CN1663962A - Recombinant human granulocyte colony stimulating factor and one-step purifying process for chemical modifications thereof - Google Patents
Recombinant human granulocyte colony stimulating factor and one-step purifying process for chemical modifications thereof Download PDFInfo
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- CN1663962A CN1663962A CN 200410007171 CN200410007171A CN1663962A CN 1663962 A CN1663962 A CN 1663962A CN 200410007171 CN200410007171 CN 200410007171 CN 200410007171 A CN200410007171 A CN 200410007171A CN 1663962 A CN1663962 A CN 1663962A
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Abstract
The invention relates to a recombinant human granulocyte colony stimulating factor and one-step purifying process for chemical modifications, which employs the method of cation exchange chromatography, wherein one-step chromatography is utilized for obtaining high activity, high purity and high recovery ratio rhG-CSF and polyethylene glycol chemically modified rhG-CSF. The process is especially suitable for industrial production.
Description
One, technical field
The present invention relates to protein and modifier purifying field thereof, particularly is exactly the field that simply prepares high purity, highly active medical protein solution efficiently.The present invention relates to quite simple method, be purified into the method for highly purified rhG-CSF or its polyoxyethylene glycol (PEG) modifier solution by a step, be particularly suitable for this albumen and carbowax modifier thereof in medicine industry large-scale production process.
Two, background technology
Development along with genetic engineering technique, the application of genetically engineered recombinant protein technology in medicine industry is more and more, much in the prevention of human diseases and treatment, there is the protein of obvious effect all to be produced out, and is effectively applied in the prevention and treatment of many human diseasess by the means of genetically engineered reorganization.Interferon, rabbit (α-IFN, β-IFN, γ-IFN), interleukin-(IL-2 for example, IL-11), tumour necrosis factor, hemopoieticgrowth factor (G-CSF, GM-CSF and EPO), all kinds of thrombolytic drug (SK, tPA, hirudin) and various hormone (insulin, PTH) etc.
Be applied to both be suitable for large-scale production in nearly 20 years evolution of medicine industry in genetically engineered recombinant protein technology, its products obtained therefrom meets the research of recombinant protein production process route of the quality standard of medicine again and is paid attention to by people gradually.For example be used for the affinity chromatography method of recombinant protein purifying in early days, more and more be not suitable for needs of scale production; The recombinant protein purification processing method complexity that has, step is various, though purity has reached necessary requirement, the rate of recovery and proteic biological activity are all very influenced.G-CSF was put on market by drugs approved by FDA in 1991, was used for the treatment of neutrophilic granulocytopenia after the radiotherapy, and domestic have producer at present in production and sales.Its polyethyleneglycol modified prolonged action preparation PEG-G-CSF (commodity are called Neulasta) was also put on market by drugs approved by FDA in 2002.According to the report of document and relevant patent, the purifying process route of existing rhG-CSF adopts renaturing inclusion bodies-hydrophobic chromatography-ion exchange chromatography etc. mostly; The purifying process route of PEG-G-CSF adopts gel-filtration to cooperate ion exchange chromatography etc. mostly.
Three, summary of the invention
The present invention is the processing method that a kind of advantages of simplicity and high efficiency is produced rhG-CSF and PEG modifier thereof, obtains highly purified rhG-CSF or PEG modifier by primary column chromatography.
The present invention is on the basis of some purification process of domestic and international comparatively sophisticated rhG-CSF of research and PEG modifier thereof, some characteristics by experimental study target protein (rhG-CSF and PEG modifier thereof) and with some special roles relations of positively charged ion chromatography medium, sum up and invent this method.
G-CSF is the treatment protein that is used for the treatment of the leukopenia (as chemotherapy) that hematopoietic disorders causes.G-CSF of the present invention can be from the separation and purification of Mammals organism, also chemosynthesis product, or the product that obtains of the exogenous dna sequence dna by prokaryotic organism or eukaryote host expresses.Wherein DNA can be synthesized into from genome or cDNA.Suitable prokaryotic organism host comprises various bacteriums (as intestinal bacteria); Suitable eukaryote host comprise yeast and mammalian cell (as, CHO).Because of host's difference, gained G-CSF may be by glycosylation or non-glycosylated.G-CSF also can comprise an initial methionine residues (first).Simultaneously, the G-CSF analogue can comprise having aminoacid addition, disappearance, substitute and amalgamation mode etc.Above-mentioned G-CSF and analogue thereof are fit to the present invention.
Wherein adopt the rhG-CSF of escherichia coli expression mostly to be the inclusion body form, the present invention to preparation, washing, dissolving and the refolding method of rhG-CSF inclusion body is: add N,O-Diacetylmuramidase and be aided with the broken bacterium of high-pressure homogenization, broken bacterium buffer strip 5mM EDTA and 1.0% Triton X-100; Directly with the damping fluid washing inclusion body that contains 4M urea, can reach 80% behind centrifugal the inclusion body with inclusion body protein purity after the method washed twice; Centrifugal inclusion body precipitation is directly dissolved inclusion body with the damping fluid that contains 8M urea, and the dissolving back adopts progressively dilution to be aided with ultrafiltration inclusion body is carried out renaturation and to concentrate, and the gained sample carries out cationic exchange.Sample and level pad pH are 4.0 on the chromatography, and rhG-CSF can be combined on the medium well, uses 100mM, and pH is 7.0 sodium phosphate buffer (PB) wash-out, and gained rhG-CSF purity is greater than 95%, and the rate of recovery is greater than 80%.
It is the method for preparing PEG and protein connector that PEG of the present invention modifies, and has mainly adopted alkylation methods and acidylate method.Under the reduced form alkylation reaction condition, will contain the protein molecule and the PEG reaction of a unnecessary amino, the pH value of reaction solution should be suitable for selecting to activate the a-amino acid of said protein molecule amino-terminal end, so PEG is attached on the described a-amino-acid residue.The acidylate method be PEG with the Argine Monohydrochloride residue in Lys the acidylate condensation reaction takes place and combine.Alkylation methods and acidylate method are comparatively sophisticated at present two kinds of PEG modifying method.
Gained rhG-CSF is carried out PEG modify, contain the rhG-CSF of unmodified in the gained sample, modify the rhG-CSF of N-terminal and the rhG-CSF that non-N end (Lys site) is modified.Use same chromatography media and buffering condition, reaction product can be combined on the medium well, and free PEG directly sees through.Fully after the balance, change the pH to 5.6 of damping fluid, the rhG-CSF that only modifies N-terminal is eluted; Change the pH to 6.2 of damping fluid, the rhG-CSF that non-N end (Lys site) is modified is eluted; Change the pH to 7.0 of damping fluid again, the rhG-CSF of unmodified is eluted.Wherein, gained PEG-G-CSF purity is greater than 95%, and the rate of recovery is greater than 80%.
Four, description of drawings
Accompanying drawing 1:SP Sepharose F.F. purifying rhG-CSF synoptic diagram
The rhG-CSF synoptic diagram that accompanying drawing 2:SP Sepharose F.F. purifying N end PEG modifies
The rhG-CSF synoptic diagram that accompanying drawing 3:SP Sepharose F.F. purifying Lys PEG modifies
Accompanying drawing 4: the SDS-PAGE electrophoretic analysis of the rhG-CSF of purifying and PEG-G-CSF
Five, concrete embodiment
Example one rhG-CSF structure, expression and renaturation
The human G-CSF aminoacid sequence is as follows:
NH
2-Met?Thr?Pro?Leu?Gly?Pro?Ala?Ser?Ser?Leu?Pro?Gln?Ser?Phe?Leu?Leu?LysCys?Leu?Glu?Gln?Val?Arg?Lys?Ile?Gln?Gly?Asp?Gly?Ala?Ala?Leu?Gln?Glu?Lys?Leu?CysAla?Thr?Tyr?Lys?Leu?Cys?His?Pro?Glu?Glu?Leu?Val?Leu?Leu?Gly?His?Ser?Leu?Gly?IlePro?Trp?Ala?Pro?Leu?Ser?Ser?Cys?Pro?Ser?Gln?Ala?Leu?Gln?Leu?Ala?Gly?Cys?Leu?SerGln?Leu?His?Ser?Gly?Leu?Phe?Leu?Tyr?Gln?Gly?leu?Leu?Gln?Ala?Leu?Glu?Gly?Ile?SerPro?Glu?Leu?Gly?Pro?Thr?Leu?Asp?Thr?Leu?Gln?Leu?Asp?Vla?Ala?Asp?Phe?Ala?Thr?ThrIle?Trp?Gln?Gln?Met?Glu?Glu?Leu?Gly?Met?Ala?Pro?Ala?Leu?Gln?Pro?Thr?Gln?Gly?AlaMet?Pro?Ala?Phe?Ala?Ser?Ala?Phe?Gln?Arg?Arg?Ala?Gly?Gly?Val?Leu?Val?Ala?Ser?HisLeu?Gln?Ser?Phe?Leu?Glu?Val?Ser?Tyr?Arg?Val?Leu?Arg?His?Leu?Ala?Gln?Pro-COOH
The synthetic human G-CSF cDNA gene fragment of full gene is used the recurrence PCR method and is obtained 3 ' end and contain the insertion fragment that NdeI and 5 ' end contain BamHI, and this sequence plasmid pET-3a that recombinates transforms host bacterium BL21 (DE
3) pLysS expresses, and expresses 37 ℃ of temperature, IPTG final concentration 0.2mmol/L, gained target protein are the inclusion body form.The broken bacterium damping fluid of preparation is as follows:
50mmol/L?Tris.CL，pH10.0
5mmol/L?EDTA
0.1mg/ml N,O-Diacetylmuramidase
1.0％?Triton
With the wet bacterium of 100g: 1L damping fluid ratio suspension thalline, high-pressure homogenization break bacterium (30-40MP/2-3 time), and 10, the centrifugal collection inclusion body of 000rpm/15min precipitates.Preparation inclusion body washings is as follows:
50mmol/L?Tris.CL，pH9.0
5mmol/L?EDTA
4mmol/L Urea is with the 10g inclusion body: 100ml damping fluid ratio suspension inclusion body, and agitator treating 30min, 10, the centrifugal supernatant of abandoning of 000rpm/15min, preparation dissolving damping fluid:
50mmol/L?Tris.CL，pH9.0
5mmol/L?EDTA
8mmol/L?Urea
0.05% (v/v) β-ME is with the 10g inclusion body: 100ml damping fluid ratio suspension inclusion body, stirring and dissolving 1hr, 10, the centrifugal collection supernatant of 000rpm/15min.The preparation renaturation buffer:
20mmol/L?Tris.CL，pH9.0
20umol/L CuSO
4Get inclusion body solution, add 1 times of volume renaturation buffer, the urea final concentration is 4mmol/L, stirring at room 30min; Add 1 times of volume renaturation buffer again, the urea final concentration is 2.7mmol/L, stirring at room 30min; Add 1 times of volume renaturation buffer again, the urea final concentration is 2mmol/L, stirring at room 30min; Add 1 times of volume renaturation buffer again, the urea final concentration is 1.6mmol/L, stirring at room 30min; Add 1 times of volume renaturation buffer again, the urea final concentration is 0.8mmol/L, stirred overnight at room temperature (about 12hr).With the molecular interception amount is hollow fiber ultrafiltration post ultrafiltration and concentration to 1/10 volume of 3KD.Prepared and diluted liquid:
25mmol/L?Tris.CL，pH9.0
1mmol/L?EDTA
10umol/L CuSO
4With 4 times of diluted concentrated solutions, ultrafiltration and concentration to 1/4 volume gets rhG-CSF protein renaturation liquid again.
Example two rhG-CSF single step purifications
Sample preparation is promptly regulated renaturation solution to pH4.0 with acetate, the centrifugal precipitation of going.Get positively charged ion chromatography medium SP Sepharose F.F., (10mmol/L acetate-sodium acetate, pH4.0) balance goes up all product to dress post back balance damping fluid to baseline, uses the level pad balance to baseline again.Get elution buffer (100mmol/L Sodium phosphate dibasic/phosphate sodium dihydrogen buffer solution, pH7.0) rhG-CSF target protein peak under the wash-out.Press medium specification sheets method regeneration chromatography media, remove other impurity albumen.Measure gained target protein purity with SDS-PAGE electrophoretic method and RP-HPLC method.
The PEG of example three rhG-CSF modifies
1. alkylation methods N-is end modified
Get purifying gained rhG-CSF, be diluted to 2.0mg/ml, to 0.1M NaH
2PO
4, pH5.0 regulates pH to 5.0 in 4 ℃ of dialysed overnight.Getting molecular weight is the mPEG-ButyrALD (NEKTAR of 20KD
TM), in mPEG-ButyrALD: 5: 1 ratios of rhG-CSF mol ratio add mPEG-ButyrALD, fully stirring and dissolving.Get 1M NaCNBH again
3Add reaction solution, final concentration is 20mM.Fully the rearmounted 4 ℃ of reactions of stirring and evenly mixing are spent the night.
2. acidylate method N-is end modified
Get purifying gained rhG-CSF, be diluted to 2.0mg/ml, to 0.1M NaH
2PO
4, pH6.5 regulates pH to 6.5 in 4 ℃ of dialysed overnight.Get the mPEG-NH that molecular weight is 20KD
2(NEKTAR
TM), press mPEG-NH
2: 5: 1 ratios of rhG-CSF mol ratio add, fully stirring and dissolving.Put 25 ℃ again and stir 1hr, add azanol to final concentration 2M, fully stirring and evenly mixing afterreaction 1hr.
The single step purification of example four alkylation methods PEG-G-CSF
Sample preparation is got and is modified the back sample, after the distilled water diluting two volumes, is adjusted to pH4.0 with acetate again, the centrifugal precipitation of going.Get positively charged ion chromatography medium SP Sepharose F.F., and the weighing apparatus damping fluid I that makes even behind the dress post (10mmol/L acetate-sodium acetate, pH4.0) balance goes up all product to baseline, and (10mmol/L acetate-sodium acetate, pH5.0) balance is to baseline to use level pad II again.Get elution buffer I (10mmol/L acetate-sodium acetate, pH5.6) PEG-G-CSF target protein peak under the wash-out.Get elution buffer II (10mmol/L acetate-sodium acetate, pH7.0) the rhG-CSF protein peak of unmodified under the wash-out.
The single step purification of example five acidylate method PEG-G-CSF
Sample preparation is got and is modified the back sample, after the distilled water diluting two volumes, is adjusted to pH4.0 with acetate again, the centrifugal precipitation of going.Get positively charged ion chromatography medium SP Sepharose F.F., and the weighing apparatus damping fluid I that makes even behind the dress post (10mmol/L acetate-sodium acetate, pH4.0) balance goes up all product to baseline, and (10mmol/L acetate-sodium acetate, pH5.0) balance is to baseline to use level pad II again.Get elution buffer I (10mmol/L acetate-sodium acetate, pH6.2) PEG-G-CSF target protein peak under the wash-out.Get elution buffer II (10mmol/L acetate-sodium acetate, pH7.0) the rhG-CSF protein peak of unmodified under the wash-out.
Example six product purity analyses and external biological determination of activity
Get PEG-G-CSF that PEG-G-CSF that the rhG-CSF, alkylation methods of unmodified modify and acidylate method modify as trial-product.With irreducibility SDS-PAGE method and RP-HPLC method product is carried out purity check respectively.With reference to the described method of " Chinese biological goods rules (version in 2000) " appendix " rhG-CSF titration (NFS-60 relies on strain/MTT colorimetry) ", product is carried out the external biological determination of activity simultaneously.
1. irreducibility SDS-PAGE method is measured purity
With reference to " Chinese biological goods rules " (2000 editions) " SDS-polyacrylamide gel electrophoresis " described method, resolving gel concentration is 15%, gets the PEG-G-CSF of rhG-CSF, alkylation methods modification and the PEG-G-CSF mensuration that the acidylate method is modified then.Sample 10ug on every hole.Gel scanning back protein gelatin analytical system analysis purposes purity of protein.
2.RP-HPLC method is measured purity
Chromatographic condition is as follows:
The high performance liquid phase system is waters (600E)
Chromatographic column: Hypersil C18 (300A, 5u, 4.6 * 250mm)
Flow velocity: 1.0ml/min
Applied sample amount: 20ul.
Detect wavelength: 280nm
Elution requirement: 100% A liquid wash-out 5 minutes, 0-100%B liquid gradient elution 30 minutes
A liquid: 5% acetonitrile, 0.1%TFA
B liquid: 90% acetonitrile, 0.1%TFA
3. external biological determination of activity
Prepare basic culture solution earlier:
RPMI-1640
2.5% calf serum (FBS, v/v)
12.5% horse serum (v/v)
The 10ml/L Sodium.alpha.-ketopropionate
The 10ml/L glutaminase
Get the cell strain NFS-60 that G-CSF is relied on,, be suspended in again and be made into 2.0 * 10 in the basic medium with basic culture solution washing 3 times
5The cell suspension of individual cell/ml, put 37 ℃ standby.Get rhG-CSF with reference to product (U.S. Amgen company) and trial-product, be diluted to 1.0ng/ml with basic medium.Get 96 porocyte culture plates,, do 7 extent of dilution altogether, 2 multiple holes of each gradient doing gradient dilution with 2 times of extent of dilution with reference to product and trial-product.Every then hole adds the 50ul cell suspension, in 37 ℃, and 5%CO
2Cultivated 48 hours, every hole adds 20ulMTT solution, 37 ℃, 5%CO
2Cultivated 5 hours, every hole adds 200ul lysate (1% concentrated hydrochloric acid, the Virahol of 10%TritonX-100), behind the mixing on microplate reader colorimetric, measure wavelength 570nm, reference wavelength 630nm, the record measurement result, calculate the trial-product specific activity according to following formula:
4. measurement result is summarized as follows table:
SDS-PAGE purity RP-HPLC purity specific activity
Trial-product
(％) (％) (IU/mg)
rhG-CSF 97.7 98.5 1.1×10
8
PEG-G-CSG (alkylation methods) 98.1 98.7 4.7 * 10
7
PEG-G-CSG (acidylate method) 97.2 97.9 4.1 * 10
7
Electronically readable sequence-peggcsf patent
The single step purification process sequence table of recombinant methionyl human G-CSF and chemical modification object thereof
Organization?Applicant
Street: No. 70, four streets, stone bridge shop, Chongqing City section garden
City: Chongqing City
State: Chongqing City
Country: the People's Republic of China (PRC)
PostalCode：400041
PhoneNumber：023-68888852
FaxNumber：023-68699676
EmailAddress：[email protected]
＜110〉OrganizationName: Chongqing Fujin Biological Medicine Co. Ltd
Application?Project
＜120〉Title: the single step purification technology that is recombined into granulocyte colony-stimulating factor and chemical modification object thereof
<130>AppFileReference：
<140>CurrentAppNumber：
<141>CurrentFilingDate：
<160>1
<170>PatentIn?version?3.2
Sequence：Seq1
＜213〉OrganismName: native sequences
<400>PreSequenceString：
Met?Thr?Pro?Leu?Gly?Pro?Ala?Ser?Ser?Leu?Pro?Gln?Ser?Phe?Leu 15
Leu?Lys?Cys?Leu?Glu?Gln?Val?Arg?Lys?Ile?Gln?Gly?Asp?Gly?Ala 30
Ala?Leu?Gln?Glu?Lys?Leu?Cys?Ala?Thr?Tyr?Lys?Leu?Cys?His?Pro 45
Glu?Glu?Leu?Val?Leu?Leu?Gly?His?Ser?Leu?Gly?Ile?Pro?Trp?Ala 60
Pro?Leu?Ser?Ser?Cys?Pro?Ser?Gln?Ala?Leu?Gln?Leu?Ala?Gly?Cys 75
Leu?Ser?Gln?Leu?His?Ser?Gly?Leu?Phe?Leu?Tyr?Gln?Gly?leu?Leu 90
Gln?Ala?Leu?Glu?Gly?Ile?Ser?Pro?Glu?Leu?Gly?Pro?Thr?Leu?Asp 105
Thr?Leu?Gln?Leu?Asp?Val?Ala?Asp?Phe?Ala?Thr?Thr?Ile?Trp?Gln 120
Gln?Met?Glu?Glu?Leu?Gly?Met?Ala?Pro?Ala?Leu?Gln?Pro?Thr?Gln 135
Gly?Ala?Met?Pro?Ala?Phe?Ala?Ser?Ala?Phe?Gln?Arg?Arg?Ala?Gly 150
Gly?Val?Leu?Val?Ala?Ser?His?Leu?Gln?Ser?Phe?Leu?Glu?Val?Ser 165
Tyr?Arg?Val?Leu?Arg?His?Leu?Ala?Gln?Pro 175
<212>Type：PRT
<211>Length：10
SequenceName: human G-CSF aminoacid sequence
Feature
<221>FeatureKey：mat_peptide
<222>LocationFrom：1
<222>LocationTo：10
Other Information: human G-CSF aminoacid sequence
CDSJoin：Yes
Claims (10)
1. the method with cation-exchange chromatography purification of Recombinant Filgrastim and polyoxyethylene glycol chemistry modified outcome thereof is characterized in that single step purification, purity height, active height and rate of recovery height.
2. the described step cation-exchange chromatography method of claim 1 is applicable to the purifying of recombinant methionyl human G-CSF.
3. the described step cation-exchange chromatography method of claim 1 is equally applicable to the purifying through the recombinant methionyl human G-CSF of polyoxyethylene glycol chemistry modification.
4. the described cation-exchange chromatography of claim 1 refers to weak ionic exchang medium and strong ionic exchang medium.
5. the described cation-exchange chromatography dielectric polymers of claim 4 comprises Sephadex, Sepharose, Cellulose and Source etc.
6. the described recombinant methionyl human G-CSF of claim 2 refers to the natural type of using gene engineering means preparations or through disappearance, sudden change and chimeric form.
7. the described recombinant methionyl human G-CSF polyoxyethylene glycol chemistry of claim 3 modified outcome refers to the polyethylene glycol modified product of recombinant methionyl human G-CSF N-terminal and non-N-terminal.
8. the condition of the described cation-exchange chromatography of claim 1 is:
(1). the level pad ionic strength is 5-50mmol/L, and potential of hydrogen is 2.0-5.0.
(2). the elution buffer ionic strength is 10-200mmol/L, and potential of hydrogen is 5.0-9.0.
(3). elution buffer salt ion gradient is a 0-500mmol/L sodium-chlor.
9. the described buffer solution system of claim 8 mainly refers to sodium acetate, sodium phosphate, yellow soda ash, the buffering of Trisodium Citrate and glycine.
10. the described elution requirement of claim 8 can be separately with pH or sodium-chlor gradient, but also both use simultaneously.
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| CN101585864B (en) * | 2009-01-05 | 2011-11-09 | 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 |
| 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 |
| CN103908660A (en) * | 2013-01-05 | 2014-07-09 | 石药集团百克(山东)生物制药有限公司 | Polyethylene glycol modified rhG-CSF pharmaceutical composition and preparation method thereof |
| WO2014155365A3 (en) * | 2013-03-29 | 2015-02-19 | Dr.Reddy's Laboratories Limited | Purification method |
| CN104491843A (en) * | 2015-01-23 | 2015-04-08 | 石药集团百克(山东)生物制药有限公司 | RhG-CSF (recombinant human granulocyte colony-stimulating factor) active drug composition modified by polyethylene glycol |
| WO2016009451A2 (en) | 2014-07-14 | 2016-01-21 | Gennova Biopharmaceuticals Limited | A novel process for purification of rhu-gcsf |
| CN107129531A (en) * | 2016-04-15 | 2017-09-05 | 江苏恒瑞医药股份有限公司 | A kind of purification process of PEG-rhG-CSF |
| 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 |
| US9815879B2 (en) | 2005-07-15 | 2017-11-14 | Sandoz Ag | Method for the purification of G-CSF |
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2004
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9815879B2 (en) | 2005-07-15 | 2017-11-14 | Sandoz Ag | Method for the purification of G-CSF |
| US10844103B2 (en) | 2005-07-15 | 2020-11-24 | Mylan Pharmaceuticals Inc. | Method for the purification of G-CSF |
| EP1904522B2 (en) † | 2005-07-15 | 2020-05-27 | Mylan Pharmaceuticals Inc. | Method for the purification of g-csf |
| CN101585864B (en) * | 2009-01-05 | 2011-11-09 | 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 |
| 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 |
| CN103908660A (en) * | 2013-01-05 | 2014-07-09 | 石药集团百克(山东)生物制药有限公司 | Polyethylene glycol modified rhG-CSF pharmaceutical composition and preparation method thereof |
| CN103908660B (en) * | 2013-01-05 | 2015-02-04 | 石药集团百克(山东)生物制药有限公司 | Polyethylene glycol modified rhG-CSF pharmaceutical composition and preparation method thereof |
| WO2014155365A3 (en) * | 2013-03-29 | 2015-02-19 | Dr.Reddy's Laboratories Limited | Purification method |
| JP2017526649A (en) * | 2014-07-14 | 2017-09-14 | ジェンノヴァ バイオファーマシューティカルズ リミテッド | New process for purification of rHu-GCSF |
| EP3169697A4 (en) * | 2014-07-14 | 2017-11-08 | Gennova Biopharmaceuticals Ltd. | A novel process for purification of rhu-gcsf |
| WO2016009451A2 (en) | 2014-07-14 | 2016-01-21 | Gennova Biopharmaceuticals Limited | A novel process for purification of rhu-gcsf |
| CN104491843A (en) * | 2015-01-23 | 2015-04-08 | 石药集团百克(山东)生物制药有限公司 | RhG-CSF (recombinant human granulocyte colony-stimulating factor) active drug composition modified by polyethylene glycol |
| CN107129531A (en) * | 2016-04-15 | 2017-09-05 | 江苏恒瑞医药股份有限公司 | A kind of purification process of PEG-rhG-CSF |
| CN107129531B (en) * | 2016-04-15 | 2020-09-11 | 江苏恒瑞医药股份有限公司 | Purification method of pegylated recombinant human granulocyte stimulating factor |
| 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 |
| CN107188952B (en) * | 2016-05-17 | 2020-07-28 | 江苏恒瑞医药股份有限公司 | Method for purifying recombinant human granulocyte colony stimulating factor |
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