CN110041423A - A kind of renaturation and purification process of recombinant human granulocyte colony stimulating factor - Google Patents
A kind of renaturation and purification process of recombinant human granulocyte colony stimulating factor Download PDFInfo
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Abstract
The present invention provides a kind of renaturation of recombinant human granulocyte colony stimulating factor and purification process, comprising: 1) prepares solubilization of inclusion bodies liquid；2) solubilization of inclusion bodies liquid is added in renaturation buffer, cystine and cysteine is added, carry out renaturation；3) the weak cation chromatographic column of loading to composite ligand is purified.Purifying process of the present invention is easy to operate, the used time is short, while equipment requirement is not high, save the cost, is suitble to the production scale of amplification, and forgives purity of protein height after high albumen bulk concentration, renaturation yield height, chromatographic purifying when renaturation.
Description
Technical field
The present invention relates to biological medicine and bioengineering downstream protein purification arts, and in particular to a kind of recombinant humangranulocyte
Colony stimulating factor (recombinant human granulocyte colony stimulating factor, rhG-CSF)
Renaturation and purification process.
Background technique
Filgrastim (human granulocyte colony stimulating factor, hG-
It CSF) is a kind of hemopoieticgrowth factor for adjusting granulocyte (especially neutrophil leucocyte) maturation and growth in mammals.It
By stimulation granulocyte macrophage colony forming units (CFU-GM) to granular leukocyte colony formed unit (CFU-G) differentiation and
Maturation promotes release of the mature neutrophil leucocyte to peripheral blood, to promote the growth of neutrophil leucocyte.Clinically it is used to control
Neutrophilic granulocytopenia caused by a variety of causes is treated, there is very big economy and society meaning.
Welte.K in 1985 is successfully purified from the culture supernatant of 5637 cell line and is refined out people's grain
Colony-stimulating factor (hG-CSF), then the Souza.L.M of the AMGEN company in Welte.K and the U.S. is further determined
The N-terminal amino acid of this hG-CSF puts in order, and the hG-CSF gene cloning that will derive from 5637 cell strains, using gene
Gene insertion Escherichia coli are obtained hG-CSF by engineering technology, have developed recombinant human granulocyte colony stimulating factor (rhG-
CSF), trade name Filgratin (G-CSF).
Since the source of hG-CSF is very limited, the method that large-scale industrial production is all made of genetic engineering is obtained
?.Pharmacy corporation mostly uses greatly widely used E. coli system to express rhG-CSF 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, aggregation is easy, substantially with inactive
Inclusion bodies be present in cell, need by denaturation and renaturation could obtain active rhG-CSF.After renaturation
Sample in there are a large amount of host cell proteins and other related impurities, therefore, the protein purification after renaturation is most important.
Currently, patented technology reports much the refolding method of rhG-CSF, but all it cannot be guaranteed that purifying process operation letter
Just, while the time is short, renaturation yield is high, renaturation when forgive that albumen bulk concentration is high, purity is high.
CN1718739A is reported using SP Sepharose High Performance (SP HP) directly to rhG-CSF
The method of renaturation solution purifying, but in renaturing inclusion bodies liquid other than destination protein, also containing a certain amount of host protein, lipid and
DNA (DNA).The partial size very little of SP HP filler is not suitable for the renaturation containing more impurity, situation complexity
Liquid, and flow velocity is slow, and processing operation time is long.
CN107188952A reports thick inclusion body and carries out renaturation after denaturation dissolution is using displacement steps such as ultrafiltration, most
Capto MMC chromatographic purifying is carried out afterwards.But this method implementation needs displacement step, complicated for operation, the time is long, and forgives when renaturation
Albumen bulk concentration is low, renaturation yield is low.
In view of the above problems, it develops that a kind of rhG-CSF renaturation manipulation is easy, the time is short, and forgives when renaturation
The Dipurification process high, with high purity, that renaturation yield is high of albumen bulk concentration is very necessary.
Summary of the invention
A kind of recombinant human granulocyte colony thorn is provided the purpose of the present invention is to solve the above the deficiencies in the prior art
Swash the renaturation and purification process of the factor.
The technical solution of the present invention is as follows: the renaturation and purification process of a kind of recombinant human granulocyte colony stimulating factor, comprising:
1) recombinant human granulocyte colony stimulating factor inclusion body is dissolved with lysate, DTT is added, obtains solubilization of inclusion bodies
Liquid；
2) solubilization of inclusion bodies liquid is added in renaturation buffer, cystine and cysteine is added, carried out renaturation, obtain
Recombinant human granulocyte colony stimulating factor renaturation solution；
3) the weak cation chromatographic column of recombinant human granulocyte colony stimulating factor renaturation solution loading to composite ligand is carried out
Purifying.
The solid-to-liquid ratio of recombinant human granulocyte colony stimulating factor inclusion body and lysate described in step 1) be 1g:(5~
10)mL。
Renaturation buffer described in step 2) is made of water, arginine, urea, EDTA-2Na, Tris, pH adjusting agent；
Or it is made of water, arginine, urea, EDTA-2Na, Tris, sorbierite, pH adjusting agent.
Arginic content is 5wt%~25wt% in renaturation buffer；Preferably, arginic content be 5wt%~
10wt% or 8wt%~13wt% or 10wt%~15wt% or 13wt%~18wt% or 15wt%~20wt% or 18wt%
~23wt% or 20wt%~25wt%.
The concentration of urea is 1~3mol/L in renaturation buffer；Preferably, the concentration of urea be 1~2mol/L or 1.5~
2.5mol/L or 2~3mol/L.
The concentration of EDTA-2Na is 1~3mmol/L in renaturation buffer；Preferably, the concentration of EDTA-2Na be 1~
2mmol/L or 1.5~2.5mmol/L or 2~3mmol/L.
The concentration of Tris is 0.05~0.15mol/L in renaturation buffer；Preferably, the concentration of Tris be 0.05~
0.08mol/L or 0.07~0.10mol/L or 0.09~0.12mol/L or 0.11~0.14mol/L or 0.13~0.15mol/
L。
Renaturation buffer pH adjusting agent is selected from HCl and/or NaOH.The pH of renaturation buffer is 8.0~9.5.When renaturation is slow
When containing sorbierite in fliud flushing, the content of sorbierite is not higher than 10wt%；Preferably, the content of sorbierite is not higher than 8wt%；More
Preferably, the content of sorbierite is not higher than 5wt%；It is further preferred that the content of sorbierite is 0.01wt%~5wt%.
Illustratively, the composition of renaturation buffer can be (aqueous solution):
8wt% arginine, 2mol/L urea, 2mmol/L EDTA-2Na, 0.1mol/L Tris, pH9.0；Or
15wt% arginine, 5wt% sorbierite, 1mol/L urea, 3mmol/L EDTA-2Na, 0.05mol/L Tris,
pH8.0；Or
8.5wt% arginine, 2mol/L urea, 2mmol/L EDTA-2Na, 0.1mol/L Tris, pH 9.0；Or
5wt% arginine, 1mol/L urea, 2mmol/L EDTA-2Na, 0.1mol/L Tris, pH9.0；Or
8wt% arginine, 3mol/L urea, 1mmol/L EDTA-2Na, 0.15mol/L Tris, pH9.0；Or
13wt% arginine, 1mol/L urea, 3mmol/L EDTA-2Na, 0.05mol/L Tris, pH9.0.
Cysteine is added to final concentration of to final concentration of 0.8~2mmol/L in addition cystine described in step 2)
0.1~1mmol/L.
Preferably, cysteine is added to final concentration of 0.9~1.5mmol/L in addition cystine described in step 2)
To final concentration of 0.2~0.7mmol/L.
In step (2), before solubilization of inclusion bodies liquid is added to renaturation buffer, the temperature of renaturation buffer is adjusted to 2
~12 DEG C；Preferably, the temperature of renaturation buffer is adjusted to 2~6 DEG C or 4~8 DEG C or 6~10 DEG C or 8~12 DEG C.
Lysate described in step 1) is made of water, guanidine hydrochloride, Tris, pH adjusting agent.
The concentration of guanidine hydrochloride is 4~8mol/L in lysate；Preferably, the concentration of guanidine hydrochloride be 4~6mol/L or 5~
7mol/L or 6~8mol/L.
The concentration of Tris is 0.05~0.15mol/L in lysate；Preferably, the concentration of Tris is 0.05~0.08mol/
L or 0.07~0.10mol/L or 0.09~0.12mol/L or 0.11~0.14mol/L or 0.13~0.15mol/L.
PH adjusting agent is selected from HCl and/or NaOH in lysate.The pH of lysate is 7.0~9.5；Preferably, pH value is
7.0~8.0 or 7.5~8.5 or 8.0~9.0 or 8.5~9.5.
Illustratively, the composition of lysate can be (aqueous solution):
6mol/L guanidine hydrochloride, 0.15mol/L Tris, pH 8.0；Or
5mol/L guanidine hydrochloride, 0.1mol/L Tris, pH9.0；Or
6mol/L guanidine hydrochloride, 0.1mol/L Tris, pH8.0.
In step 1), DTT to final concentration of 1~20mmol/L is added；Preferably, be added DTT to final concentration of 5~
10mmol/L or 9~14mmol/L.
The weak cation filler of composite ligand described in step 3) is selected from Capto MMC filler, Generic MC-CM filler
Or Eshmuno HCX filler；It is preferred that Capto MMC filler.
The method of the invention further includes carrying out before chromatographing loading to recombinant human granulocyte colony stimulating factor renaturation solution
PH is adjusted, filtration step.The renaturation solution pH is adjusted to 3~5；Preferably, pH is adjusted to 3.5~4.5；It is highly preferred that pH
It is adjusted to 3.6~4.0 or 3.8~4.2 or 4.0~4.4.
The method of the invention further includes by chromatographic column before chromatographing loading through equilibrium liquid equilibrium step.Side of the present invention
Method further includes carrying out first time elution with equilibrium liquid after chromatographing loading, carries out second with leacheate and elutes, with eluent into
Row elution step.Equilibrium liquid is made of water, sodium chloride, phosphate, pH adjusting agent in step 3).Sodium chloride concentration in equilibrium liquid
For 100~300mmol/L；Preferably, sodium chloride concentration be 100~200mmol/L or 150~250mmol/L or 200~
300mmol/L.Phosphate concn is 0.05~0.15mol/L in equilibrium liquid；Preferably, phosphate concn be 0.05~
0.10mol/L or 0.08~0.12mol/L.PH adjusting agent is selected from HCl and/or NaOH in equilibrium liquid.The pH of equilibrium liquid be 3.5~
4.5；Preferably, pH is 3.5~4 or 3.8~4.3 or 4~4.5.
Leacheate is made of water, sodium chloride, phosphate, pH adjusting agent in step 3).Sodium chloride concentration is in leacheate
180~200mmol/L；Preferably, sodium chloride concentration 200mmol/L.In leacheate phosphate concn be 0.08~
0.12mol/L；Preferably, phosphate concn 0.1mol/L.PH adjusting agent is selected from HCl and/or NaOH in leacheate.Leacheate
PH be 5~6；Preferably, pH is 5~5.5 or 5.2~5.8 or 5.5~6.
Eluent is made of water, sodium chloride, phosphate, pH adjusting agent in step 3).Sodium chloride concentration is in eluent
280~320mmol/L；Preferably, sodium chloride concentration 300mmol/L.In eluent phosphate concn be 0.08~
0.12mol/L；Preferably, phosphate concn 0.1mol/L.PH adjusting agent is selected from HCl and/or NaOH in eluent.Eluent
PH be 6~7；Preferably, pH is 6~6.5 or 6.2~6.8 or 6.5~7.0.
In the present invention, phosphate is the sodium salt of phosphoric acid or the sylvite of phosphoric acid；Preferably, phosphate is the sodium salt of phosphoric acid.Phosphorus
The sodium salt of acid is selected from one of sodium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate or a variety of；The sylvite of phosphoric acid be selected from potassium phosphate,
One of dipotassium hydrogen phosphate, potassium dihydrogen phosphate are a variety of.
Specifically, the present invention provides a kind of renaturation of recombinant human granulocyte colony stimulating factor and purification process, packet
It includes:
A) recombinant human granulocyte colony stimulating factor inclusion body is dissolved with lysate, addition DTT to final concentration of 1~
20mmol/L obtains solubilization of inclusion bodies liquid；
B) solubilization of inclusion bodies liquid is added in the pre- renaturation buffer for being cooled to 2 DEG C~12 DEG C, it is dense to end that cystine is added
Degree is 0.8~2mmol/L, and cysteine is added to final concentration of 0.1~1mmol/L, carries out renaturation, obtains recombinant humangranulocyte
Colony stimulating factor renaturation solution；
C) recombinant human granulocyte colony stimulating factor renaturation solution is subjected to pH adjusting, filtering；
D) chromatographic column is balanced with equilibrium liquid；
E) the filtered recombinant human granulocyte colony stimulating factor renaturation solution loading for obtaining step c) is to composite ligand
Weak cation chromatographic column purified；
F) it carries out eluting for the first time again and second elutes, finally elute, collect eluting peak；
Wherein, the renaturation buffer in step b) are as follows: 5wt%~25wt% arginine, 1~3mol/L urea, 1~
3mmol/L EDTA-2Na, 0.05~0.15mol/L Tris, pH are 8.0~9.5.
Lysate described in step a) are as follows: 5~7mol/L guanidine hydrochloride, 0.05~0.15mol/L Tris, pH be 7.0~
9.5。
The solid-to-liquid ratio of recombinant human granulocyte colony stimulating factor inclusion body and lysate described in step a) be 1g:(5~
10)mL。
The weak cation filler of the composite ligand be selected from Capto MMC filler, Generic MC-CM filler or
Eshmuno HCX filler；It is preferred that Capto MMC filler.
The elution of first time described in step f) is eluted for the second time to elute chromatographic column with equilibrium liquid to be eluted with leacheate
Chromatographic column.
The equilibrium liquid is 150~250mmol/L sodium chloride, 0.08~0.12mol/L disodium hydrogen phosphate, pH 3.5
~4.5, the leacheate is 180~200mmol/L sodium chloride, and 0.08~0.12mol/L phosphate, pH is 5~6, described
Eluent be 280~320mol/L sodium chloride, 0.08~0.12mol/L phosphate, pH be 6~7.
The entitled disodium ethylene diamine tetraacetate of EDTA-2Na Chinese in the present invention, entitled three (methylol) the amino first of Tris Chinese
Alkane, the entitled dithiothreitol (DTT) of DTT Chinese.
The utility model has the advantages that
1, the implementation of the method for the present invention is easy to operate, the technique used time is short without other steps such as displacements, while equipment requirement
Not high, save the cost is suitble to the production scale of amplification；
2, it is high to forgive albumen bulk concentration when renaturation, solubilization of inclusion bodies liquid is added in renaturation buffer, can make to forgive egg
Lean type concentration reaches 1.2~1.5g/L, is 4~5 times that existing method forgives albumen bulk concentration；
3, purity of protein is high after the method for the present invention chromatographic purifying, and SEC-HPLC method measures rhG-CSF stoste purity and reaches
99.7% or more；
4, the method for the present invention recombinant human granulocyte colony stimulating factor renaturing inclusion bodies rate is high, and renaturation yield is higher than 45%, can
To reach renaturation yield for 65%~85%.
Detailed description of the invention
Fig. 1 is the plasmid map of pET-9a-G-CSF prepared by embodiment one；
Fig. 2 is the biological activity figure of the rhG-CSF of embodiment one after purification, and wherein RS is standard items.
Specific embodiment
Following embodiment is used for detailed description of the present invention content, rather than limiting the scope of the invention.It is practical
RhG-CSF engineered strain is more in, and construction method can refer to the " system of G-CSF-pJGW1/pGP1-2/DH5 α engineering bacteria
Stability study " (liberation army Guangzhou cure college journals, Huang tree its etc., 1997.12.25), " G-CSF engineering bacteria is in IPTG and cream
Sugar induces lower expression of results to compare " report such as (Heilungkiang medicine, Li Zhengwu etc., 1998.4.30).The rhG- that the present invention uses
CSF engineered strain is that applicant constructs bacterial strain.
The building of one: pET-9a-G-CSF prokaryotic expression bacterial strain of embodiment
Human G-CSF gene order is as shown in SEQ ID No.1.According to common molecular cloning process composition sequence such as SEQ ID
Human G-CSF gene shown in No.1 simultaneously introduces terminator TAA and target gene both ends NdeI and BamHI restriction enzyme site；Use NdeI
Double digestion is distinguished to prokaryotic expression plasmid vector pET-9a and human G-CSF genetic fragment with BamHI, by the pET-9a after double digestion
It is connected with human G-CSF genetic fragment, is transformed into E.coli DH5 α bacterial strain after obtaining pET-9a-G-CSF recombinant plasmid (such as Fig. 1)
In.
E.coli DH5 α bacterial strain containing pET-9a-G-CSF recombinant plasmid is inoculated into 1% inoculum concentration containing 50 μ
In the LB culture medium of g/mL kanamycins, shaking table (180rpm) overnight incubation at 37 DEG C, using proposing examination in plasmid endotoxin-free
Agent box (being purchased from Biomega company) extraction purification plasmid, plasmid after purification are imported according to the method for transformation in " molecular cloning "
E.coli BL21 (DE3) (is purchased from Invitrogen company), reuses the LB agar plate sieve containing 50 μ g/mL kanamycins
Select positive colony.10 positive colony single colonies are selected, SuperBroth culture medium is inoculated in after label respectively and (contains 50 μ g/
ML kanamycins, 4.1%SuperBroth, 1g/L lactose) in, shaking table (180rpm) is cultivated 20~24 hours at 37 DEG C.It takes
500 μ L bacterium solutions collect bacterium mud after 3500rpm is centrifuged and abandons supernatant, and bacterium mud is resuspended with 1mL PBS, takes 15 μ of bacteria suspension after being resuspended
L is added 15 2 × sample-loading buffers of μ L (Loading Buffer), sets boiling water bath 5min, is centrifuged 5min in 10000rpm, takes
Clear 15 μ L carries out the whole bacterium electrophoresis of SDS-PAGE, deposition condition are as follows: 4.5% concentration glue 100v electrophoresis 10min, 15% separation gel 120v
Electrophoresis stops electrophoresis to bromophenol blue to gel bottom.After, with coomassie brilliant blue staining 30min, then the 3h that decolourizes, upper BIO-
RAD gel imaging system detects G-CSF expression quantity.The strain that selection expression quantity is up to 40% carries out PCR and sequencing identification, and
It is named as E.coliBL21 (DE3)-pET-9a-G-CSF.
Embodiment two:
1, rhG-CSF renaturation solution is prepared
Technique bacterial strain uses therefor is the e.colistraindh5α of pET9a/G-CSF plasmid conversion in embodiment one.It is fermented
Afterwards, thallus to be collected, carries out clasmatosis using high pressure homogenizer, centrifugation obtains thick inclusion body, then thick inclusion body is washed,
Centrifugation obtains the inclusion body of preliminary purification.
The inclusion body 60g for taking preliminary purification, in ratio inclusion body lysate (the 6mol/L salt of solid-to-liquid ratio 1:10 (g/mL)
Sour guanidine, 0.15mol/L Tris, pH8.0) denaturation dissolution is carried out, DTT (two sulphur threoses are added after being stirred at room temperature to clear
Alcohol) to final concentration of 7mmol/L, continue to stir 30min or more, after 0.22 μm of filter filters, obtains solubilization of inclusion bodies liquid.
Solubilization of inclusion bodies liquid is slowly pumped into renaturation buffer (8wt% arginine, the 2mol/L urine that 10L is cooled to 8 DEG C in advance
Element, 2mmol/L EDTA-2Na, 0.1mol/L Tris, pH9.0) in, (by forgiving proteosome in measurement solubilization of inclusion bodies liquid
Concentration calculates the volume that solubilization of inclusion bodies liquid is added) to forgive albumen bulk concentration to be 1.2~1.5g/L, cystine is added extremely
Final concentration of 1.2mmol/L and cysteine stir, renaturation to final concentration of 0.3mmol/L under the conditions of 12 DEG C, 120rpm
18h.2, the Capto MMC chromatography of rhG-CSF
RhG-CSF solution after renaturation glacial acetic acid is adjusted into pH to 4.0, after standing 30min, via hole diameter is (8+1.2) μ
The filtering of m filter；
Disinfection: chromatography media 30min or more, flow velocity 60cm/h are sterilized with 0.8mol/L sodium hydroxide；
Balance: chromatographic column, stream are balanced with equilibrium liquid (200mmol/L sodium chloride, 0.1mol/L disodium hydrogen phosphate, pH4.5)
100~200cm/h of speed, until pH, conductance are steady；
Loading: by filtered renaturation solution loading, 100~200cm/h of flow velocity, retention time >=4min；
It elutes for the first time: after completion of the sample, eluting chromatographic column again with equilibrium liquid, until ultraviolet steady；
Second elutes: with leacheate (200mmol/L sodium chloride, 0.1mol/L disodium hydrogen phosphate/sodium dihydrogen phosphate,
PH6 chromatographic column) is eluted, until pH6；
Elution: it is eluted with eluent (300mol/L sodium chloride, 0.1mol/L disodium hydrogen phosphate/sodium dihydrogen phosphate, pH6.5)
Destination protein collects eluting peak, 100~200cm/h of flow velocity.
3, sample detection
(1)SEC-HPLC
Chromatographic column: hydrophilic silica gels molecular sieve column (TSK-Gel G3000SWXL 7.8 × 300mm, 5 μm, 250A)
Mobile phase: phosphate buffer adjusts pH to 6.9, with 0.22 μm of membrane filtration
Wavelength: 214nm；Column temperature: 25 DEG C；Flow velocity: 0.5ml/min；Sample cell temperature: 2~8 DEG C；Applied sample amount: 30 μ g
Using SEC-HPLC method carry out rhG-CSF stoste purity detecting, analysis detection as a result, rhG-CSF stoste purity
It is 99.7%.
(2) determination of activity
With recombinant human granulocyte colony stimulating factor national standard for activity criteria's product, dissolve to specifications.
The rhG-CSF stoste for taking standard items (RS) and the present invention to obtain will be marked in 96 well culture plates with basic culture solution
Quasi- product are diluted to 2000IU/mL, and sample is diluted to 20000pg/mL, then carries out 3 times of gradient dilutions.Take culture M-NFS-60 ( CRL-1838TM) cell, it is centrifuged 7 minutes, is repeated 3 times, finally with basis training with RPMI-1640 culture medium 1200rpm
It supports base suspension cell and counts, and be configured to 2 × 105The cell suspension of a/mL.In every 50 μ L standard items of hole/rhG-CSF stoste
50 μ L cell suspensions of middle addition, in 37 DEG C, 5%CO2(c/c) 36~48h is cultivated under the conditions of.Then 10 μ L CCK- are added in every hole
8, in 37 DEG C, 5%CO2(c/c) continue to cultivate 4h under the conditions of.
It is measurement wavelength (reference wavelength 630nm, according to microplate reader type that microplate reader (BioTekSynergyH1) 450nm, which is arranged,
Number it is configured) absorption value is measured, calculate the specific activity for obtaining rhG-CSF stoste.
Using M-NFS-60 cell/CCK-8 measuring method measurement rhG-CSF stoste biological activity, calculates and obtain than living
Property be 8 × 107IU/mg.The specific activity that the present invention measures is higher than " Pharmacopoeia of People's Republic of China 2015 editions " third portion " recombined human
The requirement that stoste is examined and determine under granulocyte stimulating factor injection " item.
Embodiment three:
1, rhG-CSF renaturation solution is prepared
Technique bacterial strain uses therefor is the e.colistraindh5α of pET9a/G-CSF plasmid conversion.After fermented, bacterium is collected
Body carries out clasmatosis using high pressure homogenizer, and centrifugation obtains thick inclusion body, then is washed to thick inclusion body, is centrifuged acquisition
The inclusion body of preliminary purification.
The inclusion body 60g for taking preliminary purification, in ratio inclusion body lysate (the 5mol/L salt of solid-to-liquid ratio 1:10 (g/mL)
Sour guanidine, 0.1mol/L Tris, pH9.0) denaturation dissolution is carried out, DTT (dithiothreitol (DTT)) is added after being stirred at room temperature to clear
To final concentration of 10mmol/L, continue to stir 30min or more, after 0.22 μm of filter filters, obtains solubilization of inclusion bodies liquid.
Solubilization of inclusion bodies liquid is slowly pumped into renaturation buffer (15wt% arginine, the mountain 5wt% that 10L is cooled to 10 DEG C in advance
Pears alcohol, 1mol/L urea, 3mmol/L EDTA-2Na, 0.05mol/L Tris, pH8.0) in, so that forgiving albumen bulk concentration and being
Cystine is added to final concentration of 1.5mmol/L and cysteine to final concentration of 0.5mmol/L, in 10 in 1.2~1.5g/L
DEG C, stir under the conditions of 120rpm, renaturation 18h.
2, the Capto MMC chromatography of rhG-CSF
RhG-CSF solution after renaturation glacial acetic acid is adjusted into pH to 4.0, after standing 30min, via hole diameter is (8+1.2) μ
The filtering of m filter；
Disinfection: chromatography media 30min or more, flow velocity 60cm/h are sterilized with 1mol/L sodium hydroxide；
Balance: chromatographic column, flow velocity are balanced with equilibrium liquid (180mmol/L sodium chloride, 0.08mol/L disodium hydrogen phosphate, pH4)
100~200cm/h, until pH, conductance are steady；
Loading: by filtered renaturation solution loading, 100~200cm/h of flow velocity, retention time >=4min；
It elutes for the first time: after completion of the sample, eluting chromatographic column again with equilibrium liquid, until ultraviolet steady；
Second elutes: with leacheate (180mmol/L sodium chloride, 0.12mol/L disodium hydrogen phosphate/sodium dihydrogen phosphate,
PH5.5 chromatographic column) is eluted, until pH5.5；
Elution: mesh is eluted with eluent (280mol/L sodium chloride, 0.1mol/L disodium hydrogen phosphate/sodium dihydrogen phosphate, pH7)
Albumen, collect eluting peak, 100~200cm/h of flow velocity.
3, sample detection
(1)SEC-HPLC
For detection method with embodiment two, the purity for being detected rhG-CSF stoste is 99.8%.
(2) determination of activity
For measuring method with embodiment two, being computed and obtaining specific activity is 9 × 107IU/mg。
Example IV:
1, technique bacterial strain uses therefor is the e.colistraindh5α of pET9a/G-CSF plasmid conversion.After fermented, bacterium is collected
Body carries out clasmatosis using high pressure homogenizer, and centrifugation obtains thick inclusion body, then is washed to thick inclusion body, is centrifuged acquisition
The inclusion body of preliminary purification takes the inclusion body 60g of preliminary purification, be dissolved in 600mL inclusion body lysate (6mol/L guanidine hydrochloride,
0.1mol/L Tris, pH8.0), it is placed on magnetic stirring apparatus and stirs to clarify transparent yellow；1mL solution is taken, for forgiving
Proteosome purity testing (i.e. ratio shared by destination protein, HPLC method)
2, then two sulphur are added for forgiving proteosome purity testing in the solubilization of inclusion bodies liquid for taking out the preparation of 1mL step 1
Threitol (DTT) continues to stir at least 30min to final concentration of 5mmol/L；
3, after 0.22 μm of filtering, measurement solubilization of inclusion bodies liquid protein concentration (content of total protein, UV in ie in solution
Method, λ=280nm), it takes out and contains 12g destination protein solubilization of inclusion bodies liquid, be pumped into the renaturation buffer (8.5wt% of 10L pre-cooling
Arginine, 2mol/L urea, 2mmol/L EDTA-2Na, 0.1mol/L Tris, pH9.0) in；
4,24mL cystine solution is then first added, 3mL cysteine solution is added after waiting 3min, then after waiting 3min
5mL cystine solution is added, under the conditions of revolving speed 120rpm, 10~12 DEG C of temperature, starts renaturation；
5, after renaturation 18h, renaturation solution is pumped out from renaturation tank, takes out 1mL renaturation solution and 40 μ L glacial acetic acid are added, measures renaturation
Rate (such as active method, rp-hplc method).
Through detecting: forgiving proteosome purity is 86%, and renaturing inclusion bodies rate is 80%.
Embodiment five:
The renaturation buffer that the present embodiment has studied different proportion is multiple to recombinant human granulocyte colony stimulating factor inclusion body
The influence of property rate, renaturation yield detection process such as example IV.The present embodiment experimental design and it the results are shown in Table 1.
Table 1
It is to be considered as qualification that renaturation yield, which is greater than 45%, in the present invention.
Purity of protein is high after the method for the present invention chromatographic purifying, and SEC-HPLC method measures rhG-CSF stoste purity up to 99.7%
More than, renaturing inclusion bodies rates is high, while it is easy to operate, the technique used time is short, equipment requirement is not high, save the cost, is suitble to amplification
Production scale.
Sequence table
<110>Jiangsu Aosaikang Pharmaceutical Co., Ltd.
<120>renaturation and purification process of a kind of recombinant human granulocyte colony stimulating factor
<141> 2018-01-16
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 525
<212> DNA
<213> Homo sapiens
<400> 1
acccccctgg gccctgccag ctccctgccc cagagcttcc tgctcaagtg cttagagcaa 60
gtgaggaaga tccagggcga tggcgcagcg ctccaggaga agctgtgtgc cacctacaag 120
ctgtgccacc ccgaggagct ggtgctgctc ggacactctc tgggcatccc ctgggctccc 180
ctgagcagct gccccagcca ggccctgcag ctggcaggct gcttgagcca actccatagc 240
ggccttttcc tctaccaggg gctcctgcag gccctggaag ggatctcccc cgagttgggt 300
cccaccttgg acacactgca gctggacgtc gccgactttg ccaccaccat ctggcagcag 360
atggaagaac tgggaatggc ccctgccctg cagcccaccc agggtgccat gccggccttc 420
gcctctgctt tccagcgccg ggcaggaggg gtcctagttg cctcccatct gcagagcttc 480
ctggaggtgt cgtaccgcgt tctacgccac cttgcccagc cctga 525
Claims (14)
1. the renaturation and purification process of a kind of recombinant human granulocyte colony stimulating factor, comprising:
1) recombinant human granulocyte colony stimulating factor inclusion body is dissolved with lysate, DTT is added, obtains solubilization of inclusion bodies liquid；
2) solubilization of inclusion bodies liquid is added in renaturation buffer, cystine and cysteine is added, carried out renaturation, recombinated
Filgrastim's renaturation solution；
3) the weak cation chromatographic column of recombinant human granulocyte colony stimulating factor renaturation solution loading to composite ligand is purified.
2. the renaturation and purification process of recombinant human granulocyte colony stimulating factor according to claim 1, it is characterised in that
Renaturation buffer described in step 2 is made of water, arginine, urea, EDTA-2Na, Tris, pH adjusting agent；Or by water, essence
Propylhomoserin, urea, EDTA-2Na, Tris, sorbierite, pH adjusting agent composition.
3. the renaturation and purification process of recombinant human granulocyte colony stimulating factor according to claim 2, the renaturation is slow
Fliud flushing has following one or more features:
Arginic content is 5wt% ~ 25wt%；It is preferred that arginic content is 8wt% ~ 13wt% or 10wt% ~ 15wt% or 13wt%
~ 18wt% or 15wt% ~ 20wt% or 18wt% ~ 23wt% or 20wt% ~ 25wt%；Or
The concentration of urea is 1 ~ 3mol/L；It is preferred that the concentration of urea is 1 ~ 2mol/L or 1.5 ~ 2.5mol/L or 2 ~ 3mol/L；Or
The concentration of EDTA-2Na is 1 ~ 3mmol/L；It is preferred that the concentration of EDTA-2Na is 1 ~ 2mmol/L or 1.5 ~ 2.5mmol/L or 2
~3mmol/L；Or
The concentration of Tris is 0.05 ~ 0.15mol/L；It is preferred that the concentration of Tris is 0.05 ~ 0.08mol/L or 0.07 ~ 0.10mol/L
Or 0.09 ~ 0.12mol/L or 0.11 ~ 0.14mol/L or 0.13 ~ 0.15mol/L；Or
PH is 8.0 ~ 9.5；Or
When containing sorbierite, the content of sorbierite is not higher than 10wt%；It is preferred that the content of sorbierite is not higher than 8wt%；More preferably
The content of sorbierite is not higher than 5wt%；Still more preferably the content of sorbierite is 0.01wt%~5wt%.
4. the renaturation and purification process of recombinant human granulocyte colony stimulating factor according to claim 1, it is characterised in that
Addition cystine described in step 2 to final concentration of 0.8 ~ 2mmol/L, be added cysteine to final concentration of 0.1 ~
1mmol/L。
5. the renaturation and purification process of recombinant human granulocyte colony stimulating factor according to claim 1, it is characterised in that
Step 1) has following one or more features:
The solid-to-liquid ratio of the recombinant human granulocyte colony stimulating factor inclusion body and lysate is 1g:(5 ~ 10) mL；Or
The amount of the DDT is that DTT to final concentration of 1 ~ 20mmol/L is added；DTT is preferably added to final concentration of 5 ~ 10mmol/L
Or 9~14mmol/L.
6. the renaturation and purification process of recombinant human granulocyte colony stimulating factor according to claim 1, it is characterised in that
Lysate described in step 1) is made of water, guanidine hydrochloride, Tris, pH adjusting agent.
7. the renaturation and purification process of recombinant human granulocyte colony stimulating factor according to claim 6, the lysate
Have following one or more features:
The concentration of guanidine hydrochloride is 4~8mol/L；It is preferred that the concentration of guanidine hydrochloride is 4~6mol/L or 5~7mol/L or 6~8mol/
L；Or
The concentration of Tris is 0.05 ~ 0.15mol/L；It is preferred that the concentration of Tris is 0.05 ~ 0.08mol/L or 0.07 ~ 0.10mol/L
Or 0.09 ~ 0.12mol/L or 0.11 ~ 0.14mol/L or 0.13 ~ 0.15mol/L；Or
PH is 7.0 ~ 9.5；Preferable ph is 7.0 ~ 8.0 or 7.5~8.5 or 8.0~9.0 or 8.5~9.5.
8. the renaturation and purification process of recombinant human granulocyte colony stimulating factor according to claim 1, it is characterised in that
The weak cation filler of composite ligand described in step 3) be selected from Capto MMC filler, Generic MC-CM filler or
Eshmuno HCX filler；It is preferred that Capto MMC filler.
9. the renaturation and purification process of recombinant human granulocyte colony stimulating factor according to claim 1, which is characterized in that
The weak cation chromatographic column of loading described in step 3) to composite ligand carries out purifying and has following one or more features:
PH adjusting is carried out to recombinant human granulocyte colony stimulating factor renaturation solution before chromatographing loading；Or
Chromatographic column is balanced through equilibrium liquid before chromatography loading；Or
First time elution is carried out with equilibrium liquid after chromatographing loading, second is carried out with leacheate and elutes, washed with eluent
De- step.
10. the renaturation and purification process of recombinant human granulocyte colony stimulating factor according to claim 9, feature exist
In recombinant human granulocyte colony stimulating factor renaturation solution pH is adjusted to 3~5 before chromatographing loading；It is preferred that pH be adjusted to 3.5~
4.5；More preferable pH is adjusted to 3.6~4.0 or 3.8 ~ 4.2 or 4.0~4.4.
11. the renaturation and purification process of recombinant human granulocyte colony stimulating factor according to claim 9, it is characterised in that
The equilibrium liquid is made of water, sodium chloride, phosphate, pH adjusting agent, the leacheate by water, sodium chloride, phosphoric acid
Salt, pH adjusting agent composition, the eluent are made of water, sodium chloride, phosphate, pH adjusting agent.
12. the renaturation and purification process of recombinant human granulocyte colony stimulating factor according to claim 11, described is flat
Weighing apparatus liquid has following one or more features:
Sodium chloride concentration be 100 ~ 300mmol/L, preferably sodium chloride concentration be 100 ~ 200mmol/L or 150 ~ 250mmol/L or
200~300mmol/L；Or
Phosphate concn is 0.05 ~ 0.15mol/L, and preferably phosphate concentration is 0.05 ~ 0.10mol/L or 0.08 ~ 0.12mol/
L, preferably phosphate are the sodium salt of phosphoric acid or the sylvite of phosphoric acid；Or
PH is 3.5~4.5, and preferably pH is 3.5~4 or 3.8~4.3 or 4~4.5.
13. the renaturation and purification process of recombinant human granulocyte colony stimulating factor according to claim 11, the leaching
Washing lotion has following one or more features:
Sodium chloride concentration is 180 ~ 200mmol/L, and preferably sodium chloride concentration is 200mmol/L；Or
Phosphate concn is 0.08 ~ 0.12mol/L, and preferably phosphate concentration is 0.1mol/L, and preferably phosphate is the sodium of phosphoric acid
The sylvite of salt or phosphoric acid；Or
PH is 5~6, and preferably pH is 5~5.5 or 5.2~5.8 or 5.5~6.
14. the renaturation and purification process of recombinant human granulocyte colony stimulating factor according to claim 11, described washes
De- liquid has following one or more features:
Sodium chloride concentration is 280 ~ 320mmol/L, and preferably sodium chloride concentration is 300mmol/L；Or
Phosphate concn is 0.08 ~ 0.12mol/L, and preferably phosphate concentration is 0.1mol/L, and preferably phosphate is the sodium of phosphoric acid
The sylvite of salt or phosphoric acid；Or
PH is 6~7, and preferably pH is 6~6.5 or 6.2~6.8 or 6.5~7.0.
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| CN115141247A (en) * | 2022-07-10 | 2022-10-04 | 湖北博大生物股份有限公司 | Disulfide bond remodeling reagent for proteins |
| CN116874552A (en) * | 2023-09-08 | 2023-10-13 | 成都华任康生物科技有限公司 | Purification method of target protein, kit and related application thereof |
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Address after: 211112 Kejian Road, Jiangning Science Park, Nanjing City, Jiangsu Province, 699 Applicant after: JIANGSU AOSAIKANG PHARMACEUTICAL Co.,Ltd. Address before: 211112 Kejian Road, Jiangning Science Park, Nanjing City, Jiangsu Province, 699 Applicant before: JIANGSU AOSAIKANG PHARMACEUTICAL Co.,Ltd. |
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Application publication date: 20190723 |