. The protein content of the producer strain is 30 ng / mg rhG-CSF. The DNA content is less than 10 pg / mg rhG-CSF (General requirements for medical immunobiological preparations taught by genetic engineering methods. RD 42-68-9-89, M., 1989).

The proposed scheme allows to obtain on an industrial scale stable preparations of rhG-CSF with a large quantitative yield, high purity, homogeneity and activity that exceeds the activity of CSF obtained by the prototype, which allows them to be used, including as a substance for creating new drugs generations.

New in comparison with the prototype method is:

- introduction of an industrial NFC washing scheme with obtaining a generalized pool of washed inclusion bodies; the implementation of a laboratory approach to washing inclusion bodies on an industrial scale is impossible;

- an increase in the number of inclusion bodies involved in the biotechnological process to 290-350 g, which corresponds to the industrial level of protein production;

- change in the volume and conditions of renaturation in relation to a large-scale process:

- an increase in the volume of the solution of the target protein at the stage of renaturation up to 350-450 l

- dilution of the reconstituted solution of inclusion bodies with a pH 7-9 buffer solution containing EDTA by adding the protein solution to the total volume at a fixed speed with stirring.

- increase the renaturation time to 70-90 hours at a temperature of 10-15 ° C.

- an increase in the incubation time of the protein solution with a decrease in the pH of the free fraction after anion exchange chromatography up to 24 hours at 10-15 ° C.

- concentration of the target protein 40-50 times using cation exchangers in order to reduce the working volume of the solutions.

- conducting cation-exchange chromatography in the mode of fraction recycling to increase the degree of extraction of the target protein, the degree of purity and homogeneity of rhG-CSF by HPLC.

- reducing the time for the implementation of technological stages by including filters in industrial chromatographic systems that allow precipitates to be separated during the application of protein solutions to sorbents.

- the use of membrane technologies to stabilize the properties of the target protein at the final stage of obtaining the drug substance.

The changes introduced allow us to scale the process of obtaining rG-CSF to an industrial level while maintaining the product yield characteristics of the prototype claimed in the laboratory method. Moreover, the total yield of the target protein with a homogeneity of more than 97% by HPLC is 15-20 g.

The present invention is illustrated by a figure of a graphic image (see figure 1), which presents:

1. Stage renaturation 72 hours.

2. The stage of concentration

3. 1st chromatographic cycle

4. 2nd cycle of chromatographic purification

5. 3rd cycle of chromatographic purification Examples of a specific implementation of the method are given below.

EXAMPLE 1

(approximation to the industrial method)

1.1. Obtaining and washing inclusion bodies from 150 grams of cells of the E. coli SGK25 / pA3GF producer strain.

A part of the biomass (150 g) obtained in a fermenter with a working volume of 250 liters is suspended in a lysing buffer of 1000 ml until a homogeneous mass is obtained and destroyed using a French press at 1100 bar, cooling the cell lysate. The resulting suspension is separated on a separator. The precipitate is washed using ultrafiltration membranes with a cut-off threshold of 10 kDa in the wash buffer containing Tween-20, Triton X-100 and urea.

The weight of the inclusion bodies is 20 g, the content of the target protein is 72%.

1.2. Solubilization of inclusion bodies. Protein Recovery

The resulting inclusion bodies are suspended in a urea containing pH 7-9 buffer solution of 2500 ml volume, 2-mercaptoethanol is added to a final concentration of 20 mM and stirred for 20 hours at room temperature. The precipitate formed is filtered off.

1.3. Protein renaturation.

The reconstituted protein solution is diluted 10 times with renaturation buffer cooled to 8 ° C containing 10 mM sodium phosphate and 10 mM EDTA pH 8.0. Renaturation is carried out for 72 hours at 8 ° C, the precipitate formed during renaturation is separated by filtration. Renaturation efficacy was evaluated by HPLC. The homogeneity of the renatured rhG-CSF by HPLC is 73%.

1.4 Removal of impurity bacterial proteins.

The resulting protein solution was applied to an XC 45/50 column (Pharmacia Sweden) containing 100 ml of DEAE of Sepharose, equilibrated with Tris HCl buffer, pH 8.0 ± 0.05, at a rate of 50 ml / min at 8 ° C. Unbound fractions containing rhG-CSF are collected as target. In a solution of partially purified protein, the pH is reduced to a value of 4.0 ± 0.05. The solution was incubated for 20 hours at 8 ° C, the precipitate was separated by filtration.

1.5. Concentration of rhG-CSF.

The resulting protein solution was sequentially applied to HC 45/50 columns (Pharmacia Sweden) with anion exchange (DEAE sepharose (100 ml)) and cation exchange (SO ₃ fractogel (85 ml)) sorbents balanced with acetate buffer with pH 4.5 ± 0.05, with 10 ml / min. The target protein is eluted with SO ₃ fractogel in isocratic mode with a balancing buffer containing 0.9 M sodium chloride. The eluate is collected in a total volume of 1750 ml. The homogeneity of the target protein by HPLC is 83%.

1.6. Chromatographic purification on cation exchangers in the mode of fraction recycling.

A solution of partially purified protein is applied to a column with a SO ₃ -fractogel cation exchanger (85 ml) at a rate of 5 ml / min, balanced with acetate buffer with a pH of 4.5 ± 0.05. The sorbent is washed with a balancing buffer containing 0.2 M sodium chloride and the protein is eluted in a gradient of sodium chloride from 0.2 to 1.0 M in 20 column volumes. Fractions with target protein homogeneity by HPLC 92% are combined.

The combined fractions containing the target protein under the same conditions are again applied to a column with SO ₃ fractogel cation exchanger. The sorbent is washed with equilibration buffer and the protein is eluted in a similar manner. Fractions with the target protein homogeneity by HPLC more than 95% are combined as the target product.

1.7. Stabilization of purified rhG-CSF.

The pooled fractions are dialyzed against 10 volumes of dialysis buffer 5 mM sodium acetate pH 4.0. The yield of the target protein is 1003 mg. The protein solution is subjected to sterilizing filtration and stored at 8 ° C.

EXAMPLE 2

2.1. Obtaining cells of the producer strain of E. coli SGK25 / pA3GF is carried out according to claim 1.1. Described in Example 1. The culture fluid from the fermenter with a working volume of 300 l was concentrated using membranes to a volume of 20-30 l. The cell suspension was treated twice with a French press at 1200 bar. The content of the target protein in the NFC is according to gel electrophoresis in PAAG 40-60%.

2.2. The solubilization of inclusion bodies and protein recovery is carried out according to claim 1.2. The volume of the solution of the recovered protein is 40 liters.

2.3. Protein renaturation is carried out according to 1.3. The volume of the solution of the renaturing protein is 400 l. The homogeneity of the renatured rhG-CSF is 70% according to HPLC.

2.4. Removal of impurity bacterial proteins is carried out according to 1.4. The resulting protein solution is applied to a column with a diameter of 300 mm containing 6000 ml of DEAE sepharose at a rate of 600-1000 ml / min at a temperature of 20 ° C. The acidified protein solution is incubated for 24 hours at 12 ° C.

2.5. Concentration of rhG-CSF is carried out at 1.5. The resulting protein solution is successively applied to columns with a diameter of 300 mm with DEAE Sepharose (6000 ml) and SO ₃ fractogel (6000 ml), at a rate of 200-300 ml / min. The volume of the eluate is 34,000 ml. The homogeneity of the target protein by HPLC is 82%.

2.6. Chromatographic purification on cation exchangers in the mode of fraction recycling is carried out in 1.6. The protein solution is applied to a column with a diameter of 170 mm with a cation exchanger CMsepharose (5000 ml) at a rate of 200-250 ml / min, balanced with acetate buffer. The sorbent is washed with equilibration buffer. The protein is eluted in isocratic mode with a solution of 0.13 M sodium chloride. Fractions with target protein homogeneity by HPLC 92% are combined.

The combined target protein fractions under the same conditions are again applied to a cation exchanger column. Fractions with the target protein homogeneity by HPLC more than 95% are combined as the target product.

2.7. Stabilization of purified rhG-CSF as a substance is carried out according to 1.7. The combined target fractions are diafiltered using ultrafiltration membranes with a cutoff threshold of 10 kDa. The protein solution is diafiltered to equalize its conductivity with the conductivity of the protein storage buffer. The yield of the target protein is 17840 mg. The protein solution is subjected to sterilizing filtration and stored at 8 ° C.

Thus, the proposed method allows to obtain in one cycle a stable preparation of rhG-CSF with a homogeneity of more than 97% by HPLC in an amount of 15-20 grams, which is 60-100 times more compared to the prototype method. In this case, the relative yield of protein is 60-80 mg / per liter of culture fluid, its activity is 1000-1400%, which exceeds the activity values of the prototype, and the duration of the production process practically corresponds to the laboratory method for producing G-CSF in the prototype method.

Claims (5)

1. The method of obtaining, isolation and purification of rhG-CSF, including
a) obtaining cells of a recombinant strain,
b) the destruction of bacterial cells during the isolation of inclusion bodies,
c) preliminary removal of impurity proteins,
g) dissolution of inclusion bodies,
d) recovery
e) renaturation,
g) anion exchange and cation exchange chromatographic purifications
h) and stabilization of rhG-KSF
characterized in that the concentration of the culture fluid after stage a) is carried out, double treatment with high pressure during destruction of the cells of the producer strain in stage b) to obtain a generalized pool of washed inclusion bodies, solubilization of inclusion bodies, as well as renaturation by diluting the protein solution to 400 l and 70-90 h duration, anion-exchange chromatographic purification in LC 300 mm in diameter with a linear application rate of 600-1000 ml / min, followed by concentration on chromatographic columns with anion-exchange and thion exchange sorbents and chromatographic purification on a cation exchanger in the mode of fraction recycling in stage g) and stabilization of rhG-CSF in stage h) by dialysis. 2. The method according to claim 1, in which the E. coli strain SGK25 / pA3GF deposited in VKPM under the number B-8685 is used as the producer strain. 3. The method according to claim 1, characterized in obtaining the target protein at the stage of concentration of the rhG-CSF solution by elution in isocratic mode in the presence of 0.9 M-0.13 M sodium chloride. 4. The method according to claim 1, characterized in that Fractogel SO _{3 is} used as a cation exchange sorbent for chromatographic purification of the protein in the mode of fraction recycling. 5. The method according to claim 1, characterized in that SM sepharose is used as a cation exchange sorbent for chromatographic purification of the protein in the mode of fraction recycling.

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