TWI758285B - A method for renaturation and purification of recombinant human granulocyte colony stimulating factor - Google Patents

Abstract

The invention discloses a method for renaturation and purification of Recombinant Human Granulocyte Colony-stimulating Factor. In particular, the invention removes the reducing agent in the form of buffer replacement prior to renaturation, thereby improving the controllability and renaturation yield of the renaturation process. In addition, the renaturation and purification method of the invention optimizes the composition of the renaturation buffer. Compared with the prior art, the invention is more suitable for large-scale industrialization production, low requirement on equipment, simple operation, can obtain protein with high purity, good stability, can reduce the side effects of the final product in clinical practice, can improve the safety and effectiveness of drugs.

Description

A kind of renaturation and purification method of recombinant human granulocyte stimulating factor

The invention relates to the field of biomedicine and downstream protein purification of bioengineering, in particular to a method for renaturation and purification of recombinant human granulocyte colony stimulating factor (rhG-CSF). , the operation is simple, and it is suitable for industrial scale production.

Human granulocyte colony stimulating factor (human granulocyte colony stimulating factor, hG-CSF) belongs to the family of hematopoietic growth factors. hG-CSF is a protein synthesized by mononuclear macrophages, vascular endothelial cells and fibroblasts. It consists of 174 amino acids and has a molecular weight of 19KD. Its sequence is well known, as shown in SEQ ID NO: 1. The main mechanisms of action of hG-CSF are: (1) specifically acting on the precursor cells of bone marrow granulocytes and macrophages to promote their differentiation and proliferation into mature granulocytes; (2) acting on bone marrow mature neutrophils to promote It is released from bone marrow to peripheral blood; (3) activates the function of mature granulocytes, enhances their ability to migrate, phagocytose and kill bacteria, and prolongs their survival time; (4) stimulates the release of bone marrow hematopoietic stem cells to peripheral blood.

hG-CSF has a very wide range of clinical uses in the treatment of neutropenia caused by various reasons. At present, it is mainly used for the prevention and treatment of myelosuppression caused by chemotherapy and radiotherapy; Leukemia, AIDS leukopenia and aplastic anemia, etc., all show significant curative effect.

The natural sources of hG-CSF are limited and the yield is very small, which cannot meet the needs of clinical application. At the same time, polyethylene glycol-modified hG-CSF achieves the purpose of increasing the number of neutrophils in peripheral blood in a long-term manner. Its large-scale production also requires a large amount of hG-CSF stock solution, so the large-scale production of hG-CSF stock solution has a significant impact. economic and social significance.

E. coli is the most widely used expression system for expressing foreign proteins. However, rhG-CSF is basically expressed in the form of inclusion bodies in the E. coli system, and the expression product has no biological activity. After dissolving and restoring its natural conformation through the renaturation process, a highly active protein sample can be obtained, and then a high-purity, functional product can be obtained through a purification step. Taking advantage of the fact that rhG-CSF can exert the corresponding biological activities of natural molecules independent of glycosylation modification, it is the most convenient and feasible method to express this protein in E. coli with high yield.

In the process of process research and development, the main step that affects the yield is renaturation. If the renaturation effect is good, the yield must be high. Refolding is completely spontaneous and random, resulting in slow renaturation and low recovery rates. At present, the protein renaturation methods used at home and abroad are all based on the exclusion of the environment that denatures the protein. Specific methods include dialysis, ultrafiltration renaturation, column renaturation, and dilution methods.

The dialysis method takes a long time and requires multiple changes of the dialysis solution, while The solution inside the dialysis bag is not uniform during the dialysis process. The concentration of the denaturant in the solution near the dialysis membrane decreases rapidly while the inner decreases slowly. Only some proteins are in an environment suitable for renaturation, and other proteins are easily aggregated, flocculated, and precipitated. The recovery rate of renaturation decreases, and more importantly, it is difficult to scale up the dialysis method to the production scale due to the limitation of the size of the dialysis bag.

US Patent No. 5,849,883 of Amgen Corporation in 1998 disclosed a G-CSF purification method, wherein the renaturation of rhG-CSF adopts the addition of copper sulfate under stirring conditions, and the protein renaturation is carried out by means of metal ion oxidation. The disadvantage of this method is that there is a risk of trace metal ions remaining, which in turn affects drug-related detection and drug safety, and is rarely used in industrial production at present.

Chinese patent CN1167150A reported the use of hollow fiber ultrafiltration to renature rhG-CSF. Although it can be used for large-scale production, it has the following disadvantages: The internal concentration is not uniform, and it is easy to cause protein flocculation, precipitation or even blockage of the hollow fiber column; ② In large-scale production, a hollow fiber column with a length of more than 90 cm is often required to provide sufficient membrane area, and the length of the hollow fiber column in the research and development stage is all In the range of 30-60cm, the operation of hollow fiber ultrafiltration cannot be linearly scaled up, and it is difficult to carry out detailed process research for the complex renaturation process to ensure the stability of the process.

Chinese patent CN102344931A provides a method for renaturation of nickel affinity chromatography column, which is complicated to operate and difficult to scale up. Chinese patent CN104120159 A provides a method for renaturing Sephadex G-25 column chromatography. The loading protein concentration is 1.0-2.0 mg/ml, the loading volume is less than 20% of the column bed volume, and the loading and elution flow rates are 5-10 cm/h; It is difficult to meet the needs of commercial production.

Compared with other methods, dilution renaturation has the advantages of simple equipment requirements, convenient operation, low cost, and easy amplification. 20% or so.

The renaturation method of rhG-CSF reported in Chinese patent CN1718739 A is: the first dilution and renaturation for more than 20 hours→ultrafiltration concentration→the second dilution and renaturation for more than 96 hours→ultrafiltration and concentration, that is, carry out two dilution and renaturation operation and two ultrafiltration treatments, although this method uses a combination of dilution renaturation and ultrafiltration treatment, the renaturation rate is high, but there are many renaturation steps, long renaturation time, and low efficiency, which will inevitably affect the overall yield and increase the difficulty of process scale-up.

In view of the above problems, considering the whole process of inclusion body denaturation, dissolution and renaturation, a reducing agent needs to be added to reduce the inclusion body protein in the process of denaturation and dissolution, usually using dithiothreitol (DL-Dithiotheitol, DTT). However, , because DTT is easily oxidized, has poor stability, and is easy to volatilize, its effect is easily affected by the environment, and it is difficult to control. Therefore, it is a safe practice to add a step to remove DTT after the dissolution of inclusion bodies and then dilute and renature.

Chinese patent CN101045742A provides a method for renaturing and purifying recombinant human granulocyte stimulating factor. Before renaturation, the lysate is separated by Sephadex G-25 and the excess reducing agent DTT is removed. The renaturation quality yield is higher than 50%, the RP-HPLC purity of rhG-CSF after purification is higher than 97%, although this method can achieve the purpose of removing excess reducing agent in the lysate by Sephadex G-25 chromatography, the salt concentration of the lysate containing Urea is extremely high, and there is a large back pressure during Sephadex G-25 chromatography. There are disadvantages of slow flow rate of chromatography, long processing time, and difficulty in industrial scale-up.

At the same time, it is necessary to provide an appropriate redox environment for the correct formation of disulfide bonds in the protein molecule during the renaturation process. The redox agent usually used is GSH/GSSG, and the ratio of GSH and GSSG will also affect the renaturation efficiency of the protein. . By studying the effect of different addition ratios of GSH and GSSG on the renaturation effect, the present invention innovatively develops a method for rhG-CSF dilution and renaturation only by adding GSSG, and develops a simpler and more efficient renaturation method for rhG-CSF. and purification process.

The present invention provides a preparation method of recombinant human granulocyte stimulating factor, comprising the following steps: a) fermenting and culturing rhG-CSF genetically engineered bacteria, separating and washing to obtain refined inclusion bodies; b) denaturing the inclusion bodies by denaturing liquid Dissolving the denatured protein solution; c) performing buffer replacement on the denatured protein solution to obtain a denatured solution after replacement; d) diluting and renaturing the denatured solution after replacement; e) purifying the renatured rhG-CSF to obtain rhG - CSF stock solution.

Wherein, in step a, the rhG-CSF genetically engineered bacteria are Escherichia coli transformed by a recombinant plasmid with rhG-CSF gene, such as Escherichia coli DH5α strain transformed by recombinant plasmid pBV220/G-CSF.

Inclusion body washing buffer can be: buffer A (5-100mmol/L Tris-HCl, 2-20mmol/L EDTA, 100-500mmol/L NaCl, 2-4mol/L Urea, pH 7-9), buffer B (5-100mmol/L Tris-HCl, 2-20mmol/L EDTA , pH 8-10); the washing mode is buffer A→buffer B to wash sequentially.

The denaturing and dissolving conditions of this step b may be: the inclusion bodies are added to a denaturing solution (6-10mol/L Urea, 2-20mmol/L DTT, 2-20mmol/L EDTA, 1:20 to 1:25 (W/V), 5-100mmol/L Tris-HCl, pH 7-9), stirring and cracking for 10-18h.

The denaturing and dissolving conditions of this step b are preferably as follows: the inclusion bodies are added to a denaturing solution (8mol/L Urea, 10mmol/L DTT, 5mmol/L EDTA, 20mmol/L Tris- HCl, pH 8.2), stirring and lysing for 10-18 h.

The buffer used for replacement in this step c can comprise 6-10mol/L Urea, 2-20mmol/L EDTA, 5-100mmol/L Tris-HCl, pH 7-9, preferably 8mol/L Urea, 5mmol/L EDTA, 20mmol/L Tris-HCl, pH 8.2; the temperature of the buffer solution used for replacement is controlled to be 2 to 30°C, preferably 10 to 20°C; the methods of buffer replacement include but are not limited to ultrafiltration, desalting column chromatography ; Wherein ultrafiltration includes but is not limited to hollow fiber ultrafiltration and membrane package ultrafiltration; if ultrafiltration is used for buffer exchange, ultrafiltration membrane pore size includes but not limited to 3KD, 5KD and 10KD, preferably 5KD; if GE Healthcare is used The company's hollow fiber column is used for ultrafiltration buffer replacement, and the shear rate can be controlled below 16000sec ^-1 , more preferably about 8000sec ^-1 ; if the hollow fiber ultrafiltration is used for buffer replacement, the ultrafiltration conditions can be: ① Denaturation The protein solution is properly concentrated in the ultrafiltration system; ②Constant volume replacement is adopted, and the sample volume is kept constant in the ultrafiltration system, so that the flow rate of the filtrate is the same as that of the permeate; ③Transmembrane pressure (TMP) is not greater than 50PSI , more preferably 10-18PSI; ④ co-ultrafiltration replaces more than 3 times the volume of the sample, taking into account the reasonable process time, it is more preferably 7 times the volume, that is, the volume of the co-flow and washing filtrate is 3 times the volume of the initial sample to replace more than 7 times the volume.

The renaturation buffer in this step d may contain GSSG, and the concentration of the GSSG may be 0.1-1 mmol/L. Preferably, the renaturation buffer can be 0.5mmol/L GSSG, 20mmol/L Tris-HCl, pH 8.2, and the temperature of the renaturation buffer can be controlled to be 2 to 8°C before the denaturation buffer is added to the renaturation buffer after replacement. ; The conditions for dilution and renaturation in this step d may be: the final protein concentration of the renaturing liquid is 0.1-0.4 g/L, preferably, the final protein concentration of the renaturing liquid is 0.3 g/L, and the denaturing liquid is slowly added to the renaturing liquid after replacement. Buffer solution, continue stirring for 30min, stop stirring, and stand at 2 to 8°C for renaturation for 10-18 hours; in this step e, the rhG-CSF renaturation solution can be subjected to salting out, hydrophobic column chromatography, and desalting column in sequence. Chromatography for purification, wherein salting out can be ammonium sulfate precipitation or sodium chloride precipitation, more preferably ammonium sulfate precipitation; if using ammonium sulfate precipitation, the conditions can be: under stirring conditions, slowly add (NH ₄ ) _{2 SO4} to make the final concentration 0.9mol/L, and add EDTA to make the final concentration 5mmol/L, stop stirring and let stand for 30min; Membrane filtration in the process of salting out can use hollow fiber tangential flow filtration, membrane bag Filtration or dead end filtration, preferably hollow fiber tangential flow filtration; if hollow fiber tangential flow filtration is used, the optimized conditions are: membrane pore size 0.2μm, transmembrane pressure (TMP) 3-8PSI, shear rate About 8000sec ^-1 ; If the column chromatography in this step e adopts the chromatographic medium of GE Healthcare company, the optimized chromatographic purification strategy is: firstly by the Phenyl Sepharose FF column chromatography of Phenyl Sepharose Sephadex (eg Sephadex G-25) column chromatography with low adsorption.

，其中m選自 50-2500的整數，較佳自400-500的整數，G為Met-G-CSF。 The recombinant human granulocyte-stimulating factor stock solution obtained by the preparation method of the recombinant human granulocyte-stimulating factor provided by the present invention can be used to prepare corresponding preparation products, and can also be used to prepare polyethylene glycol-modified recombinant human granulocyte-stimulating factor, such as The polyethylene glycol-modified recombinant human granulocyte-stimulating factor described in WO9611953 and CN101172161A, the preparation method thereof may include the steps of preparing the recombinant human granulocyte-stimulating factor according to the method for preparing the recombinant human granulocyte-stimulating factor, and the The step of conjugating recombinant human granulocyte stimulating factor with polyethylene glycol. Wherein, the structure of the recombinant human granulocyte stimulating factor modified by polyethylene glycol can be as shown in formula I,

, wherein m is selected from an integer of 50-2500, preferably an integer of 400-500, and G is Met-G-CSF.

The beneficial effects of the present invention are:

(1) The optimized inclusion body washing process laid the foundation for the subsequent purification steps.

(2) The step of removing the reducing agent is added before the dilution and renaturation, which improves the renaturation rate.

(3) By determining the acceptable residual amount of DTT in the denaturing solution before renaturation, the process operation is significantly shortened on the premise that the renaturation rate is not affected. time, and is conducive to the maintenance of protein activity.

(4) The concentration of renatured protein is higher than that of general dilution and renaturation, which reduces the sample processing volume.

(5) The renaturation efficiency is greatly increased to more than 70%.

(6) The renaturation method involved in the present invention optimizes the renaturation conditions, greatly reduces the production cost, is easy to control, and improves the stability of the process operation.

(7) After renaturation, the protein purity reaches more than 85%, which reduces the pressure of subsequent purification steps.

(8) The renaturation solution remained clear, and no precipitation occurred.

(9) There are no difficult-to-scale methods such as centrifugation and dialysis in the process flow, and the process scale is feasible and successfully scaled up to a production scale.

(10) The overall process cost is low and the purification cycle is short.

(11) High-purity, high-activity rhG-CSF protein stock solution can be obtained. After testing, its SDS-PAGE electrophoresis purity reaches more than 99%, RP-HPLC purity reaches more than 98%, and the specific activity range is (10.0±4.0)×10 ⁷ IU/mg, which is higher than the requirements of the original solution test under the item "Recombinant Human Granulocyte Stimulating Factor Injection" in Part III of "Pharmacopoeia of the People's Republic of China 2015 Edition".

Figure 1 is the SDS-PAGE electrophoresis purity detection map of the washed inclusion bodies in Example 1, wherein electrophoresis lane 1: washed inclusion bodies, electrophoresis lane 2: rhG-CSF reference substance (self-made).

Figure 2 is the SDS-PAGE electrophoresis purity detection map of the rhG-CSF stock solution in Example 1, wherein electrophoresis lane 1: molecular weight standard protein Mark 12, and electrophoresis lane 2: rhG-CSF stock solution.

The technical solutions of the present invention will be clearly and completely described below by means of specific embodiments. The described embodiments are only a part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, the common All other embodiments obtained by the skilled person without creative work fall within the protection scope of the present invention.

Example 1

The method for renaturation and purification of recombinant human granulocyte-stimulating factor inclusion bodies mainly includes the following steps:

Step 1 Preparation of high-purity rhG-CSF inclusion bodies 1) Process steps:

30%)後啟動補料：補料培養基1(蛋白腖20%，酵母粉10%)30-40g/min，補料培養基2(葡萄糖50%)20-60g/min；當OD600

30，升溫至42℃開始誘導，誘導4小時後，降溫至15-20℃，發酵結束。 The Escherichia coli DH5α strain transformed by pBV220/G-CSF was inserted into the primary seed medium (10 g/L of protein, 5 g/L of yeast powder, 5 g/L of NaCl), and cultured at 30°C and 220 rpm for 7 hours; the primary seeds were inserted into the secondary seed medium ( Protein gluten 10g/L, yeast powder 5g/L, NaCl 5g/L, glucose 5g/L), cultured at 30°C 220rpm for 17 hours; secondary seeds were inserted into fermentation medium (protein gluten 10g/L, yeast powder 5g/L, NaCl 5g) /L, glucose 5g/L, KH ₂ PO ₄ 2.7g/L, Na ₂ HPO ₄ 11g/L, MgSO ₄ 0.3g/L), cultured at 30℃ to pH, dissolved oxygen double rebound (pH 7.0, dissolved oxygen

30%), start feeding: feed medium 1 (20% protein, 10% yeast powder) 30-40g/min, feed medium 2 (50% glucose) 20-60g/min; when OD600

30. The temperature is raised to 42°C to start the induction, and after 4 hours of induction, the temperature is lowered to 15-20°C, and the fermentation is completed.

After fermentation, take 1kg of wet thalline and buffer with 5 times the volume of thalli weight TE liquid (20mmol/L Tris-HCl, 5mmol/L EDTA, pH8.2) to suspend the bacterial cells, mix well, and pump into a high-pressure homogenizer for homogenization and crushing. After that, 500 g of wet crude inclusion bodies were obtained by centrifugation.

The crude inclusion bodies obtained above were treated with buffer solution A (20mmol/L Tris-HCl, 5mmol/L EDTA, 4mol/L Urea, 0.25mol/L NaCl, pH8.2) and buffer solution B (20mmol/L Tris-HCl). , 5mmol/L EDTA, pH8.2) washed sequentially to obtain high-purity inclusion bodies.

2) Sample detection:

The inclusion bodies were tested for purity by SDS-PAGE electrophoresis. The conditions and results of electrophoresis were as follows:

SDS Polyacrylamide Gel: ^NuPAGE® 4-12% Bis-Tris Gel, Life technologies

Sample buffer: ^NuPAGE® LDS Sample Buffer (4×), Life technologies

Running buffer: ^NuPAGE® MOPS SDS Running Buffer (20×), Life technologies

Staining solution: Gelcode ^TM Blue safe protein Stain, Thermo scientific

Decolorization solution: purified water, self-made

After loading, the samples were electrophoresed at a constant voltage of 150V to the bromophenol blue migration gel bottom, stained by Coomassie brilliant blue fast staining, and the purity was analyzed by a gel imager.

It can be seen from the electrophoresis lane 1 in Figure 1 of the manual that the obtained inclusion body has a purity of 72.5% by SDS-PAGE electrophoresis.

Step 2 Denaturation and dissolution of rhG-CSF inclusion bodies

Take 20g of preliminarily purified inclusion bodies, denature and dissolve them with a denaturing solution (20mmol/L Tris-HCl, 5mmol/L EDTA, 8mol/L Urea, 10mmol/L DTT, pH8.2) at a ratio of solid-liquid ratio of 1:20, Stir at room temperature for 14 to 16 hours to obtain 400 ml of denatured protein solution.

Step 3 Buffer exchange of denatured protein solution 1) Process steps:

Sample clarification and filtration: the denatured protein solution in the above step 2 was clarified by 10 μm and 0.45 μm two-stage filtration; equilibrium hollow fiber: 5KD hollow fiber column was selected, and the equilibrium solution (20mmol/L Tris-HCl, 5mmol/L EDTA, 8mol/L) was used. Urea, pH8.2) to balance the hollow fiber and the system; concentration: add the clarified sample to the hollow fiber system, run the system at a shear rate of 8000sec ^-1 , control the transmembrane pressure (TMP) to 10-18PSI, and concentrate to 300ml; Constant volume displacement: Add displacement buffer (20mmol/L Tris-HCl, 5mmol/L EDTA, 8mol/L Urea, pH 8.2) to the hollow fiber system continuously, and control the flow acceleration to be the same as the flow rate at the permeate end to keep the denatured protein The liquid volume was constant. During the ultrafiltration process, 2100ml of replacement buffer (20mmol/L Tris-HCl, 5mmol/L EDTA, 8mol/L Urea, pH 8.2) was continuously added to the denatured protein solution, and the transmembrane pressure (TMP) was controlled during the process. For 10-18 PSI, replace the buffer temperature at 10 to 20 °C.

Sample collection: collect the sample from the bottom valve of the hollow fiber system, rinse the hollow fiber system with 100ml of replacement buffer, and incorporate the rinse into the sample to obtain 400ml of denatured protein solution after replacement.

2) Sample detection:

Take 20 μl of the denatured protein solution after replacement and use RP-HPLC to detect the protein concentration of rhG-CSF in the sample. The RP-HPLC conditions are as follows:

rhG-CSF reference substance: homemade, protein concentration of 0.85mg/ml

Chromatography column: Symmetry ShieldTM RP18, 3.5μm, 100mm×4.6mm

Phase A: trifluoroacetic acid-water solution (take 1.0 ml of trifluoroacetic acid and add water to 1000 ml, fully mix and ultrasonically degas for 20 minutes)

Phase B: trifluoroacetic acid-acetonitrile solution (take 1.0 ml of trifluoroacetic acid, add chromatographically pure acetonitrile to 1000 ml, and ultrasonically degas for 20 min)

At room temperature, gradient elution was performed according to the following table, and the detection wavelength was 214 nm.

According to the peak area of rhG-CSF in the test sample and the reference substance, the concentration of rhG-CSF in the denatured protein solution after replacement was calculated to be 7.5 mg/ml by the area normalization method.

Step 4 Dilution and renaturation of rhG-CSF 1) Process steps

Prepare 9.6L of buffer solution (20mmol/L Tris-HCl, pH8.2), control its temperature at 2 to 8°C, and slowly add the denatured protein solution after replacement into the buffer solution, that is, the final protein concentration is 0.3g/L, and at the same time Add GSSG to a final concentration of 0.5 mmol/L, stir for 30 minutes, stop stirring, and let stand at 2 to 8 °C for renaturation for 16 to 18 hours.

2) Sample detection:

Take 50 μl of the renaturation solution to detect the protein concentration of rhG-CSF in the sample by RP-HPLC (the conditions are the same as the RP-HPLC conditions in step 3), and normalize the area according to the peak area of rhG-CSF in the detected sample and reference substance. The concentration of rhG-CSF in the renaturation solution was calculated to be 0.23 mg/ml.

Step 5 Column chromatography purification of rhG-CSF 1) Process steps:

Ammonium sulfate was added to the renatured rhG-CSF solution to a final concentration of 0.9 mol/L, and EDTA was added to a final concentration of 5 mmol/L. After stirring and mixing, let it stand for 30 min, and 0.2 μm hollow fiber tangential flow clarification and filtration were carried out. Column chromatography purification.

Phenyl Sepharose FF column chromatography:

(1) Equilibrate the column with 20 mmol/L Tris-HCl buffer containing 0.9 mol/L (NH ₄ ) ₂ SO ₄ , pH 8.2, flow rate 150 cm/hour, and equilibrate 3 times the column volume; (2) Clarify (3) Rinse the column with 20 mmol/L Tris-HCl containing 0.65mol/L (NH ₄ ) ₂ SO ₄ , pH 8.2, at a flow rate of 150 cm/h, rinse 5 double the column volume; (4) Elute with 20 mmol/L Tris-HCl containing 0.1 mol/L (NH ₄ ) ₂ SO ₄ , pH 8.2, flow rate 150 cm/hour, and collect the target protein peak.

Sephadex G-25 column chromatography:

(1) Equilibrate the column with 50 mmol/L acetate buffer at pH 5.0, the flow rate is 150 cm/hour, and equilibrate 2 times the column volume.

(2) Phenyl Sepharose FF column chromatography elution collection solution was used for sample loading, and the single sample loading volume did not exceed 1/4 of the column volume, and the flow rate was 150 cm/hour.

(3) 50 mmol/L acetate buffer at pH 5.0 was eluted, and the flow rate was 150 cm/hour, and the target protein peak was collected.

The recombinant human granulocyte-stimulating factor stock solution is obtained after sterilization and filtration of protein samples.

2) Sample detection:

The purity of rhG-CSF stock solution was analyzed by SDS-PAGE and RP-HPLC.

(1) The conditions for SDS-PAGE to detect the purity of rhG-CSF stock solution are the same as the conditions for SDS-PAGE electrophoresis analysis in step 1

The SDS-PAGE method for the detection and analysis of the purity of the rhG-CSF stock solution is shown in Figure 2. It can be seen from the analysis results that the SDS-PAGE electrophoresis purity of the final rhG-CSF stock solution is 100%.

(2) The conditions and results of the RP-HPLC method to detect the purity of the rhG-CSF stock solution are as follows:

Chromatography column: Symmetry ShieldTM RP18, 3.5μm, 100mm×4.6mm

Phase A: trifluoroacetic acid-water solution (take 1.0 ml of trifluoroacetic acid and add water to 1000 ml, fully mix and ultrasonically degas for 20 minutes)

Phase B: trifluoroacetic acid-acetonitrile solution (take 1.0 ml of trifluoroacetic acid, add chromatographically pure acetonitrile to 1000 ml, and ultrasonically degas for 20 min)

At room temperature, gradient elution was performed according to the following table, and the detection wavelength was 280 nm.

The RP-HPLC method was used to detect the purity of the rhG-CSF stock solution. From the analysis of the results, it can be seen that the RP-HPLC purity of the final rhG-CSF stock solution was 99.14%.

It can be seen from the electrophoresis lane 2 in Figure 2 of the manual that the electrophoretic purity of the obtained rhG-CSF stock solution is 100%.

Using the national standard for the determination of recombinant human granulocyte colony-stimulating factor activity as the activity standard, the biological activity of the rhG-CSF stock solution was determined by the NFS-60 cell/MTT colorimetric method, and the specific activity of the rhG-CSF stock solution was calculated according to the sample protein concentration. 1.35×10 ⁸ IU/mg.

Embodiment 2

The method for renaturation and purification of recombinant human granulocyte-stimulating factor inclusion bodies mainly includes the following steps:

Steps 1, 2, and 3 are the same as steps 1, 2, and 3 in the first embodiment.

Step 4 Dilution and renaturation of rhG-CSF

1) Process steps

Prepare 9.6L buffer solution (20mmol/L Tris-HCl, pH8.2), control its temperature at 2 to 8°C, and slowly add the denatured protein solution after replacement into the buffer solution, that is, the final protein concentration is 0.3g/L, and at the same time Add GSSG to a final concentration of 0.3 mmol/L, add GSH to a final concentration of 0.1 mmol/L, stir for 30 minutes, stop stirring, and stand at 2 to 8 °C for renaturation for 16 to 18 hours.

2) Sample detection:

Take 50 μl of the renaturation solution to detect the protein concentration of rhG-CSF in the sample by RP-HPLC (the conditions are the same as the RP-HPLC conditions in step 3), and normalize the area with the peak area of rhG-CSF in the detected sample and reference substance The concentration of rhG-CSF in the renaturation solution was calculated to be 0.20 mg/ml.

Step 5 Column chromatography purification of rhG-CSF 1) Process steps

The process steps are the same as those of step 5 in the first embodiment.

2) Sample detection:

The purity of the rhG-CSF stock solution was analyzed by SDS-PAGE electrophoresis and RP-HPLC. The detection method was the same as the sample detection method in step 5 of Example 1. The activity of rhG-CSF was measured by NFS-60 cell/MTT colorimetric method, and the obtained rhG - The SDS-PAGE electrophoresis purity of the CSF stock solution was 100%, the RP-HPLC purity was 98.61%, and the specific activity was 1.26×10 ⁸ IU/mg.

Comparative Example 1

Refolding and purification of recombinant human granulocyte-stimulating factor was carried out using the methods described in Example 1 and Example 2 to prepare rhG-CSF stock solution, and the method disclosed in the prior art CN101045742A was used for comparison. The comparison results are as follows:

It can be seen that the use of the rhG-CSF renaturation and purification method of the present invention simplifies the operation steps, the technological process is simple, easy to control, the renatured protein concentration is higher than the prior art, the sample processing volume is reduced, and the loss of the target protein is small at the same time, The protein yield and quality are significantly improved, and it is suitable for large-scale industrial production.

Although the preferred embodiments of the present invention have been disclosed above, this is not intended to limit the content of the present invention, and the protection scope of the present invention is subject to the actual scope of the patent application.

<110> JIANGSU HENGRUI MEDICINE CO.,LTD.

<120> A kind of renaturation and purification method of recombinant human granulocyte stimulating factor

<130> 770026CPCT

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 175

<212> PRT

<213> Homo sapiens

<400> 1

Since the graph of this case is experimental data, it is not a representative graph of this case. Therefore, there is no designated representative map in this case.

Claims (15)

A method for preparing recombinant human granulocyte stimulating factor, comprising the following steps: a) fermenting and culturing rhG-CSF genetically engineered bacteria, and separating and washing to obtain refined inclusion bodies; wherein, the rhG-CSF genetically engineered bacteria are made of Escherichia coli transformed with the recombinant plasmid with rhG-CSF gene; the buffer used for inclusion body washing is: buffer A (5 to 100mmol/L Tris-HCl, 2 to 20mmol/L EDTA, 100 to 500mmol/L NaCl , 2 to 4 mol/L Urea, pH 7 to 9), buffer B (5 to 100 mmol/L Tris-HCl, 2 to 20 mmol/L EDTA, pH 8 to 10); the washing method is buffer A→buffer B Wash successively; b) inclusion bodies are dissolved in denaturing liquid to obtain denatured protein liquid; wherein, inclusion body denaturing liquid also contains 5 to 100 mmol/L Tris-HCl (pH 7-9), 6 to 10 mol/L Urea, 2 to 20 mmol/L L EDTA and 2 to 20 mmol/L DTT; c) performing buffer replacement on the denatured protein solution to obtain a denatured solution after replacement; wherein, the buffer used in the buffer replacement comprises 6 to 10 mol/L Urea, 2 to 20 mmol/L EDTA, 5 to 100 mmol/L Tris-HCl, pH 7 to 9; d) dilute and renature the denaturing solution after replacement; wherein, the buffer used in the diluting and renaturing process contains 0.1 to 1 mmol/L GSSG and does not contain GSH ; e) Purify the renatured rhG-CSF to obtain the rhG-CSF stock solution. Recombinant human granulocyte stimulating factor as described in item 1 of the patent application scope The preparation method of , wherein, in step b, the inclusion body denaturation solution contains 20mmol/L Tris-HCl (pH 8.2), 8mol/L Urea, 5mmol/L EDTA, and 10mmol/L DTT. The method for preparing recombinant human granulocyte-stimulating factor as described in item 1 of the patent application scope, wherein, in step c, the mode of buffer replacement is selected from ultrafiltration or desalting column chromatography. The method for preparing recombinant human granulocyte-stimulating factor as described in item 3 of the patent application scope, wherein the buffer replacement method in step c is ultrafiltration. The method for preparing recombinant human granulocyte-stimulating factor according to item 4 of the patent application scope, wherein the ultrafiltration is hollow fiber ultrafiltration. The method for preparing recombinant human granulocyte-stimulating factor as described in item 5 of the patent application scope, wherein the hollow fiber material of the hollow fiber ultrafiltration is selected from polysaccharide, modified polysaccharide, cellulose acetate or nitrocellulose one or more of. The method for preparing recombinant human granulocyte-stimulating factor according to item 6 of the patent application scope, wherein the hollow fiber has a molecular weight cut-off of 3,000 to 10,000. The method for preparing recombinant human granulocyte-stimulating factor according to item 6 of the patent application scope, wherein the hollow fiber has a molecular weight cut-off of 5,000. The method for preparing recombinant human granulocyte-stimulating factor as described in item 1 of the claimed scope, wherein the buffer used for the buffer replacement in step c comprises 8mol/L Urea, 5mmol/L EDTA, 20mmol/L Tris-HCl , pH 8.2. The method for preparing recombinant human granulocyte-stimulating factor as described in item 1 of the claimed scope, wherein the buffer used in the dilution and renaturation process in step d comprises 0.5mmol/L GSSG, 20mmol/L Tris-HCl, pH8.2 . The method for preparing recombinant human granulocyte-stimulating factor as described in item 1 of the patent application scope, wherein in step e, salting out, hydrophobic column chromatography, and desalting column chromatography are sequentially performed on the rhG-CSF renaturing solution. The method for preparing recombinant human granulocyte-stimulating factor as described in item 11 of the patent application scope, wherein the salting out is in the form of ammonium sulfate precipitation, the hydrophobic column packing of the hydrophobic column chromatography is a Phenyl-based hydrophobic medium, The filler of the desalting column chromatography is a low-adsorption Sephadex series chromatography medium. A method for preparing a polyethylene glycol-modified recombinant human granulocyte-stimulating factor, including the method for preparing a recombinant human granulocyte-stimulating factor described in any one of items 1 to 12 of the patent application scope. step, and the step of coupling recombinant human granulocyte stimulating factor with a water-soluble polymer. The method for preparing a polyethylene glycol-modified recombinant human granulocyte-stimulating factor as described in item 13 of the patent application scope, wherein the structure of the polyethylene glycol-modified recombinant human granulocyte-stimulating factor is shown in formula I

, where m is selected from an integer from 50 to 2500, and G is Met-G-CSF. The polyethylene glycol-modified recombinant human as described in item 14 of the patent application scope The preparation method of granulocyte stimulating factor, wherein, m is selected from the integer of 400-500.

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