CN114853872A - Preparation method of polyethylene glycol modified rhG-CSF - Google Patents

Preparation method of polyethylene glycol modified rhG-CSF Download PDF

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CN114853872A
CN114853872A CN202210466641.0A CN202210466641A CN114853872A CN 114853872 A CN114853872 A CN 114853872A CN 202210466641 A CN202210466641 A CN 202210466641A CN 114853872 A CN114853872 A CN 114853872A
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csf
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CN114853872B (en
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刘忠
王磊
刘文财
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Shandong New Time Pharmaceutical Co Ltd
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    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52Cytokines; Lymphokines; Interferons
    • C07K14/53Colony-stimulating factor [CSF]
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Abstract

The invention belongs to the field of biomedicine, and particularly relates to a preparation method of polyethylene glycol modified rhG-CSF. The method specifically comprises the following steps: the rhG-CSF protein is loaded and adsorbed on a chromatography filler by taking a composite chromatography medium as a solid-phase reaction carrier, a modification reaction is completed by taking a polyethylene glycol modifier as a mobile phase under the catalysis of a catalyst, and after the reaction is completed, the polyethylene glycol modified protein mPEG-rhG-CSF is obtained by adopting gradient elution. By adopting the preparation method, the mPEG-rhG-CSF modified protein with higher single modification rate and purity can be obtained, the single modification rate of the obtained mPEG modified protein reaches more than 91 percent, and the purity reaches more than 99.3 percent; meanwhile, the preparation method simplifies modification and purification reaction operations, greatly reduces production cost, provides a new process for realizing mPEG modification of protein, and has good application prospect.

Description

Preparation method of polyethylene glycol modified rhG-CSF
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to a preparation method of polyethylene glycol modified rhG-CSF.
Background
At the end of the 50 s of the 20 th century, the study of the structure and function of protein molecules by chemical modification has become a hot spot in the fields of biochemistry and molecular biology. Protein modifications are intended for use in biomedical and biotechnological applications. In biomedical applications, chemical modifications can reduce immunogenicity or immunoreactivity, inhibit immunoglobulin E production, and the like; in the field of biotechnology, enzymes can efficiently play a catalytic role in organic solvents after being chemically modified, and show novel catalytic performance.
The nature of the reaction between protein modifiers and proteins can be largely classified into four types: (1) acylation and related reactions, and the modification agent can perform acylation reaction with side chain groups of protein; (2) the modifier is characterized by containing active halogen atoms, and because of the electronegativity of the halogen atoms, alkyl groups have partial positive charges, so that nucleophilic groups of protein molecules are easily alkylated; (3) oxidation and reduction reactions, wherein the modifier has oxidizing property and can oxidize side chain groups; (4) aromatic ring substitution reactions, iodination and nitration reactions in which the phenolic hydroxyl groups of protein amino acid residues are readily electrophilically substituted at the 3 and 5 positions. Representative modifying agents include N-ethylmaleimide, carbodiimide, carboxymethylcellulose, polysialic acid, dextran, cyclodextrin, polyethylene glycol (PEG), and the like.
Among them, polyethylene glycol (PEG) is a linear, uncharged polymer that can curl freely in solution, and has no toxicity, weak antigenicity and good biocompatibility. The covalent modification of protein can increase the in vivo circulation half-life of protein, reduce its antigenicity, increase the solubility of protein and change the biological distribution of protein in human body. Since the modification of proteins with PEG was first reported by Abuchowski, Davis (J.biol.chem.1977,252: 3578-.
At present, a plurality of PEG modified medicines are applied to clinic at home and abroad, and remarkable social and economic benefits are obtained. Pfizer PEG-modified human growth hormone receptor antagonists for the treatment of acromegaly and hyperproliferative disorders; PEG modified alpha interferon of Hoffman-LaRoche company is used for treating hepatitis C; PEG-modified vascular endothelial (cell) growth factor aptamer for treating senile macular degeneration.
The recombinant human granulocyte colony-stimulating factor (rhG-CSF) can remarkably improve the severity and duration of neutropenia caused by chemotherapy and is widely used for treating bone marrow suppression caused by cancer chemotherapy since 1991, but the plasma half-life of the recombinant human granulocyte colony-stimulating factor is only 3-4 hours, the recombinant human granulocyte colony-stimulating factor needs to be taken every day for 7-14 days, and the treatment convenience and the patient compliance are not good. The PEG modification of the rhG-CSF can better realize the prolongation of the half-life period, the increase of the stability and the reduction of the immunogenicity.
The PEG modification of rhG-CSF can be divided into N-terminal modification, C-terminal modification and on-chain modification, but the difficulty of C-terminal modification is large; the mature rhG-CSF protein has four Lys residues and also has a sulfydryl of free cysteine at 18 positions, and cannot be singly modified at fixed points; thus, more research has focused on specific site-directed modification of the N-terminus of PEG proteins. The main strategy is realized by improving the selectivity of PEG active group to N-terminal alpha amino.
Initially, site-directed modification was achieved by controlling the modification reaction pH. However, this modification is not optimal for rhG-CSF because, in addition to the N-terminal α -amino group, the amino groups of the remaining four Lys residues in the rhG-CSF structure may also be bound to activated PEG and may also be attached to the hydroxyl group of a serine, tyrosine or threonine residue via an ester bond. Resulting in non-uniformity of modification and various problems such as isomers, which are not easy to be solved, and are not beneficial to the subsequent purification and clinical application of rhG-CSF. Specific site-directed modification can also be achieved by certain enzymes, such as subtilases, glutaminases, etc., but this will undoubtedly increase the cost and difficulty of downstream processing, and is more disadvantageous for scale-up production.
CN200680021159 discloses a new isomer of human granulocyte colony stimulating factor obtained by substituting or adding cysteine at specific site, adding amino acid to two ends of human granulocyte colony stimulating factor polypeptide chain and simultaneously pegylating at the addition site. The process is complicated and is inevitably accompanied by many problems associated with unnatural amino acids.
CN201410272925 discloses a preparation method of a pegylated recombinant human granulocyte stimulating factor, which is also a PEG-rhG-CSF classical modification method which is marketed at home and abroad at present. The new ruibai of zilu pharmacy, jin you li of Shi Yao group and Shenlida of Lunan pharmacy all adopt similar liquid phase modification methods. However, the method has low single modification rate, more unmodified and multiple modified products, difficulty for subsequent purification, more required modifying agents and increased production cost.
CN202011019986 discloses a preparation method of fixed-point pegylation modification of protein, which realizes the specific coupling of PEG and N-terminal alpha-amino by closing lysine-epsilon amino on rhG-CSF molecule through ion exchange chromatography, and simultaneously realizes the purification of PEG-rhG-CSF, and the method realizes higher modification rate by adding vitamin B6 and coenzyme during modification. However, this method has disadvantages in that it increases the cost of modification, increases process-related impurities and the pressure for subsequent purification, is not advantageous in terms of cost after commercial scale-up, and has a possibility that the thiol group of cysteine at position 18 may be modified.
Therefore, the modification method which has good universality and simple and convenient process is provided, is used for PEG modification of the rhG-CSF, improves the component uniformity of the rhG-CSF, and has important significance in the technical field of medicines.
Disclosure of Invention
The invention aims to make up the defects of the existing PEG modification process of rhG-CSF and provides a preparation method of rhG-CSF modified by polyethylene glycol.
The purpose of the invention is realized by the following technical scheme: the rhG-CSF protein is loaded and adsorbed on a chromatography filler by taking a composite chromatography medium as a solid-phase reaction carrier, a modification reaction is completed by taking a polyethylene glycol modifier as a mobile phase under the catalysis of a catalyst, and after the reaction is completed, the polyethylene glycol modified protein mPEG-rhG-CSF is obtained by adopting gradient elution.
A preparation method of polyethylene glycol modified rhG-CSF comprises the following steps:
a. column mounting: dissolving rhG-CSF protein in buffer solution A to obtain rhG-CSF protein solution, then loading and adsorbing the rhG-CSF protein solution on chromatographic packing;
b. modification reaction: dissolving a polyethylene glycol modifier in the buffer solution A, adding a catalyst to obtain a modified buffer solution serving as a mobile phase, and circulating the modified buffer solution through a chromatographic column;
c. and (3) elution: and eluting with a buffer solution B and the buffer solution A to obtain the single modified target protein mPEG-rhG-CSF.
Preferably, the chromatography packing of step a is selected from, but not limited to, Capto MMC packing, HCX packing and Bestarose Diamond MMC packing, preferably Capto MMC packing.
In a preferred embodiment, the rhG-CSF protein solution of step a has a concentration of 2-3 mg/mL.
In a preferred embodiment, the polyethylene glycol modifier in step b is selected from mPEG-butyraldehyde, mPEG-propionaldehyde, mPEG-succinate and the like, and has a molecular weight of 10-50 kDa.
In a preferred embodiment, the concentration of the polyethylene glycol modifier in the modification buffer solution in the step b is 1-5 mg/mL, preferably 2-3 mg/mL.
Preferably, the mass ratio of the rhG-CSF protein to the polyethylene glycol modifier is 1: 2-10; further preferably, the mass ratio of the rhG-CSF protein to the polyethylene glycol modifier is 1: 3-6.
In a preferred embodiment, the buffer a in steps a, b and c is a sodium acetate buffer containing sodium chloride.
Preferably, the concentration of sodium chloride in the buffer solution A is 0.1-0.8M; further preferably, the concentration of sodium chloride in the buffer solution A is 0.2-0.5M.
Preferably, the concentration of sodium acetate in the buffer solution A is 10-50 mM; further preferably, the concentration of sodium acetate in the buffer solution A is 20-30 mM.
Preferably, the pH value of the buffer solution A is 4.0-5.0.
In a preferred embodiment, the catalyst in step b is sodium cyanoborohydride, and the final concentration is 10-30 mM.
In a preferred embodiment, the cyclic reaction time in step b is 4 to 8 hours.
In a preferred embodiment, the modification reaction of step b is carried out at room temperature and the column is protected from light.
In a preferred embodiment, the buffer B in step c is a sodium acetate buffer containing sodium chloride.
Preferably, the concentration of sodium chloride in the buffer solution B is 10-50 mM; further preferably, the concentration of sodium chloride in the buffer solution B is 20-30 mM.
Preferably, the concentration of sodium acetate in the buffer B is 10-50 mM; further preferably, the concentration of sodium acetate in the buffer B is 20-30 mM.
Preferably, the pH value of the buffer B is 5.5-6.0.
In a preferred embodiment, gradient elution is carried out by different mixture ratios of buffer B and buffer A to obtain the polyethylene glycol modified rhG-CSF.
Preferably, the gradient elution method is that the flow rate is 5mL/min, firstly 10% -30% of buffer solution B and 3CV are used for gradient elution of the multi-modified rhG-CSF, then 30% -80% of buffer solution B and 10CV are used for gradient elution of the single-modified rhG-CSF, and the single-modified target protein mPEG-rhG-CSF is obtained.
In a preferred embodiment, a method for preparing polyethylene glycol modified rhG-CSF comprises the following steps:
a. loading: loading a proper amount of chromatographic filler (one of Capto MMC filler, HCX filler or Bestarose Diamond MMC filler) into a chromatographic column, and balancing the chromatographic column by using a buffer solution A (a sodium acetate buffer solution containing sodium chloride, wherein the sodium acetate is 10-50 mM, the sodium chloride is 0.1-0.8M, and the pH is adjusted to be 4.0-5.0); and preparing rhG-CSF into a solution with the concentration of 2-3mg/mL by using the buffer solution A, loading the solution onto a chromatographic column, and washing the chromatographic column by using the buffer solution A until no protein passes out.
b. Modification reaction: according to the molar ratio rhG-CSF: weighing the modifying agent according to the proportion of 1: 2-10, preparing a 1-5 mg/mL solution by using the buffer solution A (sodium acetate buffer solution containing sodium chloride, wherein the sodium acetate is 10-50 mM, the sodium chloride is 0.1-0.8M, and the pH is adjusted to 4.0-5.0), adding 10-30 mM of sodium cyanoborohydride to adjust the pH to 4.0-5.0 to obtain the modifying buffer solution, taking the modifying buffer solution as a mobile phase, circulating the modifying buffer solution to a chromatographic column at the flow rate of 2-3 mL/min, controlling the circulating reaction time to be 4-8 h, maintaining the column temperature at room temperature, and carrying out light-shielding treatment on the chromatographic column; at the end of the cycle, the column was washed again with buffer A as described above and 5 column volumes were washed at a flow rate of 10 mL/min.
c. And (3) elution: gradient elution is carried out by using a buffer solution B (sodium acetate buffer solution containing sodium chloride, wherein the pH value of the sodium acetate buffer solution is 10-50 mM, and the pH value of the sodium chloride buffer solution is adjusted to 5.5-6.0) and the buffer solution A (sodium acetate buffer solution containing sodium chloride, wherein the pH value of the sodium acetate buffer solution is 10-50 mM, the sodium chloride is 0.1-0.8M, and the pH value of the sodium acetate buffer solution is adjusted to 4.0-5.0) in different proportions, and the method comprises the following steps: and (3) eluting the multi-modified rhG-CSF in a gradient manner by using 10-30% of buffer solution B and 3CV at the flow rate of 5mL/min, and eluting the single-modified rhG-CSF in a gradient manner by using 30-80% of buffer solution B and 10CV to obtain the single-modified target protein mPEG-rhG-CSF.
The solution used for pH adjustment in the invention adopts acid or alkali aqueous solution which is conventional in the field, and acetic acid solution and sodium hydroxide solution are preferred for reducing the introduction of other substances in the reaction system.
Compared with the prior art, the invention has the following advantages and remarkable progress:
(1) the mPEG-rhG-CSF modified protein with high single modification rate is obtained under the conditions of low PH and high salt by taking a composite chromatography medium as a solid phase reaction carrier, and the single modification rate of the obtained polyethylene glycol modified protein is more than 91%;
(2) the process carries out gradient elution with higher pH and low salt concentration to obtain the high-purity mPEG-rhG-CSF modified protein, and the purity reaches more than 99.6 percent;
(3) the process simplifies the modification and purification reaction operations, greatly reduces the production cost and provides a new process for realizing PEG modification of the protein.
Detailed Description
The advantageous effects of the present invention will now be further described by the following examples, which are for illustrative purposes only and do not limit the scope of the present invention, and variations and modifications apparent to those skilled in the art according to the present invention are also included in the scope of the present invention.
Example 1
a. Loading: loading a proper amount of chromatographic filler Capto MMC into an XK26 chromatographic column, wherein the column height is 20cm, and connecting the chromatographic filler Capto a protein purification system; balancing the chromatographic column by using a buffer solution A (sodium acetate buffer solution containing sodium chloride, wherein the sodium acetate is 25mM, the sodium chloride is 400mM, and the pH value is adjusted to be 4.5), wherein the column volume is 3-5, and the flow rate is 10 mL/min; and preparing rhG-CSF into a solution with the concentration of 2mg/mL by using the buffer solution A, loading the rhG-CSF into a chromatographic column with the loading capacity of 20mg/mL, and flushing 3-5 column volumes by using the buffer solution A after the loading is finished until no protein penetrates out.
b. Modification reaction: according to the molar ratio rhG-CSF: weighing mPEG-butyraldehyde (20kDa) according to the proportion of 1:4 of the mPEG modifier, preparing a solution with the concentration of 2mg/mL by using the buffer solution A, adding sodium cyanoborohydride with the final concentration of 20mM, adjusting the pH value to 4.5 to obtain a modified buffer solution serving as a mobile phase, circulating the modified buffer solution to flow through a chromatographic column at the flow rate of 2-3 mL/min, controlling the circulating reaction time to be 5h, maintaining the column temperature at room temperature, and simultaneously carrying out light-shielding treatment on the chromatographic column; at the end of the cycle, the column was washed again with buffer A as described above and 5 column volumes were washed at a flow rate of 10 mL/min.
c. And (3) elution: gradient elution is carried out by using buffer solution B (sodium acetate buffer solution containing sodium chloride, wherein the sodium acetate is 25mM, the sodium chloride is 20mM, and the pH value is 5.8) and the buffer solution A in different proportions, and the specific operation is as follows: and (3) eluting the multi-modified rhG-CSF in a gradient manner by using 10-30% of buffer solution B and 3CV at the flow rate of 5mL/min, eluting the singly-modified rhG-CSF in a gradient manner by using 30-80% of buffer solution B and 10CV to obtain the singly-modified target protein mPEG-rhG-CSF, and sampling to detect the purity and calculate the yield, wherein the results are shown in table 1.
Example 2
a. Loading: loading a proper amount of chromatographic filler Capto MMC in an XK26 chromatographic column, wherein the column height is 20cm, and connecting the chromatographic filler Capto a protein purification system; balancing the chromatographic column by using a buffer solution A (sodium acetate buffer solution containing sodium chloride, wherein the sodium acetate is 20mM, the sodium chloride is 400mM, and the pH is adjusted to 4), wherein the flow rate is 10mL/min for 3-5 column volumes; and (3) preparing rhG-CSF into a solution with the concentration of 3mg/mL by using the buffer solution A, loading the solution on a chromatographic column with the load of 20mg/mL, and washing 3-5 column volumes by using the buffer solution A until no protein penetrates out.
b. Modification reaction: according to the molar ratio rhG-CSF: weighing mPEG-propionaldehyde (20kDa) according to the proportion of 1:2, preparing a solution with the concentration of 3mg/mL by using the buffer solution A, adding sodium cyanoborohydride with the final concentration of 10mM, adjusting the pH value to 4 to obtain a modified buffer solution serving as a mobile phase, circulating the modified buffer solution to flow through a chromatographic column at the flow rate of 2-3 mL/min, controlling the circulating reaction time to be 5h, maintaining the column temperature at room temperature, and simultaneously carrying out light-shielding treatment on the chromatographic column; at the end of the cycle, the column was washed again with buffer A as described above and 5 column volumes were washed at a flow rate of 10 mL/min.
c. And (3) elution: gradient elution is carried out by using buffer solution B (sodium acetate buffer solution containing sodium chloride, wherein the sodium acetate is 30mM, the sodium chloride is 20mM, and the pH value is 6) and the buffer solution A in different proportions, and the specific operation is as follows: and (3) eluting the multi-modified rhG-CSF in a gradient manner by 10-30% of buffer solution B and 3CV at the flow rate of 5mL/min, eluting the single-modified rhG-CSF in a gradient manner by 30-80% of buffer solution B and 10CV to obtain the single-modified target protein PEG-rhG-CSF, and sampling to detect the purity and calculate the yield, wherein the results are shown in table 1.
Example 3
a. Loading: loading a proper amount of chromatographic filler Capto MMC in an XK26 chromatographic column, wherein the column height is 20cm, and connecting the chromatographic filler Capto a protein purification system; balancing the chromatographic column by using a buffer solution A (sodium acetate buffer solution containing sodium chloride, wherein the sodium acetate is 30mM, the sodium chloride is 400mM, and the pH is adjusted to 5), wherein the column volume is 3-5, and the flow rate is 10 mL/min; and preparing rhG-CSF into a solution with the concentration of 2mg/mL by using the buffer solution A, loading the solution on a chromatographic column with the loading capacity of 20mg/mL, and washing 3-5 column volumes by using the buffer solution A until no protein passes out.
b. Modification reaction: according to the molar ratio rhG-CSF: weighing mPEG-succinate (20kDa) according to the proportion of 1:10, preparing a solution with the concentration of 1mg/mL by using the buffer solution A, adding sodium cyanoborohydride with the final concentration of 10mM, adjusting the pH value to 5 to obtain a modified buffer solution serving as a mobile phase, circulating the modified buffer solution to flow through a chromatographic column at the flow rate of 2-3 mL/min, controlling the circulating reaction time to be 5h, maintaining the temperature of the column at room temperature, and simultaneously carrying out light-shielding treatment on the chromatographic column; at the end of the cycle, the column was washed again with buffer A as described above and 5 column volumes were washed at a flow rate of 10 mL/min.
c. And (3) elution: gradient elution is carried out by using buffer solution B (sodium acetate buffer solution containing sodium chloride, wherein the sodium acetate is 20mM, the sodium chloride is 20mM, and the pH value is 5.5) and the buffer solution A in different proportions, and the specific operation is as follows: and (3) eluting the multi-modified rhG-CSF in a gradient manner by 10-30% of buffer solution B and 3CV at the flow rate of 5mL/min, eluting the single-modified rhG-CSF in a gradient manner by 30-80% of buffer solution B and 10CV to obtain the single-modified target protein PEG-rhG-CSF, and sampling to detect the purity and calculate the yield, wherein the results are shown in table 1.
Example 4
a. Loading: loading a proper amount of chromatographic filler Capto MMC in an XK26 chromatographic column, wherein the column height is 20cm, and connecting the chromatographic filler Capto a protein purification system; balancing the chromatographic column by using a buffer solution A (a sodium acetate buffer solution containing sodium chloride, wherein the sodium acetate is 10mM, the sodium chloride is 100mM, and the pH value is adjusted to 4.5), wherein the column volume is 3-5, and the flow rate is 10 mL/min; and preparing rhG-CSF into a solution with the concentration of 3mg/mL by using the buffer solution A, loading the solution on a chromatographic column with the loading capacity of 20mg/mL, and washing 3-5 column volumes by using the buffer solution A until no protein passes out.
b. Modification reaction: according to the molar ratio rhG-CSF: weighing mPEG-butyraldehyde (20kDa) according to the proportion of 1:3 of the mPEG modifier, preparing a solution with the concentration of 5mg/mL by using the buffer solution A, adding sodium cyanoborohydride with the final concentration of 30mM, adjusting the pH value to 4.5 to obtain a modified buffer solution serving as a mobile phase, circulating the modified buffer solution to flow through a chromatographic column at the flow rate of 2-3 mL/min, controlling the circulating reaction time to be 4h, maintaining the column temperature at room temperature, and simultaneously carrying out light-shielding treatment on the chromatographic column; at the end of the cycle, the column was washed again with buffer A as described above and 5 column volumes were washed at a flow rate of 10 mL/min.
c. And (3) elution: gradient elution is carried out by using buffer solution B (sodium acetate buffer solution containing sodium chloride, wherein the sodium acetate is 10mM, the sodium chloride is 50mM, and the pH value is 5.8) and the buffer solution A in different proportions, and the specific operation is as follows: and (3) eluting the multi-modified rhG-CSF in a gradient manner by 10-30% of buffer solution B and 3CV at the flow rate of 5mL/min, eluting the single-modified rhG-CSF in a gradient manner by 30-80% of buffer solution B and 10CV to obtain the single-modified target protein PEG-rhG-CSF, and sampling to detect the purity and calculate the yield, wherein the results are shown in table 1.
Example 5
a. Loading: loading a proper amount of chromatographic filler Capto MMC in an XK26 chromatographic column, wherein the column height is 20cm, and connecting the chromatographic filler Capto a protein purification system; balancing the chromatographic column by using a buffer solution A (a sodium acetate buffer solution containing sodium chloride, wherein the sodium acetate is 50mM, the sodium chloride is 800mM, and the pH value is adjusted to 4.5), wherein the column volume is 3-5, and the flow rate is 10 mL/min; and preparing rhG-CSF into a solution with the concentration of 2mg/mL by using the buffer solution A, loading the solution on a chromatographic column with the loading capacity of 20mg/mL, and washing 3-5 column volumes by using the buffer solution A until no protein passes out.
b. Modification reaction: according to the molar ratio rhG-CSF: weighing mPEG-butyraldehyde (20kDa) according to the proportion of 1:6, preparing a solution with the concentration of 2mg/mL by using the buffer solution A, adding sodium cyanoborohydride with the final concentration of 30mM, adjusting the pH value to 4.5 to obtain a modified buffer solution serving as a mobile phase, circulating the modified buffer solution to flow through a chromatographic column at the flow rate of 2-3 mL/min, controlling the circulating reaction time to be 6h, maintaining the column temperature at room temperature, and simultaneously carrying out light-shielding treatment on the chromatographic column; at the end of the cycle, the column was washed again with buffer A as described above and 5 column volumes were washed at a flow rate of 10 mL/min.
c. And (3) elution: gradient elution is carried out by using buffer solution B (sodium acetate buffer solution containing sodium chloride, wherein the sodium acetate is 50mM, the sodium chloride is 10mM, and the pH value is 5.8) and the buffer solution A in different proportions, and the specific operation is as follows: and (3) eluting the multi-modified rhG-CSF in a gradient manner by 10-30% of buffer solution B and 3CV at the flow rate of 5mL/min, eluting the single-modified rhG-CSF in a gradient manner by 30-80% of buffer solution B and 10CV to obtain the single-modified target protein PEG-rhG-CSF, and sampling to detect the purity and calculate the yield, wherein the results are shown in table 1.
Example 6
a. Loading: loading a proper amount of chromatographic filler Capto MMC in an XK26 chromatographic column, wherein the column height is 20cm, and connecting the chromatographic filler Capto a protein purification system; balancing the chromatographic column by using a buffer solution A (sodium acetate buffer solution containing sodium chloride, wherein the sodium acetate is 25mM, the sodium chloride is 500mM, and the pH value is adjusted to be 4.5), wherein the column volume is 3-5, and the flow rate is 10 mL/min; and preparing rhG-CSF into a solution with the concentration of 2mg/mL by using the buffer solution A, loading the solution on a chromatographic column with the loading capacity of 20mg/mL, and washing 3-5 column volumes by using the buffer solution A until no protein passes out.
b. Modification reaction: according to the molar ratio rhG-CSF: weighing mPEG-butyraldehyde (20kDa) according to the proportion of 1:4 of the mPEG modifier, preparing a solution with the concentration of 2mg/mL by using the buffer solution A, adding sodium cyanoborohydride with the final concentration of 20mM, adjusting the pH value to 4.5 to obtain a modified buffer solution serving as a mobile phase, circulating the modified buffer solution to flow through a chromatographic column at the flow rate of 2-3 mL/min, controlling the circulating reaction time to be 8h, maintaining the column temperature at room temperature, and simultaneously carrying out light-shielding treatment on the chromatographic column; at the end of the cycle, the column was washed again with buffer A as described above and 5 column volumes were washed at a flow rate of 10 mL/min.
c. And (3) elution: gradient elution is carried out by using buffer B (sodium acetate buffer solution containing sodium chloride, wherein the sodium acetate is 25mM, the sodium chloride is 30mM, and the pH value is 5.8) and the buffer A in different proportions, and the specific operation is as follows: and (3) eluting the multi-modified rhG-CSF in a gradient manner by 10-30% of buffer solution B and 3CV at the flow rate of 5mL/min, eluting the single-modified rhG-CSF in a gradient manner by 30-80% of buffer solution B and 10CV to obtain the single-modified target protein PEG-rhG-CSF, and sampling to detect the purity and calculate the yield, wherein the results are shown in table 1.
Example 7
a. Loading: taking appropriate amount of chromatography filler
Figure BDA0003619130630000081
HCX, loaded in XK26 chromatography column, 20cm high, connected to protein purification system; balancing the chromatographic column by using a buffer solution A (sodium acetate buffer solution containing sodium chloride, wherein the sodium acetate is 25mM, the sodium chloride is 400mM, and the pH value is adjusted to be 4.5), wherein the column volume is 3-5, and the flow rate is 10 mL/min; preparing rhG-CSF into a solution with the concentration of 2mg/mL by using the buffer solution A, loading the solution on a chromatographic column with the load of 20mg/mL, and washing 3-5 column volumes by using the buffer solution A until no protein penetrates out.
b. Modification reaction: according to the molar ratio rhG-CSF: weighing mPEG-butyraldehyde (20kDa) according to the proportion of 1:4 of the mPEG modifier, preparing a solution with the concentration of 2mg/mL by using the buffer solution A, adding sodium cyanoborohydride with the final concentration of 20mM, adjusting the pH value to 4.5 to obtain a modified buffer solution serving as a mobile phase, circulating the modified buffer solution to flow through a chromatographic column at the flow rate of 2-3 mL/min, controlling the circulating reaction time to be 6h, maintaining the column temperature at room temperature, and simultaneously carrying out light-shielding treatment on the chromatographic column; at the end of the cycle, the column was washed again with buffer A as described above and 5 column volumes were washed at a flow rate of 10 mL/min.
c. And (3) elution: gradient elution is carried out by using buffer solution B (sodium acetate buffer solution containing sodium chloride, wherein the sodium acetate is 25mM, the sodium chloride is 20mM, and the pH value is 5.8) and the buffer solution A in different proportions, and the specific operation is as follows: and (3) eluting the multi-modified rhG-CSF in a gradient manner by 10-30% of buffer solution B and 3CV at the flow rate of 5mL/min, eluting the single-modified rhG-CSF in a gradient manner by 30-80% of buffer solution B and 10CV to obtain the single-modified target protein PEG-rhG-CSF, and sampling to detect the purity and calculate the yield, wherein the results are shown in table 1.
Example 8
a. Loading: loading a proper amount of chromatographic filler Capto MMC into an XK26 chromatographic column, wherein the column height is 20cm, and connecting the chromatographic filler Capto a protein purification system; balancing the chromatographic column by using a buffer solution A (sodium acetate buffer solution containing sodium chloride, wherein the sodium acetate is 100mM, the sodium chloride is 400mM, and the pH value is adjusted to 4.5), wherein the column volume is 3-5, and the flow rate is 10 mL/min; and preparing rhG-CSF into a solution with the concentration of 2mg/mL by using the buffer solution A, loading the solution on a chromatographic column with the loading capacity of 20mg/mL, and washing 3-5 column volumes by using the buffer solution A until no protein passes out.
b. Modification reaction: according to the molar ratio rhG-CSF: weighing mPEG-butyraldehyde (20kDa) according to the proportion of 1:20, preparing a solution with the concentration of 10mg/mL by using the buffer solution A, adding sodium cyanoborohydride with the final concentration of 20mM, adjusting the pH value to 4.5 to obtain a modified buffer solution serving as a mobile phase, circulating the modified buffer solution to flow through a chromatographic column at the flow rate of 2-3 mL/min, controlling the flow time to be 7h, maintaining the column temperature at room temperature, and simultaneously carrying out light-shielding treatment on the chromatographic column; at the end of the cycle, the column was washed again with buffer A as described above and 5 column volumes were washed at a flow rate of 10 mL/min.
c. And (3) elution: gradient elution is carried out by using buffer solution B (sodium acetate buffer solution containing sodium chloride, wherein the sodium acetate is 100mM, the sodium chloride is 20mM, and the pH value is 5.8) and the buffer solution A in different proportions, and the specific operation is as follows: and (3) eluting the multi-modified rhG-CSF in a gradient manner by 10-30% of buffer solution B and 3CV at the flow rate of 5mL/min, eluting the single-modified rhG-CSF in a gradient manner by 30-80% of buffer solution B and 10CV to obtain the single-modified target protein PEG-rhG-CSF, and sampling to detect the purity and calculate the yield, wherein the results are shown in table 1.
Example 9
a. Loading: loading a proper amount of chromatographic filler Capto MMC in an XK26 chromatographic column, wherein the column height is 20cm, and connecting the chromatographic filler Capto a protein purification system; balancing the chromatographic column by using a buffer solution A (sodium acetate buffer solution containing sodium chloride, wherein the sodium acetate is 25mM, the sodium chloride is 400mM, and the pH is adjusted to 3), wherein the column volume is 3-5, and the flow rate is 10 mL/min; and preparing rhG-CSF into a solution with the concentration of 2mg/mL by using the buffer solution A, loading the solution on a chromatographic column with the loading capacity of 20mg/mL, and washing 3-5 column volumes by using the buffer solution A until no protein passes out.
b. Modification reaction: according to the molar ratio rhG-CSF: weighing mPEG-butyraldehyde (20kDa) according to the proportion of 1:4 of mPEG modifier, preparing a solution with the concentration of 2mg/mL by using the buffer solution A, adding sodium cyanoborohydride with the final concentration of 20mM, adjusting the pH value to 3 to obtain a modified buffer solution as a mobile phase, circularly flowing through a chromatographic column at the flow rate of 2-3 mL/min, controlling the circular reaction time to be 5h, maintaining the column temperature at room temperature, and simultaneously carrying out photophobic treatment on the chromatographic column; at the end of the cycle, the column was washed again with buffer A as described above and 5 column volumes were washed at a flow rate of 10 mL/min.
c. And (3) elution: gradient elution is carried out by using buffer solution B (sodium acetate buffer solution containing sodium chloride, wherein the sodium acetate is 25mM, the sodium chloride is 20mM, and the pH value is 5) and the buffer solution A in different proportions, and the specific operation is as follows: and (3) eluting the multi-modified rhG-CSF in a gradient manner by 10-30% of buffer solutions B and 3CV at the flow rate of 5mL/min, eluting the single-modified rhG-CSF in a gradient manner by 30-80% of buffer solutions B and 10CV to obtain the single-modified target protein PEG-rhG-CSF, and sampling to detect the purity and calculate the yield, wherein the results are shown in a table 1.
Example 10
a. Loading: loading a proper amount of chromatographic filler Capto MMC in an XK26 chromatographic column, wherein the column height is 20cm, and connecting the chromatographic filler Capto a protein purification system; balancing the chromatographic column by using a buffer solution A (sodium acetate buffer solution containing sodium chloride, wherein the sodium acetate is 25mM, the sodium chloride is 400mM, and the pH is adjusted to 6), wherein the column volume is 3-5, and the flow rate is 10 mL/min; and preparing rhG-CSF into a solution with the concentration of 2mg/mL by using the buffer solution A, loading the solution on a chromatographic column with the loading capacity of 20mg/mL, and washing 3-5 column volumes by using the buffer solution A until no protein passes out.
b. Modification reaction: according to the molar ratio rhG-CSF: weighing mPEG-butyraldehyde (20kDa) according to the proportion of 1:4 of the mPEG modifier, preparing a solution with the concentration of 2mg/mL by using the buffer solution A, adding sodium cyanoborohydride with the final concentration of 20mM, adjusting the pH value to 6 to obtain a modified buffer solution serving as a mobile phase, circulating the modified buffer solution to flow through a chromatographic column at the flow rate of 2-3 mL/min, controlling the circulating reaction time to be 5h, maintaining the column temperature at room temperature, and simultaneously carrying out light-shielding treatment on the chromatographic column; at the end of the cycle, the column was washed again with buffer A as described above and 5 column volumes were washed at a flow rate of 10 mL/min.
c. And (3) elution: gradient elution is carried out by using buffer solution B (sodium acetate buffer solution containing sodium chloride, wherein the sodium acetate is 25mM, the sodium chloride is 20mM, and the pH value is 6.3) and the buffer solution A in different proportions, and the specific operation is as follows: and (3) eluting the multi-modified rhG-CSF in a gradient manner by 10-30% of buffer solution B and 3CV at the flow rate of 5mL/min, eluting the single-modified rhG-CSF in a gradient manner by 30-80% of buffer solution B and 10CV to obtain the single-modified target protein PEG-rhG-CSF, and sampling to detect the purity and calculate the yield, wherein the results are shown in table 1.
Example 11
a. Loading: loading a proper amount of chromatographic filler Capto MMC in an XK26 chromatographic column, wherein the column height is 20cm, and connecting the chromatographic filler Capto a protein purification system; balancing the chromatographic column by using a buffer solution A (sodium acetate buffer solution containing sodium chloride, wherein the sodium acetate is 25mM, the sodium chloride is 1.2M, and the pH value is adjusted to be 4.5), and carrying out column volume of 3-5 at the flow rate of 10 mL/min; and preparing rhG-CSF into a solution with the concentration of 2mg/mL by using the buffer solution A, loading the solution on a chromatographic column with the loading capacity of 20mg/mL, and washing 3-5 column volumes by using the buffer solution A until no protein passes out.
b. Modification reaction: according to the molar ratio rhG-CSF: weighing mPEG-butyraldehyde (20kDa) according to the proportion of 1:4 of the mPEG modifier, preparing a solution with the concentration of 2mg/mL by using the buffer solution A, adding sodium cyanoborohydride with the final concentration of 20mM, adjusting the pH value to 4.5 to obtain a modified buffer solution serving as a mobile phase, circulating the modified buffer solution to flow through a chromatographic column at the flow rate of 2-3 mL/min, controlling the circulating reaction time to be 5h, maintaining the column temperature at room temperature, and simultaneously carrying out light-shielding treatment on the chromatographic column; at the end of the cycle, the column was washed again with buffer A as described above and 5 column volumes were washed at a flow rate of 10 mL/min.
c. And (3) elution: gradient elution is carried out by using buffer solution B (sodium acetate buffer solution containing sodium chloride, wherein the sodium acetate is 25mM, the sodium chloride is 5mM, and the pH value is 5.8) and the buffer solution A in different proportions, and the specific operation is as follows: and (3) eluting the multi-modified rhG-CSF in a gradient manner by 10-30% of buffer solution B and 3CV at the flow rate of 5mL/min, eluting the single-modified rhG-CSF in a gradient manner by 30-80% of buffer solution B and 10CV to obtain the single-modified target protein PEG-rhG-CSF, and sampling to detect the purity and calculate the yield, wherein the results are shown in table 1.
Example 12
a. Loading: loading a proper amount of chromatographic filler Capto MMC in an XK26 chromatographic column, wherein the column height is 20cm, and connecting the chromatographic filler Capto a protein purification system; balancing the chromatographic column by using a buffer solution A (sodium acetate buffer solution containing sodium chloride, wherein the sodium acetate is 25mM, the sodium chloride is 50mM, and the pH value is adjusted to be 4.5), wherein the column volume is 3-5, and the flow rate is 10 mL/min; and preparing rhG-CSF into a solution with the concentration of 2mg/mL by using the buffer solution A, loading the solution on a chromatographic column with the loading capacity of 20mg/mL, and washing 3-5 column volumes by using the buffer solution A until no protein passes out.
b. Modification reaction: according to the molar ratio rhG-CSF: weighing mPEG-butyraldehyde (20kDa) according to the proportion of 1:4 of the mPEG modifier, preparing a solution with the concentration of 2mg/mL by using the buffer solution A, adding sodium cyanoborohydride with the final concentration of 20mM, adjusting the pH value to 4.5 to obtain a modified buffer solution serving as a mobile phase, circulating the modified buffer solution to flow through a chromatographic column at the flow rate of 2-3 mL/min, controlling the circulating reaction time to be 5h, maintaining the column temperature at room temperature, and simultaneously carrying out light-shielding treatment on the chromatographic column; at the end of the cycle, the column was washed again with buffer A as described above and 5 column volumes were washed at a flow rate of 10 mL/min.
c. And (3) elution: gradient elution is carried out by using buffer solution B (sodium acetate buffer solution containing sodium chloride, wherein the sodium acetate is 25mM, the sodium chloride is 100mM, and the pH value is 5.8) and the buffer solution A in different proportions, and the specific operation is as follows: and (3) eluting the multi-modified rhG-CSF in a gradient manner by 10-30% of buffer solution B and 3CV at the flow rate of 5mL/min, eluting the single-modified rhG-CSF in a gradient manner by 30-80% of buffer solution B and 10CV to obtain the single-modified target protein PEG-rhG-CSF, and sampling to detect the purity and calculate the yield, wherein the results are shown in table 1.
Comparative example 1
a. Loading: taking a proper amount of chromatographic packing Phenyl Sepharose 6Fast Flow (high sub), loading the chromatographic packing Phenyl Sepharose 6Fast Flow (high sub) in an XK26 chromatographic column, wherein the height of the column is 20cm, and connecting the chromatographic packing Phenyl Sepharose 6Fast Flow (high sub) to a protein purification system; balancing the chromatographic column by using a buffer solution A (sodium acetate buffer solution containing sodium chloride, wherein the sodium acetate is 25mM, the sodium chloride is 400mM, and the pH value is adjusted to be 4.5), wherein the column volume is 3-5, and the flow rate is 10 mL/min; and preparing rhG-CSF into a solution with the concentration of 2mg/mL by using the buffer solution A, loading the solution on a chromatographic column with the loading capacity of 20mg/mL, and washing 3-5 column volumes by using the buffer solution A until no protein passes out.
b. Modification reaction: according to the molar ratio rhG-CSF: weighing mPEG-butyraldehyde (20kDa) according to the proportion of 1:4 of the mPEG modifier, preparing a solution with the concentration of 2mg/mL by using the buffer solution A, adding sodium cyanoborohydride with the final concentration of 20mM, adjusting the pH value to 4.5 to obtain a modified buffer solution serving as a mobile phase, circulating the modified buffer solution to flow through a chromatographic column at the flow rate of 2-3 mL/min, controlling the circulating reaction time to be 6h, maintaining the column temperature at room temperature, and simultaneously carrying out light-shielding treatment on the chromatographic column; at the end of the cycle, the column was washed again with buffer A as described above and 5 column volumes were washed at a flow rate of 10 mL/min.
c. And (3) elution: gradient elution is carried out by using buffer solution B (sodium acetate buffer solution containing sodium chloride, wherein the sodium acetate is 25mM, the sodium chloride is 20mM, and the pH value is 5.8) and the buffer solution A in different proportions, and the specific operation is as follows: and (3) eluting the multi-modified rhG-CSF in a gradient manner by 10-30% of buffer solution B and 3CV at the flow rate of 5mL/min, eluting the single-modified rhG-CSF in a gradient manner by 30-80% of buffer solution B and 10CV to obtain the single-modified target protein PEG-rhG-CSF, and sampling to detect the purity and calculate the yield, wherein the results are shown in table 1.
Comparative example 2
a. Loading: loading a proper amount of chromatographic filler Generic MC60-SP into an XK26 chromatographic column with the column height of 20cm, and connecting to a protein purification system; balancing the chromatographic column by using a buffer solution A (sodium acetate buffer solution containing sodium chloride, wherein the sodium acetate is 25mM, the sodium chloride is 400mM, and the pH value is adjusted to be 4.5), wherein the column volume is 3-5, and the flow rate is 10 mL/min; and preparing rhG-CSF into a solution with the concentration of 2mg/mL by using the buffer solution A, loading the solution on a chromatographic column with the loading capacity of 20mg/mL, and washing 3-5 column volumes by using the buffer solution A until no protein passes out.
b. Modification reaction: according to the molar ratio rhG-CSF: weighing mPEG-butyraldehyde (20kDa) according to the proportion of 1:4 of the mPEG modifier, preparing a solution with the concentration of 2mg/mL by using the buffer solution A, adding sodium cyanoborohydride with the final concentration of 20mM, adjusting the pH value to 4.5 to obtain a modified buffer solution serving as a mobile phase, circulating the modified buffer solution to flow through a chromatographic column at the flow rate of 2-3 mL/min, controlling the circulating reaction time to be 5h, maintaining the column temperature at room temperature, and simultaneously carrying out light-shielding treatment on the chromatographic column; at the end of the cycle, the column was washed again with buffer A as described above and 5 column volumes were washed at a flow rate of 10 mL/min.
c. And (3) elution: gradient elution is carried out by using buffer solution B (sodium acetate buffer solution containing sodium chloride, wherein the sodium acetate is 25mM, the sodium chloride is 20mM, and the pH value is 5.8) and the buffer solution A in different proportions, and the specific operation is as follows: and (3) eluting the multi-modified rhG-CSF in a gradient manner by 10-30% of buffer solutions B and 3CV at the flow rate of 5mL/min, eluting the single-modified rhG-CSF in a gradient manner by 30-80% of buffer solutions B and 10CV to obtain the single-modified target protein PEG-rhG-CSF, and sampling to detect the purity and calculate the yield, wherein the results are shown in a table 1.
Comparative example 3
a. Loading: loading a proper amount of chromatographic filler Capto MMC in an XK26 chromatographic column, wherein the column height is 20cm, and connecting the chromatographic filler Capto a protein purification system; balancing the chromatographic column by using a buffer solution A (sodium acetate buffer solution containing sodium chloride, wherein the sodium acetate is 25mM, the sodium chloride is 400mM, and the pH value is adjusted to be 4.5), wherein the column volume is 3-5, and the flow rate is 10 mL/min; and preparing rhG-CSF into a solution with the concentration of 2mg/mL by using the buffer solution A, loading the solution on a chromatographic column with the loading capacity of 20mg/mL, and washing 3-5 column volumes by using the buffer solution A until no protein passes out.
b. Modification reaction: according to the molar ratio rhG-CSF: weighing mPEG-butyraldehyde (20kDa) according to the proportion of 1:4 of the mPEG modifier, preparing a solution with the concentration of 2mg/mL by using the buffer solution A, adding sodium cyanoborohydride with the final concentration of 20mM, adjusting the pH value to 4.5 to obtain a modified buffer solution serving as a mobile phase, circulating the modified buffer solution to flow through a chromatographic column at the flow rate of 2-3 mL/min, controlling the circulating reaction time to be 5h, maintaining the column temperature at room temperature, and simultaneously carrying out light-shielding treatment on the chromatographic column; at the end of the cycle, the column was washed again with buffer A as described above and 5 column volumes were washed at a flow rate of 10 mL/min.
c. And (3) elution: eluting with buffer B (sodium acetate buffer containing sodium chloride, wherein sodium acetate is 25mM, sodium chloride is 20mM, pH5.8) and buffer A at flow rate of 5mL/min, and specifically performing: eluting the multi-modified rhG-CSF by 30% buffer solution B and 3CV, eluting the mono-modified rhG-CSF by 50% buffer solution B and 10CV to obtain the mono-modified target protein PEG-rhG-CSF, sampling, detecting the purity and calculating the yield, wherein the results are shown in Table 1.
Comparative example 4
rhG-CSF was prepared in a concentration of 2mg/mL using buffer A (sodium acetate buffer containing sodium chloride, wherein sodium acetate was 25mM, sodium chloride was 400mM, adjusted to pH4.5) at a molar ratio of rhG-CSF: mPEG-butyraldehyde (20kDa) was added to the mPEG modifier at a ratio of 1:4, the mixture was dissolved with stirring, sodium cyanoborohydride was added to the mixture at a final concentration of 20mM, and the reaction mixture was reacted with stirring for 5 hours. Loading a proper amount of chromatographic filler Capto MMC in an XK26 chromatographic column, wherein the column height is 20cm, and connecting the chromatographic filler Capto a protein purification system; balancing a chromatographic column by using the buffer solution A, wherein the volume of the chromatographic column is 3-5, and the flow rate is 10 mL/min; circularly loading a modification liquid according to the loading capacity of 20 g/L; finally, washing 3-5 column volumes by using the buffer solution A;
gradient elution is carried out by using buffer solution B (sodium acetate buffer solution containing sodium chloride, wherein the sodium acetate is 25mM, the sodium chloride is 20mM, and the pH value is 5.8) and the buffer solution A in different proportions, and the specific operation is as follows: and (3) eluting the multi-modified rhG-CSF in a gradient manner by 10-30% of buffer solution B and 3CV at the flow rate of 5mL/min, eluting the single-modified rhG-CSF in a gradient manner by 30-80% of buffer solution B and 10CV to obtain the single-modified target protein PEG-rhG-CSF, and sampling to detect the purity and calculate the yield, wherein the results are shown in table 1.
Comparative example 5
Taking 1L of mother liquor (containing 1g of rhG-CSF, 20mM NaAc and 50Mm NaCl) of the recombinant human granulocyte stimulating factor, cooling to 4 ℃, and adding 0.5M/L NaOH solution to adjust the pH to 6. Adding 11g of methoxy-polyethylene glycol-propionaldehyde (mPEG-PAL, the molecular weight is about 20KD), stirring for dissolution, adding nitrile sodium borohydride (NaBH3CN) to enable the final concentration of the nitrile sodium borohydride to be 15mM/L, and stirring the reaction mixture at a low speed for reaction for 15 hours; to the reaction mixture was added 0.5M/L hydrochloric acid solution to adjust the pH of the reaction mixture to 3. The reaction solution was purified by passing through a column packed with SP Sepharose FF, equilibrated in an equilibration solution (an aqueous solution containing 20mM/L NaAc and 150mM/L NaCl), and then eluted with PEG-rhG-CSF by passing through an eluent (an aqueous solution containing 20mM/L NaAc and 200mM/L NaCl). Sampling to detect purity and calculating yield, and the results are shown in table 1.
Comparative example 6
The rhG-CSF reaction solution (40mL, concentration 3mg/mL) containing 100mM sodium phosphate, pH5.0, and 40mM NADPH (nicotinamide adenine dinucleotide phosphate) was stirred well and frozen (4 ℃ C.), and methoxypolyethylene glycol propionaldehyde (MPEG-PALD) (molecular weight 20kDa) of 5 times the protein mass was added. The reaction mixture is continuously stirred and reacted for 10 hours at the same temperature; the reaction mixture was adjusted to pH4.0 with 0.5mol/L hydrochloric acid and left to stand for 10 minutes. Filtration was carried out using a filter element having a pore size of 0.22. mu.m. Equilibrating the Capto MMC column with buffer (20mmol/L citric acid/disodium hydrogen phosphate, pH4.0) at a flow rate of 300 cm/hr until the pH stabilizes at 4.0; loading the filtered sample at the flow rate of 300 cm/h; after the loading, the column was equilibrated again with a buffer (20mmol/L citric acid/disodium hydrogen phosphate, pH4.0) at a flow rate of 300 cm/h; eluting with 10mmol/L citric acid/disodium hydrogen phosphate buffer (pH7.5) to remove impurities at a flow rate of 180 cm/hr; : eluting the target protein with 50mmol/L citric acid/disodium hydrogen phosphate buffer solution (pH8.0) at flow rate of 300 cm/hr, and collecting the target protein peak. Sampling to detect purity and calculating yield, and the results are shown in table 1.
Comparative example 7
rhG-CSF (5mg/mL) was dissolved in 100mM sodium acetate solution containing 20mM sodium cyanoborohydride, pH5, stirred in an ice bath, 5-fold molar excess mPEG-acetaldehyde (20KD) was added, and the reaction mixture was stirred under the same conditions for 10 h; the reaction mixture was diluted 5-fold and the pH was adjusted to 4. The single mPEG-rhG-CSF conjugate was isolated by ion exchange chromatography using a HiLoad 16/10SP Sepharose HP column equilibrated with 20mM sodium acetate buffer, pH4, and eluted with a linear 0-1M NaCl gradient, sampled for purity, and the yield calculated, the results are shown in Table 1.
Verification examples
1. Determination of purity and yield
The reversed phase purity is determined according to 0512 of the four-part general regulation of the 2020 edition of Chinese pharmacopoeia, and the SEC purity is determined according to 0514 of the four-part general regulation of the 2020 edition of Chinese pharmacopoeia. The purity and yield results of examples 1 to 12 and comparative examples 1 to 7 are shown in Table 1.
TABLE 1 purity and yield test results
Figure BDA0003619130630000141
Figure BDA0003619130630000151
It can be seen that comparative example 1 and comparative example 2 have lower sample purity and yield by replacing the column; the isocratic elution in the comparative example 3 has poor elution effect and lower purity and yield; comparative example 4 reaction first and column last, resulting in lower yield; comparative examples 5-7 are reported in the prior art with lower purity and lower yield than example 1.
2. Determination of modification Rate
Respectively referring to the preparation methods of examples 1-12 and comparative examples 1-3, repeating the step a and the step b, eluting the chromatographic column with 20mM Tris and pH9.0 at the flow rate of 5mL/min, collecting all elution peaks, and determining the modification rate according to 0514 of the general rules of four departments in the 2020 edition of Chinese pharmacopoeia; in comparative examples 4 to 7, the reaction mixture after the reaction was stirred was measured for modification rate according to 0514, a fourth general guideline of 2020 edition, China pharmacopoeia. The results are shown in Table 2.
TABLE 2 detection of modification Rate
Figure BDA0003619130630000152
Figure BDA0003619130630000161
Thus, it can be seen that the column was replaced in comparative example 1 and comparative example 2, and the amount of the multi-modified product increased; comparative example 3 isocratic elution affects product purity and yield, and has no effect on single modification rate; comparative example 4 reaction first and column last, resulting in an increase in multiple modification products; comparative examples 5 to 7 are the methods reported in the prior art, and the single modification rate is low.

Claims (10)

1. A preparation method of polyethylene glycol modified rhG-CSF is characterized by comprising the following steps: the rhG-CSF protein is loaded and adsorbed on a chromatography filler by taking a composite chromatography medium as a solid-phase reaction carrier, a modification reaction is completed by taking a polyethylene glycol modifier as a mobile phase under the catalysis of a catalyst, and after the reaction is completed, the polyethylene glycol modified protein mPEG-rhG-CSF is obtained by adopting gradient elution.
2. The method of claim 1, comprising the steps of:
a. loading: dissolving rhG-CSF protein in buffer solution A to obtain rhG-CSF protein solution, then loading and adsorbing the rhG-CSF protein solution on chromatographic packing;
b. modification reaction: dissolving a polyethylene glycol modifier in the buffer solution A, adding a catalyst to obtain a modified buffer solution serving as a mobile phase, and circulating the modified buffer solution through a chromatographic column;
c. and (3) elution: and carrying out gradient elution by using a buffer solution B and the buffer solution A to obtain the single modified target protein mPEG-rhG-CSF.
3. The process according to claim 2, characterized in that the chromatography packing is selected from Capto MMC packing, HCX packing and Bestarose Diamond MMC packing, preferably Capto MMC packing.
4. The method as claimed in claim 2, wherein the polyethylene glycol modifier is selected from mPEG-butyraldehyde, mPEG-propionaldehyde and mPEG-succinate, and has a molecular weight of 10-50 kDa.
5. The method according to claim 2, wherein the mass ratio of the rhG-CSF protein to the polyethylene glycol modifier is 1: 2-10, preferably 1: 3-6.
6. The method of claim 2, wherein the rhG-CSF protein solution of step a is in a concentration of 2-3 mg/mL; the concentration of the polyethylene glycol modifier in the modification buffer solution in the step b is 1-5 mg/mL, preferably 2-3 mg/mL.
7. The method according to claim 2, wherein the buffer A in steps a, b and c is sodium acetate buffer containing sodium chloride, wherein the concentration of the sodium chloride is 0.1-0.8M, and the concentration of the sodium acetate is 10-50 mM; preferably, the concentration of sodium chloride is 0.2-0.5M, and the concentration of sodium acetate is 20-30 mM; the pH value of the buffer solution A is 4.0-5.0.
8. The method according to claim 2, wherein the catalyst in the step b is sodium cyanoborohydride, and the final concentration is 10-30 mM; the circulating reaction time is 4-8 hours.
9. The method according to claim 2, wherein the buffer B in step c is a sodium acetate buffer containing sodium chloride, wherein the concentration of sodium chloride is 10-50 mM, and the concentration of sodium acetate is 10-50 mM; preferably, the concentration of sodium chloride is 20-30 mM, and the concentration of sodium acetate is 20-30 mM; the pH value of the buffer solution B is 5.5-6.0.
10. The method according to claim 2, wherein the gradient elution in step c is that the buffer solution B and the buffer solution A are mixed in different ratios and are subjected to gradient elution at a flow rate of 5mL/min, the multi-modified rhG-CSF is eluted in a gradient manner by using 10% -30% of the buffer solution B and 3CV, and the single-modified rhG-CSF is eluted in a gradient manner by using 30% -80% of the buffer solution B and 10CV, so that the single-modified target protein mPEG-rhG-CSF is obtained.
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