WO2017177981A1 - 一种重组人粒细胞刺激因子的复性及纯化方法 - Google Patents

一种重组人粒细胞刺激因子的复性及纯化方法 Download PDF

Info

Publication number
WO2017177981A1
WO2017177981A1 PCT/CN2017/080656 CN2017080656W WO2017177981A1 WO 2017177981 A1 WO2017177981 A1 WO 2017177981A1 CN 2017080656 W CN2017080656 W CN 2017080656W WO 2017177981 A1 WO2017177981 A1 WO 2017177981A1
Authority
WO
WIPO (PCT)
Prior art keywords
mmol
recombinant human
stimulating factor
human granulocyte
granulocyte stimulating
Prior art date
Application number
PCT/CN2017/080656
Other languages
English (en)
French (fr)
Inventor
张晨光
王宏伟
徐峰
汪军
齐艳艳
Original Assignee
江苏恒瑞医药股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 江苏恒瑞医药股份有限公司 filed Critical 江苏恒瑞医药股份有限公司
Priority to CN201780004941.4A priority Critical patent/CN108368162B/zh
Publication of WO2017177981A1 publication Critical patent/WO2017177981A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF

Definitions

  • the invention relates to the field of biomedical and bioengineering downstream protein purification, in particular to a method for refolding and purifying a recombinant human granulocyte colony stimulating factor (rhG-CSF), and the renaturation rate of the invention High, easy to operate, suitable for industrial scale-up production.
  • rhG-CSF human granulocyte colony stimulating factor
  • hG-CSF Human granulocyte colony stimulating factor
  • hG-CSF Human granulocyte colony stimulating factor
  • hG-CSF acts on precursor cells of bone marrow granulocytes and macrophages to promote differentiation and proliferation into mature granulocytes; (2) acts on mature neutrophils of bone marrow and promotes It is released from the bone marrow to the peripheral blood; (3) activates the function of mature granulocytes, enhances its ability to migrate, phagocytosis and bactericidal, prolongs its survival time; (4) stimulates the release of bone marrow hematopoietic stem cells to the peripheral blood.
  • hG-CSF has a wide range of clinical uses for the treatment of neutropenia caused by various causes. It is mainly used for prevention and treatment of bone marrow suppression caused by chemotherapy and radiotherapy. It is also used for self-marrow transplantation, some types. Leukemia, leukopenia of AIDS, and aplastic anemia have all shown significant effects.
  • hG-CSF The natural source of hG-CSF is limited, and the yield is very small, which cannot meet the needs of clinical application.
  • polyethylene glycol modified hG-CSF achieves the purpose of long-term increase of peripheral blood neutrophil count, and its large-scale production also requires a large amount of hG-CSF stock solution, so the large-scale production of hG-CSF stock solution is significant. Economic and social significance.
  • Escherichia coli is the most widely used expression system for the expression of foreign proteins.
  • rhG-CSF is expressed in the form of inclusion bodies in the E. coli system.
  • the expression product has no biological activity. To use its biological activity, it must be fully transcercerized. Dissolving, and then restoring its natural conformation through the renaturation process, can obtain high-activity protein samples, and then obtain a high-purity, functional product through the purification step.
  • the use of rhG-CSF to achieve the corresponding biological activity of natural molecules without relying on glycosylation modification is the most convenient and feasible method for selecting this protein in E. coli.
  • the main steps affecting the yield are renaturation, good renaturation effect, and high yield.
  • the renaturation is completely spontaneous and random, so the renaturation rate is slow and the recovery rate is low.
  • the protein renaturation methods used at home and abroad are based on the exclusion of denatured proteins. The specific methods include dialysis, ultrafiltration renaturation, on-column renaturation, and dilution methods.
  • the dialysis method takes a long time and requires multiple replacements of the dialysis solution.
  • the solution inside the dialysis bag is not uniform during the dialysis process.
  • the concentration of the solution denaturant near the dialysis membrane decreases rapidly and the internal decline is slow.
  • Only part of the protein is suitable for complex In the sexual environment, other proteins are easy to aggregate, flocculate, and precipitate, which reduces the recovery rate of renaturation. More importantly, it is limited by the size of the dialysis bag, and it is difficult to enlarge the dialysis method to the scale of production.
  • a G-CSF purification process is disclosed in U.S. Patent No. 5,849,883, the entire disclosure of which is incorporated herein by reference.
  • 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 currently rarely used in industrial production.
  • Chinese patent CN1167150A reports the renaturation of rhG-CSF by means of hollow fiber ultrafiltration. Although it can be used for large-scale production, it has the following disadvantages: 1 Due to the concentration polarization phenomenon, the refolding protein solution is in the hollow fiber column. The internal concentration is not uniform, prone to protein flocculation, sedimentation or even block the hollow fiber column; 2 large-scale production often requires a hollow fiber column with a length of more than 90cm to provide sufficient membrane area, and the length of the hollow fiber column in the development stage is In the range of 30-60 cm, the operation of hollow fiber ultrafiltration cannot be linearly amplified, 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 on a nickel affinity chromatography column, which is cumbersome to operate and difficult to amplify.
  • Chinese patent CN 104120159 A provides a method of renaturation of Sephadex G-25 column chromatography, the column diameter is 5-20cm, the height of the column bed is 60-100cm, and the chromatographic protein concentration is 1.0-2.0mg/ml.
  • the loading volume is less than 20% of the volume of the bed, the loading and elution flow rate is 5-10 cm/h; according to the reported scale, the batch is about 10-20 g, which is difficult to meet the needs of commercial production.
  • the renaturation method of rhG-CSF reported by Chinese patent CN1718739A is: the first dilution doubling for more than 20 hours ⁇ ultrafiltration concentration ⁇ the second dilution doubling for more than 96 hours ⁇ ultrafiltration concentration, ie two dilution renaturation operations And two ultrafiltration treatments, although the method uses a combination of dilution renaturation and ultrafiltration treatment, the renaturation rate is higher, but there are many disadvantages of multiple renaturation steps, long renaturation time and low efficiency, which will inevitably affect the overall income. Rate and increase the difficulty of process amplification.
  • DTT DL-Dithiotheitol
  • Chinese patent CN101045742A provides a refolding purification method of recombinant human granulocyte stimulating factor, which increases Sephadex G-25 separation before renaturation to remove excess reducing agent DTT
  • the renaturation mass yield is higher than 50%, and the purity of the rhG-CSF after purification is higher than 97%.
  • the method can remove the excess reducing agent in the lysate by Sephadex G-25 chromatography.
  • the concentration of the lysate containing Urea is extremely high.
  • Sephadex G-25 is used for chromatography, there is a large back pressure, and there is a disadvantage that the chromatographic flow rate is slow, the treatment time is long, and the industrialization amplification is difficult.
  • the present invention innovatively developed a method of adding GSSG for the refolding of rhG-CSF, and developed a simpler and more efficient renaturation of rhG-CSF. And purification process.
  • the invention provides a preparation method of recombinant human granulocyte stimulating factor, comprising the following steps:
  • the inclusion body is dissolved in a denatured liquid to obtain a denatured protein solution
  • the rhG-CSF genetically engineered strain in the step a is an Escherichia coli transformed with a recombinant plasmid carrying the rhG-CSF gene, such as the recombinant plasmid pBV220/G-CSF transformed Escherichia coli DH5 ⁇ strain.
  • the inclusion body washing buffer may be: buffer A (5-100 mmol/L Tris-HCl, 2-20 mmol/L EDTA, 100-500 mmol/L NaCl, 2-4 mol/L Urea, pH 7-9), buffer B (5-100 mmol/L Tris-HCl, 2-20 mmol/L EDTA, pH 8-10); washing method was buffer A ⁇ buffer B followed by washing.
  • the denaturing dissolution condition of the step b may be: the inclusion body is added with a denaturation solution at a ratio of 1:20 to 1:25 (W/V) (6-10 mol/L Urea, 2-20 mmol/L DTT, 2-20 mmol/L EDTA). In 5-100 mmol/L Tris-HCl, pH 7-9), the mixture was lysed by stirring for 10-18 h.
  • the denatured dissolution condition of the step b is preferably: the inclusion body is added with a denaturation solution (8 mol/L Urea, 10 mmol/L DTT, 5 mmol/L EDTA, 20 mmol/L Tris-HCl) at 1:20 to 1:25 (W/V). In pH 8.2), stir and lyse for 10-18 h.
  • a denaturation solution 8 mol/L Urea, 10 mmol/L DTT, 5 mmol/L EDTA, 20 mmol/L Tris-HCl
  • the buffer for replacement in the step c may comprise 6-10 mol/L Urea, 2-20 mmol/L EDTA, 5-100 mmol/L Tris-HCl, pH 7-9, preferably 8 mol/L Urea, 5 mmol/L EDTA.
  • buffer replacement means include, but are not limited to, ultrafiltration, desalting column chromatography; Ultrafiltration includes, but is not limited to, hollow fiber ultrafiltration and membrane-package ultrafiltration; if ultrafiltration is used for buffer displacement, ultrafiltration membrane pore sizes include, but are not limited to, 3KD, 5KD and 10KD, more preferably 5KD; if GE Healthcare is used
  • the hollow fiber column is subjected to ultrafiltration buffer replacement, and the shear rate can be controlled to be 16,000 sec -1 or less, more preferably about 8000 sec -1 ;
  • the ultrafiltration conditions may be: 1 suitable concentration of the denatured protein solution in the ultrafiltration system; 2 constant volume replacement, constant sample volume in the ultrafiltration system, and flow washing
  • the filtrate speed is the same as the permeate velocity; 3 transmembrane pressure (TMP) is not more than 50 PSI, more preferably 10-18 PSI; 4 total ultrafiltration displacement sample volume is more than 3 times, more preferably 7 when considering a reasonable process.
  • TMP transmembrane pressure
  • the double volume that is, the volume of the co-flowing washing filtrate is more than 3 times, more preferably 7 times, the volume of the replacement starting sample.
  • the renaturation buffer in the step d may comprise GSSG, and the concentration of the GSSG may be 0.1-1 mmol/L.
  • the renaturation buffer may be 0.5 mmol/L GSSG, 20 mmol/L Tris-HCl, pH 8.2, and the temperature of the refolding buffer may be 2-8 ° C before the replacement denaturant is added to the refolding buffer. ;
  • the condition for dilution and renaturation in the step d may be: a final concentration of the refolding liquid protein of 0.1-0.4 g/L, preferably, the final concentration of the refolding liquid protein is 0.3 g/L, and the denatured liquid is slowly added to the renaturation after the replacement.
  • the buffer solution was stirred for 30 minutes, the stirring was stopped, and the renaturation was carried out at 2-8 ° C for 10-18 hours;
  • the rhG-CSF refolding solution can be purified by sequential salting out, hydrophobic column chromatography and desalting column chromatography, wherein the salting out can be precipitated by ammonium sulfate or precipitated by sodium chloride, more preferably ammonium sulfate.
  • the conditions may be: under stirring, slowly add (NH 4 ) 2 SO 4 to a final concentration of 0.9 mol / L, and add EDTA to a final concentration of 5 mmol / L, Stop stirring and let stand for 30 min;
  • Membrane filtration during salting out can be carried out by hollow fiber tangential flow filtration, membrane filtration or dead end filtration, more preferably hollow fiber tangential flow filtration; if hollow fiber tangential flow filtration is used, the optimized conditions are: membrane The pore size is 0.2 ⁇ m, the transmembrane pressure (TMP) is 3-8 PSI, and the shear rate is about 8000 sec -1 ;
  • the column chromatography in the step e is as follows: using the chromatography medium of GE Healthcare, the optimized chromatographic purification strategy is: firstly chromatographic chromatography from phenyl sepharose Phenyl Sepharose FF column to low adsorption dextran Gel Sephadex (such as Sephadex G-25) column chromatography.
  • the recombinant human granulocyte stimulating factor stock solution obtained by the preparation method of the recombinant human granulocyte stimulating factor provided by the invention can be used for preparing the corresponding preparation product, and can also be used for preparing polyethylene glycol modified recombinant human granulocyte stimulating factor, such as The method for preparing a polyethylene glycol-modified recombinant human granulocyte stimulating factor described in WO9611953 and CN101172161A, which comprises the method for preparing a recombinant human granulocyte stimulating factor according to the present invention, for preparing a recombinant human granulocyte stimulating factor, and A step of coupling a recombinant human granulocyte stimulating factor to polyethylene glycol.
  • the structure of the polyethylene glycol-modified recombinant human granulocyte stimulating factor may be as shown in Formula I,
  • n is selected from an integer of from 50 to 2500, preferably from an integer of from 400 to 500, and G is Met-G-CSF.
  • the refolding protein concentration is higher than the general dilution renaturation, which reduces the sample processing volume.
  • the renaturation method according to the present invention optimizes the renaturation condition, greatly reduces the production cost, is easy to control, and improves the stability of the process operation.
  • the purity of the protein is more than 85%, which reduces the pressure of the subsequent purification step.
  • Figure 1 SDS-PAGE electrophoresis purity detection map of the inclusion bodies after washing in Example 1, wherein lane 1: washed inclusion bodies, lane 2: rhG-CSF reference (home made);
  • Figure 2 SDS-PAGE electrophoresis purity detection map of rhG-CSF stock solution in Example 1, wherein lane 1: molecular weight standard protein Mark 12, lane 2: rhG-CSF stock solution.
  • the renaturation and purification method of the recombinant human granulocyte stimulating factor inclusion body mainly includes the following steps:
  • pBV220/G-CSF transformed E. coli DH5 ⁇ strain was inserted into primary seed medium (protein ⁇ 10g/L, yeast powder 5g/L, NaCl 5g/L), cultured at 30°C220rpm for 7 hours;
  • the first-stage seed is connected to the secondary seed culture medium (peptone 10g/L, yeast powder 5g/L, NaCl 5g/L, glucose 5g/L), and cultured at 30° C. and 220 rpm for 17 hours;
  • the secondary seed culture medium peptone 10g/L, yeast powder 5g/L, NaCl 5g/L, glucose 5g/L
  • Secondary seed is connected to fermentation medium (peptone 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 0.3 g/L), 30 ° C culture to pH, double oxygen rebound (pH 7.0, dissolved oxygen ⁇ 30%), start feeding: feed medium 1 (peptone 20%, yeast powder 10%) 30-40g / min Feed medium 2 (glucose 50%) 20-60 g/min; when OD600 ⁇ 30, the temperature was raised to 42 ° C to start the induction, after 4 hours of induction, the temperature was lowered to 15-20 ° C, and the fermentation was finished.
  • fermentation medium peptone 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 0.3 g/L
  • feed medium 1 peptone 20%, yeast
  • the crude inclusion body obtained above was used with buffer A (20 mmol/L Tris-HCl, 5 mmol/L EDTA, 4 mol/L Urea, 0.25 mol/L NaCl, pH 8.2) and buffer B (20 mmol/L Tris-HCl). , 5 mmol/L EDTA, pH 8.2) was washed successively to obtain inclusion bodies of high purity.
  • the inclusion body was subjected to SDS-PAGE electrophoresis purity detection.
  • the conditions and results of electrophoresis detection were as follows:
  • SDS polyacrylamide gel 4-12% Bis-Tris Gel
  • Life Technologies Sample Buffer LDS Sample Buffer (4 ⁇ )
  • Life technologies running buffer MOPS SDS Running Buffer (20 ⁇ )
  • Decolorizing solution purified water, homemade
  • Step 2 degeneration and dissolution of rhrG-CSF inclusion bodies
  • Sample clarification filtration the denatured protein solution of the above step 2 is clarified by two-stage filtration of 10 ⁇ m and 0.45 ⁇ m;
  • Balance hollow fiber select 5KD hollow fiber column, balance the hollow fiber and system with balance solution (20mmol/L Tris-HCl, 5mmol/L EDTA, 8mol/L Urea, pH8.2);
  • the clarified sample was added to the hollow fiber system, the system was operated at a shear rate of 8000 sec -1 , the transmembrane pressure (TMP) was controlled to 10-18 PSI, and concentrated to 300 ml;
  • Constant volume replacement continuous replacement of buffer buffer (20mmol / L Tris-HCl, 5mmol / L EDTA, 8mol / L Urea, pH 8.2) into the hollow fiber system, control flow acceleration and flow rate of the same end, maintain denatured protein
  • the volume of the liquid was constant, and 2100 ml of replacement buffer (20 mmol/L Tris-HCl, 5 mmol/L EDTA, 8 mol/L Urea, pH 8.2) was continuously added to the denatured protein solution during the ultrafiltration, during which the transmembrane pressure (TMP) was controlled.
  • TMP transmembrane pressure
  • the displacement buffer temperature is between 10 and 20 °C.
  • Sample collection Samples were collected from the bottom valve of the hollow fiber system, the hollow fiber system was rinsed with 100 ml of replacement buffer, and the rinse was incorporated into the sample to obtain 400 ml of the denatured protein solution after replacement.
  • RP-HPLC 20 ⁇ l of the denatured protein solution after replacement was used to detect the concentration of rhG-CSF protein in the sample by RP-HPLC.
  • the RP-HPLC conditions were as follows:
  • rhG-CSF reference homemade, protein concentration of 0.85mg / ml
  • Phase A trifluoroacetic acid-water solution (take 1.0 ml of trifluoroacetic acid and add water to 1000 ml, thoroughly mix and deaerate for 20 min)
  • Phase B trifluoroacetic acid-acetonitrile solution (take 1.0 ml of trifluoroacetic acid into chromatographic pure acetonitrile to 1000 ml, ultrasonic degassing for 20 min)
  • the gradient elution was carried out according to the following table, and the detection wavelength was 214 nm.
  • Step 4 Dilute renaturation of rhG-CSF
  • rhG-CSF protein concentration in the sample 50 ⁇ l was used to detect the rhG-CSF protein concentration in the sample by RP-HPLC (conditions are the same as the RP-HPLC conditions in step 3), and the area was normalized according to the peak area of rhG-CSF in the test sample and the reference substance.
  • concentration of rhG-CSF in the reconstituted solution was calculated to be 0.23 mg/ml.
  • the recombinant human granulocyte stimulating factor stock solution is filtered after protein collection and sterilization.
  • the rhG-CSF stock solution was subjected to purity analysis by SDS-PAGE electrophoresis and RP-HPLC method.
  • Phase A trifluoroacetic acid-water solution (take 1.0 ml of trifluoroacetic acid and add water to 1000 ml, thoroughly mix and deaerate for 20 min)
  • Phase B trifluoroacetic acid-acetonitrile solution (take 1.0 ml of trifluoroacetic acid into chromatographic pure acetonitrile to 1000 ml, ultrasonic degassing for 20 min)
  • the gradient elution was carried out according to the following table, and the detection wavelength was 280 nm.
  • the purity of rhG-CSF stock solution was determined by RP-HPLC method. It can be seen from the results analysis that the final RP-HPLC purity of the obtained rhG-CSF stock solution was 99.14%.
  • rhG-CSF stock solution has an electrophoretic purity of 100%.
  • the national standard was used as the active standard for the determination of recombinant human granulocyte colony-stimulating factor activity.
  • the biological activity of rhG-CSF stock solution was determined by NFS-60 cell/MTT colorimetric method, and the specific activity of rhG-CSF stock solution was calculated according to the sample protein concentration. It is 1.35 ⁇ 10 8 IU / mg.
  • the renaturation and purification method of the recombinant human granulocyte stimulating factor inclusion body mainly includes the following steps:
  • Steps 1, 2, and 3 are the same as steps 1, 2, and 3 of the first embodiment.
  • rhG-CSF protein concentration in the sample 50 ⁇ l was used to detect the rhG-CSF protein concentration in the sample by RP-HPLC (conditions are the same as the RP-HPLC conditions in step 3), and the area was normalized according to the peak area of rhG-CSF in the test sample and the reference substance.
  • concentration of rhG-CSF in the reconstituted solution was calculated to be 0.20 mg/ml.
  • the purity of rhG-CSF was analyzed by SDS-PAGE electrophoresis and RP-HPLC.
  • the detection method was the same as that of the sample of step 5 in Example 1.
  • the activity of rhG-CSF was determined by NFS-60 cell/MTT colorimetric method, and the obtained rhG was obtained.
  • the SDS-PAGE of the CSF stock solution had a purity of 100%, an RP-HPLC purity of 98.61%, and a specific activity of 1.26 ⁇ 10 8 IU/mg.
  • the renaturation and purification of recombinant human granulocyte stimulating factor were carried out by the method described in Example 1 and Example 2.
  • the rhG-CSF stock solution was prepared and compared by the method disclosed in the prior art CN101045742A. The comparison results were as follows:
  • the rhG-CSF renaturation and purification method of the invention simplifies the operation steps, the process is simple and easy to control, the refolding protein concentration is higher than the prior art, the sample processing volume is reduced, and the target protein loss is small. The protein yield and quality are obviously improved, which is suitable for large-scale industrial production.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

一种重组人粒细胞刺激因子的复性及纯化方法，通过在复性前以缓冲液置换方式去除还原剂，提高了复性工艺的可控性和复性率，优化了复性缓冲液的组成。上述方法适合大规模工业化放大生产，对设备要求低，操作简单，得到的蛋白纯度高，稳定性好，可降低最终产品在临床中的副作用，提高药品的安全性和有效性。

Description

一种重组人粒细胞刺激因子的复性及纯化方法 技术领域
本发明涉及的是生物医药及生物工程下游蛋白纯化领域，具体的涉及一种重组人粒细胞刺激因子(recombinant human granulocyte colony stimulating factor，rhG-CSF)的复性及纯化方法，本发明复性率高，操作简单，适合于工业化放大生产。
背景技术
人粒细胞集落刺激因子(human granulocyte colony stimulating factor，hG-CSF)属于造血生长因子家族。hG-CSF是由单核巨噬细胞、血管内皮细胞及成纤维细胞合成的蛋白，由174个氨基酸组成，分子量为19KD，其序列是公知的，如序列1所示。hG-CSF的主要作用机理有：(1)特异地作用于骨髓粒细胞和巨噬细胞的前驱细胞，促进其向成熟粒细胞分化、增殖；(2)作用于骨髓成熟中性粒细胞，促进其从骨髓向外周血释放；(3)激活成熟粒细胞的功能，增强其游走、吞噬及杀菌能力，延长其存活时间；(4)刺激骨髓造血干细胞向外周血释放。
hG-CSF在临床上有非常广泛的用途，治疗各种原因引起的中性粒细胞减少症，目前主要用于防治化疗及放疗后引起的骨髓抑制；此外还用于自身骨髓移植、某些类型的白血病、艾滋病的白细胞减少和再生障碍性贫血等，均显示具有显著的疗效。
hG-CSF的天然来源有限，产量甚微，不能满足临床应用的需要。同时聚乙二醇修饰的hG-CSF实现了长效增加外周血中性粒细胞数的目的，其规模化生产同样需要大量的hG-CSF原液，因此hG-CSF原液的大规模生产有重大的经济意义和社会意义。
大肠杆菌是目前最为广泛用来表达外源蛋白的表达系统，然而rhG-CSF在大肠杆菌系统中基本以包涵体形式表达，表达产物没有生物活性，欲利用其生物活性，必须先经变性剂充分溶解，然后经复性过程恢复其天然构象，才能得到高活性的蛋白样品，尔后通过纯化步骤得到高纯度，有功能的产品。利用rhG-CSF不依赖于糖基化修饰即可发挥天然分子相应的生物学活性的特点，选择在大肠杆菌中表达该蛋白是最为简便可行而且产率较高的方法。
工艺研发过程中，影响得率的主要步骤在于复性，复性效果好，得率必然高。复性完全是自发和随机的，因此复性速度慢，回收率低。目前国内外所用的蛋白质复性方法均是依据排除使蛋白变性的环境，具体方法有透析法、超滤复性、柱上复性、稀释法等。
透析法需要时间长，且需要多次更换透析溶液，同时透析袋内部溶液在透析过程中是不均一的，靠近透析膜的溶液变性剂浓度下降快而内部下降慢，只有部分蛋白处于适于复性的环境，其他蛋白很容易聚集、絮凝、沉淀，使复性回收率下降，更主要的是受透析袋规格的限制，透析法难以放大至生产规模。
Amgen公司的美国专利5,849,883在1998年公开了一种G-CSF纯化方法，其中rhG-CSF的复性采用了在搅拌条件下加入硫酸铜，通过金属离子氧化的的方式进行蛋白复性。该方法缺点是有痕量金属离子残留的风险，进而影响药品相关检测及药品安全性，目前在工业化生产中已很少采用。
中国专利CN1167150A报道了使用中空纤维超滤的方式进行rhG-CSF的复性，虽然可以用于大规模生产，但存在以下缺点：①受浓差极化现象影响，复性蛋白溶液在中空纤维柱内浓度并不均一，容易出现蛋白絮集、沉淀甚至堵塞中空纤维柱的情况；②大规模生产中往往需要长度超过90cm的中空纤维柱提供足够的膜面积，研发阶段的中空纤维柱长度都是在30-60cm范围内，因此中空纤维超滤的操作无法线性放大，对于复杂的复性过程难以进行细致的工艺研究来保障工艺的稳定性。
中国专利CN102344931A提供了一种镍亲和层析柱上复性的方法，该方法操作繁琐，难以放大。中国专利CN 104120159 A提供了一种Sephadex G-25柱层析复性的方式，层析柱直径在5-20cm，柱床高度60-100cm，层析上样蛋白浓度1.0-2.0mg/ml，上样体积小于柱床体积的20％，上样和洗脱流速为5-10cm/h；按其报道的规模计算，批量约为10-20g，难以满足商业化生产的需要。
稀释复性同其他方法相比而言具有设备要求简单、操作方便、成本低、容易放大的优点，但是也存在大量错误的折叠和聚合，以至蛋白沉淀，使复性收率大大降低，平均只有20％左右。
中国专利CN1718739A报道的rhG-CSF的复性方法是：第一次稀释复性20小时以上→超滤浓缩→第二次稀释复性96小时以上→超滤浓缩，即进行两次稀释复性操作和两次超滤处理，该方法虽然结合使用了稀释复性和超滤处理，复性率较高，但是存在复性步骤多、复性时间过长、效率低的缺点，这势必影响整体收率并增加工艺放大的难度。
针对上述问题，从包涵体变性溶解及复性的整个过程考虑，在包涵体蛋白变性溶解过程中需加入还原剂对其进行还原，通常使用二硫苏糖醇(DL-Dithiotheitol,DTT)，然而，由于DTT容易被氧化，稳定性较差，而且容易挥发，作用易受环境影响，难以控制，因此在包涵体溶解后增加去除DTT的步骤再稀释复性是稳妥的做法。
中国专利CN101045742A提供了一种重组人粒细胞刺激因子的复性纯化方法，在复性前增加了将裂解液进行Sephadex G-25分离，去除多余的还原剂DTT 的步骤，复性质量收率高于50％，纯化后rhG-CSF的RP-HPLC纯度高于97％，该方法通过Sephadex G-25层析虽然可以达到除去裂解液中过量还原剂的目的，但是含有Urea的裂解液盐浓度极高，进行Sephadex G-25层析时存在反压大，存在层析流速慢，处理时间长，工业化放大难度大的缺点。
同时，在复性过程中需要为蛋白分子内二硫键的正确形成提供适当的氧化还原环境，通常所用的氧化还原剂为GSH/GSSG，其中GSH和GSSG的比例也会影响蛋白的复性效率。通过研究不同GSH和GSSG的添加比例对复性效果的影响，本发明创新性地开发了仅添加GSSG进行rhG-CSF稀释复性的方法，开发了一种rhG-CSF更简单、高效的复性和纯化工艺。
发明内容
本发明提供了一种重组人粒细胞刺激因子的制备方法，包括以下步骤：
a)对rhG-CSF基因工程菌进行发酵培养，经分离和洗涤，获得精制的包涵体；
b)包涵体经变性液溶解得变性蛋白液；
c)对变性蛋白液进行缓冲液置换，得置换后变性液；
d)将置换后变性液进行稀释复性；
e)对复性后的rhG-CSF进行纯化，获得rhG-CSF原液。
其中，所述步骤a中rhG-CSF基因工程菌系由带有rhG-CSF基因的重组质粒转化的大肠杆菌，如重组质粒pBV220/G-CSF转化的大肠杆菌DH5α菌株。
包涵体洗涤缓冲液可以是：缓冲液A(5-100mmol/L Tris-HCl，2-20mmol/L EDTA，100-500mmol/L NaCl，2-4mol/L Urea，pH 7-9)，缓冲液B(5-100mmol/L Tris-HCl，2-20mmol/L EDTA，pH 8-10)；洗涤方式为缓冲液A→缓冲液B依次洗涤。
所述步骤b的变性溶解条件可以是：包涵体按1:20～1:25(W/V)加入变性液(6-10mol/L Urea，2-20mmol/L DTT，2-20mmol/L EDTA，5-100mmol/L Tris-HCl，pH 7-9)中，搅拌裂解10-18h。
所述步骤b的变性溶解条件优选：包涵体按1:20～1:25(W/V)加入变性液(8mol/L Urea，10mmol/L DTT，5mmol/L EDTA，20mmol/L Tris-HCl，pH 8.2)中，搅拌裂解10-18h。
所述步骤c中置换用的缓冲液可包含6-10mol/L Urea，2-20mmol/L EDTA，5-100mmol/L Tris-HCl，pH 7-9，优选8mol/L Urea，5mmol/L EDTA，20mmol/L Tris-HCl，pH 8.2；置换用的缓冲液温度控制为2～30℃，更优选为10～20℃；缓冲液置换的方式包括但不限于超滤、脱盐柱层析；其中超滤包括但不限于中空纤维超滤和膜包超滤；如若采用超滤进行缓冲液置换，超滤膜孔径包括但不限于3KD，5KD和10KD，更优选为5KD；如若采用GE Healthcare公司的中空纤维 柱进行超滤缓冲液置换，剪切速率可以控制在16000sec-1以下，更优选为约8000sec-1；
如若采用中空纤维超滤进行缓冲液置换，超滤条件可以是：①变性蛋白液在超滤系统中适当浓缩；②采用恒体积置换，在超滤系统内保持样品体积恒定，使流加的洗滤液速度与透出液速度相同；③跨膜压力(TMP)不大于50PSI，更优选为10-18PSI；④共超滤置换样品体积的3倍以上，考虑到合理的工艺用时，更优选为7倍体积，即共流加洗滤液体积为置换起始样品体积的3倍以上，更优选为7倍体积。
所述步骤d中的复性缓冲液可包含GSSG，所述GSSG的浓度可以是0.1-1mmol/L。优选地，所述复性缓冲液可以是0.5mmol/L GSSG，20mmol/L Tris-HCl，pH8.2，置换后变性液加入复性缓冲液前控制复性缓冲液温度可为2～8℃；
所述步骤d中稀释复性的条件可以是：复性液蛋白终浓度0.1-0.4g/L，优选地，所述复性液蛋白终浓度0.3g/L，置换后变性液缓慢加入复性缓冲液，继续搅拌30min后停止搅拌，2～8℃静置复性10-18小时；
所述步骤e中对rhG-CSF复性液可采用依次进行盐析、疏水柱层析、脱盐柱层析进行纯化，其中盐析可采用硫酸铵沉淀或氯化钠沉淀，更优选为硫酸铵沉淀；如若采用硫酸铵沉淀的方式，条件可为：搅拌条件下，缓慢加入(NH4)2SO4使其终浓度为0.9mol/L，并补加EDTA使其终浓度为5mmol/L，停止搅拌并静置30min；
盐析过程中的膜过滤可采用中空纤维切向流过滤、膜包过滤或死端过滤，更优选为中空纤维切向流过滤；如若采用中空纤维切向流过滤，优化后的条件为：膜孔径0.2μm，跨膜压(TMP)为3-8PSI，剪切速率约8000sec-1；
所述步骤e中的柱层析如若采用GE Healthcare公司的层析介质，优化后的层析纯化策略是：先由苯基琼脂糖凝胶Phenyl Sepharose FF柱层析再到低吸附的葡聚糖凝胶Sephadex(如Sephadex G-25)柱层析。
使用本发明所提供的重组人粒细胞刺激因子的制备方法得到的重组人粒细胞刺激因子原液可用于制备相应的制剂产品，也可用于制备聚乙二醇修饰的重组人粒细胞刺激因子，如WO9611953以及CN101172161A中描述的聚乙二醇修饰的重组人粒细胞刺激因子，其制备方法可包括本发明所述的重组人粒细胞刺激因子的制备方法制备重组人粒细胞刺激因子的步骤，以及将重组人粒细胞刺激因子与聚乙二醇偶联的步骤。其中，聚乙二醇修饰的重组人粒细胞刺激因子的结构可以是如式Ⅰ所示，
Figure PCTCN2017080656-appb-000001
其中m选自 50-2500的整数，优选自400-500的整数，G为Met-G-CSF。
本发明的有益效果是：
(1)优化的包涵体洗涤工艺，为后续的纯化步骤奠定了基础。
(2)稀释复性前增加去除还原剂的步骤，提高了复性率。
(3)通过确定复性之前变性液中DTT的可接受残留量，在保证复性率不受影响的前提下，显著缩短了工艺操作时间，且有利于蛋白活性的维持。
(4)复性蛋白浓度高于一般的稀释复性，降低了样品处理体积。
(5)复性效率大幅提高至70％以上。
(6)本发明涉及的复性方法，优化了复性条件，大大降低了生产成本，且易于控制，提高了工艺操作的稳定性。
(7)复性后蛋白纯度即达到85％以上，降低了后续纯化步骤的压力。
(8)复性液保持澄清，未出现沉淀现象。
(9)工艺流程中没有离心、透析等难以放大的方法，工艺放大的可行性好，成功放大至生产规模。
(10)整体工艺成本低，纯化周期短。
(11)可获得高纯度，高活性的rhG-CSF蛋白原液，经检测其SDS-PAGE电泳纯度达到99％以上，RP-HPLC纯度达到98％以上，比活性范围在(10.0±4.0)×107IU/mg，高于《中华人民共和国药典2015版》第三部“重组人粒细胞刺激因子注射液”项下原液检定的要求。
附图说明
图1：实施例一中洗涤后包涵体的SDS-PAGE电泳纯度检测图谱，其中泳道1：洗涤后的包涵体，泳道2：rhG-CSF对照品(自制)；
图2：实施例一中rhG‐CSF原液的SDS‐PAGE电泳纯度检测图谱，其中泳道1：分子量标准蛋白Mark 12，泳道2：rhG-CSF原液。
具体实施方式
下面通过具体实施例，对本发明的技术方案进行清晰、完整的描述，所描述的实施例仅仅是本发明的一部分实施例，而不是全部的实施例，基于本发明的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例一
重组人粒细胞刺激因子包涵体的复性及纯化方法，主要包括以下步骤：
步骤1制备高纯度的rhG-CSF包涵体
1)工艺步骤：
pBV220/G-CSF转化的大肠杆菌DH5α菌株接入一级种子培养基(蛋白 胨10g/L，酵母粉5g/L，NaCl 5g/L)，30℃220rpm培养7小时；
一级种子接入二级种子培养基(蛋白胨10g/L，酵母粉5g/L，NaCl 5g/L，葡萄糖5g/L)，30℃220rpm培养17小时；
二级种子接入发酵培养基(蛋白胨10g/L，酵母粉5g/L，NaCl 5g/L，葡萄糖5g/L，KH2PO4 2.7g/L，Na2HPO4 11g/L，MgSO4 0.3g/L)，30℃培养至pH、溶氧双反弹(pH 7.0、溶氧≥30％)后启动补料：补料培养基1(蛋白胨20％，酵母粉10％)30-40g/min，补料培养基2(葡萄糖50％)20-60g/min；当OD600≥30，升温至42℃开始诱导，诱导4小时后，降温至15-20℃，发酵结束。
发酵后，取湿菌体1kg，用5倍体积菌体重量TE缓冲液(20mmol/L Tris-HCl,5mmol/L EDTA,pH8.2)悬浮菌体，混合均匀，泵入高压匀质机进行匀浆破碎。之后，离心分离获得湿的包涵体粗品500g。
将上述得到的粗包涵体用缓冲液A(20mmol/L Tris-HCl,5mmol/L EDTA,4mol/L Urea,0.25mol/L NaCl,pH8.2)和缓冲液B(20mmol/L Tris-HCl,5mmol/L EDTA,pH8.2)依次洗涤，获得高纯度的包涵体。
2)样品检测：
对包涵体进行SDS-PAGE电泳纯度检测，电泳检测的条件和结果如下：
SDS聚丙烯酰胺凝胶：
Figure PCTCN2017080656-appb-000002
4-12％Bis-Tris Gel，Life technologies样品缓冲液：
Figure PCTCN2017080656-appb-000003
LDS Sample Buffer(4×)，Life technologies电泳缓冲液：
Figure PCTCN2017080656-appb-000004
MOPS SDS Running Buffer(20×)，Life technologies
染色液：GelcodeTM Blue safe protein Stain，Thermo scientific
脱色液：纯化水，自制
上样后150V恒压电泳至溴酚蓝迁移胶底处，考马斯亮蓝快速染色法染色，凝胶成像仪进行纯度分析。
从说明书图1泳道1可以看出获得的包涵体SDS-PAGE电泳纯度为72.5％。
步骤2rhG-CSF包涵体的变性溶解
取20g初步纯化的包涵体，以固液比1:20的比例用变性液(20mmol/L Tris-HCl,5mmol/L EDTA,8mol/L Urea,10mmol/L DTT,pH8.2)变性溶解，室温搅拌14～16小时，得变性蛋白液400ml。
步骤3变性蛋白液的缓冲液置换
1)工艺步骤：
样品澄清过滤：上述步骤2的变性蛋白液经10μm和0.45μm两级过滤澄清；
平衡中空纤维：选用5KD中空纤维柱，用平衡液(20mmol/L Tris-HCl,5mmol/L EDTA,8mol/L Urea,pH8.2)平衡中空纤维及系统；
浓缩：将澄清后样品加入中空纤维系统，以8000sec-1的剪切速率运行系统，控制跨膜压(TMP)为10-18PSI，浓缩至300ml；
恒体积置换：向中空纤维系统中连续流加置换缓冲液(20mmol/L Tris-HCl,5mmol/L EDTA,8mol/L Urea,pH 8.2)，控制流加速度与透出端流速相同，保持变性蛋白液体积恒定，超滤过程中共向变性蛋白液中连续补加2100ml置换缓冲液(20mmol/L Tris-HCl,5mmol/L EDTA,8mol/L Urea,pH 8.2)，期间控制跨膜压(TMP)为10-18PSI，置换缓冲液温度在10～20℃。
样品收集：从中空纤维系统底阀处收集样品，用100ml置换缓冲液冲洗中空纤维系统，冲洗液并入样品中，得置换后变性蛋白液400ml。
2)样品检测：
取置换后变性蛋白液20μl用RP-HPLC法检测样品中rhG-CSF蛋白浓度，RP-HPLC条件如下：
rhG-CSF对照品：自制，蛋白浓度为0.85mg/ml
色谱柱：Symmetry ShieldTM RP18，3.5μm，100mm×4.6mm
A相：三氟乙酸－水溶液(取1.0ml三氟乙酸加水至1000ml，充分混匀超声脱气20min)
B相：三氟乙酸－乙腈溶液(取1.0ml三氟乙酸加入色谱纯乙腈至1000ml，超声脱气20min)
室温条件下，按下表进行梯度洗脱，检测波长214nm。
[根据细则26改正31.05.2017]　
Figure WO-DOC-TABLE-1
[根据细则26改正31.05.2017]　
根据检测样品及对照品中rhG-CSF的峰面积，用面积归一化法计算得置换后变性蛋白液中rhG-CSF的浓度为7.5mg/ml。
步骤4 rhG-CSF的稀释复性
1)工艺步骤
配制缓冲液(20mmol/L Tris-HCl,pH8.2)9.6L，控制其温度在2～8℃， 将置换后变性蛋白液缓慢加入缓冲液中，即蛋白终浓度为0.3g/L，同时加入GSSG至终浓度为0.5mmol/L，搅拌30min混匀后停止搅拌，2～8℃静置复性16～18小时。
2)样品检测：
取复性液50μl用RP-HPLC法检测样品中rhG-CSF蛋白浓度(条件同步骤3中的RP-HPLC条件)，根据检测样品及对照品中rhG-CSF的峰面积，用面积归一化法计算得复性液中rhG-CSF的浓度为0.23mg/ml。
步骤5 rhG-CSF的柱层析纯化
1)工艺步骤：
复性后的rhG-CSF溶液中加入硫酸铵至终浓度为0.9mol/L，加入EDTA至终浓度为5mmol/L，搅拌混匀后静置30min，0.2μm中空纤维切向流澄清过滤后进行柱层析纯化。
Phenyl Sepharose FF柱层析：
⑴以含0.9mol/L(NH4)2SO4，pH8.2的20mmol/L Tris-HCl缓冲液平衡层析柱，流速150cm/小时，平衡3倍柱体积；
⑵澄清液样品上样，流速150cm/小时；
⑶以含0.65mol/L(NH4)2SO4，pH8.2的20mmol/L Tris-HCl冲洗层析柱，流速150cm/小时，冲洗5倍柱体积；
⑷用含0.1mol/L(NH4)2SO4，pH8.2的20mmol/L Tris-HCl洗脱，流速150cm/小时，收集目标蛋白峰。
Sephadex G-25柱层析：
⑴以pH 5.0的50mmol/L醋酸盐缓冲液平衡层析柱，流速150cm/小时，平衡2倍柱体积。
⑵Phenyl Sepharose FF柱层析洗脱收集液上样，单次上样体积不超过柱体积的1/4，流速150cm/小时。
⑶pH 5.0的50mmol/L醋酸盐缓冲液洗脱，流速150cm/小时，收集目标蛋白峰。
蛋白收样除菌过滤后即为重组人粒细胞刺激因子原液。
2)样品检测：
rhG-CSF原液用SDS-PAGE电泳、RP-HPLC方法进行纯度分析。
⑴SDS-PAGE法检测rhG-CSF原液纯度的条件同步骤1中的SDS-PAGE电泳分析条件
SDS-PAGE法进行rhG-CSF原液纯度的检测分析结果如图2示，从分析结果可以看出，最终所得rhG-CSF原液的SDS-PAGE电泳纯度为100％。
⑵RP-HPLC法检测rhG-CSF原液纯度的条件和结果如下：
色谱柱：Symmetry ShieldTM RP18，3.5μm，100mm×4.6mm
A相：三氟乙酸－水溶液(取1.0ml三氟乙酸加水至1000ml，充分混匀超声脱气20min)
B相：三氟乙酸－乙腈溶液(取1.0ml三氟乙酸加入色谱纯乙腈至1000ml，超声脱气20min)
室温条件下，按下表进行梯度洗脱，检测波长280nm。
时间(min) A(％) B(％)
0 100 0
15 30 70
25 30 70
26 100 0
RP-HPLC法进行rhG-CSF原液纯度的检测，从结果分析可以看出，最终所得rhG-CSF原液的RP-HPLC纯度为99.14％。
从说明书图2泳道2可以看出获得的rhG-CSF原液电泳纯度为100％。以重组人粒细胞集落刺激因子活性测定国家标准品为活性标准品，用NFS-60细胞/MTT比色法测定rhG-CSF原液的生物学活性，并根据样品蛋白浓度计算rhG-CSF原液比活性为1.35×108IU/mg。
实施例二
重组人粒细胞刺激因子包涵体的复性及纯化方法，主要包括以下步骤：
步骤1、2、3同实施例一的步骤1、2、3。
步骤4rhG-CSF的稀释复性
1)工艺步骤
配制缓冲液(20mmol/L Tris-HCl,pH8.2)9.6L，控制其温度在2～8℃，将置换后变性蛋白液缓慢加入缓冲液中，即蛋白终浓度为0.3g/L，同时加入GSSG至终浓度为0.3mmol/L，加入GSH至终浓度为0.1mmol/L，搅拌30min混匀后停止搅拌，2～8℃静置复性16～18小时。
2)样品检测：
取复性液50μl用RP-HPLC法检测样品中rhG-CSF蛋白浓度(条件同步骤3中的RP-HPLC条件)，根据检测样品及对照品中rhG-CSF的峰面积，用面积归一化法计算得复性液中rhG-CSF的浓度为0.20mg/ml。
步骤5rhG-CSF的柱层析纯化
1)工艺步骤
同实施例一步骤5的工艺步骤。
2)样品检测：
rhG-CSF原液用SDS-PAGE电泳、RP-HPLC方法进行纯度分析，检测方法同实施例一步骤5的样品检测方法，用NFS-60细胞/MTT比色法测定rhG-CSF的活性，所得rhG-CSF原液的SDS-PAGE电泳纯度为100％，RP-HPLC纯度为98.61％，比活性为1.26×108IU/mg。
比较例一
采用实施例一、实施例二所述的方法进行重组人粒细胞刺激因子的复性及纯化，制备rhG-CSF原液，同时采用现有技术CN101045742A公开的方法进行对比，对比结果如下：
Figure PCTCN2017080656-appb-000006
可见，采用本发明所述的rhG-CSF复性和纯化方法，简化了操作步骤，工艺过程简单，易于控制，复性蛋白浓度高于现有技术，降低了样品处理体积，同时目的蛋白损失少，蛋白收率和质量明显提高，适合于大规模工业生产。
虽然本发明已将较佳实施例揭示如上，但是这并非用以限制本发明的内容，本发明的保护范围以申请专利的实际权利要求范围为准。

Claims (16)

  1. 一种重组人粒细胞刺激因子的制备方法，其特征在于包括以下步骤：
    a)对rhG-CSF基因工程菌进行发酵培养，经分离和洗涤，获得精制的包涵体；
    b)包涵体经变性液溶解得变性蛋白液；
    c)对变性蛋白液进行缓冲液置换，得置换后变性液；
    d)将置换后变性液进行稀释复性；
    e)对复性后的rhG-CSF进行纯化，获得rhG-CSF原液。
  2. 根据权利要求1所述的重组人粒细胞刺激因子的制备方法，其特征在于步骤a中包涵体洗涤所使用的缓冲液为：缓冲液A(5-100mmol/L Tris-HCl，2-20mmol/L EDTA，100-500mmol/L NaCl，2-4mol/L Urea，pH 7-9)，缓冲液B(5-100mmol/L Tris-HCl，2-20mmol/L EDTA，pH 8-10)；洗涤方式为缓冲液A→缓冲液B依次洗涤。
  3. 根据权利要求1所述的重组人粒细胞刺激因子的制备方法，其特征在于步骤b中的变性液含有DTT，浓度优选2-20mmol/L。
  4. 根据权利要求3所述的重组人粒细胞刺激因子的制备方法，其特征在于步骤b中包涵体变性液还含有5-100mmol/L Tris-HCl(pH 7-9)，6-10mol/L Urea以及2-20mmol/L EDTA。
  5. 根据权利要求3所述的重组人粒细胞刺激因子的制备方法，其特征在于步骤b中包涵体变性液含有20mmol/L Tris-HCl(pH 8.2)，8mol/L Urea，5mmol/L EDTA，10mmol/L DTT。
  6. 根据权利要求1所述的重组人粒细胞刺激因子的制备方法，其特征在于步骤c中所述缓冲液置换的方式选自超滤或脱盐柱层析，优选超滤，所述超滤优选中空纤维超滤。
  7. 根据权利要求6所述的重组人粒细胞刺激因子的制备方法，其特征在于所述的中空纤维超滤的中空纤维材质选自聚砜、改良型聚砜、醋酸纤维素酯或硝酸纤维素酯中的一种或多种，中空纤维截留分子量优选3000-10000，更优选5000。
  8. 根据权利要求1所述的重组人粒细胞刺激因子的制备方法，其特征在于步骤c中所述缓冲液置换使用的缓冲液包含6-10mol/L Urea，2-20mmol/L EDTA， 5-100mmol/L Tris-HCl，pH 7-9；优选8mol/L Urea，5mmol/L EDTA，20mmol/L Tris-HCl，pH 8.2。
  9. 根据权利要求1所述的重组人粒细胞刺激因子的制备方法，其特征在于步骤d中稀释复性过程使用的缓冲液包含GSSG。
  10. 根据权利要求1所述的重组人粒细胞刺激因子的制备方法，其特征在于步骤d中稀释复性过程使用的缓冲液不包含GSH。
  11. 根据权利要求9所述的重组人粒细胞刺激因子的制备方法，其特征在于所述GSSG浓度为0.1-1mmol/L。
  12. 根据权利要求1所述的重组人粒细胞刺激因子的制备方法，其特征在于步骤d中稀释复性过程使用的缓冲液包含0.5mmol/L GSSG，20mmol/L Tris-HCl，pH8.2。
  13. 根据权利要求1所述的重组人粒细胞刺激因子的制备方法，其特征在于步骤e中对rhG-CSF复性液依次进行盐析、疏水柱层析、脱盐柱层析。
  14. 根据权利要求13所述的重组人粒细胞刺激因子的制备方法，其特征在于所述盐析优选硫酸铵沉淀的方式，所述疏水柱层析的疏水柱填料优选Phenyl-基的疏水介质，所述脱盐柱层析的填料优选低吸附的Sephadex系列层析介质。
  15. 一种聚乙二醇修饰的重组人粒细胞刺激因子的制备方法，其特征在于包括权利要求1-14任一项所述的重组人粒细胞刺激因子的制备方法制备重组人粒细胞刺激因子的步骤，以及将重组人粒细胞刺激因子与水溶性聚合物偶联的步骤。
  16. 根据权利要求15所述的聚乙二醇修饰的重组人粒细胞刺激因子的制备方法，其特征在于所述的聚乙二醇修饰的重组人粒细胞刺激因子的结构如式Ⅰ所示
    Figure PCTCN2017080656-appb-100001
    其中m选自50-2500的整数，优选自400-500的整数，G为Met-G-CSF。
PCT/CN2017/080656 2016-04-15 2017-04-14 一种重组人粒细胞刺激因子的复性及纯化方法 WO2017177981A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201780004941.4A CN108368162B (zh) 2016-04-15 2017-04-14 一种重组人粒细胞刺激因子的复性及纯化方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201610237240 2016-04-15
CN201610237240.2 2016-04-15

Publications (1)

Publication Number Publication Date
WO2017177981A1 true WO2017177981A1 (zh) 2017-10-19

Family

ID=60042312

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/080656 WO2017177981A1 (zh) 2016-04-15 2017-04-14 一种重组人粒细胞刺激因子的复性及纯化方法

Country Status (3)

Country Link
CN (1) CN108368162B (zh)
TW (1) TWI758285B (zh)
WO (1) WO2017177981A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110041423A (zh) * 2018-01-16 2019-07-23 江苏奥赛康药业股份有限公司 一种重组人粒细胞集落刺激因子的复性及纯化方法
CN116874555A (zh) * 2023-09-08 2023-10-13 成都华任康生物科技有限公司 一种置换液及其试剂盒和相关应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114224853B (zh) * 2022-01-04 2022-09-23 山东新时代药业有限公司 聚乙二醇化重组人粒细胞刺激因子注射用冻干制剂

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH02234692A (ja) * 1989-03-08 1990-09-17 Kyowa Hakko Kogyo Co Ltd ヒト顆粒球コロニー刺激因子活性を有する蛋白質の精製法
US6322779B1 (en) * 1987-04-16 2001-11-27 Chiron Corporation Recombinant human CSF-1 dimer and compositions thereof
CN101045742A (zh) * 2006-03-27 2007-10-03 华北制药集团新药研究开发有限责任公司 重组人粒细胞集落刺激因子的复性纯化工艺
CN101220082A (zh) * 2008-01-28 2008-07-16 江苏吴中医药集团有限公司苏州中凯生物制药厂 重组蛋白提取纯化方法
CN103233053A (zh) * 2013-04-03 2013-08-07 北京四环生物制药有限公司 一种重组人粒细胞刺激因子的生产方法
WO2015047062A1 (en) * 2013-09-27 2015-04-02 Uab Profarma Fused proteins of granulocyte colony-stimulating factor with other partners of growth factor, preferably with stem cell factor, and method of preparation thereof

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101079993B1 (ko) * 2006-11-17 2011-11-04 동아제약주식회사 폴리에틸렌글리콜 과립구 콜로니 자극인자 접합체
CN103014100B (zh) * 2012-12-25 2014-10-01 哈药集团生物工程有限公司 一种重组人粒细胞刺激因子的纯化方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6322779B1 (en) * 1987-04-16 2001-11-27 Chiron Corporation Recombinant human CSF-1 dimer and compositions thereof
JPH02234692A (ja) * 1989-03-08 1990-09-17 Kyowa Hakko Kogyo Co Ltd ヒト顆粒球コロニー刺激因子活性を有する蛋白質の精製法
CN101045742A (zh) * 2006-03-27 2007-10-03 华北制药集团新药研究开发有限责任公司 重组人粒细胞集落刺激因子的复性纯化工艺
CN101220082A (zh) * 2008-01-28 2008-07-16 江苏吴中医药集团有限公司苏州中凯生物制药厂 重组蛋白提取纯化方法
CN103233053A (zh) * 2013-04-03 2013-08-07 北京四环生物制药有限公司 一种重组人粒细胞刺激因子的生产方法
WO2015047062A1 (en) * 2013-09-27 2015-04-02 Uab Profarma Fused proteins of granulocyte colony-stimulating factor with other partners of growth factor, preferably with stem cell factor, and method of preparation thereof

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110041423A (zh) * 2018-01-16 2019-07-23 江苏奥赛康药业股份有限公司 一种重组人粒细胞集落刺激因子的复性及纯化方法
CN116874555A (zh) * 2023-09-08 2023-10-13 成都华任康生物科技有限公司 一种置换液及其试剂盒和相关应用
CN116874555B (zh) * 2023-09-08 2023-11-28 成都华任康生物科技有限公司 一种置换液及其试剂盒和相关应用

Also Published As

Publication number Publication date
CN108368162B (zh) 2021-11-12
CN108368162A (zh) 2018-08-03
TW201738264A (zh) 2017-11-01
TWI758285B (zh) 2022-03-21

Similar Documents

Publication Publication Date Title
WO2017177981A1 (zh) 一种重组人粒细胞刺激因子的复性及纯化方法
CN103233053B (zh) 一种重组人粒细胞刺激因子的生产方法
JP2011078421A (ja) 封入体における高い量の正しく折りたたまれた異種タンパク質の生産方法
US20070293660A1 (en) Method for purifying granulocyte-colony stimulating factor
JP6742300B2 (ja) ｒＨｕ−ＧＣＳＦの精製のための新規プロセス
EA022821B1 (ru) Способ получения биологически активного рекомбинантного г-ксф человека
CN102850450B (zh) 聚乙二醇化重组人粒细胞集落刺激因子的纯化方法
WO2024087860A1 (zh) 一种制备高纯度高稳定性蛋白质的方法
JP5908496B2 (ja) トランスジェニックイネの子実からヒト血清アルブミンを抽出する方法
CN107188952B (zh) 一种重组人粒细胞集落刺激因子的纯化方法
EP2341061B1 (en) A novel process for preparing G-CSF (granulocyte colony stimulating factor)
CN107129531A (zh) 一种聚乙二醇化重组人粒细胞刺激因子的纯化方法
DE3876843T2 (de) Verfahren zur reinigung des granulocyt-koloniestimulierungsfaktors.
CN106986928B (zh) 一种聚乙二醇化重组人粒细胞刺激因子的纯化方法
CN1718739A (zh) 重组人粒细胞集落刺激因子的制备方法
CN101921820B (zh) 具有活性重组肿瘤特异性凋亡因子的制备方法及其制品的应用
CN104120159A (zh) 一种重组人粒细胞集落刺激因子的制备方法
CN110041423A (zh) 一种重组人粒细胞集落刺激因子的复性及纯化方法
CN112209999B (zh) 一种快速分离重组表皮生长因子发酵液中色素的方法
EP2978770B1 (en) Refolding of proteins
RU2242516C1 (ru) Способ получения рекомбинантного человеческого интерферона альфа-2b, рекомбинантная плазмида и штамм продуцент для его осуществления
CN118834283A (zh) 一种重组人il-3包涵体纯化复性方法
RU2319502C1 (ru) РАСТВОР ДЛЯ ИНЪЕКЦИЙ, РЕКОМБИНАНТНАЯ ПЛАЗМИДНАЯ ДНК pSX50, КОДИРУЮЩАЯ СИНТЕЗ РЕКОМБИНАНТНОГО ЧЕЛОВЕЧЕСКОГО АЛЬФА-2b ИНТЕРФЕРОНА, ШТАММ Escherichia coli SX50 - ПРОМЫШЛЕННЫЙ ШТАММ-ПРОДУЦЕНТ РЕКОМБИНАНТНОГО ЧЕЛОВЕЧЕСКОГО АЛЬФА-2b ИНТЕРФЕРОНА И СПОСОБ ПРОМЫШЛЕННОГО ПОЛУЧЕНИЯ ИНТЕРФЕРОНА АЛЬФА-2b
RU2487885C2 (ru) Способ крупномасштабного получения, выделения и очистки рекомбинантного гранулоцитарного колониестимулирующего фактора человека
CN111793124A (zh) 一种去除重组白介素-2内毒素的方法

Legal Events

Date Code Title Description
NENP Non-entry into the national phase

Ref country code: DE

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17781957

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 17781957

Country of ref document: EP

Kind code of ref document: A1