CN107188952A - A kind of purification process of recombinant human granulocyte colony stimulating factor - Google Patents
A kind of purification process of recombinant human granulocyte colony stimulating factor Download PDFInfo
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- C07K14/52—Cytokines; Lymphokines; Interferons
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Abstract
The present invention provides a kind of purification process of recombinant human granulocyte colony stimulating factor.Specifically, the present invention contains recombinant human granulocyte colony stimulating factor (rhG CSF) renaturation solution from cation column chromatography, the rhG CSF stostes that appropriate buffer exchange prepares high-purity are carried out again, wherein filler is the cationic of mixed mode in cationic layer analysis post.Purifying process of the present invention simplifies process route, saves the production time, and protein recovery is high, and the albumen stoste purity obtained is high, and pharmaceutical preparation can be prepared into by adding appropriate pharmaceutic adjuvant, is very suitable for heavy industrialization amplification production.The present invention also provides a kind of method for preparing PEG-rhG-CSF simultaneously.
Description
Technical field
The present invention relates to biological medicine and bioengineering downstream protein purification art, and in particular to a kind of recombined human grain
Colony-stimulating factor (recombinant human granulocyte colony stimulating factor, rhG-
CSF purification process), the simple to operate, time of the invention is short, is suitable for industrial amplification production.
Background technology
Filgrastim (human granulocyte colony stimulating factor, hG-
CSF grain system progenitor cells) can be specifically acted on, promotes it to breed and is divided into the neutrophil leucocyte of maturation, are regulation marrow
One of major cytokine of middle grain system hematopoietic cell.It is for by cancer chemotherapy, chemotherapy of acute leukemia, myeloproliferative disorder
Neutrophil leucocyte when neutrophil leucocyte deficiency disease caused by the disease such as syndrome and alpastic anemia and bone-marrow transplantation increases
Plus wait and have obvious curative effect.
HG-CSF source is very limited amount of, and therefore, large-scale industrial production is obtained using the method for genetic engineering
.Pharmacy corporation expresses rhG-CSF using widely used E. coli system mostly both at home and abroad at present.Due to Escherichia coli
Intracellular reducing environment is unfavorable for the formation of disulfide bond, and rhG-CSF folds mistake, easily aggregation, substantially with inactive
Inclusion bodies be present in cell.Inclusion body could obtain active rhG-CSF by denaturation and renaturation.At present, specially
It is numerous to rhG-CSF refolding method report in profit, study extremely clear.
No matter use which kind of refolding method, undeniably, after renaturation in sample still containing substantial amounts of host cell proteins with
And product related impurities, therefore, the purifying process after renaturation be determine qualified product whether important step, and one into
The purifying process of work(will take into account many factors such as product purity, yield, process cycle, production cost, operability simultaneously.
US5849883, CN1167150, CN1206716, CN1814779 etc. are reported to be arranged in pairs or groups using more chromatographic enrichment method,
RhG-CSF for purifying Recombinant protein expression.If US5849883, CN1206716, CN1814779 are simultaneously using weak
Cation (e.g., CM Sepharose) and weak anionic (e.g., DEAE Sepharose) displacement chromatography.In production technology, layer
Process certification, the workload of cleaning checking can be greatly increased more than analysis step, while increase the time of processing sample, meanwhile, multistep
Rapid handling process can also influence the rate of recovery of albumen.Therefore, one step chromatography method of exploitation is used to purify rhG-CSF with good
Application prospect.
And being related to filler used in the step chromatographic purification methods of rhG-CSF mono- more strong cation exchange chromatography (e.g., SP fillers) and
Weak cation exchange chromatographs (e.g., CM fillers).
CN1718739A is reported using SP Sepharose High Performance (SP HP) directly to rhG-CSF
The method of renaturation solution purifying, under conditions of pH5.4, destination protein is eluted using NaCl concentration.But in renaturing inclusion bodies liquid
In addition to destination protein, also containing a certain amount of host protein, lipid and DNA (DNA).The particle diameter of SP HP fillers
Very little, is 30 μm, is generally used for polishing purification, is not suitable for containing the complicated renaturation solution of more impurity, situation.And particle diameter
The small pressure for causing the upper chromatographic column of production is very big, and flow velocity is slack-off, increases processing operation time.
It is pure using SP Sepharose Fast Flow (SP FF) to the rhG-CSF after renaturation that CN1663962A is reported
The method of change：The loading under conditions of pH4.0 (10mmol/L acetic acid-sodium acetate), pH7.0 (100mmol/L disodium hydrogen phosphates/
Sodium dihydrogen phosphate) elution destination protein.But SP FF are required to sample ions intensity, are usually no more than 5mS/cm.Wherein,
Urea containing higher concentration in sample renaturation solution, the sequence of operations such as is concentrated, is diluted, being concentrated using doughnut, with
Obtain the sample for meeting the requirement of SP FF loadings.Therefore, this method is cumbersome, the difficulty of increase technique amplification.
It is pure that CN104120109A provides a kind of weak cation exchange chromatography CM Sepharose Fast Flow (CM FF)
Change rhG-CSF method, condition is similar to CN1718739A, the loading under conditions of pH5.4, purpose is eluted using NaCl concentration
Albumen.But the loading of the purification process, balance, elution flow rate are 10cm/h.So low velocity potential must greatly increase work
The operating time of skill, it may can also influence the stability of protein sample.
In view of the above-mentioned problems, exploitation one kind is adapted in rhG-CSF renaturation solutions complicated ingredient, pollutant be more, renaturation solution from
The features such as sub- intensity is high, and enable to the purifying process that process flow rate is big, the operating time is short particularly important.With this end in view,
The invention provides rhG-CSF after the chromatography column chromatography purification renaturation of the cation with mixed mode filler, this method is to sample
Strong applicability, carrying capacity are big, high income, and operate simpler, efficient.
The content of the invention
The invention provides a kind of purification process of recombinant human granulocyte colony stimulating factor, including：Recombined human grain is thin
Born of the same parents' colony stimulating factor renaturation solution loading is extremely purified with the cation chromatographic column of mixed mode filler.
Wherein, the filler of the cation-exchange chromatography post is the cation chromatographic stuffing of mixed mode, is selected from but does not limit
In Capto MMC fillers,In HCX fillers, Bestarose Diamond MMC fillers and UniMSP-30S fillers
At least one, preferably Capto MMC fillers andHCX fillers.
Furthermore, the purification process of the recombinant human granulocyte colony stimulating factor is first washed using buffer solution progress impurity
It is de-, then carry out elution collection sample peak.Buffer is selected from acetate, citrate, phosphorus in buffer solution used in the Impurity elution
Buffer is selected in one or more in hydrochlorate, carbonate, Tris, glycine, histidine, buffer solution used in the sample elution
One or more from acetate, citrate, phosphate, carbonate, Tris, glycine, histidine.
Described Impurity elution buffer solution ionic strength is 5~20mmol/L, and pH is 7.2~7.8, in a particular embodiment
Impurity elution buffer solution ionic strength can for 7,9,11,13,15,17,19mmol/L, pH is chosen as 7.2,7.4,7.6,7.8.
Described elution buffer ionic strength is 20~100mmol/L, and pH is 8.0~9.0 wherein.In specific embodiment
Middle elution buffer ionic strength can for 40,55,65,80,90mmol/L, pH is chosen as 8.0,8.2,8.4,8.6,8.8,9.0.
Further, the method for the invention also cation chromatographic column including mixed mode filler is buffered before loading
Liquid equilibrium step, the level pad ionic strength is 5~50mmol/L, and pH is 3.0~6.5, and pH can in specific embodiment
Elect 4.0,4.5,5.0,6.0 as.
There are various ways in usual sample elution, sample elution of the present invention is mainly using pH elutions or salt ionic concentration
Type of elution, in order to ensure the effect of sample elution, preferably pH types of elution, wherein the pH types of elution are to utilize eluent
Different pH value conventional elution program is carried out to stoste containing protein.Furthermore, Impurity elution buffer solution of the present invention
PH is differed with the elution buffer pH numerical value.
Further, the method for the invention also includes the step by obtained purification of samples progress buffer exchange is collected
Suddenly, the buffer exchange can use the one or more in dialysis, ultrafiltration, desalting column chromatography scheme, and the ultrafiltration preferably is selected from
Hollow Fiber Ultrafiltration or film bag ultrafiltration, more preferably from film bag ultrafiltration.Wherein described displacement buffer solution is sodium acetate buffer, in acid
Property.If the G-CSF stostes after displacement are produced for preparation, pH is 2.0~5.0, preferably pH=3.5~4.5, and specific pH is optional
3.6、3.7、3.8、3.9、4.0、4.1、4.2、4.3、4.4.If G-CSF stostes are used for follow-up modification reaction, long-acting G- is prepared
CSF, pH are 3.0~8.0, preferably pH=4.0~7.0, in a particular embodiment pH=4.5,5.0,5.5,6.0,6.5.
Buffer exchange wherein is carried out according to the ultrafiltration of film bag, ultra-filtration conditions are：1. using ultrafiltration system to compound sun
Sample is suitably concentrated ion filler after purification；2. keep sample volume constant in ultrafiltration system, carried out using displacement liquid
Displacement, the displacement liquid speed for adding stream is identical with peritoneal effluent speed；3. transmembrane pressure (TMP) is not more than 50PSI, more preferably
10-30PSI；4. more than 3 times of ultrafiltration displacement sample volume are total to, it is contemplated that rational technique used time, more preferably 5 times volumes.
The film bag ultrafiltration membrane aperture is 1KD~20KD, preferably 3KD, 5KD or 10KD, more preferably 5KD.
Film packaging material is in Kynoar, modified cellulose, polyether sulfone or improvement polyether sulfone in the film bag ultrafiltration
It is one or more.
Further, the method for the invention is also included in before loading and carries out pH regulations, filtration step to renaturation solution.
It is 2.0~6.0 that the protein renaturation liquid, which carries out value after pH regulations, preferably is selected from 3.5~4.5, for example pH=3.6,
3.8th, 4.0,4.2,4.4, the one or more that pH adjusting agent used includes but is not limited in acetic acid, citric acid, phosphoric acid.
The filtering can use the one or more in hollow fibre filtering, film packet filtering or dead-end filtration mode, preferably
Dead-end filtration.Doughnut bore including but not limited to 0.5mm, 0.75mm, 1mm that wherein Hollow Fiber Ultrafiltration is used and
1.75mm, more preferably 1mm；Ultrafiltration buffer displacement such as is carried out according to the hollow fiber column of GE Healthcare companies, is cut
Cutting speed rate should be controlled in 16000sec-1Hereinafter, even more preferably about 8000sec-1。
Sediment is removed according to the dead-end filtration, filter sizes are filter membrane material in 0.22 μm, the dead-end filtration
It is Kynoar or polytetrafluoroethylene (PTFE).
Specifically, the invention provides a kind of purification process of recombinant human granulocyte colony stimulating factor, including：
A) pH regulations, filtering are carried out before loading to recombinant human granulocyte colony stimulating factor renaturation solution；
B) the cation chromatographic column of mixed mode filler buffered liquid balance before loading；
C) loading to the cation chromatographic column of mixed mode filler carries out chromatographic purifying, and first carrying out impurity using buffer solution washes
It is de-, then carry out elution collection sample peak.
D) obtained purification of samples progress buffer exchange will be collected；
Wherein, b) and c) described in buffer solution buffer be selected from acetate, citrate, phosphate, carbonate,
One or more in Tris, glycine, histidine, the level pad ionic strength be 5-50mmol/L, pH 3.0~
6.5, the Impurity elution buffer solution ionic strength is 5~20mmol/L, and pH is 7.0~8.0, and the elution buffer ion is strong
Spend for 20~100mmol/L, pH is 8.0~9.0.
The filler of the cation-exchange chromatography post is the cation chromatographic stuffing of mixed mode, is selected from, but not limited to,
Capto MMC fillers,In HCX fillers, Bestarose Diamond MMC fillers and UniMSP-30S fillers extremely
Few one kind, preferably Capto MMC fillers andHCX fillers.
One kind that wherein a) step filtering can be using hollow fibre filtering, film packet filtering or in dead-end filtration mode or
It is a variety of, preferred dead-end filtration, filter membrane material is Kynoar or polytetrafluoroethylene (PTFE) in the dead-end filtration, and wherein aperture is
0.22μm。
PH adjusting agent used in wherein described a) step includes but is not limited to acetic acid, hydrochloric acid and phosphoric acid, acetate, citric acid
It is one or more in salt, phosphate.
Wherein described displacement buffer solution is sodium acetate buffer, in acidity.If the G-CSF stostes after displacement are given birth to for preparation
Production, pH is 2.0~5.0, preferred pH=3.5~4.5, specific pH optional 3.6,3.7,3.8,3.9,4.0,4.1,4.2,4.3,
4.4.If G-CSF stostes are used for follow-up modification reaction, long-acting G-CSF is prepared, pH is 3.0~8.0, preferably pH=4.0~
7.0, pH=4.5,5.0,5.5,6.0,6.5 in a particular embodiment.
The present invention also provides a kind of method for preparing PEG-rhG-CSF, including foundation
Recombinant human granulocyte colony stimulating factor purification process described above, and by obtained recombinant human granulocyte colony stimulating factor
Polyethyleneglycol modified step is carried out, the structure of its PEG-rhG-CSF is as shown in formula I
Wherein m is selected from 50-2500 integer, preferably is selected from 400-500 integer, G is Met-G-CSF.
The beneficial effects of the invention are as follows：
(1) renaturation solution adjusts pH to acidity so that a large amount of Bacillus coli cells albumen precipitations, the step is easy and effective, holds
The separation of foreign protein and destination protein is easily realized, is that follow-up chromatographic purifying technique eases off the pressure.
(2) the high carrying capacity of cation exchange filler of composite ligand, high flow rate processing bulk sample, the complexity to renaturation solution
Buffer system strong applicability, greatly shortens the process time, and the feasibility of technique amplification is good, is successfully amplified to production scale.
(3) purification process of the present invention, is only chromatographed comprising a step, and processing step is few, and purification cycle is short, it is easy to control
System, improves the stability of technological operation.
(4) this technique can obtain high-purity, the rhG-CSF albumen stostes of high activity.After testing, its SDS-PAGE purity reaches
More than 98% is reached to more than 99%, RP-HPLC purity, specific activity is not less than 8.0 × 107IU/mg.These are above《China
People's republic's pharmacopeia 2015 editions》The requirement that stoste is examined and determine under 3rd " recombined human granulocyte stimulating factors parenteral solution " item.
Term is explained：
Brief description of the drawings
Fig. 1：The SDS-PAGE purity detecting collection of illustrative plates of rhG-CSF stostes, wherein swimming lane M：Molecular weight marker proteins Mark
12, swimming lane 1：RhG-CSF stostes.
Embodiment
With reference to embodiment, technical scheme is expanded on further, it will be appreciated that described embodiment is only
For the scope illustrated the present invention and not to limit the present invention.
Embodiment 1 prepares rhG-CSF renaturation solutions
Technique bacterial strain uses therefor is the e.colistraindh5α that pBV220/G-CSF plasmids are converted.After fermentation, thalline is collected,
Clasmatosis is carried out using high pressure homogenizer, thick inclusion body is obtained, then thick inclusion body is washed, the bag of preliminary purification is obtained
Contain body.
The inclusion body 30g of preliminary purification is taken, according to solid-to-liquid ratio 1:20 (m/v) ratio denaturing liquid (20mmol/L
Tris-HCl, 5mmol/L EDTA, 8mol/L Urea, 10mmol/L DTT, pH8.2) denaturation dissolving is carried out, it is stirred at room temperature 15
~18 hours, obtain albuminate liquid 600ml.Afterwards, the protein liquid is carried out 10 μm and 0.45 μm of double-filtration is clarified, then profit
Buffer exchange is carried out with 5KD hollow fiber columns, to remove reducing agent DTT, replacement process control transmembrane pressure (TMP) is 10-
18PSI, displacement buffer solution is 20mmol/L Tris-HCl, 5mmol/L EDTA, 8mol/L Urea, pH 8.2.Replaced
Albuminate liquid 600ml afterwards.
Albuminate liquid after displacement is slowly added in about 15L renaturation buffers (20mmol/L Tris-HCl, pH8.2),
So that final concentration of protein is 0.3g/L, while GSSG to final concentration of 0.5mmol/L is added, after stirring 30min is mixed, 2~25
DEG C stand renaturation 16~18 hours.
The rhG-CSF of embodiment 2 Capto MMC purifying
By the rhG-CSF solution 0.5mol/L salt acid for adjusting pH to 4.0 after renaturation, 10 minutes are stood.It is using aperture
0.22 μm of filter core is filtered.
Balance：Chromatographic column, flow velocity are balanced with level pad (20mmol/L citric acids/disodium hydrogen phosphate, pH4.0)
300cm/ hours, until pH is stable 4.0；
Loading：Renaturation solution sample loading, flow velocity 300cm/ hours after filtering；
Releveling：After end of the sample, put down again with level pad (20mmol/L citric acids/disodium hydrogen phosphate, pH4.0)
Weigh chromatographic column, flow velocity 300cm/ hours；
Wash miscellaneous：With 10mmol/L citric acids/disodium hydrogen phosphate buffer solution (pH7.5) elution impurity, flow velocity 180cm/ hours；
Elution：With 50mmol/L citric acids/disodium hydrogen phosphate buffer solution (pH8.0) elution destination protein, flow velocity 300cm/
Hour, collect target protein peak.
The screening of the chromatographic stuffing of embodiment 3
Prepare the process such as embodiment 1 of rhG-CSF renaturation solutions.The chromatography purified to obtain suitable rhG-CSF renaturation solutions is filled out
Material, this research compares the ability of three kinds of filler purification renaturation liquid with weak cation aglucon, and purge process is with reference to embodiment
2, as a result as shown in table 1.CM FF obtain sample HPLC purity less than mixed mode Capto MMC and
HCX, and flow velocity is slow, yield is low.Comparatively speaking, Capto MMC andHCX can more meet rhG-CSF pharmacopeia mark
Standard, and it is adapted to heavy industrialization amplification.
Table 1：Three kinds of fillers compare the purification effect of rhG-CSF renaturation solutions
The screening of the Capto MMC impurity of embodiment 4 and sample elution pH of buffer
The present embodiment have studied Capto MMC purifying rhG-CSF renaturation solutions Impurity elutions and sample elution pH of buffer
Can selection range.Prepare the process such as embodiment 1 of rhG-CSF renaturation solutions.The present embodiment experimental design and result are as shown in table 2.
The pH that Impurity elution buffer solution can be chosen is 7.2-7.8.Impurity elution pH occurs failing elution impurity or mesh at 7.0 and 8.0
Albumen the problem of walk elution herein, influence the purity or yield of final elution samples.Equally, sample elution buffer solution can be chosen
PH be 8.0-9.0.When pH is less than 8.0 (experimental example 3), sample fails elution.
Table 2：The influence that impurity and sample elution pH of buffer are purified to rhG-CSF renaturation solutions
The ultrafiltration displacement of the rhG-CSF buffer solutions of embodiment 5
Buffer exchange is carried out from 5KD films bag.
Balance：With level pad (20mmol/L sodium acetates, pH4.0) balance film bag；
Constant volume is replaced：Capto MMC column chromatographies elution collection liquid adds film packet system.Into film bag, continuous stream adds displacement
Buffer solution (20mmol/L sodium acetates, pH4.0), control flow acceleration is identical with appearing end flow velocity, keeps sample volume constant, the phase
Between control transmembrane pressure to be 18-22PSI, whole process is co-continuous to add 5 times of displacement buffer solutions (20mmol/L sodium acetates, pH4.0).
The sample detection of embodiment 6：
RhG-CSF stostes carry out purity analysis with SDS-PAGE, RP-HPLC method.
1)SDS-PAGE
Polyacrylamide gel：4-12%Bis-Tris Gel, Life technologies；
Sample buffer：LDS Sample Buffer(4×),Life technologi；
Electrophoretic buffer：MOPS SDS Running Buffer(20×),Life technologies；
Dyeing liquor：GelcodeTMBlue safe protein Stain,Thermo scientific；
Destainer：Purified water, self-control.
After recombined human granulocyte stimulating factors stoste loading, 150V constant pressure electrophoresis makes bromophenol blue migrate to glue bottom.Using
Coomassie brilliant blue fast staining is dyed, and purifies water decolorization, and gel imager carries out purity analysis, testing result such as explanation
Shown in book accompanying drawing 1.
It was found from analysis result, the SDS-PAGE purity of the rhG-CSF stostes finally obtained is 99.2%.
2)RP-HPLC
Chromatographic column：SHISEIDO CAPCELL PAK C18SG300 5μm 150mm×4.6mm；
A phases：Trifluoroacetic acid-the aqueous solution (takes 1.0ml trifluoroacetic acids to add water to 1000ml, fully to mix)；
B phases：Trifluoroacetic acid-acetonitrile solution (take 1.0ml trifluoroacetic acids to add trifluoroacetic acid aqueous solution to 1000ml, it is fully mixed
It is even)；
Table 3：Under room temperature condition, according to the form below carries out gradient elution, Detection wavelength 280nm
| Time (min) | A (%) | B (%) |
| 0 | 100 | 0 |
| 15 | 30 | 70 |
| 25 | 30 | 70 |
| 26 | 100 | 0 |
RhG-CSF stoste purity detectings are carried out using RP-HPLC methods, testing result is analyzed, the rhG-CSF finally obtained is former
The purity of liquid is 99.21%.
3)SEC-HPLC
Chromatographic column：Hydrophilic silica gels molecular sieve column (7.8 × 300mm of TSK-Gel G3000SWXL, 5 μm, 250A)；
Mobile phase：50mM ammonium hydrogen carbonate, pH=7.0；
Wavelength：215nm；Column temperature：25℃；Flow velocity：0.5ml/min；
RhG-CSF stoste purity detectings are carried out using SEC-HPLC methods, testing result, the purity of rhG-CSF stostes is analyzed
For 99.32%.
4) determination of activity
Using recombinant human granulocyte colony stimulating factor determination of activity national standard as activity criteria's product.Take standard items and this
The rhG-CSF stostes obtained are invented, in 96 porocyte culture plates, 1.0ng/ml are diluted to basal medium, then carry out 2 times
Gradient dilution.The NFS-60 cell lines of culture are taken, are washed with RPM1640 nutrient solutions 3 times, cell suspension is taken before being centrifuged at the 3rd time
For cell count.After counting, cell is resuspended with basic culture solution, 2.0 × 10 are configured to5Cell/ml.In every μ l of hole 50 marks
50 μ l cell suspensions, 37 DEG C, 5%CO are added in quasi- product/rhG-CSF stostes2Under the conditions of cultivate 40-48 hours.Then, add per hole
Enter 20 μ l MTT solution (5mg/ml), 37 DEG C, 5%CO2Under the conditions of cultivate about 5 hours, add 100 μ l lysates per hole, mix
ELIASA colorimetric is utilized after even, measure wavelength/reference wavelength is 570nm/630nm.Calculate the ratio work for obtaining rhG-CSF stostes
Property.
Using the biological activity of NFS-60 cells/MTT colorimetric method for determining rhG-CSF stostes, calculating acquisition specific activity is
9.0×107IU/mg。
Claims (18)
1. a kind of purification process of recombinant human granulocyte colony stimulating factor, including：By recombinant human granulocyte colony stimulating factor
Renaturation solution loading is extremely purified with the cation chromatographic column of mixed mode filler.
2. the purification process of recombinant human granulocyte colony stimulating factor according to claim 1, it is characterised in that described mixed
Syntype filler is selected from Capto MMC fillers；HCX fillers；Bestarose Diamond MMC fillers；
UniMSP-30S fillers, preferably Capto MMC fillers andHCX fillers.
3. the purification process of recombinant human granulocyte colony stimulating factor according to claim 1 or 2, it is characterised in that also wrap
Include and Impurity elution is first carried out using buffer solution, then carry out the step of sample elution collects sample peak.
4. the purification process of recombinant human granulocyte colony stimulating factor according to claim 3, it is characterised in that described miscellaneous
Matter elution buffer solution ionic strength used is 5~20mmol/L, and pH is 7.2~7.8, buffer solution ion used in the sample elution
Intensity is 20~100mmol/L, and pH is 8.0~9.0.
5. according to the purification process of any described recombinant human granulocyte colony stimulating factors of claim 1-4, it is characterised in that
The also buffered liquid equilibrium step before loading of the cation chromatographic column including mixed mode filler, the level pad ion is strong
Spend for 5~50mmol/L, pH is 3.0~6.5.
6. the purification process of recombinant human granulocyte colony stimulating factor according to claim 3, it is characterised in that described miscellaneous
Buffer is selected from acetate, citrate, phosphate, carbonate, Tris, glycine, histidine in matter elution buffer solution used
In one or more, buffer is selected from acetate, citrate, phosphate, carbonic acid in buffer solution used in the sample elution
One or more in salt, Tris, glycine, histidine.
7. the purification process of recombinant human granulocyte colony stimulating factor according to claim 5, it is characterised in that described flat
The one kind of buffer in acetate, citrate, phosphate, carbonate, Tris, glycine, histidine in weighing apparatus buffer solution
Or it is a variety of.
8. the purification process of recombinant human granulocyte colony stimulating factor according to claim 3, it is characterised in that the sample
Product elution is using pH elutions or salt ionic concentration type of elution, preferably pH types of elution.
9. according to the purification process of any described recombinant human granulocyte colony stimulating factors of claim 1-8, it is characterised in that
The step of also including obtained purification of samples progress buffer exchange is collected.
10. the purification process of recombinant human granulocyte colony stimulating factor according to claim 9, it is characterised in that described slow
Fliud flushing displacement can use the one or more in dialysis, ultrafiltration, desalting column chromatography scheme, and the ultrafiltration preferably is selected from doughnut and surpassed
Filter or film bag ultrafiltration, more preferably from film bag ultrafiltration.
11. the purification process of recombinant human granulocyte colony stimulating factor according to claim 10, it is characterised in that described
Film bag ultrafiltration membrane aperture is 1KD~20KD, preferably 3KD, 5KD or 10KD, more preferably 5KD.
12. the purification process of recombinant human granulocyte colony stimulating factor according to claim 11, it is characterised in that described
One or more of the film packaging material in Kynoar, modified cellulose, polyether sulfone, improvement polyether sulfone in film bag ultrafiltration.
13. according to the purification process of any described recombinant human granulocyte colony stimulating factors of claim 1-12, its feature exists
In to renaturation solution progress pH regulations, filtration step also before loading.
14. the purification process of recombinant human granulocyte colony stimulating factor according to claim 13, it is characterised in that described
It is 2.0~6.0 that renaturation solution, which is carried out after pH regulations, preferably is selected from 3.5~4.5, the filtering is using hollow fibre filtering, film bag mistake
One or more in filter, dead-end filtration mode, preferably dead-end filtration.
15. according to the purification process of any described recombinant human granulocyte colony stimulating factors of claim 1-14, including：
A) pH regulations, filtering are carried out before loading to recombinant human granulocyte colony stimulating factor renaturation solution；
B) the cation chromatographic column of mixed mode filler buffered liquid balance before loading；
C) loading to the cation chromatographic column of mixed mode filler carries out chromatographic purifying, first carries out Impurity elution using buffer solution,
Elution is carried out again collects sample peak；
D) obtained purification of samples progress buffer exchange will be collected；
Wherein, b) and c) described in buffer solution buffer be selected from acetate, it is citrate, phosphate, carbonate, Tris, sweet
One or more in propylhomoserin, histidine, the level pad ionic strength is 5-50mmol/L, and pH is 3.0~6.5, institute
Impurity elution buffer solution ionic strength is stated for 5~20mmol/L, pH is 7.2~7.8, the elution buffer ionic strength is 20
~100mmol/L, pH are 8.0~9.0.
16. the purification process of recombinant human granulocyte colony stimulating factor according to claim 15, it is characterised in that described
PH is 2.0~6.0 after the regulation of step a) renaturation solutions, preferably is selected from 3.5~4.5.
17. the purification process of recombinant human granulocyte colony stimulating factor according to claim 16, it is characterised in that described
Step a) filterings can use the one or more in hollow fibre filtering, film packet filtering, dead-end filtration mode, preferably dead end mistake
Filter membrane material is Kynoar or polytetrafluoroethylene (PTFE) in filter, the dead-end filtration, and wherein aperture is 0.22 μm.
18. a kind of method for preparing PEG-rhG-CSF, including described in claim 1-17
Recombinant human granulocyte colony stimulating factor purification step, and obtained recombinant human granulocyte colony stimulating factor is subjected to poly- second
Glycol modification step, the structure of preferably described PEG-rhG-CSF is as shown in formula I
Wherein m is selected from 50-2500 integer, preferably is selected from 400-500 integer, G is Met-G-CSF.
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| CN110041423A (en) * | 2018-01-16 | 2019-07-23 | 江苏奥赛康药业股份有限公司 | A kind of renaturation and purification process of recombinant human granulocyte colony stimulating factor |
| CN114224853A (en) * | 2022-01-04 | 2022-03-25 | 山东新时代药业有限公司 | Freeze-dried preparation for injection of polyethylene glycol recombinant human granulocyte stimulating factor |
| CN114853872A (en) * | 2022-04-27 | 2022-08-05 | 山东新时代药业有限公司 | Preparation method of polyethylene glycol modified rhG-CSF |
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| CN1663962A (en) * | 2004-03-01 | 2005-09-07 | 重庆富进生物医药有限公司 | Recombinant human granulocyte colony stimulating factor and one-step purifying process for chemical modifications thereof |
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| KR0160934B1 (en) * | 1996-01-15 | 1998-11-16 | 성재갑 | Process for the purification of recombinant human granulocyte-colony stimulating factor in the form of inclusion body from yeast |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110041423A (en) * | 2018-01-16 | 2019-07-23 | 江苏奥赛康药业股份有限公司 | A kind of renaturation and purification process of recombinant human granulocyte colony stimulating factor |
| CN114224853A (en) * | 2022-01-04 | 2022-03-25 | 山东新时代药业有限公司 | Freeze-dried preparation for injection of polyethylene glycol recombinant human granulocyte stimulating factor |
| CN114224853B (en) * | 2022-01-04 | 2022-09-23 | 山东新时代药业有限公司 | Freeze-dried preparation for injection of polyethylene glycol recombinant human granulocyte stimulating factor |
| CN114853872A (en) * | 2022-04-27 | 2022-08-05 | 山东新时代药业有限公司 | Preparation method of polyethylene glycol modified rhG-CSF |
| CN114853872B (en) * | 2022-04-27 | 2023-11-17 | 山东新时代药业有限公司 | Preparation method of polyethylene glycol modified rhG-CSF |
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