CN107188952B

CN107188952B - Method for purifying recombinant human granulocyte colony stimulating factor

Info

Publication number: CN107188952B
Application number: CN201710343246.2A
Authority: CN
Inventors: 陈磊; 王宏伟; 马宣宣; 翟紫凝; 朱壮志; 张晨光; 孙金星
Original assignee: Jiangsu Hengrui Medicine Co Ltd
Current assignee: Jiangsu Hengrui Medicine Co Ltd
Priority date: 2016-05-17
Filing date: 2017-05-16
Publication date: 2020-07-28
Anticipated expiration: 2037-05-16
Also published as: CN107188952A

Abstract

The invention provides a method for purifying a recombinant human granulocyte colony stimulating factor. Specifically, the invention selects a cation chromatographic column to purify renaturation solution containing recombinant human granulocyte colony-stimulating factor (rhG-CSF), and then proper buffer solution replacement is carried out to prepare high-purity rhG-CSF stock solution, wherein the filler in the cation chromatographic column is mixed-mode cation. The purification process simplifies the process route, saves the production time, has high protein recovery rate and high purity of the obtained protein stock solution, can be prepared into a pharmaceutical preparation by adding proper pharmaceutical excipients, and is very suitable for large-scale industrial scale-up production. Meanwhile, the invention also provides a method for preparing the PEGylated recombinant human granulocyte colony stimulating factor.

Description

Method for purifying recombinant human granulocyte colony stimulating factor

Technical Field

The invention relates to the field of biological medicine and biological engineering downstream protein purification, in particular to a purification method of recombinant human granulocyte colony stimulating factor (rhG-CSF).

Background

Human granulocyte colony stimulating factor (hG-CSF) is one of the major cytokines that regulate the hematopoietic cells of myeloid lineage, and can specifically act on the granulocytic progenitor cells to promote their proliferation and differentiation into mature neutrophils. It has obvious curative effect on neutrophilic granulocytopenia caused by cancer chemotherapy, acute leukemia chemotherapy, myelodysplastic syndrome, aplastic anemia and other diseases, and neutrophilic granulocytopenia in bone marrow transplantation.

The source of hG-CSF is very limited, and therefore, large-scale industrial production is achieved by genetic engineering methods. At present, pharmaceutical enterprises at home and abroad mostly adopt an escherichia coli system with wide application to express rhG-CSF. Since the reducing environment in the Escherichia coli cells is not favorable for the formation of disulfide bonds, rhG-CSF is folded incorrectly and is easy to aggregate, and basically exists in the cells in the form of inclusion bodies without biological activity. The inclusion bodies are denatured and renatured to obtain active rhG-CSF. At present, the renaturation method of rhG-CSF in the patent is reported in many ways, and the research is extremely clear.

Regardless of the renaturation method, admittedly, the sample after renaturation still contains a large amount of host cell protein and product-related impurities, so that the purification process after renaturation is an important step for determining whether the product quality is qualified or not, and a successful purification process simultaneously considers various factors such as product purity, yield, process period, production cost, operability and the like.

US5849883, CN1167150, CN1206716, CN1814779, etc. all report the use of multi-step chromatography method for purifying rhG-CSF expressed by recombinant Escherichia coli. For example, US5849883, CN1206716, CN1814779 all utilize exchange chromatography with weak cations (e.g., CM Sepharose) and weak anions (e.g., DEAE Sepharose). In the production process, the multiple chromatography steps greatly increase the workload of process verification and cleaning verification, and increase the time for processing samples, and meanwhile, the multiple step processing process also influences the recovery rate of the protein. Therefore, the development of a one-step chromatography method for purifying rhG-CSF has good application prospect.

The rhG-CSF one-step chromatographic purification method uses fillers which mostly relate to strong cation exchange chromatography (such as SP fillers) and weak cation exchange chromatography (such as CM fillers).

CN1718739A reports a method for directly purifying rhG-CSF renaturation solution by using SP Sepharose High Performance (SP HP), and the target protein is eluted by NaCl concentration under the condition of pH5.4. However, the inclusion body renaturation solution contains a certain amount of host protein, lipid and deoxyribonucleic acid (DNA) besides the target protein. The SP HP packing has a small particle size of 30 mu m, is generally used for refining and purification, and is not suitable for renaturation liquid containing more impurities and being complex in condition. And the small particle size causes the pressure of the produced upper chromatographic column to be large, the flow rate to be slow, and the process operation time to be prolonged.

CN1663962A reports a method for purifying rhG-CSF by using SP Sepharose Fast Flow (SP FF), wherein the rhG-CSF is subjected to sample loading under the condition of pH4.0(10 mmol/L acetic acid-sodium acetate) and the target protein is eluted at pH7.0(100 mmol/L disodium hydrogen phosphate/sodium dihydrogen phosphate), but the SP FF has requirements on the ionic strength of a sample and generally does not exceed 5 mS/cm., wherein the sample renaturation solution contains urea with higher concentration, and a series of operations such as concentration, dilution, concentration and the like are carried out by using hollow fibers to obtain a sample meeting the SP FF sample loading requirements.

CN104120109A provides a method for purifying rhG-CSF by weak cation exchange chromatography CM Sepharose Fast Flow (CM FF), the conditions are similar to CN1718739A, the sample is loaded under the condition of pH5.4, and the target protein is eluted by NaCl concentration. However, the loading, equilibration and elution flow rates of the purification method are all 10 cm/h. Such low flow rates tend to greatly increase the process run time and may also affect the stability of the protein sample.

Aiming at the problems, the development of a purification process which can adapt to the characteristics of complex components, more pollutants, high ionic strength of the renaturation liquid and the like of the rhG-CSF renaturation liquid and can ensure that the process flow rate is large and the operation time is short is particularly important. Therefore, the invention provides the rhG-CSF purified by cation chromatographic column chromatography with mixed mode packing, and the method has strong applicability to samples, large carrying capacity, high yield and simpler and more efficient operation.

Disclosure of Invention

The invention provides a method for purifying a recombinant human granulocyte colony stimulating factor, which comprises the following steps: and (3) loading the renaturation solution of the recombinant human granulocyte colony stimulating factor to a cation chromatographic column filled with a mixed mode for purification.

Wherein the filler of the cation exchange chromatography column is mixed-mode cation chromatography filler selected from but not limited to Capto MMC filler,

At least one of HCX filler, Bestarose Diamond MMC filler and UniMSP-30S filler, preferably Capto MMC filler and

HCX filler.

Furthermore, the purification method of the recombinant human granulocyte colony stimulating factor firstly adopts buffer solution to elute impurities, and then elutes and collects sample peaks. The buffer used for eluting the impurities is one or more selected from acetate, citrate, phosphate, carbonate, Tris, glycine and histidine, and the buffer used for eluting the samples is one or more selected from acetate, citrate, phosphate, carbonate, Tris, glycine and histidine.

The ionic strength of the impurity elution buffer solution is 5-20 mmol/L, the pH value is 7.2-7.8, in a specific embodiment, the ionic strength of the impurity elution buffer solution can be 7, 9, 11, 13, 15, 17 and 19 mmol/L, and the pH value can be selected from 7.2, 7.4, 7.6 and 7.8.

The ionic strength of the elution buffer solution is 20-100 mmol/L, and the pH value is 8.0-9.0, wherein in a specific embodiment, the ionic strength of the elution buffer solution can be 40, 55, 65, 80 and 90 mmol/L, and the pH value can be 8.0, 8.2, 8.4, 8.6, 8.8 and 9.0.

Further, the method also comprises a buffer solution balancing step before loading the cation chromatographic column filled with the mixed mode filler, wherein the ionic strength of the balancing buffer solution is 5-50 mmol/L, the pH is 3.0-6.5, and the pH can be selected from 4.0, 4.5, 5.0 and 6.0 in specific embodiments.

Generally, a plurality of modes exist in sample elution, the sample elution mainly adopts a pH elution mode or a salt ion concentration elution mode, and in order to ensure the effect of the sample elution, the pH elution mode is preferably selected, wherein the pH elution mode is a routine elution procedure of the protein-containing stock solution by utilizing different pH values of an eluent. Further, the pH of the impurity elution buffer of the present invention is different from the pH of the elution buffer.

Further, the method of the present invention further comprises the step of subjecting the collected purified sample to buffer replacement, wherein the buffer replacement can be performed by one or more of dialysis, ultrafiltration, and desalting column chromatography, and the ultrafiltration is preferably hollow fiber ultrafiltration or membrane ultrafiltration, and more preferably membrane ultrafiltration. Wherein the replacement buffer solution is sodium acetate buffer solution and is acidic. If the G-CSF stock solution after replacement is used for preparation production, the pH is 2.0-5.0, preferably 3.5-4.5, and the specific pH can be 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3 and 4.4. If the G-CSF stock solution is used for the subsequent modification reaction, a long-acting G-CSF is prepared with a pH of 3.0 to 8.0, preferably 4.0 to 7.0, in specific examples 4.5, 5.0, 5.5, 6.0, 6.5.

Wherein if membrane-packed ultrafiltration is adopted for buffer solution replacement, the ultrafiltration conditions are ① that an ultrafiltration system is utilized to properly concentrate a sample after purifying the composite cationic filler, ② that the volume of the sample is kept constant in the ultrafiltration system and replacement is carried out by a replacement solution, so that the velocity of the fed replacement solution is the same as that of a permeate solution, ③ transmembrane pressure (TMP) is not more than 50PSI, more preferably 10-30PSI, and ④ that ultrafiltration replaces more than 3 times of the volume of the sample, more preferably 5 times of the volume when reasonable process usage is considered.

The pore diameter of the ultrafiltration membrane of the membrane pack is 1 KD-20 KD, preferably 3KD, 5KD or 10KD, more preferably 5 KD.

The membrane material in the membrane ultrafiltration is selected from one or more of polyvinylidene fluoride, modified cellulose, polyether sulfone or modified polyether sulfone.

Further, the method also comprises the steps of pH adjustment and filtration of the renaturation solution before loading.

The protein renaturation solution has a pH value of 2.0-6.0, preferably 3.5-4.5, such as pH 3.6, 3.8, 4.0, 4.2, 4.4 after being subjected to pH adjustment, and the pH adjusting agent comprises one or more of acetic acid, citric acid and phosphoric acid.

The filtration may be one or more of hollow fiber filtration, membrane filtration or dead-end filtration, preferably dead-end filtration. The inner diameter of the hollow fiber tube used in the hollow fiber ultrafiltration includes, but is not limited to, 0.5mm, 0.75mm, 1mm and 1.75mm, more preferably 1 mm; if the hollow fiber column of GE Healthcare company is used for ultrafiltration buffer replacement, the shear rate should be controlled at 16000sec-¹Hereinafter, more preferably about 8000sec^-1。

If the dead-end filtration is adopted to remove the precipitate, the aperture of the filter membrane is 0.22 μm, and the filter membrane material in the dead-end filtration is polyvinylidene fluoride or polytetrafluoroethylene.

Specifically, the invention provides a method for purifying recombinant human granulocyte colony-stimulating factor, which comprises the following steps:

a) before loading, carrying out pH adjustment on the recombinant human granulocyte colony stimulating factor renaturation solution, and filtering;

b) balancing a cation chromatographic column filled with a mixed mode filler by a buffer solution before loading;

c) and (3) loading the sample to a cation chromatographic column filled with the mixed mode filler for chromatographic purification, eluting impurities by adopting a buffer solution, and then eluting to collect a sample peak.

d) Performing buffer solution replacement on the collected purified sample;

wherein, the buffering agent in the buffer solution in b) and c) is selected from one or more of acetate, citrate, phosphate, carbonate, Tris, glycine and histidine, the ionic strength of the equilibrium buffer solution is 5-50 mmol/L, the pH value is 3.0-6.5, the ionic strength of the impurity elution buffer solution is 5-20 mmol/L, the pH value is 7.0-8.0, the ionic strength of the elution buffer solution is 20-100 mmol/L, and the pH value is 8.0-9.0.

The filler of the cation exchange chromatographic column is mixed-mode cation chromatographic filler selected fromBut are not limited to Capto MMC filler,

HCX filler.

Wherein the filtration in the step a) can adopt one or more of hollow fiber filtration, membrane filtration or dead-end filtration, preferably dead-end filtration, the material of the filtration membrane in the dead-end filtration is polyvinylidene fluoride or polytetrafluoroethylene, and the pore diameter is 0.22 μm.

Wherein the pH regulator used in step a) includes but is not limited to one or more of acetic acid, hydrochloric acid and phosphoric acid, acetate, citrate and phosphate.

Wherein the replacement buffer solution is sodium acetate buffer solution and is acidic. If the G-CSF stock solution after replacement is used for preparation production, the pH is 2.0-5.0, preferably 3.5-4.5, and the specific pH can be 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3 and 4.4. If the G-CSF stock solution is used for the subsequent modification reaction, a long-acting G-CSF is prepared with a pH of 3.0 to 8.0, preferably 4.0 to 7.0, in specific examples 4.5, 5.0, 5.5, 6.0, 6.5.

The invention also provides a method for preparing the PEGylated recombinant human granulocyte colony stimulating factor, which comprises the steps of purifying the recombinant human granulocyte colony stimulating factor according to the recombinant human granulocyte colony stimulating factor and performing polyethylene glycol modification on the obtained recombinant human granulocyte colony stimulating factor, wherein the structure of the PEGylated recombinant human granulocyte colony stimulating factor is shown as the formula I

Wherein m is an integer from 50 to 2500, preferably an integer from 400-500, and G is Met-G-CSF.

The invention has the beneficial effects that:

(1) the pH value of the renaturation solution is adjusted to be acidic, so that a large amount of escherichia coli cell protein is precipitated, the step is simple and effective, the separation of the hybrid protein and the target protein is easy to realize, and the pressure is reduced for the subsequent chromatography purification process.

(2) The cation exchange filler of the composite ligand has high loading capacity, high flow rate for processing large-volume samples, strong applicability to complex buffer systems of renaturation liquid, greatly shortened process time, good feasibility of process amplification and successful amplification to production scale.

(3) The purification method provided by the invention only comprises one step of chromatography, so that the process steps are few, the purification period is short, the control is easy, and the stability of the process operation is improved.

(4) The process can obtain high purity and high activity rhG-CSF protein stock solution, and its SDS-PAGE purity can be up to above 99%, RP-HP L C purity can be up to above 98%, and its specific activity is not less than 8.0 × 10⁷IU/mg. These are all higher than the requirements of the original liquid detection under the item of ' recombinant human granulocyte stimulating factor injection ' in the third pharmacopoeia 2015 edition of the people's republic of China.

Interpretation of terms:

drawings

FIG. 1: SDS-PAGE purity detection profile of rhG-CSF stock, wherein lane M: molecular weight standard protein Mark12, lane 1: rhG-CSF stock solution.

Detailed Description

The technical solutions of the present invention are further illustrated below with reference to examples, and it should be understood that the described examples are only for illustrating the present invention and do not limit the scope of the present invention.

EXAMPLE 1 preparation of rhG-CSF renaturation solution

The strain used in the process is Escherichia coli DH5 α strain transformed by pBV220/G-CSF plasmid, after fermentation, the strain is collected, a high-pressure homogenizer is used for cell disruption to obtain crude inclusion bodies, and the crude inclusion bodies are washed to obtain the preliminarily purified inclusion bodies.

Taking 30g of the primarily purified inclusion bodies, performing denaturation and dissolution by using a denaturation liquid (20 mmol/L Tris-HCl,5 mmol/L EDTA,8 mol/L Urea,10 mmol/L DTT, pH8.2) according to the solid-to-liquid ratio of 1:20(m/v), stirring at room temperature for 15-18 hours to obtain 600ml of the denatured protein liquid, performing two-stage filtration and clarification on the protein liquid at 10 mu m and 0.45 mu m, performing buffer solution replacement by using a 5KD hollow fiber column to remove a reducing agent DTT, controlling transmembrane pressure (TMP) to be 10-18PSI in the replacement process, and controlling the replacement buffer solution at 20 mmol/L Tris-HCl,5 mmol/L EDTA,8 mol/L Urea and pH8.2 to obtain 600ml of the replaced denatured protein liquid.

Slowly adding the replaced denatured protein solution into about 15L renaturation buffer solution (20 mmol/L Tris-HCl, pH8.2) to enable the final concentration of the protein to be 0.3 g/L, simultaneously adding GSSG to the final concentration to be 0.5 mmol/L, stirring for 30min, uniformly mixing, and standing at the temperature of 2-25 ℃ for renaturation for 16-18 hours.

Example 2 Capto MMC purification of rhG-CSF

The pH of the renatured rhG-CSF solution was adjusted to 4.0 with 0.5 mol/L hydrochloric acid, left for 10 minutes and filtered using a filter element with a pore size of 0.22. mu.m.

Equilibrating the column with equilibration buffer (20 mmol/L citric acid/disodium phosphate, pH4.0) at a flow rate of 300 cm/hr until the pH stabilizes at 4.0;

loading: loading the filtered renaturation liquid sample at the flow rate of 300 cm/h;

reequilibration, after the sample loading is finished, the chromatographic column is reequilibrated by an equilibration buffer solution (20 mmol/L citric acid/disodium hydrogen phosphate, pH4.0), and the flow rate is 300 cm/h;

impurity washing, namely eluting impurities by using a 10 mmol/L citric acid/disodium hydrogen phosphate buffer solution (pH7.5) at the flow rate of 180 cm/h;

eluting with 50 mmol/L citric acid/disodium hydrogen phosphate buffer solution (pH8.0) at flow rate of 300 cm/hr to collect target protein peak.

EXAMPLE 3 screening of chromatography packing

The procedure for preparing rhG-CSF renaturation solution is as in example 1. In order to obtain chromatographic packing suitable for purifying rhG-CSF renaturation liquid, the research compares the capacity of three kinds of packing with weak cation ligand for purifying renaturation liquid, and the purification process refers to the embodiment2, results are shown in Table 1. sample HP L C obtained from CM FF was less pure than the mixed mode Capto MMC and

HCX, low flow rate and low yield. In contrast, Capto MMC and

HCX can better meet the pharmacopoeia standard of rhG-CSF and is suitable for large-scale industrial amplification.

Table 1: comparing the purification effect of the three fillers on the rhG-CSF renaturation solution

Example 4 Capto MMC impurity and sample elution buffer pH screening

This example investigates the selectable range of impurity elution and sample elution buffer pH for Capto MMC purified rhG-CSF renaturation. The procedure for preparing rhG-CSF renaturation solution is as in example 1. The experimental design and results of this example are shown in Table 2. The pH of the impurity elution buffer is preferably 7.2 to 7.8. When the pH of impurity elution is between 7.0 and 8.0, the problem that impurities cannot be eluted or target protein cannot be eluted at the step occurs, and the purity or yield of the final eluted sample is influenced. Similarly, the sample elution buffer may be selected to have a pH of 8.0 to 9.0. At a pH below 8.0 (Exp. 3), the sample failed to elute.

Table 2: effect of impurities and sample elution buffer pH on purification of rhG-CSF renaturation solutions

EXAMPLE 5 Ultrafiltration Displacement of rhG-CSF buffer

Buffer exchange was performed using a 5KD membrane pack.

Equilibrating the membrane with equilibration buffer (20 mmol/L sodium acetate, pH 4.0);

constant volume displacement, namely adding the elution collected liquid of Capto MMC column chromatography into a membrane package system, continuously flowing a displacement buffer solution (20 mmol/L sodium acetate, pH4.0) into the membrane package, controlling the flow acceleration to be the same as the flow velocity of a permeation end, keeping the volume of a sample constant, controlling the transmembrane pressure to be 18-22PSI during the period, and continuously adding 5 times of the displacement buffer solution (20 mmol/L sodium acetate, pH4.0) in the whole process.

Example 6 sample testing:

the purity of the rhG-CSF stock was analyzed by SDS-PAGE and RP-HP L C.

1)SDS-PAGE

Polyacrylamide gel:

4-12％Bis-Tris Gel,Life technologies；

sample buffer:

LDS Sample Buffer(4×),Life technologi；

electrophoresis buffer solution:

MOPS SDS Running Buffer(20×),Life technologies；

dyeing liquid: GelcodeTMBlue safe protein Stain, Thermo scientific;

decoloring liquid: purified water, self-made.

After the recombinant human granulocyte stimulating factor stock solution is loaded, 150V constant voltage electrophoresis is carried out to ensure that bromophenol blue migrates to the position of the colloidal bottom. Dyeing by adopting a Coomassie brilliant blue rapid dyeing method, decoloring purified water, and analyzing the purity by using a gel imager, wherein the detection result is shown in the attached figure 1 of the specification.

As is clear from the analysis results, the purity of the rhG-CSF stock solution finally obtained was 99.2% by SDS-PAGE.

2)RP-HPLC

Chromatographic column SHISEIDO CAPCE LL PAK C18SG 3005 μm 150mm × 4.6.6 mm;

phase A: trifluoroacetic acid-water solution (1.0 ml trifluoroacetic acid added with water to 1000ml, fully mixing);

phase B: trifluoroacetic acid-acetonitrile solution (1.0 ml of trifluoroacetic acid is added into chromatographic pure acetonitrile to 1000ml, and the mixture is fully and evenly mixed);

table 3: at room temperature, gradient elution was carried out according to the following table, with a detection wavelength of 280nm

Time (min)	A(％)	B(％)
			0	100	0
15	30	70
			25	30	70
26	100	0

And (3) performing purity detection on the rhG-CSF stock solution by adopting an RP-HP L C method, and analyzing a detection result to obtain the final rhG-CSF stock solution with the purity of 99.21%.

3)SEC-HPLC

The chromatographic column is a hydrophilic silica Gel molecular sieve column (TSK-Gel G3000SWX L7.8.8 7.8 × 300mm, 5 mu m, 250A);

mobile phase: 50mM ammonium bicarbonate, pH 7.0;

wavelength: 215 nm; column temperature: 25 ℃; flow rate: 0.5 ml/min;

and (3) performing purity detection on the rhG-CSF stock solution by using an SEC-HP L C method, and analyzing and detecting results to obtain the rhG-CSF stock solution with the purity of 99.32%.

4) Activity assay

Taking the standard substance and rhG-CSF stock solution obtained by said invention, diluting it to 1.0ng/ml by using basic culture medium in 96-hole cell culture plate, then making 2-fold gradient dilution, taking the cultured NFS-60 cell strain, washing it for 3 times by using RPM1640 culture solution, taking cell suspension before third centrifugation for cell counting, after counting, using basic culture solution to make cell be resuspended and preparing it into 2.0 × 10⁵Cells/ml. Mu.l of cell suspension was added to 50. mu.l of standard/rhG-CSF stock solution per well at 37 ℃ with 5% CO₂Culturing under the condition for 40-48 hours. Then, 20. mu.l of MTT solution (5mg/ml), 37 ℃ C., 5% CO was added to each well₂Culturing for about 5 hours under the condition, adding 100 mul of lysis solution into each hole, mixing uniformly, and carrying out color comparison by using an enzyme-labeling instrument, wherein the determination wavelength/reference wavelength is 570nm/630 nm. The specific activity of the obtained rhG-CSF stock solution was calculated.

The NFS-60 cell/MTT colorimetric method is adopted to measure the biological activity of the rhG-CSF stock solution, and the specific activity obtained by calculation is 9.0 × 10⁷IU/mg。

Claims

1. A method for purifying a recombinant human granulocyte colony stimulating factor, comprising: loading the renaturation solution of the recombinant human granulocyte colony stimulating factor to a cation chromatographic column filled with a mixed mode filler for purification, eluting impurities by adopting a buffer solution, eluting a sample, and collecting a sample peak, wherein the mixed mode filler is selected from Capto MMC filler;

HCX filler, Bestarose Diamond MMC filler and UniMSP-30S filler, wherein the ionic strength of the buffer solution for eluting impurities is 5-20 mmol/L, pH is 7.2-7.8, the ionic strength of a buffer solution used for eluting the sample is 20-100 mmol/L, and the pH value is 8.0-9.0.

2. The method for purifying recombinant human granulocyte colony-stimulating factor of claim 1, wherein the mixed mode filler is selected from the group consisting of Capto MMC filler and Capto MMC filler

HCX filler.

3. The method for purifying human granulocyte colony-stimulating factor as claimed in claim 1-2, wherein the cation chromatographic column with mixed mode packing is subjected to a buffer solution equilibration step before loading, the ionic strength of the equilibration buffer solution is 5-50 mmol/L, and the pH is 3.0-6.5.

4. The method of claim 1, wherein the buffer of the buffer for eluting impurities is selected from the group consisting of acetate, citrate, phosphate, carbonate, Tris, glycine, histidine, and one or more of acetate, citrate, phosphate, carbonate, Tris, glycine, histidine.

5. The method of claim 3, wherein the buffer in the equilibration buffer is selected from one or more of acetate, citrate, phosphate, carbonate, Tris, glycine, and histidine.

6. The method of claim 1, wherein the sample is eluted by pH elution or salt ion concentration elution.

7. The method of claim 6, wherein the sample is eluted by pH elution.

8. The method of claim 1-7, further comprising the step of buffer exchange of the collected purified sample.

9. The method of claim 8, wherein the buffer exchange is performed by one or more of dialysis, ultrafiltration, and desalting column chromatography.

10. The method of claim 9, wherein the ultrafiltration is selected from the group consisting of hollow fiber ultrafiltration and membrane ultrafiltration.

11. The method of claim 9, wherein the ultrafiltration is selected from the group consisting of membrane-based ultrafiltration.

12. The method for purifying recombinant human granulocyte colony-stimulating factor as claimed in claim 10, wherein the pore size of the ultrafiltration membrane is 1-20 KD.

13. The method of claim 10, wherein the membrane-packed ultrafiltration membrane has a pore size of 3KD, 5KD, or 10 KD.

14. The method of claim 10, wherein the pore size of the membrane-packed ultrafiltrate is 5 kD.

15. The method for purifying human granulocyte colony stimulating factor as claimed in claim 11, wherein the membrane ultrafiltration is performed by membrane ultrafiltration, wherein the membrane material is selected from one or more of polyvinylidene fluoride, modified cellulose, polyethersulfone and modified polyethersulfone.

16. The method of claim 1-15, further comprising the steps of adjusting the pH of the renaturation solution and filtering before loading.

17. The method for purifying recombinant human granulocyte colony stimulating factor as claimed in claim 16, wherein the renaturation solution is adjusted to pH 2.0-6.0, and the filtration is performed by one or more of hollow fiber filtration, membrane filtration and dead-end filtration.

18. The method of claim 17, wherein the renaturation solution is adjusted to pH 3.5-4.5.

19. The method of claim 17, wherein the filtration is dead-end filtration.

20. The method for purifying recombinant human granulocyte colony-stimulating factor of any one of claims 1-19, comprising:

c) loading the sample to a cation chromatographic column filled with a mixed mode filler for chromatographic purification, eluting impurities by adopting a buffer solution, and then eluting to collect a sample peak;

d) performing buffer solution replacement on the collected purified sample;

wherein, the buffering agent in the buffer solution in b) and c) is selected from one or more of acetate, citrate, phosphate, carbonate, Tris, glycine and histidine, the ionic strength of the equilibrium buffer solution is 5-50 mmol/L, the pH value is 3.0-6.5, the ionic strength of the impurity elution buffer solution is 5-20 mmol/L, the pH value is 7.2-7.8, the ionic strength of the elution buffer solution is 20-100 mmol/L, and the pH value is 8.0-9.0.

21. The method of claim 20, wherein the pH of the renaturation solution of step a) is 2.0-6.0 after adjustment.

22. The method of claim 21, wherein the pH of the renaturation solution of step a) is 3.5-4.5 after adjustment.

23. The method for purifying recombinant human granulocyte colony stimulating factor as claimed in claim 21, wherein the step a) is performed by one or more of hollow fiber filtration, membrane filtration and dead-end filtration, wherein the membrane material of the dead-end filtration is polyvinylidene fluoride or polytetrafluoroethylene, and the pore size is 0.22 μm.

24. The method for purifying recombinant human granulocyte colony-stimulating factor of claim 23, wherein the step a) is performed by dead-end filtration.

25. A method for preparing pegylated recombinant human granulocyte colony stimulating factor, comprising the steps of purifying the recombinant human granulocyte colony stimulating factor as claimed in any one of claims 1-24, and subjecting the obtained recombinant human granulocyte colony stimulating factor to a polyethylene glycol modification.

26. The method of claim 25, wherein the PEGylated recombinant human granulocyte colony stimulating factor has the structure of formula I

Wherein m is an integer of 50 to 2500, and G is Met-G-CSF.

27. The method according to claim 26, wherein m is an integer selected from 400-500.