CN114853872B - Preparation method of polyethylene glycol modified rhG-CSF - Google Patents
Preparation method of polyethylene glycol modified rhG-CSF Download PDFInfo
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- 239000002202 Polyethylene glycol Substances 0.000 title claims abstract description 47
- 229920001223 polyethylene glycol Polymers 0.000 title claims abstract description 47
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 238000010828 elution Methods 0.000 claims abstract description 64
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 51
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 51
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- 238000012986 modification Methods 0.000 claims abstract description 35
- 238000000034 method Methods 0.000 claims abstract description 26
- 238000006011 modification reaction Methods 0.000 claims abstract description 23
- 239000012071 phase Substances 0.000 claims abstract description 22
- 238000006243 chemical reaction Methods 0.000 claims abstract description 12
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- 238000006555 catalytic reaction Methods 0.000 claims abstract description 3
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- 239000011541 reaction mixture Substances 0.000 description 8
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- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- GHAZCVNUKKZTLG-UHFFFAOYSA-N N-ethyl-succinimide Natural products CCN1C(=O)CCC1=O GHAZCVNUKKZTLG-UHFFFAOYSA-N 0.000 description 1
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- 239000003102 growth factor Substances 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 230000003463 hyperproliferative effect Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000026045 iodination Effects 0.000 description 1
- 238000006192 iodination reaction Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
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- 208000004235 neutropenia Diseases 0.000 description 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
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- 231100000956 nontoxicity Toxicity 0.000 description 1
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- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical class C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- RADKZDMFGJYCBB-UHFFFAOYSA-N pyridoxal hydrochloride Natural products CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 description 1
- 229910000033 sodium borohydride Inorganic materials 0.000 description 1
- 239000012279 sodium borohydride Substances 0.000 description 1
- MNWBNISUBARLIT-UHFFFAOYSA-N sodium cyanide Chemical compound [Na+].N#[C-] MNWBNISUBARLIT-UHFFFAOYSA-N 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000011191 terminal modification Methods 0.000 description 1
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000011726 vitamin B6 Substances 0.000 description 1
- 235000019158 vitamin B6 Nutrition 0.000 description 1
- 229940011671 vitamin b6 Drugs 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/53—Colony-stimulating factor [CSF]
- C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention belongs to the field of biological medicine, and in particular 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 chromatographic packing by taking a composite chromatographic medium as a solid-phase reaction carrier, a polyethylene glycol modifier is taken as a mobile phase, modification reaction is completed under the condition of catalysis of a catalyst, and the polyethylene glycol modified protein mPEG-rhG-CSF is obtained by gradient elution after the reaction is completed. 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 mPEG modified protein is more than 91%, and the purity is more than 99.3%; meanwhile, the preparation method simplifies the modification and purification reaction operation, greatly reduces the production cost, provides a new process for realizing mPEG modification of the protein, and has good application prospect.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to a preparation method of polyethylene glycol modified rhG-CSF.
Background
The relationship between the structure and function of protein molecules studied by chemical modification methods at the end of the 50 s of the 20 th century has become a hotspot in the fields of biochemistry and molecular biology. Protein modification is intended for biomedical and biotechnological applications. In biomedical aspects, chemical modifications can reduce immunogenicity or immunoreactivity, inhibit the production of immunoglobulin E, etc.; in the field of biotechnology, enzymes can efficiently perform catalytic action in organic solvents after chemical modification, and exhibit novel catalytic properties.
The nature of the protein modifier in reacting with the protein can be largely divided into four types: (1) Acylation and related reactions, wherein the modifier can be subjected to acylation reaction with a side chain group of the protein; (2) Alkylation reaction, the modifier is characterized by having active halogen atoms, and the alkyl groups have partial positive charges due to electronegativity of the halogen atoms, so that nucleophilic groups of protein molecules are easily alkylated; (3) Oxidation and reduction reactions, such modifiers are oxidative and can oxidize side chain groups; (4) Aromatic ring substitution reaction, iodination and nitration reactions of electrophilic substitution of phenolic hydroxyl groups of amino acid residues of proteins are easy to occur at positions 3 and 5. Representative modifiers are N-ethylmaleimide, carbodiimide, carboxymethyl cellulose, polysialic acid, dextran, cyclodextrin, polyethylene glycol (PEG), and the like.
Among them, polyethylene glycol (PEG) is a linear, uncharged polymer that can be curled freely in solution, and has no toxicity, weak antigenicity and good biocompatibility. Covalent modification of proteins with it increases the in vivo circulation half-life and reduces antigenicity of the protein, increases protein solubility and alters the biological distribution of the protein in humans. Since Abuchowski, davis (J.biol. Chem.1977,252: 3578-3581.) et al reported modification of proteins with PEG for the first time in 1977, PEG has been widely used in the modification studies of protein polypeptides, and protein PEGylation technology has become one of the most effective methods for reducing the immunogenicity and improving the pharmacokinetic/pharmacodynamic properties of protein biopharmaceuticals.
At present, a plurality of PEG modified medicines are applied to clinic at home and abroad, and remarkable social and economic benefits are obtained. PEG-modified human growth hormone receptor antagonists from Pfizer company are useful for the treatment of acromegaly and hyperproliferative disorders; PEG modified interferon alpha from Hoffman-LaRoche company is used for treating hepatitis C; PEG modified vascular endothelial (cell) growth factor aptamer for treating senile macular degeneration.
Recombinant human granulocyte colony-stimulating factor (rhG-CSF) can significantly improve the severity and duration of neutropenia caused by chemotherapy, and has been widely used for treating myelosuppression caused by cancer chemotherapy since 1991, but has a plasma half-life of only 3-4 hours, requires daily administration for 7-14 days, and is disadvantageous for convenience of treatment and patient compliance. PEG modification and reconstruction of rhG-CSF can better realize the prolongation of half life, the increase of stability and the reduction of immunogenicity.
PEG modification of rhG-CSF can be classified into N-terminal, C-terminal and on-chain modification, but the difficulty of C-terminal modification is great; the mature rhG-CSF protein has four Lys residues and a sulfhydryl group of 18-site free cysteine, and cannot be subjected to site-directed single modification; 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 groups to N-terminal alpha amino groups.
Initially, site-directed modification was achieved by controlling the pH of the modification reaction. However, this modification is not optimal for rhG-CSF because, in addition to the alpha-amino group at the N-terminus, 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 linked to the hydroxyl groups of serine, tyrosine or threonine residues by ester linkages. The modified heterogeneity, isomers and the like are not easy to solve, and the subsequent purification, clinical application and the like of the rhG-CSF are not facilitated. Specific site-directed modifications, such as subtilases, glutaminase, etc., can also be achieved by certain enzymes, but this undoubtedly increases the cost and difficulty of downstream processing, which is more detrimental to scale-up production.
CN200680021159 discloses a novel isomer of human granulocyte colony stimulating factor obtained by adding amino acids to both ends of the polypeptide chain of human granulocyte colony stimulating factor by substituting or adding cysteines at specific sites and simultaneously pegylating at the addition sites. The method has complex process and inevitably brings about a plurality of related problems of unnatural amino acid.
CN201410272925 discloses a preparation method of polyethylene glycol recombinant human granulocyte stimulating factor, which is also a classical modification method of PEG-rhG-CSF which has been marketed at home and abroad. Similar liquid phase modification methods are adopted for the new Rui Bai of Qilu pharmacy and the Jinyou Li of Shi medicine group and the Shen Li Dai of Lu nan pharmacy. However, the method has low single modification rate, more unmodified and multi-modified, brings difficulty to subsequent purification work, requires more modifier and increases production cost.
CN202011019986 discloses a preparation method of protein site-directed polyethylene glycol modification, which realizes specific coupling of PEG and N-terminal alpha-amino by "blocking" lysine-epsilon amino on rhG-CSF molecule by ion exchange chromatography, and simultaneously realizes purification of PEG-rhG-CSF, and the method realizes higher modification rate by adding vitamin B6 and coenzyme during modification. However, the method increases the modification cost, increases the pressure of the impurities related to the process and the subsequent purification, has no advantage in commercialized scale-up of the post-production cost, and has the possibility of modification of the sulfhydryl group of the 18-cysteine.
Therefore, the modification method with good universality and simple process 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 prior PEG modification process of rhG-CSF and provides a preparation method of polyethylene glycol modified rhG-CSF.
The invention aims at realizing the following technical scheme: the rhG-CSF protein is loaded and adsorbed on a chromatographic packing by taking a composite chromatographic medium as a solid-phase reaction carrier, a polyethylene glycol modifier is taken as a mobile phase, modification reaction is completed under the condition of catalysis of a catalyst, and the polyethylene glycol modified protein mPEG-rhG-CSF is obtained by gradient elution after the reaction is completed.
A preparation method of polyethylene glycol modified rhG-CSF comprises the following steps:
a. and (3) column loading: dissolving rhG-CSF protein in buffer A to obtain rhG-CSF protein solution, and loading the rhG-CSF protein solution onto chromatographic packing;
b. modification reaction: dissolving polyethylene glycol modifier in the buffer solution A, adding a catalyst to obtain a modified buffer solution which is taken as a mobile phase and circularly flows through a chromatographic column;
c. eluting: eluting with buffer B and buffer A to obtain single modified target protein mPEG-rhG-CSF.
Preferably, the chromatography packing material 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 in step a has a concentration of 2-3 mg/mL.
In a preferred embodiment, the polyethylene glycol modifier of step b is selected from the group consisting of mPEG-butyraldehyde, mPEG-propionaldehyde, mPEG-succinate and the like, and has a molecular weight of from 10 kDa to 50kDa.
In a preferred embodiment, the concentration of polyethylene glycol modifier in the modification buffer of step b is 1 to 5mg/mL, preferably 2 to 3mg/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 of 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 to 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 A is 20 to 30mM.
Preferably, the pH value of the buffer solution A is 4.0-5.0.
In a preferred embodiment, the catalyst of step b is sodium cyanoborohydride in a final concentration of 10 to 30mM.
In a preferred embodiment, the cyclic reaction time of step b is from 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 of 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 B is 20 to 30mM.
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 to 30mM.
Preferably, the pH of the buffer B is 5.5-6.0.
In a preferred embodiment, polyethylene glycol modified rhG-CSF is obtained by gradient elution with different ratios of buffer B to buffer A.
Preferably, the gradient elution method comprises the steps of carrying out gradient elution on multi-modified rhG-CSF by using 10% -30% of buffer B and 3CV at a flow rate of 5mL/min, and then carrying out gradient elution on single-modified rhG-CSF by using 30% -80% of buffer B and 10CV to obtain the single-modified target protein mPEG-rhG-CSF.
In a preferred embodiment, a method for preparing polyethylene glycol modified rhG-CSF comprises the steps of:
a. loading: loading a proper amount of chromatographic packing (Capto MMC packing, HCX packing or Bestarose Diamond MMC packing) into a chromatographic column, and balancing the chromatographic column by using a 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 value is adjusted to be 4.0-5.0); preparing rhG-CSF into a solution with the concentration of 2-3mg/mL by using the buffer solution A, loading the solution to be adsorbed on a chromatographic column, and then flushing the chromatographic column by using the buffer solution A until no protein passes out.
b. Modification reaction: according to the mol ratio of rhG-CSF: PEG modifier=1:2-10 ratio weighing modifier, using the buffer A (sodium acetate buffer containing sodium chloride, wherein sodium acetate is 10-50 mM, sodium chloride is 0.1-0.8M, pH is adjusted to 4.0-5.0) to prepare 1-5 mg/mL solution, adding 10-30 mM sodium cyanoborohydride, pH is adjusted to 4.0-5.0, obtaining modified buffer and taking the modified buffer as mobile phase, circulating through a chromatographic column, controlling the circulation reaction time to be 4-8 h, maintaining the column temperature at room temperature, and performing light-proof treatment on the chromatographic column; after the end of the cycle, the column was further washed with buffer A, above, and 5 column volumes were washed at a flow rate of 10 mL/min.
c. Eluting: the buffer B (sodium acetate buffer containing sodium chloride, wherein the sodium acetate is 10-50 mM, the sodium chloride is 10-50 mM, and the pH is adjusted to be 5.5-6.0) and the buffer A (sodium acetate buffer 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) are used for gradient elution according to different proportions, and the method comprises the following steps: the flow rate is 5mL/min, 10-30% of buffer B and 3CV are used for gradient elution of multi-modified rhG-CSF, and then 30-80% of buffer B and 10CV are used for gradient elution of single-modified rhG-CSF, so that the single-modified target protein mPEG-rhG-CSF is obtained.
The solution for pH adjustment in the invention adopts an aqueous solution of acid or alkali which is conventional in the art, and acetic acid solution and sodium hydroxide solution are preferred in order to reduce the introduction of other substances in the reaction system.
Compared with the prior art, the invention has the following advantages and remarkable progress:
(1) Taking a composite chromatographic medium as a solid phase reaction carrier, obtaining the mPEG-rhG-CSF modified protein with high single modification rate under the low pH and high salt state, wherein 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 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 operation, greatly reduces the production cost, and provides a new process for realizing the PEG modification of the protein.
Detailed Description
The advantages of the invention will now be further described by the following examples, which are given for illustrative purposes only and do not limit the scope of the invention, while variations and modifications apparent to those skilled in the art in light of the present invention are included within the scope of the invention.
Example 1
a. Loading: taking a proper amount of chromatographic packing Capto MMC, loading the MMC into an XK26 chromatographic column with the column height of 20cm, and connecting the MMC to a protein purification system; the chromatographic column is balanced by buffer A (sodium acetate buffer containing sodium chloride, wherein sodium acetate is 25mM, sodium chloride is 400mM, and pH is adjusted to 4.5), the volume of the column is 3-5, and the flow rate is 10mL/min; preparing rhG-CSF into a solution with the concentration of 2mg/mL by using the buffer solution A, loading the solution into a chromatographic column with the loading capacity of 20mg/mL, and flushing the column with the buffer solution A for 3-5 column volumes after the loading is completed until no protein passes out.
b. Modification reaction: according to the mol ratio of rhG-CSF: mPEG modifier=1:4, weighting mPEG-butyraldehyde (20 kDa), preparing a solution with the concentration of 2mg/mL by using the buffer solution A, adding sodium cyanoborohydride with the final concentration of 20mM, regulating the pH value to 4.5, obtaining a modified buffer solution which is taken as a mobile phase, circularly flowing through a chromatographic column, controlling the circulation reaction time to be 5h, maintaining the column temperature at room temperature, and simultaneously, carrying out light-shielding treatment on the chromatographic column; after the end of the cycle, the column was further washed with buffer A, above, and 5 column volumes were washed at a flow rate of 10 mL/min.
c. Eluting: the gradient elution is carried out by using a buffer B (sodium acetate buffer containing sodium chloride, wherein the sodium acetate is 25mM, the sodium chloride is 20mM, and the pH is 5.8) and the buffer A in different proportions, and the specific operation is as follows: the flow rate is 5mL/min, 10% -30% of buffer B and 3CV are used for gradient elution of multi-modified rhG-CSF, then 30% -80% of buffer B and 10CV are used for gradient elution of single-modified rhG-CSF, and the single-modified target protein mPEG-rhG-CSF is obtained, the purity is detected by sampling, and the yield is calculated, and the result is shown in Table 1.
Example 2
a. Loading: taking a proper amount of chromatographic packing Capto MMC, loading the MMC into an XK26 chromatographic column with the column height of 20cm, and connecting the MMC 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 20mM, the sodium chloride is 400mM, and the pH is adjusted to 4), wherein the volume of the chromatographic column is 3-5, and the flow rate is 10mL/min; preparing rhG-CSF into a solution with the concentration of 3mg/mL by using the buffer solution A, loading the solution into a chromatographic column with the loading capacity of 20mg/mL, and flushing the column volume for 3-5 times by using the buffer solution A until no protein passes out.
b. Modification reaction: according to the mol ratio of rhG-CSF: mPEG modifier=1:2, weighting mPEG-propionaldehyde (20 kDa), 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, obtaining a modified buffer solution which is used as a mobile phase and circularly flows through a chromatographic column, the flow rate is 2-3 mL/min, the circulation reaction time is controlled to be 5h, the column temperature is maintained at room temperature, and the chromatographic column is simultaneously subjected to light-proof treatment; after the end of the cycle, the column was further washed with buffer A, above, and 5 column volumes were washed at a flow rate of 10 mL/min.
c. Eluting: the gradient elution is carried out by using a buffer B (sodium acetate buffer containing sodium chloride, wherein the sodium acetate is 30mM, the sodium chloride is 20mM, and the pH is 6) and the buffer A according to different proportions, and the specific operation is as follows: the flow rate is 5mL/min, 10% -30% of buffer B and 3CV are used for gradient elution of multi-modified rhG-CSF, then 30% -80% of buffer B and 10CV are used for gradient elution of single-modified rhG-CSF, and the single-modified target protein PEG-rhG-CSF is obtained, the purity is detected by sampling, and the yield is calculated, and the result is shown in Table 1.
Example 3
a. Loading: taking a proper amount of chromatographic packing Capto MMC, loading the MMC into an XK26 chromatographic column with the column height of 20cm, and connecting the MMC to a protein purification system; the chromatographic column is balanced by buffer A (sodium acetate buffer containing sodium chloride, wherein sodium acetate is 30mM, sodium chloride is 400mM, pH is adjusted to 5), the column volume is 3-5, and the flow rate is 10mL/min; preparing rhG-CSF into a solution with the concentration of 2mg/mL by using the buffer solution A, loading the solution into a chromatographic column with the loading capacity of 20mg/mL, and flushing the column volume by 3-5 columns by using the buffer solution A until no protein passes out.
b. Modification reaction: according to the mol ratio of rhG-CSF: mPEG modifier=1:10, weighting mPEG-succinate (20 kDa), preparing a solution with the concentration of 1mg/mL by using the buffer solution A, adding sodium cyanoborohydride with the final concentration of 10mM, regulating the pH value to 5, obtaining a modified buffer solution which is taken as a mobile phase, circularly flowing through a chromatographic column, controlling the circulation reaction time to be 5h, maintaining the column temperature at room temperature, and simultaneously, performing light-shielding treatment on the chromatographic column; after the end of the cycle, the column was further washed with buffer A, above, and 5 column volumes were washed at a flow rate of 10 mL/min.
c. Eluting: the gradient elution is carried out by using a buffer B (sodium acetate buffer containing sodium chloride, wherein the sodium acetate is 20mM, the sodium chloride is 20mM, the pH is 5.5) and the buffer A in different proportions, and the specific operation is as follows: the flow rate is 5mL/min, 10% -30% of buffer B and 3CV are used for gradient elution of multi-modified rhG-CSF, then 30% -80% of buffer B and 10CV are used for gradient elution of single-modified rhG-CSF, and the single-modified target protein PEG-rhG-CSF is obtained, the purity is detected by sampling, and the yield is calculated, and the result is shown in Table 1.
Example 4
a. Loading: taking a proper amount of chromatographic packing Capto MMC, loading the MMC into an XK26 chromatographic column with the column height of 20cm, and connecting the MMC to a protein purification system; the chromatographic column is balanced by buffer A (sodium acetate buffer containing sodium chloride, wherein the sodium acetate is 10mM, the sodium chloride is 100mM, and the pH is adjusted to 4.5), the column volume is 3-5, and the flow rate is 10mL/min; preparing rhG-CSF into a solution with the concentration of 3mg/mL by using the buffer solution A, loading the solution into a chromatographic column with the loading capacity of 20mg/mL, and flushing the column volume for 3-5 times by using the buffer solution A until no protein passes out.
b. Modification reaction: according to the mol ratio of rhG-CSF: mPEG modifier=1:3, weighting mPEG-butyraldehyde (20 kDa), preparing a solution with the concentration of 5mg/mL by using the buffer solution A, adding sodium cyanoborohydride with the final concentration of 30mM, regulating the pH value to 4.5, obtaining a modified buffer solution which is taken as a mobile phase, circularly flowing through a chromatographic column, controlling the circulation reaction time to be 4h, maintaining the column temperature at room temperature, and simultaneously, carrying out light-shielding treatment on the chromatographic column; after the end of the cycle, the column was further washed with buffer A, above, and 5 column volumes were washed at a flow rate of 10 mL/min.
c. Eluting: the gradient elution is carried out by using a buffer B (sodium acetate buffer containing sodium chloride, wherein the sodium acetate is 10mM, the sodium chloride is 50mM, and the pH is 5.8) and the buffer A according to different proportions, and the specific operation is as follows: the flow rate is 5mL/min, 10% -30% of buffer B and 3CV are used for gradient elution of multi-modified rhG-CSF, then 30% -80% of buffer B and 10CV are used for gradient elution of single-modified rhG-CSF, and the single-modified target protein PEG-rhG-CSF is obtained, the purity is detected by sampling, and the yield is calculated, and the result is shown in Table 1.
Example 5
a. Loading: taking a proper amount of chromatographic packing Capto MMC, loading the MMC into an XK26 chromatographic column with the column height of 20cm, and connecting the MMC to a protein purification system; the chromatographic column is balanced by buffer A (sodium acetate buffer containing sodium chloride, wherein the sodium acetate is 50mM, the sodium chloride is 800mM, and the pH is adjusted to 4.5), the column volume is 3-5, and the flow rate is 10mL/min; preparing rhG-CSF into a solution with the concentration of 2mg/mL by using the buffer solution A, loading the solution into a chromatographic column with the loading capacity of 20mg/mL, and flushing the column volume by 3-5 columns by using the buffer solution A until no protein passes out.
b. Modification reaction: according to the mol ratio of rhG-CSF: mPEG modifier=1:6, weighting mPEG-butyraldehyde (20 kDa), preparing a solution with the concentration of 2mg/mL by using the buffer solution A, adding sodium cyanoborohydride with the final concentration of 30mM, regulating the pH value to 4.5, obtaining a modified buffer solution which is taken as a mobile phase, circularly flowing through a chromatographic column, controlling the circulation reaction time to be 6h, maintaining the column temperature at room temperature, and simultaneously, carrying out light-shielding treatment on the chromatographic column; after the end of the cycle, the column was further washed with buffer A, above, and 5 column volumes were washed at a flow rate of 10 mL/min.
c. Eluting: the gradient elution is carried out by using a buffer B (sodium acetate buffer containing sodium chloride, wherein the sodium acetate is 50mM, the sodium chloride is 10mM, and the pH is 5.8) and the buffer A according to different proportions, and the specific operation is as follows: the flow rate is 5mL/min, 10% -30% of buffer B and 3CV are used for gradient elution of multi-modified rhG-CSF, then 30% -80% of buffer B and 10CV are used for gradient elution of single-modified rhG-CSF, and the single-modified target protein PEG-rhG-CSF is obtained, the purity is detected by sampling, and the yield is calculated, and the result is shown in Table 1.
Example 6
a. Loading: taking a proper amount of chromatographic packing Capto MMC, loading the MMC into an XK26 chromatographic column with the column height of 20cm, and connecting the MMC to a protein purification system; the chromatographic column is balanced by buffer A (sodium acetate buffer containing sodium chloride, wherein sodium acetate is 25mM, sodium chloride is 500mM, and pH is adjusted to 4.5), the volume of the column is 3-5, and the flow rate is 10mL/min; preparing rhG-CSF into a solution with the concentration of 2mg/mL by using the buffer solution A, loading the solution into a chromatographic column with the loading capacity of 20mg/mL, and flushing the column volume by 3-5 columns by using the buffer solution A until no protein passes out.
b. Modification reaction: according to the mol ratio of rhG-CSF: mPEG modifier=1:4, weighting mPEG-butyraldehyde (20 kDa), preparing a solution with the concentration of 2mg/mL by using the buffer solution A, adding sodium cyanoborohydride with the final concentration of 20mM, regulating the pH value to 4.5, obtaining a modified buffer solution which is taken as a mobile phase, circularly flowing through a chromatographic column, controlling the circulation reaction time to be 8h, maintaining the column temperature at room temperature, and simultaneously, carrying out light-shielding treatment on the chromatographic column; after the end of the cycle, the column was further washed with buffer A, above, and 5 column volumes were washed at a flow rate of 10 mL/min.
c. Eluting: the gradient elution is carried out by using a buffer B (sodium acetate buffer containing sodium chloride, wherein the sodium acetate is 25mM, the sodium chloride is 30mM, and the pH is 5.8) and the buffer A according to different proportions, and the specific operation is as follows: the flow rate is 5mL/min, 10% -30% of buffer B and 3CV are used for gradient elution of multi-modified rhG-CSF, then 30% -80% of buffer B and 10CV are used for gradient elution of single-modified rhG-CSF, and the single-modified target protein PEG-rhG-CSF is obtained, the purity is detected by sampling, and the yield is calculated, and the result is shown in Table 1.
Example 7
a. Loading: taking a proper amount of chromatographic packingHCX, loaded in XK26 chromatography column, 20cm high, connected to protein purification system; the chromatographic column is balanced by buffer A (sodium acetate buffer containing sodium chloride, wherein sodium acetate is 25mM, sodium chloride is 400mM, and pH is adjusted to 4.5), the volume of the column is 3-5, and the flow rate is 10mL/min; preparing rhG-CSF into a solution with the concentration of 2mg/mL by using the buffer solution A, loading the solution into a chromatographic column with the loading capacity of 20mg/mL, and flushing the column volume by 3-5 columns by using the buffer solution A until no protein passes out.
b. Modification reaction: according to the mol ratio of rhG-CSF: mPEG modifier=1:4, weighting mPEG-butyraldehyde (20 kDa), preparing a solution with the concentration of 2mg/mL by using the buffer solution A, adding sodium cyanoborohydride with the final concentration of 20mM, regulating the pH value to 4.5, obtaining a modified buffer solution which is taken as a mobile phase, circularly flowing through a chromatographic column, controlling the circulation reaction time to be 6h, maintaining the column temperature at room temperature, and simultaneously, carrying out light-shielding treatment on the chromatographic column; after the end of the cycle, the column was further washed with buffer A, above, and 5 column volumes were washed at a flow rate of 10 mL/min.
c. Eluting: the gradient elution is carried out by using a buffer B (sodium acetate buffer containing sodium chloride, wherein the sodium acetate is 25mM, the sodium chloride is 20mM, and the pH is 5.8) and the buffer A in different proportions, and the specific operation is as follows: the flow rate is 5mL/min, 10% -30% of buffer B and 3CV are used for gradient elution of multi-modified rhG-CSF, then 30% -80% of buffer B and 10CV are used for gradient elution of single-modified rhG-CSF, and the single-modified target protein PEG-rhG-CSF is obtained, the purity is detected by sampling, and the yield is calculated, and the result is shown in Table 1.
Example 8
a. Loading: taking a proper amount of chromatographic packing Capto MMC, loading the MMC into an XK26 chromatographic column with the column height of 20cm, and connecting the MMC to a protein purification system; the chromatographic column is balanced by buffer A (sodium acetate buffer containing sodium chloride, wherein the sodium acetate is 100mM, the sodium chloride is 400mM, and the pH is adjusted to 4.5), the column volume is 3-5, and the flow rate is 10mL/min; preparing rhG-CSF into a solution with the concentration of 2mg/mL by using the buffer solution A, loading the solution into a chromatographic column with the loading capacity of 20mg/mL, and flushing the column volume by 3-5 columns by using the buffer solution A until no protein passes out.
b. Modification reaction: according to the mol ratio of rhG-CSF: mPEG modifier=1:20 ratio weighting mPEG-butyraldehyde (20 kDa), using the buffer solution a to prepare a solution with the concentration of 10mg/mL, adding cyano sodium borohydride with the final concentration of 20mM, adjusting the pH value to 4.5, obtaining modified buffer solution, taking the modified buffer solution as a mobile phase, circulating through a chromatographic column, controlling the flow rate to be 2-3 mL/min, controlling the time to be 7h, maintaining the column temperature at room temperature, and simultaneously, carrying out light-proof treatment on the chromatographic column; after the end of the cycle, the column was further washed with buffer A, above, and 5 column volumes were washed at a flow rate of 10 mL/min.
c. Eluting: the gradient elution is carried out by using a buffer B (sodium acetate buffer containing sodium chloride, wherein the sodium acetate is 100mM, the sodium chloride is 20mM, and the pH is 5.8) and the buffer A in different proportions, and the specific operation is as follows: the flow rate is 5mL/min, 10% -30% of buffer B and 3CV are used for gradient elution of multi-modified rhG-CSF, then 30% -80% of buffer B and 10CV are used for gradient elution of single-modified rhG-CSF, and the single-modified target protein PEG-rhG-CSF is obtained, the purity is detected by sampling, and the yield is calculated, and the result is shown in Table 1.
Example 9
a. Loading: taking a proper amount of chromatographic packing Capto MMC, loading the MMC into an XK26 chromatographic column with the column height of 20cm, and connecting the MMC to a protein purification system; the chromatographic column is equilibrated with buffer A (sodium acetate buffer containing sodium chloride, wherein sodium acetate is 25mM, sodium chloride is 400mM, pH3 is adjusted), 3 to 5 column volumes, and the flow rate is 10mL/min; preparing rhG-CSF into a solution with the concentration of 2mg/mL by using the buffer solution A, loading the solution into a chromatographic column with the loading capacity of 20mg/mL, and flushing the column volume by 3-5 columns by using the buffer solution A until no protein passes out.
b. Modification reaction: according to the mol ratio of rhG-CSF: mPEG modifier=1:4, weighting mPEG-butyraldehyde (20 kDa), preparing a solution with the concentration of 2mg/mL by using the buffer solution A, adding sodium cyanoborohydride with the final concentration of 20mM, regulating the pH value to 3, obtaining a modified buffer solution which is taken as a mobile phase, circularly flowing through a chromatographic column, controlling the circulation reaction time to be 5h, maintaining the column temperature at room temperature, and simultaneously, performing light-proof treatment on the chromatographic column; after the end of the cycle, the column was further washed with buffer A, above, and 5 column volumes were washed at a flow rate of 10 mL/min.
c. Eluting: the gradient elution is carried out by using a buffer B (sodium acetate buffer containing sodium chloride, wherein the sodium acetate is 25mM, the sodium chloride is 20mM, and the pH is 5) and the buffer A according to different proportions, and the specific operation is as follows: the flow rate is 5mL/min, 10% -30% of buffer B and 3CV are used for gradient elution of multi-modified rhG-CSF, then 30% -80% of buffer B and 10CV are used for gradient elution of single-modified rhG-CSF, and the single-modified target protein PEG-rhG-CSF is obtained, the purity is detected by sampling, and the yield is calculated, and the result is shown in Table 1.
Example 10
a. Loading: taking a proper amount of chromatographic packing Capto MMC, loading the MMC into an XK26 chromatographic column with the column height of 20cm, and connecting the MMC to a protein purification system; the chromatographic column is equilibrated with buffer A (sodium acetate buffer containing sodium chloride, wherein sodium acetate is 25mM, sodium chloride is 400mM, pH6 is adjusted) for 3-5 column volumes at a flow rate of 10mL/min; preparing rhG-CSF into a solution with the concentration of 2mg/mL by using the buffer solution A, loading the solution into a chromatographic column with the loading capacity of 20mg/mL, and flushing the column volume by 3-5 columns by using the buffer solution A until no protein passes out.
b. Modification reaction: according to the mol ratio of rhG-CSF: mPEG modifier=1:4, weighting mPEG-butyraldehyde (20 kDa), preparing a solution with the concentration of 2mg/mL by using the buffer solution A, adding sodium cyanoborohydride with the final concentration of 20mM, regulating the pH value to 6, obtaining a modified buffer solution which is taken as a mobile phase, circularly flowing through a chromatographic column, controlling the circulation reaction time to be 5h, maintaining the column temperature at room temperature, and simultaneously, performing light-proof treatment on the chromatographic column; after the end of the cycle, the column was further washed with buffer A, above, and 5 column volumes were washed at a flow rate of 10 mL/min.
c. Eluting: the gradient elution is carried out by using a buffer B (sodium acetate buffer containing sodium chloride, wherein the sodium acetate is 25mM, the sodium chloride is 20mM, and the pH is 6.3) and the buffer A according to different proportions, and the specific operation is as follows: the flow rate is 5mL/min, 10% -30% of buffer B and 3CV are used for gradient elution of multi-modified rhG-CSF, then 30% -80% of buffer B and 10CV are used for gradient elution of single-modified rhG-CSF, and the single-modified target protein PEG-rhG-CSF is obtained, the purity is detected by sampling, and the yield is calculated, and the result is shown in Table 1.
Example 11
a. Loading: taking a proper amount of chromatographic packing Capto MMC, loading the MMC into an XK26 chromatographic column with the column height of 20cm, and connecting the MMC to a protein purification system; the chromatographic column is balanced by buffer A (sodium acetate buffer containing sodium chloride, wherein sodium acetate is 25mM, sodium chloride is 1.2M, and pH is adjusted to 4.5), the volume of the column is 3-5, and the flow rate is 10mL/min; preparing rhG-CSF into a solution with the concentration of 2mg/mL by using the buffer solution A, loading the solution into a chromatographic column with the loading capacity of 20mg/mL, and flushing the column volume by 3-5 columns by using the buffer solution A until no protein passes out.
b. Modification reaction: according to the mol ratio of rhG-CSF: mPEG modifier=1:4, weighting mPEG-butyraldehyde (20 kDa), preparing a solution with the concentration of 2mg/mL by using the buffer solution A, adding sodium cyanoborohydride with the final concentration of 20mM, regulating the pH value to 4.5, obtaining a modified buffer solution which is taken as a mobile phase, circularly flowing through a chromatographic column, controlling the circulation reaction time to be 5h, maintaining the column temperature at room temperature, and simultaneously, carrying out light-shielding treatment on the chromatographic column; after the end of the cycle, the column was further washed with buffer A, above, and 5 column volumes were washed at a flow rate of 10 mL/min.
c. Eluting: the gradient elution is carried out by using a buffer B (sodium acetate buffer containing sodium chloride, wherein the sodium acetate is 25mM, the sodium chloride is 5mM, and the pH is 5.8) and the buffer A according to different proportions, and the specific operation is as follows: the flow rate is 5mL/min, 10% -30% of buffer B and 3CV are used for gradient elution of multi-modified rhG-CSF, then 30% -80% of buffer B and 10CV are used for gradient elution of single-modified rhG-CSF, and the single-modified target protein PEG-rhG-CSF is obtained, the purity is detected by sampling, and the yield is calculated, and the result is shown in Table 1.
Example 12
a. Loading: taking a proper amount of chromatographic packing Capto MMC, loading the MMC into an XK26 chromatographic column with the column height of 20cm, and connecting the MMC to a protein purification system; the chromatographic column is balanced by buffer A (sodium acetate buffer containing sodium chloride, wherein sodium acetate is 25mM, sodium chloride is 50mM, and pH is adjusted to 4.5), the volume of the column is 3-5, and the flow rate is 10mL/min; preparing rhG-CSF into a solution with the concentration of 2mg/mL by using the buffer solution A, loading the solution into a chromatographic column with the loading capacity of 20mg/mL, and flushing the column volume by 3-5 columns by using the buffer solution A until no protein passes out.
b. Modification reaction: according to the mol ratio of rhG-CSF: mPEG modifier=1:4, weighting mPEG-butyraldehyde (20 kDa), preparing a solution with the concentration of 2mg/mL by using the buffer solution A, adding sodium cyanoborohydride with the final concentration of 20mM, regulating the pH value to 4.5, obtaining a modified buffer solution which is taken as a mobile phase, circularly flowing through a chromatographic column, controlling the circulation reaction time to be 5h, maintaining the column temperature at room temperature, and simultaneously, carrying out light-shielding treatment on the chromatographic column; after the end of the cycle, the column was further washed with buffer A, above, and 5 column volumes were washed at a flow rate of 10 mL/min.
c. Eluting: the buffer B (sodium acetate buffer containing sodium chloride, wherein the concentration of sodium acetate is 25mM, the concentration of sodium chloride is 100mM, and the pH is 5.8) and the buffer A are used for gradient elution, and the specific operation is as follows: the flow rate is 5mL/min, 10% -30% of buffer B and 3CV are used for gradient elution of multi-modified rhG-CSF, then 30% -80% of buffer B and 10CV are used for gradient elution of single-modified rhG-CSF, and the single-modified target protein PEG-rhG-CSF is obtained, the purity is detected by sampling, and the yield is calculated, and the result is shown in Table 1.
Comparative example 1
a. Loading: taking a proper amount of chromatographic packing Phenyl Sepharose 6Fast Flow (high) and loading the chromatographic packing into an XK26 chromatographic column, wherein the column height is 20cm, and connecting the chromatographic column to a protein purification system; the chromatographic column is balanced by buffer A (sodium acetate buffer containing sodium chloride, wherein sodium acetate is 25mM, sodium chloride is 400mM, and pH is adjusted to 4.5), the volume of the column is 3-5, and the flow rate is 10mL/min; preparing rhG-CSF into a solution with the concentration of 2mg/mL by using the buffer solution A, loading the solution into a chromatographic column with the loading capacity of 20mg/mL, and flushing the column volume by 3-5 columns by using the buffer solution A until no protein passes out.
b. Modification reaction: according to the mol ratio of rhG-CSF: mPEG modifier=1:4, weighting mPEG-butyraldehyde (20 kDa), preparing a solution with the concentration of 2mg/mL by using the buffer solution A, adding sodium cyanoborohydride with the final concentration of 20mM, regulating the pH value to 4.5, obtaining a modified buffer solution which is taken as a mobile phase, circularly flowing through a chromatographic column, controlling the circulation reaction time to be 6h, maintaining the column temperature at room temperature, and simultaneously, carrying out light-shielding treatment on the chromatographic column; after the end of the cycle, the column was further washed with buffer A, above, and 5 column volumes were washed at a flow rate of 10 mL/min.
c. Eluting: the gradient elution is carried out by using a buffer B (sodium acetate buffer containing sodium chloride, wherein the sodium acetate is 25mM, the sodium chloride is 20mM, and the pH is 5.8) and the buffer A in different proportions, and the specific operation is as follows: the flow rate is 5mL/min, 10% -30% of buffer B and 3CV are used for gradient elution of multi-modified rhG-CSF, then 30% -80% of buffer B and 10CV are used for gradient elution of single-modified rhG-CSF, and the single-modified target protein PEG-rhG-CSF is obtained, the purity is detected by sampling, and the yield is calculated, and the result is shown in Table 1.
Comparative example 2
a. Loading: taking a proper amount of chromatographic packing Generik MC60-SP, loading the chromatographic packing into an XK26 chromatographic column, wherein the column height is 20cm, and connecting the chromatographic column to a protein purification system; the chromatographic column is balanced by buffer A (sodium acetate buffer containing sodium chloride, wherein sodium acetate is 25mM, sodium chloride is 400mM, and pH is adjusted to 4.5), the volume of the column is 3-5, and the flow rate is 10mL/min; preparing rhG-CSF into a solution with the concentration of 2mg/mL by using the buffer solution A, loading the solution into a chromatographic column with the loading capacity of 20mg/mL, and flushing the column volume by 3-5 columns by using the buffer solution A until no protein passes out.
b. Modification reaction: according to the mol ratio of rhG-CSF: mPEG modifier=1:4, weighting mPEG-butyraldehyde (20 kDa), preparing a solution with the concentration of 2mg/mL by using the buffer solution A, adding sodium cyanoborohydride with the final concentration of 20mM, regulating the pH value to 4.5, obtaining a modified buffer solution which is taken as a mobile phase, circularly flowing through a chromatographic column, controlling the circulation reaction time to be 5h, maintaining the column temperature at room temperature, and simultaneously, carrying out light-shielding treatment on the chromatographic column; after the end of the cycle, the column was further washed with buffer A, above, and 5 column volumes were washed at a flow rate of 10 mL/min.
c. Eluting: the gradient elution is carried out by using a buffer B (sodium acetate buffer containing sodium chloride, wherein the sodium acetate is 25mM, the sodium chloride is 20mM, and the pH is 5.8) and the buffer A in different proportions, and the specific operation is as follows: the flow rate is 5mL/min, 10% -30% of buffer B and 3CV are used for gradient elution of multi-modified rhG-CSF, then 30% -80% of buffer B and 10CV are used for gradient elution of single-modified rhG-CSF, and the single-modified target protein PEG-rhG-CSF is obtained, the purity is detected by sampling, and the yield is calculated, and the result is shown in Table 1.
Comparative example 3
a. Loading: taking a proper amount of chromatographic packing Capto MMC, loading the MMC into an XK26 chromatographic column with the column height of 20cm, and connecting the MMC to a protein purification system; the chromatographic column is balanced by buffer A (sodium acetate buffer containing sodium chloride, wherein sodium acetate is 25mM, sodium chloride is 400mM, and pH is adjusted to 4.5), the volume of the column is 3-5, and the flow rate is 10mL/min; preparing rhG-CSF into a solution with the concentration of 2mg/mL by using the buffer solution A, loading the solution into a chromatographic column with the loading capacity of 20mg/mL, and flushing the column volume by 3-5 columns by using the buffer solution A until no protein passes out.
b. Modification reaction: according to the mol ratio of rhG-CSF: mPEG modifier=1:4, weighting mPEG-butyraldehyde (20 kDa), preparing a solution with the concentration of 2mg/mL by using the buffer solution A, adding sodium cyanoborohydride with the final concentration of 20mM, regulating the pH value to 4.5, obtaining a modified buffer solution which is taken as a mobile phase, circularly flowing through a chromatographic column, controlling the circulation reaction time to be 5h, maintaining the column temperature at room temperature, and simultaneously, carrying out light-shielding treatment on the chromatographic column; after the end of the cycle, the column was further washed with buffer A, above, and 5 column volumes were washed at a flow rate of 10 mL/min.
c. Eluting: the elution was carried out with buffer B (sodium acetate buffer containing sodium chloride, wherein sodium acetate is 25mM, sodium chloride is 20mM, pH 5.8) and buffer A described above, at a flow rate of 5mL/min, and the specific procedure was: the multi-modified rhG-CSF is eluted by 30% buffer B and 3CV, then the single-modified rhG-CSF is eluted by 50% buffer B and 10CV, and the single-modified target protein PEG-rhG-CSF is obtained, the purity is detected by sampling, and the yield is calculated, and the result is shown in Table 1.
Comparative example 4
rhG-CSF was formulated in buffer A (sodium acetate buffer containing sodium chloride, wherein sodium acetate is 25mM, sodium chloride is 400mM, pH 4.5) to a concentration of 2mg/mL, at a molar ratio of rhG-CSF: mPEG modifier = 1:4 mPEG-butyraldehyde (20 kDa) was added, dissolved with stirring, sodium cyanoborohydride was added to a final concentration of 20mM and the reaction mixture was stirred for 5h. Taking a proper amount of chromatographic packing Capto MMC, loading the MMC into an XK26 chromatographic column with the column height of 20cm, and connecting the MMC to a protein purification system; balancing the chromatographic column by using the buffer solution A, wherein the volume of the chromatographic column is 3-5, and the flow rate is 10mL/min; the sample-loading modification liquid is circulated according to the loading capacity of 20 g/L; finally, flushing 3-5 column volumes by using the buffer solution A;
the gradient elution is carried out by using a buffer B (sodium acetate buffer containing sodium chloride, wherein the sodium acetate is 25mM, the sodium chloride is 20mM, and the pH is 5.8) and the buffer A according to different proportions, and the specific operation is as follows: the flow rate is 5mL/min, 10% -30% of buffer B and 3CV are used for gradient elution of multi-modified rhG-CSF, then 30% -80% of buffer B and 10CV are used for gradient elution of single-modified rhG-CSF, and the single-modified target protein PEG-rhG-CSF is obtained, the purity is detected by sampling, and the yield is calculated, and the result is shown in Table 1.
Comparative example 5
1L of mother solution of recombinant human granulocyte stimulating factor (containing 1g of rhG-CSF, 20mM NaAc and 50mM NaCl) is taken, cooled to 4 ℃, and 0.5M/L NaOH solution is added to adjust the pH to 6. 11g of methoxy-polyethylene glycol-propionaldehyde (mPEG-PAL, molecular weight about 20 KD) was added, dissolved by stirring, sodium cyanoborohydride (NaBH 3 CN) was added and the final concentration of sodium cyanoborohydride was 15mM/L, and the reaction mixture was stirred at a low speed for 15 hours; the pH of the reaction mixture was adjusted to 3 by adding a 0.5M/L hydrochloric acid solution. The reaction solution was purified by chromatography column with SP Sepharose FF as filler, equilibrated in an equilibration solution (aqueous solution containing 20mM/L NaAc and 150mM/L NaCl), and then PEG-rhG-CSF was eluted by an elution solution (aqueous solution containing 20mM/L NaAc and 200mM/L NaCl). The purity was measured by sampling and the yield was calculated, and the results are shown in Table 1.
Comparative example 6
The rhG-CSF reaction solution (40 mL, 3 mg/mL) containing 100mM sodium phosphate, pH5.0 and 40mM NADPH (nicotinamide adenine dinucleotide phosphate) was stirred and frozen sufficiently (4 ℃) and then 5-fold protein amount of methoxypolyethylene glycol propionaldehyde (MPEG-PALD) (molecular weight 20 kDa) was added. The reaction mixture was continuously stirred at the same temperature for reaction for 10 hours; the reaction mixture was adjusted to pH4.0 with 0.5mol/L hydrochloric acid and allowed to stand for 10 minutes. Filtration was carried out using a filter cartridge having a pore size of 0.22. Mu.m. Equilibrating the Capto MMC column with buffer (20 mmol/L citric acid/disodium hydrogen phosphate, pH 4.0) at a flow rate of 300 cm/hr until the pH stabilizes at 4.0; loading the filtered sample at a flow rate of 300 cm/hr; after loading, the column was equilibrated again with buffer (20 mmol/L citric acid/disodium hydrogen phosphate, pH 4.0) at a flow rate of 300 cm/hr; eluting impurities with 10mmol/L citric acid/disodium hydrogen phosphate buffer (pH 7.5) at a flow rate of 180 cm/hr; : the target protein was eluted with 50mmol/L citric acid/disodium hydrogen phosphate buffer (pH 8.0) at a flow rate of 300 cm/hr, and the target protein peak was collected. The purity was measured by sampling and the yield was calculated, and the results are shown in Table 1.
Comparative example 7
rhG-CSF (5 mg/mL) was dissolved in 100mM sodium acetate solution containing 20mM sodium cyanoborohydride, pH5, stirred in an ice bath, added with a 5-fold molar excess of mPEG-acetaldehyde (20 KD), and the reaction mixture was stirred under the same conditions for a further 10h; 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, calculated yield, and the results are presented in Table 1.
Verification embodiment
1. Purity and yield determination
The reversed phase purity is measured according to the four-part general rule 0512 of the 2020 edition of Chinese pharmacopoeia, and the SEC purity is measured according to the four-part general rule 0514 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 measurements
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From this, comparative example 1 and comparative example 2 were changed to columns, and the sample purity and yield were low; comparative example 3 shows an isocratic elution, poor elution effect, and low purity and yield; comparative example 4 was performed first and then on the column, resulting in lower yields; comparative examples 5-7 are reported in the prior art to have lower purity and lower yield than example 1.
2. Determination of modification Rate
Referring to examples 1-12, comparative examples 1-3, the preparation method of steps a and b was repeated, and then the chromatographic column was eluted with 20mM Tris, pH9.0 at a flow rate of 5mL/min, all the elution peaks were collected, and the modification ratio was determined according to the rule 0514 of the fourth edition of the Chinese pharmacopoeia 2020; comparative examples 4 to 7 the modification ratio was measured by taking the reaction mixture after the stirring reaction according to the rule 0514 of the fourth edition of the Chinese pharmacopoeia 2020. The results are shown in Table 2.
TABLE 2 modification ratio detection results
From this, it can be seen that comparative example 1 and comparative example 2 were changed to the column, and the number of the multiple-modified products was increased; comparative example 3 isocratic elution, affecting product purity and yield, no single modification; comparative example 4, first reaction and last column, increased the number of modified products; comparative examples 5 to 7 are methods reported in the prior art, and have a low single modification rate.
Claims (7)
1. The preparation method of the polyethylene glycol modified rhG-CSF is characterized by comprising the following steps: the preparation method comprises the steps of taking a composite chromatographic medium as a solid-phase reaction carrier, loading rhG-CSF protein onto a chromatographic packing, taking a polyethylene glycol modifier as a mobile phase, completing modification reaction under the condition of catalyst catalysis, and adopting gradient elution to obtain polyethylene glycol modified protein mPEG-rhG-CSF after the reaction is completed, wherein the specific steps are as follows:
a. loading: dissolving rhG-CSF protein in buffer A to obtain rhG-CSF protein solution, and loading the rhG-CSF protein solution onto chromatographic packing;
b. modification reaction: dissolving polyethylene glycol modifier in the buffer solution A, adding a catalyst to obtain a modified buffer solution which is taken as a mobile phase and circularly flows through a chromatographic column;
c. eluting: gradient elution is carried out by using a buffer B and the buffer A to obtain a single modified target protein mPEG-rhG-CSF;
wherein the chromatographic packing is selected from Capto MMC packing, HCX packing and Bestarose Diamond MMC packing; the mass ratio of the rhG-CSF protein to the polyethylene glycol modifier is 1:2-10; the buffer solution A in the steps a, b and c is sodium acetate buffer solution containing sodium chloride, wherein the concentration of the sodium chloride is 0.1-0.8M, the concentration of the sodium acetate is 10-50 mM, and the pH value of the buffer solution A is 4.0-5.0; the catalyst in the step b is sodium cyanoborohydride, and the final concentration is 10-30 mM; the cyclic reaction time is 4-8 hours; the buffer solution B is sodium acetate buffer solution containing sodium chloride, wherein the concentration of the sodium chloride is 10-50 mM, the concentration of the sodium acetate is 10-50 mM, and the pH value of the buffer solution B is 5.5-6.0; and c, performing gradient elution by using different proportions of the buffer B and the buffer A, wherein the flow rate is 5mL/min, firstly 10% -30% of the buffer B and 3CV, performing gradient elution on the multi-modified rhG-CSF, and then performing gradient elution on the single-modified rhG-CSF by using 30% -80% of the buffer B and 10CV to obtain the single-modified target protein mPEG-rhG-CSF.
2. The method of claim 1, wherein the chromatographic packing is Capto MMC packing.
3. The method of claim 1, wherein the polyethylene glycol modifier is selected from mPEG-butyraldehyde, mPEG-propionaldehyde, mPEG-succinate, and has a molecular weight of 10-50 kda.
4. The method of claim 1, wherein the mass ratio of rhG-CSF protein to polyethylene glycol modifier is 1:3-6.
5. The method of claim 1 wherein the rhG-CSF protein solution of step a has a concentration of 2-3mg/mL; and b, the concentration of the polyethylene glycol modifier in the modification buffer solution is 1-5 mg/mL.
6. The method of claim 1, wherein the concentration of sodium chloride in the buffer solution a in steps a, b and c is 0.2-0.5 m and the concentration of sodium acetate is 20-30 mm.
7. The method according to claim 1, wherein the concentration of sodium chloride in the buffer solution B in the step c is 20-30 mM, and the concentration of sodium acetate is 20-30 mM.
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Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101279999A (en) * | 2008-05-21 | 2008-10-08 | 大连理工大学 | Method for modifying hirudin by polyethyleneglycol assisted by anion exchange column |
CN101585864A (en) * | 2009-01-05 | 2009-11-25 | 天津派格生物技术有限公司 | Nitrogen-terminal fixed-point coupling method for colony stimulating factor of column chromatography granulocyte and coupled product |
WO2010146599A1 (en) * | 2009-06-16 | 2010-12-23 | Lupin Limited | Process for purification of recombinant human granulocyte colony stimulating factor |
CN102485742A (en) * | 2010-12-02 | 2012-06-06 | 山东新时代药业有限公司 | Preparation method and separation and purification method of polyethylene glycol single modified recombinant human granulocyte-colony stimulating factor |
CN102850450A (en) * | 2011-07-01 | 2013-01-02 | 齐鲁制药有限公司 | Purification method of pegylated recombinant human granulocyte colony stimulating factor |
CN103421079A (en) * | 2013-06-24 | 2013-12-04 | 温州医学院 | Modification method of polyethyleneglycol of protein |
CN107188952A (en) * | 2016-05-17 | 2017-09-22 | 江苏恒瑞医药股份有限公司 | A kind of purification process of recombinant human granulocyte colony stimulating factor |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
BRPI0611221A2 (en) * | 2005-06-01 | 2010-08-24 | Maxygen Holdings Ltd | pegylated g-csf polypeptides and their production methods |
JP2011507913A (en) * | 2007-12-29 | 2011-03-10 | バイオスティード ジーン エクスプレッション テック. カンパニー リミテッド | Y-type polyethylene glycol-modified G-CSF and its production method and use |
WO2021111470A1 (en) * | 2019-12-03 | 2021-06-10 | Indian Institute Of Technology Delhi | A process for preparation of pegylated therapeutic proteins |
-
2022
- 2022-04-27 CN CN202210466641.0A patent/CN114853872B/en active Active
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101279999A (en) * | 2008-05-21 | 2008-10-08 | 大连理工大学 | Method for modifying hirudin by polyethyleneglycol assisted by anion exchange column |
CN101585864A (en) * | 2009-01-05 | 2009-11-25 | 天津派格生物技术有限公司 | Nitrogen-terminal fixed-point coupling method for colony stimulating factor of column chromatography granulocyte and coupled product |
WO2010146599A1 (en) * | 2009-06-16 | 2010-12-23 | Lupin Limited | Process for purification of recombinant human granulocyte colony stimulating factor |
CN102485742A (en) * | 2010-12-02 | 2012-06-06 | 山东新时代药业有限公司 | Preparation method and separation and purification method of polyethylene glycol single modified recombinant human granulocyte-colony stimulating factor |
CN102850450A (en) * | 2011-07-01 | 2013-01-02 | 齐鲁制药有限公司 | Purification method of pegylated recombinant human granulocyte colony stimulating factor |
CN103421079A (en) * | 2013-06-24 | 2013-12-04 | 温州医学院 | Modification method of polyethyleneglycol of protein |
CN107188952A (en) * | 2016-05-17 | 2017-09-22 | 江苏恒瑞医药股份有限公司 | A kind of purification process of recombinant human granulocyte colony stimulating factor |
Non-Patent Citations (3)
Title |
---|
Mixed-mode chromatography in pharmaceutical and biopharmaceutical applications;Kelly Zhang等;Journal of Pharmaceutical and Biomedical Analysis;第128卷(第05期);第73-88页 * |
Solid-phase polyethylene glycol conjugation using hydrophobic interaction chromatography;Niu J等;J Chromatogr A(第1327期);第66-72页 * |
聚乙二醇定点修饰重组人粒细胞集落刺激因子突变体的研究;黄岩山等;生物工程学报(第05期);第919-923页 * |
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