CN106986928B

CN106986928B - Purification method of pegylated recombinant human granulocyte stimulating factor

Info

Publication number: CN106986928B
Application number: CN201710242953.2A
Authority: CN
Inventors: 张晨光; 王宏伟; 孙金星; 汪军; 王亚里; 孔祥林; 封海莹
Original assignee: Jiangsu Hengrui Medicine Co Ltd
Current assignee: Jiangsu Hengrui Medicine Co Ltd
Priority date: 2016-04-15
Filing date: 2017-04-14
Publication date: 2020-04-24
Anticipated expiration: 2037-04-14
Also published as: CN106986928A

Abstract

The invention relates to a purification method of a pegylated recombinant human granulocyte stimulating factor. Specifically, the invention optimizes the method for separating and purifying PEG-G-CSF by cation exchange chromatography and preparing PEG-G-CSF stock solution by appropriate buffer solution replacement, and PEG-G-CSF with HPLC and electrophoresis purity of more than 98% can be obtained by one-step column chromatography purification and buffer solution replacement. The purification process of the invention not only simplifies the process route and saves the production time, but also has high protein recovery rate and high purity of the obtained protein stock solution, and can be prepared into a pharmaceutical preparation by adding proper pharmaceutical excipients, thus being very suitable for large-scale industrial scale-up production.

Description

Purification method of pegylated recombinant human granulocyte stimulating factor

Technical Field

The invention relates to the field of purification of high-purity medicinal protein solution, in particular to purification of a PEGylation recombinant human granulocyte colony stimulating factor (PEG-G-CSF) modified product, and belongs to the technical field of medicines.

Background

In recent years, with the rapid advance of biotechnology, more and more protein and polypeptide drugs have been discovered and successfully applied to the treatment of human diseases. Among them, recombinant human granulocyte colony stimulating factor (rhG-CSF) is a very successful gene engineering drug. G-CSF is produced by monocytes and fibroblasts, stimulates granulocyte colony formation and has a stimulating effect on neutrophils, and thus is commonly used for adjuvant therapy of cancer patients receiving radiotherapy or chemotherapy and leukemia patients after bone marrow transplantation, and its sequence is well known and shown in sequence No. 1. In 1991, the rhG-CSF of Amgen company has obtained FDA approval for the treatment of neutropenia caused by chemotherapy, but due to the nature of the protein drug itself, the clinical use of the drug has been greatly limited, such as short in vivo half-life, easy in vivo enzymatic digestion to be rapidly cleared out of the body, easy production of immunogens and antigens, low solubility, etc. In order to solve these problems and improve the pharmacokinetics of protein drugs in the human body, many efforts and attempts have been made by scientists, and the most successful technology at present is polyethylene glycol chemical modification (pegylation). The Pegfilgrastin (trade name neuasta) developed by Amgen company modifies G-CSF by using polyethylene glycol modification technology, prolongs the half-life period in vivo, and improves bioavailability, thereby obtaining good clinical effect, the neuasta produced by Amgen company is approved by FDA and Europe in America and is sold on the market in 2002 and 8 months, and huge economic benefit is brought to the Amgen company.

The purification of PEG-G-CSF requires removal of excess polyethylene glycol (PEG), uncrosslinked G-CSF, and even uncrosslinked or even mostly crosslinked G-CSF from polyethylene glycol modified products, and the separation method adopted is size exclusion chromatography separation combined with ion exchange separation. Although size exclusion chromatography can remove di-and poly-crosslinked G-CSF, the method is difficult to use on an industrial production scale due to the limited loading volume. Therefore, the PEG-G-CSF purification process which is simple in process route, suitable for industrial amplification production and easy to control the product quality is urgently needed to be developed.

CN101172161A discloses a polyethylene glycol modified G-CSF conjugate, which has natural human G-CSF physiological activity, and has longer in vivo circulating half-life and better granulocyte stimulating factor activity than G-CSF.

CN1663962A discloses a process for purifying PEG-G-CSF by using SP Sepharose FF column, the average particle size of the chromatographic medium is 90 μm, the process has the advantages of fast flow rate and easy amplification, but the resolution is lower, the purity of the obtained PEG-G-CSF is low, SP Sepharose HP as the chromatographic medium with high resolution is 34 μm, we compared the effect of the SP Sepharose FF column and the SP Sepharose HP column in the invention for purifying PEG-G-CSF, and the purification effect of the SP Sepharose HP column is obviously better than that of the SP Sepharose FF column.

CN102234310A discloses a method for separating and purifying polyethylene glycol modified protein by a MacroCap SP cation chromatographic column, which is applied to the separation and purification of PEG-G-CSF, wherein the PEG-G-CSF is selected to pass through the column at pH4.0-5.0, the multi-PEG modified G-CSF is eluted by NaCl with lower concentration (5% -15% of 0.5-1.5mol/L NaCl solution), and then the single modified target peak is eluted by 0.5-1.5mol/L NaCl solution with 20% -50%, the purity of the protein purified by one step can only reach 90%, and even if the MacroCap SP column chromatography purification is carried out again, the purity can only reach 95%.

Disclosure of Invention

The invention provides a purification method of a PEGylation recombinant human granulocyte stimulating factor, which comprises the following steps: loading the PEG-G-CSF modified product to a cation exchange chromatography column for chromatographic purification, firstly adopting a buffer solution to elute impurities, then adopting a buffer solution A and a buffer solution B to elute in a linear gradient manner, and collecting a sample peak.

Wherein, the chromatography medium polymer of the cation exchange chromatography column can be Sephadex, agarose Sepharose, Cellulose or polystyrene Source, preferably Sepharose HP.

The buffer solution contains 1-100mmol/L acetic acid-sodium acetate, 30-60mmol/L NaCl, pH3.0-6.0, preferably 20mmol/L acetic acid-sodium acetate, 50mmol/L NaCl, pH 4.0. The buffer solution A comprises 1-100mmol/L acetic acid-sodium acetate and 30-60mmol/L NaCl, the pH value is 3.0-6.0, the buffer solution B comprises 1-100mmol/L acetic acid-sodium acetate and 200-300mmol/L NaCl, the pH value is 3.0-6.0, preferably, the buffer solution A comprises 20mmol/L acetic acid-sodium acetate, 50mmol/L NaCl, the pH value is 4.0, and the buffer solution B comprises 20mmol/L acetic acid-sodium acetate, 250mmol/L NaCl, and the pH value is 4.0.

The linear flow rate of the crude PEG-G-CSF applied to the column may be 10-100cm/h, preferably 40-50 cm/h.

The amount of crude PEG-G-CSF protein loaded onto the column may be 1-10mg protein per 1ml chromatography medium, preferably 2mg protein per 1ml chromatography medium.

The linear flow rate for impurity elution may be 10-100cm/h, preferably 50-80 cm/h; the impurity eluent may be 3 to 5 column volumes, preferably 5 column volumes.

The linear gradient elution condition can adopt a linear gradient from 0% B solution to 100% B solution, and the gradient volume is 10-30 times of column volume, preferably 15 times of column volume; the linear flow rate used for elution may be from 10 to 100cm/h, preferably from 60 to 80cm/h, more preferably 70 cm/h.

Further, the purification method of the present invention may further comprise the step of performing buffer exchange on the PEG-G-CSF modification product before loading.

Further, the purification method of the present invention may further comprise the step of subjecting the collected purified sample to buffer exchange.

The buffer solution replacement method can be one or more of dialysis, desalting column chromatography and ultrafiltration, and the ultrafiltration can be hollow fiber ultrafiltration or membrane ultrafiltration; if the buffer exchange is performed by ultrafiltration, the pore size of the ultrafiltration membrane for 39KD PEG-G-CSF includes, but is not limited to, 3KD-30KD, preferably 5KD or 10KD, more preferably 10 KD; hollow fiber ultrafiltration uses hollow fiber tubes with inner diameters including, but 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 hollow fiber ultrafiltration is used for buffer exchange, the ultrafiltration conditions may be that ① clarified reaction solution is appropriately concentrated in the ultrafiltration system, ② constant volume displacement is used to keep the sample volume constant in the ultrafiltration system, so that the flow rate of the added wash filtrate is the same as the permeate rate, ③ transmembrane pressure (TMP) is not more than 50PSI, more preferably 20-30PSI, ④ total ultrafiltration displaces more than 3 times the sample volume, more preferably 5 times the volume in view of reasonable process, i.e., the total flow rate of the added wash filtrate is more than 3 times the volume of the original sample volume, more preferably about 5 times the volume.

The buffer used for the buffer replacement comprises 1 to 100mmol/L acetic acid-sodium acetate, pH3.0 to 6.0, preferably 20mmol/L acetic acid-sodium acetate, pH 4.0; the temperature of the buffer solution is controlled to be 2-30 ℃, more preferably 2-8 ℃.

In the purification method provided by the invention, the cation exchange chromatography column is balanced by buffer solution before loading, and the balance buffer solution can contain 1-100mmol/L acetic acid-sodium acetate, pH3.0-6.0, preferably 20mmol/L acetic acid-sodium acetate, pH 4.0; the chromatographic column equilibrium liquid can be 3-5 times of column volume, preferably 5 times of column volume; the linear flow rate used for the column equilibration is 10-100cm/h, preferably 60-80 cm/h.

Further, the purification method of the present invention may further comprise the step of subjecting the modified product of PEG-G-CSF to clarification and filtration before buffer exchange of the modified product. The clarifying filtration can be made of cellulose acetate filter membrane or polyvinylidene fluoride (PVDF) filter core (such as Millipore CVHL71TP3), and the filter pore size is preferably 0.45 μm.

The invention also provides a purification method of the PEGylated recombinant human granulocyte stimulating factor, which comprises the following steps:

(1) clarifying and filtering the PEG-G-CSF modified product to obtain clarified reaction liquid;

(2) replacing the clarified reaction solution with a buffer solution to obtain a crude product of PEG-G-CSF;

(3) loading the PEG-G-CSF crude product on a well-balanced chromatographic column;

(4) loading the PEG-G-CSF modified product to a cation exchange chromatography column for chromatographic purification, firstly adopting a buffer solution for impurity elution, then adopting a buffer solution A and a buffer solution B for linear gradient elution, and collecting a sample peak;

(5) performing buffer solution replacement on the chromatography sample collection, sterilizing and filtering to obtain a PEG-G-CSF stock solution,

wherein, the buffer solution in the step (2) and the step (5) is 1-100mmol/L acetic acid-sodium acetate, pH is 3.0-6.0, the buffer solution in the step (4) is 1-100mmol/L acetic acid-sodium acetate, 30-60mmol/L NaCl, pH is 3.0-6.0, the buffer solution A is 1-100mmol/L acetic acid-sodium acetate, 30-60mmol/L NaCl, pH is 3.0-6.0, the buffer solution B is 1-100mmol/L acetic acid-sodium acetate, 200-300mmol/L NaCl, pH is 3.0-6.0, and the gradient volume of the linear gradient elution is 10-30 times of the column volume, preferably 15 times of the column volume.

Furthermore, the polyethylene glycol in the PEGylated recombinant human granulocyte stimulating factor of the present invention can be PEG with a molecular weight of 5-100KD, preferably 20 KD.

Furthermore, the PEGylated recombinant human granulocyte stimulating factor of the invention can have a structure shown as a formula I

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

The beneficial effects of the invention include:

(1) the PEG modified product of the G-CSF protein liquid is subjected to clarification and filtration treatment, so that a subsequent buffer solution displacement device (such as an ultrafiltration membrane) and an ion exchange chromatography medium in a chromatography purification step can be protected, and the service lives of the displacement device and the chromatography medium are prolonged;

(2) buffer solution replacement is carried out before column chromatography purification, the conductivity of a sample is reduced, and the target protein PEG-G-CSF can be combined to an ion exchange chromatography column;

(3) the purity of SDS-PAGE, SEC-HPLC and RP-HPLC detection obtained by one-step column chromatography purification is more than 98 percent, and the specific activity range is (8.6 +/-3.4) multiplied by 10⁷IU/mg protein solution;

(4) the process route is simple, and linear amplification can be realized;

(5) the purification period is short, and the process cost is low;

(6) the process is easy to control, convenient to operate and good in repeatability;

(7) the obtained protein stock solution has reliable safety and good life activity, and can be directly used for preparation production.

Drawings

FIG. 1: SDS-PAGE electrophoretic purity detection profile of PEG-G-CSF bulk, lane 1: sample a (PEG-G-CSF stock obtained in example one), lane 2: sample B (PEG-G-CSF stock obtained in example two), lane 3: sample C (PEG-G-CSF stock obtained in example three), lane 4: and (3) a molecular weight standard protein Mark 12.

Detailed Description

The present invention is further described below with reference to specific examples, which are not intended to limit the scope of the present invention.

The PEG-G-CSF modified products used in the examples were prepared by the methods of examples one to three in CN 101172161A.

The first embodiment is as follows:

step 1, PEG-G-CSF modified product buffer solution replacement

Balancing Sephadex G-25 chromatographic column with 20mmol/L acetic acid-sodium acetate and pH4.0 buffer solution at flow rate of 150cm/h for 2 times of column volume;

loading the PEG-G-CSF modified product clarified by the cellulose acetate filter membrane, wherein the single loading volume does not exceed 1/4 of the column volume, and the flow rate is 150 cm/h;

eluting with 20mmol/L acetic acid-sodium acetate and pH4.0 buffer solution, and collecting sample peak to obtain crude product of PEG-G-CSF.

Step 2, SP Sepharose HP column chromatography purification of PEG-G-CSF crude product

Balancing SP Sepharose HP chromatographic column with 20mmol/L acetic acid-sodium acetate and pH4.0 buffer solution at flow rate of 80cm/h and column volume of 5 times until ultraviolet and electric conductivity are stable;

loading the crude PEG-G-CSF at a flow rate of 50 cm/h;

after the sample loading is finished, washing the chromatographic column by using 20mmol/L acetic acid-sodium acetate and 50mmol/L NaCl buffer solution for 5 times of the column volume, wherein the flow rate is 80 cm/h;

using 20mmol/L acetic acid-sodium acetate, 50mmol/L NaCl and pH4.0 solution as A solution, using 20mmol/L acetic acid-sodium acetate, 250mmol/L NaCl and pH4.0 solution as B solution, adopting a linear gradient from 0% to 100% B solution to elute a sample, wherein the flow rate is 80cm/h, the gradient volume is 15 times of the column volume, and collecting a target protein peak.

Step 3, buffer solution replacement of purified sample

Using 20mmol/L acetic acid-sodium acetate and pH4.0 buffer solution to balance the hollow fiber column and the system;

pumping the SP Sepharose HP chromatography sample into a hollow fiber system storage tank, starting the system, adjusting a reflux valve, and controlling transmembrane pressure (TMP) at 20-30 PSI;

using 20mmol/L acetic acid-sodium acetate and pH4.0 buffer solution as displacement solution, performing constant volume displacement to make the velocity of the fed-in displacement solution identical to that of the permeate solution, keeping the volume of the sample constant in a hollow fiber system storage tank, and finishing the displacement when the amount of the displacement solution is about 5 times of the volume of the sample;

collecting a sample from the bottom valve of the hollow fiber system, and sterilizing and filtering to obtain a PEG-G-CSF stock solution.

Example two:

step 1, PEG-G-CSF modified product buffer solution replacement

Clarifying and filtering the PEG-G-CSF modified product by a 0.45 mu m filter element (CVHL71TP3) to obtain clarified reaction liquid;

20mmol/L acetic acid-sodium acetate, pH4.0 buffer solution balance hollow fiber column UFP-10-E-55 and hollow fiber system FlexStand;

pumping the clarified reaction solution into a FlexStand hollow fiber system storage tank, starting the system, adjusting a reflux valve, and controlling transmembrane pressure (TMP) to be 20-30 PSI;

samples were collected from the bottom valve of the FlexStand hollow fiber system to give crude PEG-G-CSF.

Balancing SP Sepharose HP chromatographic column with 20mmol/L acetic acid-sodium acetate and pH4.0 buffer solution at flow rate of 60cm/h and column volume of 5 times until ultraviolet and electric conductivity are stable;

loading the crude PEG-G-CSF at the flow rate of 40 cm/h;

after the sample loading is finished, washing the chromatographic column by using 20mmol/L acetic acid-sodium acetate and 50mmol/L NaCl buffer solution for 5 times of the column volume, wherein the flow rate is 70 cm/h;

using 20mmol/L acetic acid-sodium acetate, 50mmol/L NaCl and pH4.0 solution as A solution, using 20mmol/L acetic acid-sodium acetate, 250mmol/L NaCl and pH4.0 solution as B solution, eluting the sample by linear gradient from 0% to 100% B solution, the flow rate is 70cm/h, the gradient volume is 15 times of the column volume, and collecting the target protein peak.

Step 3, buffer solution replacement of purified sample

Equilibrating a Pellicon 2 membrane package and Cogent M1 ultrafiltration system with 20mmol/L acetic acid-sodium acetate, pH4.0 buffer;

pumping SP Sepharose HP chromatography sample into a storage tank of an ultrafiltration system, starting the system, adjusting a reflux valve, and controlling transmembrane pressure (TMP) at 20-30 PSI;

collecting a sample from the bottom valve of the ultrafiltration system, and sterilizing and filtering to obtain the PEG-G-CSF stock solution.

Example three:

step 1, PEG-G-CSF modified product buffer solution replacement

The same as the second embodiment: step 1

According to the purification conditions of CN101172161A example III, firstly washing 5 times volume with 0.5M NaOH solution, then washing with purified water to neutrality, then balancing SP Sepharose HP chromatographic column with 20mmol/L acetic acid-sodium acetate and pH4.0 buffer solution at flow rate of 120cm/h, balancing 5 times column volume until ultraviolet and electric conductivity are stable;

loading the crude PEG-G-CSF at the flow rate of 120 cm/h;

after the sample loading is finished, washing the chromatographic column by using 20mmol/L acetic acid-sodium acetate and buffer solution with the pH value of 4.0 for 10 times of the column volume, wherein the flow rate is 120 cm/h;

and (2) taking 20mmol/L acetic acid-sodium acetate and a solution with pH4.0 as solution A, 20mmol/L acetic acid-sodium acetate and 1mol/L NaCl and a solution with pH4.0 as solution B, eluting by adopting a linear gradient from 0% to 50% solution B, sequentially eluting impurities, products and unreacted protein at the flow rate of 120cm/h and the gradient volume of 8 times of the column volume, and collecting the target protein peak.

Step 3, buffer solution replacement of purified sample

The same as the second embodiment: step 3

Example four: purity analysis and in-vitro biological activity determination of PEG-G-CSF (polyethylene glycol-G-CSF) stock solution

Taking the PEG-G-CSF stock solution obtained in the first, second and third examples as a sample A, B, C, and performing purity analysis and activity determination on the sample by SDS-PAGE, RP-HPLC and SEC-HPLC respectively, wherein the detection method and the result are as follows:

1. detection of PEG-G-CSF stock solution purity by SDS-PAGE method

4-12% Bis-Tris gel is adopted, the loading amount is 10 mu g, electrophoresis is carried out at a constant voltage of 150V until bromophenol blue migrates to the position of a gel bottom, staining is carried out by a Coomassie brilliant blue rapid staining method, and a gel imager carries out purity analysis.

2. RP-HPLC method for detecting purity of PEG-G-CSF stock solution

A chromatographic column: symmetry shield RP18, 3.5 μm, 100 mm. times.4.6 mm

Phase A: trifluoroacetic acid-water solution (1.0 ml trifluoroacetic acid added water to 1000ml, fully mixing and ultrasonic degassing for 20min)

Phase B: trifluoroacetic acid-acetonitrile solution (1.0 ml trifluoroacetic acid added with chromatographic pure acetonitrile to 1000ml, ultrasonic degassing for 20min)

The gradient elution was performed at room temperature according to the following table. The loading amount is not less than 10 μ g, and the detection length is 220 nm.

Time (min)	A(％)	B(％)
			0	100	0
15	30	70
			25	30	70
26	100	0
			40	100	0

3. SEC-HPLC method for detecting purity of PEG-G-CSF stock solution

A chromatographic column: phenomenex SEC 2000

Mobile phase: 0.1mol/L dipotassium hydrogenphosphate 0.1mol/L aqueous sodium chloride solution (pH4.0)

The sample loading amount is 20 mul, the detection wavelength is 214nm, the flow rate is 0.5ml/min, and the column temperature is 40 ℃.

4. Biological Activity assay of PEG-G-CSF stock

The recombinant human granulocyte colony stimulating factor activity determination national standard is used as an activity standard, the NFS-60 cell/MTT colorimetric method is used for determining the biological activity of the PEG-G-CSF stock solution, and the specific activity of the stock solution is calculated according to the protein concentration of a sample.

5. Measurement results

The method described in example one, example two, and example three was used to isolate and purify PEG-G-CSF to prepare PEG-G-CSF stock solutions (each sample A, B, C), and purity and specific activity were measured, and the results are shown in the following table.

Therefore, the purity of the protein obtained by adopting the separation and purification method of the polyethylene glycol recombinant human granulocyte stimulating factor can reach more than 98 percent, the protein can be directly used for preparation production, the purification period is short, and the process cost is low; the product purified according to the method of CN101172161A has low purity, cannot meet the requirements of preparation production, can be purified repeatedly to obtain a product with qualified purity, has long purification period and high process cost, and is not beneficial to industrial amplification.

Although the preferred embodiments of the present invention have been disclosed, it should be understood that they have been presented by way of example only, and not limitation.

Sequence listing

<110> Hengrui pharmaceutical Co., Ltd of Jiangsu

<120> purification method of pegylated recombinant human granulocyte stimulating factor

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Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala

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Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala

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Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala

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Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser

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Claims

1. A method for purifying a PEGylated recombinant human granulocyte stimulating factor comprises the following steps:

loading the PEG-G-CSF modified product to a Sepharose HP cation exchange chromatography column for chromatographic purification, firstly adopting a buffer solution to elute impurities, then adopting a buffer solution A and a buffer solution B to elute in a linear gradient manner, and collecting sample peaks, wherein:

the buffer solution for eluting impurities contains 1-100mmol/L acetic acid-sodium acetate, 30-60mmol/L NaCl, and the pH value is 3.0-6.0;

the buffer solution A comprises 1-100mmol/L acetic acid-sodium acetate and 30-60mmol/L NaCl, the pH value is 3.0-6.0, the buffer solution B comprises 1-100mmol/L acetic acid-sodium acetate and 200-300mmol/L NaCl, the pH value is 3.0-6.0;

the structure of the PEG-G-CSF modified product is shown as a formula I,

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

2. The method of claim 1, wherein the buffer for eluting impurities comprises 20mmol/L acetic acid-sodium acetate, 50mmol/L NaCl, pH 4.0.

3. The method for purifying PEGylated recombinant human granulocyte stimulating factor as claimed in claim 1, wherein the buffer solution A comprises 20mmol/L acetic acid-sodium acetate, 50mmol/L NaCl, pH4.0, and the buffer solution B comprises 20mmol/L acetic acid-sodium acetate, 250mmol/L NaCl, pH 4.0.

4. The method for purifying PEGylated recombinant human granulocyte stimulating factor of claim 1, wherein the gradient volume of the linear gradient elution is 10-30 column volumes.

5. The method of claim 4, wherein the linear gradient elution is performed in a gradient volume of 15 column volumes.

6. The method of claim 1, wherein the gradient volume of the impurity elution is 3 to 5 column volumes.

7. The method of claim 6, wherein the gradient volume of the impurity elution is 5 column volumes.

8. The method of claim 1, further comprising the step of buffer exchange of the modified PEG-G-CSF product before loading.

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

10. The method for purifying PEGylated recombinant human granulocyte stimulating factor as claimed in claim 8 or 9, wherein the buffer solution is replaced by one or more methods selected from dialysis, desalting column chromatography, and ultrafiltration.

11. The method of claim 10, wherein the ultrafiltration is selected from hollow fiber ultrafiltration or membrane ultrafiltration.

12. The method for purifying PEGylated recombinant human granulocyte stimulating factor as claimed in claim 11, wherein the hollow fiber ultrafiltration membrane has a pore size of 3kD to 30 kD.

13. The method for purifying PEGylated recombinant human granulocyte stimulating factor of claim 12, wherein the hollow fiber ultrafiltration membrane has a pore size of 5kD or 10 kD.

14. The method for purifying PEGylated recombinant human granulocyte stimulating factor of claim 12, wherein the hollow fiber ultrafiltration membrane has a pore size of 10 kD.

15. The method for purifying PEGylated recombinant human granulocyte stimulating factor as claimed in claim 8 or 9, wherein the buffer used in the buffer exchange comprises 1-100mmol/L acetic acid-sodium acetate, pH 3.0-6.0.

16. The method for purifying PEGylated recombinant human granulocyte stimulating factor of claim 15, wherein the buffer used in the buffer exchange step comprises 20mmol/L acetic acid-sodium acetate, pH 4.0.

17. The method for purifying PEGylated recombinant human granulocyte stimulating factor as claimed in claim 1, wherein the cation exchange chromatography column is equilibrated with a buffer solution before loading, the equilibration buffer solution is 1-100mmol/L acetic acid-sodium acetate, and the pH value is 3.0-6.0.

18. The method for purifying PEGylated recombinant human granulocyte stimulating factor of claim 17, wherein the equilibration buffer is 20mmol/L acetic acid-sodium acetate, pH 4.0.

19. The method of claim 8, further comprising the step of clarifying and filtering the modified PEG-G-CSF before buffer exchange of the modified PEG-G-CSF.

20. The method of claim 19, wherein the clarification filtration is performed with a cellulose acetate filter or a polyvinylidene fluoride filter.

21. The method of claim 20, wherein the pore size of the clarifying filter is 0.45 μm.

22. A method for purifying a PEGylated recombinant human granulocyte stimulating factor comprises the following steps:

1) clarifying and filtering the PEG-G-CSF modified product to obtain clarified reaction liquid;

2) replacing the clarified reaction solution with a buffer solution to obtain a crude product of PEG-G-CSF;

3) loading the PEG-G-CSF crude product on a well-balanced chromatographic column;

4) loading the PEG-G-CSF modified product to a cation exchange chromatography column for chromatographic purification, firstly adopting a buffer solution for impurity elution, then adopting a buffer solution A and a buffer solution B for linear gradient elution, and collecting a sample peak;

5) performing buffer solution replacement on the chromatography sample collection, sterilizing and filtering to obtain a PEG-G-CSF stock solution,

wherein, the buffer solution in the steps 2) and 5) is 1-100mmol/L acetic acid-sodium acetate, the pH value is 3.0-6.0, the buffer solution for impurity elution in the step 4) is 1-100mmol/L acetic acid-sodium acetate, 30-60mmol/L NaCl, the pH value is 3.0-6.0, the solution A of the buffer solution is 1-100mmol/L acetic acid-sodium acetate, 30-60mmol/L NaCl, the pH value is 3.0-6.0, the solution B of the buffer solution is 1-100mmol/L acetic acid-sodium acetate, 200-300mmol/LNaCl, the pH value is 3.0-6.0, the gradient volume for linear gradient elution is 10-30 times of column volume, wherein the structure of the PEG-G-CSF modified product is shown as the formula I,

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

23. The method of claim 22, wherein the linear gradient elution is performed in a gradient volume of 15 column volumes.

24. The method for purifying PEGylated recombinant human granulocyte stimulating factor of any one of claims 1-23, wherein the molecular weight of PEG in the modified product of PEG-G-CSF is 5-100 kD.

25. The method for purifying PEGylated recombinant human granulocyte stimulating factor of any one of claims 24, wherein the molecular weight of PEG in the modified PEG-G-CSF product is 20 kD.

26. The method for purifying PEGylated recombinant human granulocyte stimulating factor as claimed in any one of claims 1-23, wherein m is an integer selected from the group consisting of 400-500.