CN102219848A - Purification method for recombinant human interferon beta-1a - Google Patents
Purification method for recombinant human interferon beta-1a Download PDFInfo
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Abstract
The invention discloses a purification method for recombinant human interferon beta-1a. The method comprises three steps: affinity chromatography, strong anion exchange chromatography and weak cation exchange chromatography. Blue dyeing affinity chromatography and interferon beta antibody affinity chromatography are adopted during affinity chromatography, Q-sepharose chromatographic column is adopted during strong anion exchange chromatography, and CM-sepharose chromatographic column is adopted during weak cation exchange chromatography. With the method, reverse phase chromatography and metal-chelating chromatography steps influencing protein activity are abandoned, two-time ionic exchange chromatography is adopted, thus obtaining the recombinant human interferon beta-1a with high purity and good bioactivity.
Description
Technical field
The present invention relates to a kind of proteic purification process.Particularly, the invention discloses the new technology for purifying of recombinant human interferon beta-1a.The present invention has abandoned influences the bigger reversed phase chromatography of protein-active and these steps of metal chelate chromatography, the purifying process that design makes new advances, adopt the purge process that comprises an affinity chromatography and twice ion exchange chromatography, obtained high purity, the good recombinant human interferon beta-1a of biological activity.
Background technology
Interferon, rabbit (interferon, IFN) be the important cytokine that a class has antiviral, antitumor and immunoregulation effect, composite factors such as generation cell, acceptor and activity according to Interferon, rabbit are divided into 2 types with it: I type and II type, interferon beta (IFN β) belongs to I type Interferon, rabbit, and IFN β is mainly produced by inoblast.People IFN β gene length 777bp is positioned at karyomit(e) 9P22, closes on IFN α gene cluster.IFN α and IFN β are incorporated into same acceptor, but the avidity of the latter and acceptor is greater than the former.Compare with IFN α, IFN β has strict species specificity, and the IFN β of other kind does not have activity on people's cell.(as removing sugar chain, molecular weight is about 18KD to interferon beta-1a for the glycoprotein of IFN β-1a) be made up of 166 amino acid, the about 22.5KD of molecular weight, and glycosylation site is positioned at Asn80.IFN β contains 3 halfcystine: Cys17, Cys31 and Cys141, wherein disulfide linkage in Cys31 and the Cys141 ingredient.IFN β-1a mainly produces by the mammalian cell of inducing human fibroblasts or process genetic modification.The Cys17 of reduced form is easy to cause the albumen instability, can improve protein stability after it is mutated into Ser, can be by intestinal bacteria with the inclusion body formal representation, obtain having active albumen by becoming renaturation, but do not have sugar chain, and lack methionine(Met) at N-terminal, therefore have only 165 amino acid, the Interferon, rabbit called after IFN β-1b that obtains thus.
IFN β relates to non-specific humoral immunization of adjusting and antiviral immunity, and its clinical treatment effect mainly shows following several respects:
1) multiple sclerosis (multiplesclerosis, MS).Multiple sclerosis is the autoimmune disorder that mainly becomes characteristics with central nervous system (CNS) white matter demyelinating disease, immune modulating treatment is main policies [the Kieseier BC of treatment MS, Hartung HP.Current disease-modifying Therapies in multiple scierosis.Semin Neurot, 2003,23 (2): 133-146.].Genetically engineered people IFN β is the biological products that approval is applied to multiple sclerosis through FDA.
2) assisting therapy of malignant tumour.IFN β has direct inhibition and therapeutic action to some malignant tumour.The U.S. and Japanese approved carry out the research that genetically engineered IFN β is applied to the assisting therapy of malignant glioma, kidney, small cell lung cancer at present, and the Rebif product of Xue Lannuo company has entered III phase clinical study.
3) multiple disease of viral infection.The same I type IFN that all belongs to of IFN β with IFN α, IFN β has the broad-spectrum disease resistance toxic action, and they can induce host cell to produce each link that the various active enzyme comes viral interference to duplicate.Clinical study result before confirms, sexual organ condyloma due to IFN β infects chronic hepatitis B, chronic hepatitis C, HPV, the epizootic disease toxicity encephalitis due to being infected by HSV and HIV infect and all have therapeutic action preferably, can be applicable to the prevention and the treatment of multiple dna virus, picornavirus infection.
IFN β is mainly used in the treatment of multiple sclerosis at present, comprises cell cultures IFN β-1a that obtains and the IFN β-1b that passes through escherichia coli expression.Recombinant human IFN β-1a class medicine has the AVONEX of Biogen IDEC company in the market
, the Rebif of Merck Xue Lannuo company
And Iran was in the imitation medicine CinnoVex in its domestic release in 2006.Present Rebif
Introduced China, Chinese commodity are called " Libiee "; Formulation comprises freeze-dried powder, and pre-filling injection liquid etc. still do not have the homemade goods listing.IFN β-the 1b of the Diichi of Japan and the development of Mochida company has ratified to be used for the clinical treatment hepatitis C, and the Rebif product of Xue Lannuo company also carries out the II phase clinical study of hepatitis C at present.
The recombinant human IFN β that utilizes mammalian cell expression to obtain has the same disulfide linkage structure with the natural IFN β of people, and more close glycosylation structure, so its biologic activity almost is equal to natural protein.And be the inclusion body form by the IFN β that escherichia coli expression obtains, do not have biologic activity, need to realize its active recovery through complicated sex change, renaturation, proteinic therebetween loss is bigger, and the protein that obtains in this way is without any glycosylation modified, its biologic activity is compared with native protein, has only the latter's about 1/10th.At present China does not also have successful exploit person IFN β listing, is escherichia coli expression and unique certain company that is in registration phase adopts, so adopt mammalian cell expression production recombinant human IFN β to remain the technological difficulties and the market vacancy.We have designed the carrier for expression of eukaryon of cance high-expression gene, have realized efficiently expressing and the homogeneity expression of recombinant human IFN β-1a, obtained highly purified IFN β-1a, and its antiviral activity is studied.Now successfully make up the eucaryotic cell strain (CHO-K1-9A3) of stably express recombinant human IFN β-1a, and stable the expressing external obtaining of energy.
Natural human IFN beta molecule amount is about 22.5KD, and molecular weight is less, but it has characteristics such as high hydrophobicity again, causes its purifying difficulty greater than other common protein.Means are caught in most use dye affinity chromatographies or the conduct of CPG controllable bore diameter glass fillers in the document at present, later stage is passed through reversed phase chromatography, ion exchange chromatography, sieve chromatography, multiple means such as metal chelate chromatography realize this protein purification [Ernest Knight, Jr.and Diana Fahey.Human Fibroblast Interferon-an Improved Purification.The Journal of Biological Chemistry, 1981,256 (8): 3609-3611.Shin Yonehara, Yuko Yanase, Toshihiko Sano, et al.Purification of Human Lymphoblastoid Interferon by a Simple Procedure with High Yields.The Journal of Biological Chemistry, 1980,256 (8): 3770-3775.Wolfgang Berthold, Celine Tan, and Y.H.Tan.Purification and in Vitro Labeling of Interferon from a Human Fibroblastoid Cell Line.The Journal of Biological Chemistry, 1977,253 (14): 5206-5212.].Wherein, reversed phase chromatography or metal chelate chromatography for these purge processes approach must be arranged.
Reversed phase chromatography needs with an organic solvent as proteinic wash-out medium, and protein is exposed to for a long time will cause molecular structure change in various degree in the organic solvent, and serious caused irreversible denaturation has a strong impact on proteic biologic activity.Metal chelate chromatography can cause high-concentration metallic ions to be introduced in the protein solution, causes the reduction of protein-active in various degree, and has increased the difficulty that metal ion is removed.For this reason, a kind of IFN β purifying process that reduces the protein-active forfeiture of exploitation has important practical significance.
Patent of invention (the production method of recombinant human IFN β, application number 200510025595.7) the chromatography purification method of recombinant human IFN β is disclosed, can use cation-exchange chromatography, anion-exchange chromatography, gel permeation chromatography, affinity chromatography, hydrophobic chromatography and combination thereof, it is how to make up that but this patent is failed specific embodiment explanation, with and effect how.
In order to obtain the good recombinant human IFN β-1a of biologic activity, and make it every index and meet requirement as drug development, the present invention attempts the purifying process of a cover recombinant human IFN β-1a, the reversed phase chromatography and the metal chelate chromatography that may cause protein inactivation have been replaced, mainly comprise ion exchange chromatography twice, comprise reinforcing yin essence ion exchange chromatography and weak cation displacement chromatography, remove and compare the foreign protein that has different electric charges with target protein, and can effectively remove host's residual protein, host's residual DNA and intracellular toxin.
Summary of the invention
The invention discloses the new technology for purifying of recombinant human IFN β-1a.
The new technology for purifying of recombinant human IFN β-1a of the present invention may further comprise the steps:
1) affinity chromatography: after the cell conditioned medium liquid concentration, go up sample behind the balance affinity column, use elutriant elution chromatography post again, collect elution peak; Concentrate affinity chromatography and collect liquid.
2) reinforcing yin essence ion exchange chromatography: balance anion chromatography column; With sample on the collection liquid in last step, collect the stream that contains recombinant human IFN β-1a and wear component.
3) weak cation displacement chromatography: balance cation chromatography column; The positively charged ion chromatography post stream of collecting is worn sample on the component; With elutriant elution chromatography post, collect the component that contains recombinant human IFN β-1a.Target protein is concentrated preservation.
Affinity chromatography process of the present invention, used affinity column is selected from one of Blue dye affinity chromatography post and antibody affinity chromatography.
Ion exchange chromatography process of the present invention, selected reinforcing yin essence ion exchange column is anion-exchange column Q-sepharose, selected weak cation displacement chromatography post is cationic exchange coloum CM-sepharose.
The invention also discloses the purification process of another kind of recombinant human IFN β-1a, comprising:
1) affinity chromatography: after the cell conditioned medium liquid concentration, go up sample behind the balance affinity column, use elutriant elution chromatography post again, collect elution peak; Concentrate affinity column and collect liquid.
2) weak cation displacement chromatography: balance cation chromatography column; With sample on the collection liquid in last step, elutriant elution chromatography post is collected the component that contains recombinant human IFN β-1a.
3) reinforcing yin essence ion exchange chromatography: balance anion chromatography column; Sample is gone up in the collection liquid dilution in last step, and collected the stream that contains recombinant human IFN β-1a and wear component.Target protein is concentrated preservation.
Affinity chromatography process of the present invention, used affinity column is selected from one of Blue dye affinity chromatography post and antibody affinity chromatography.
Ion exchange chromatography process of the present invention, selected reinforcing yin essence ion exchange column is anion-exchange column Q-sepharose.Selected weak cation displacement chromatography post is cationic exchange coloum CM-sepharose.
Ion exchange chromatography process of the present invention can at first be carried out anion-exchange chromatography with reinforcing yin essence ion chromatography post, carries out cation-exchange chromatography with the weak cation chromatography column again; Also can at first carry out cation-exchange chromatography, carry out anion-exchange chromatography with reinforcing yin essence ion chromatography post again with the weak cation chromatography column.
The new technology for purifying of recombinant human IFN β-1a of the present invention, simple and easy to do, there is not complicated change renaturation process, empirical tests can be applied to the large-scale production of IFN β-1a easily.
The new technology for purifying of recombinant human IFN β-1a of the present invention is through verifying with active, and shown good actual effect recombinant human IFN β-1a purity checking.The present invention prepares recombinant human IFN β-1a and commercially available IFN β-1a Rebif
(Libiee) shown that through contrast the present invention prepares product and has higher biologic activity.
Description of drawings
Fig. 1 recombinant human IFN β-1a purifying schema comprises an affinity chromatography and twice ion exchange chromatography.Ion exchange chromatography is respectively reinforcing yin essence ion exchange chromatography and weak cation displacement chromatography.
The Bule dye affinity chromatography figure of Fig. 2 recombinant human IFN β-1a.Wherein scheming A is AF-Blue affinity chromatography figure, and figure B is Capto-Blue Sepharose affinity chromatography figure.Wherein ordinate zou mAU represents the A280 ultraviolet absorption value, and X-coordinate is a volume.
The RP-HPLC collection of illustrative plates of Fig. 3 Blue affinity chromatography-reinforcing yin essence ion exchange chromatography-weak cation displacement chromatography purifying (embodiment 2) back IFN β-1a.Wherein, ordinate zou mAU represents the A214 ultraviolet absorption value, and X-coordinate is the time, and target protein goes out the peak in 11.8min.
The SEC-HPLC collection of illustrative plates of Fig. 4 Blue affinity chromatography-reinforcing yin essence ion exchange chromatography-weak cation displacement chromatography purifying (embodiment 2) back IFN β-1a.Wherein, ordinate zou mAU represents the A214 ultraviolet absorption value, and X-coordinate is the time, and target protein goes out the peak in 14.5min.
The RP-HPLC collection of illustrative plates of Fig. 5 Blue affinity chromatography-weak cation displacement chromatography-reinforcing yin essence ion exchange chromatography purifying (embodiment 3) back IFN β-1a.Wherein, ordinate zou mAU represents the A214 ultraviolet absorption value, and X-coordinate is the time, and target protein goes out the peak in 11.8min.
The SEC-HPLC collection of illustrative plates of Fig. 6 Blue affinity chromatography-weak cation displacement chromatography-reinforcing yin essence ion exchange chromatography purifying (embodiment 3) back IFN β-1a.Wherein, ordinate zou mAU represents the A214 ultraviolet absorption value, and X-coordinate is the time, and target protein goes out the peak in 14.5min.
The SDS-PAGE electrophoretogram of the IFN β-1a of Fig. 7 approach A and approach B purifying, standard substance (Libiee) and protein Marker, wherein:
Lane 1: the recombinant human IFN β-1a behind Blue affinity chromatography among the embodiment 2-reinforcing yin essence ion exchange chromatography-weak cation displacement chromatography purifying;
Lane 2: the recombinant human IFN β-1a behind IFN β antibody affinity chromatography among the embodiment 3-weak cation displacement chromatography-reinforcing yin essence ion exchange chromatography purifying;
Lane 3: recombinant human IFN β-1a standard substance (Libiee);
Lane 4: protein Marker.
Fig. 8 target protein and the commercially available former biologic activity comparison diagram that grinds medicine Libiee.Figure A target protein is by the recombinant human IFN β-1a behind Blue dye affinity chromatography-reinforcing yin essence ion exchange chromatography-weak cation displacement chromatography purifying.Figure B target protein is the recombinant human IFN β-1a behind IFN β antibody affinity chromatography-weak cation displacement chromatography-reinforcing yin essence ion exchange chromatography purifying.The result shows that both have the biologic activity that is equal to.Wherein zero represents Libiee, and represents other two batch sample of branch.
Embodiment
1) structure of cell strain
We adopt the codon of Mammals bias, the gene order of people IFN β among the Genbank is optimized, guaranteeing under the constant situation of encoding amino acid sequence (Sequence No.1), with the synthetic IFN beta gene sequence (Sequence No.2) of manual method.This artificial synthetic gene fragment is inserted a kind of highly effective eukaryon expression plasmid vector pCG-IFN β that we make up, transfection CHO-K1 cell, screen and set up the cell strain CHO-K1-9A3 of stable transfection with first sulfonyl sulfuric acid amine, detect proteic expression with immunoblotting and ELISA, but obtained this proteic cell strain of a strain high-level secretory expression.By the serum-free culture domestication, obtained to be adapted to the suspension growth cell strain of serum-free culture again.
2) evaluation of recombinant human IFN β-1a cell strain
All detect the IFN β albumen of this cell strain secreting, expressing with immunoblotting and ELISA, its expression amount in Tissue Culture Flask reaches 0.5-1 μ g/10
6Cell.Set up master cell bank with this cell strain, on the master cell bank basis, set up master cell bank and work storehouse.Calibrating shows that exogenous factors such as its no bacterium, fungi, mycoplasma and virus pollute to master cell bank, and working cardial cell is surpassed the cell strain calibrating of production generation more than 10 generations, shows no tumorigenicity.Synthetic primer Sequence No.3 and Sequence No.4, with PCR and real-time fluorescence quantitative PCR the cell more than 50 generations of continuous passage is carried out the evaluation and the gene copy number analysis of IFN gene, with immunoblotting and elisa assay recombinant human IFN β-1a protein expression level, the result shows that this IFN stable gene is incorporated in the Chinese hamster ovary celI karyomit(e), its copy number reaches more than 72 copies, and expression level is stable.
In vitro tests shows that this albumen has the activity that inhibition VSV virus causes the WISH cell pathology, and its specific activity is similar to the Libiee of Xue Lannuo company.
3) cultivation of recombinant human IFN β-1a cell strain
On 300L cell cultures jar scale, adopt the import serum-free cell culture medium, cooperate supplemented medium at this cell strain exploitation, realize that cell density is up to 8 * 10
6/ ml.Through cultivation in 12 days, target protein concentration reached 10～15mg/L in the nutrient solution.
We have successfully set up and can stablize the proteic CHO-K1-9A3 cell strain of recombinant human IFN β-1a that high level expression has high biologic activity, every index in master cell bank, master cell bank and the working cardial cell storehouse of setting up based on this cell strain all meet in three ones of the Pharmacopoeia of the People's Republic of China versions in 2005 for the requirement of biological products production with zooblast.This series work is for setting up this proteic production technique and carrying out researchs such as its pharmacology, pharmacodynamics and lay a good foundation.
The proteic purification route A of embodiment 2 recombinant human IFN β-1a
It is the affine filler of matrix that the affine filler of dyestuff that present embodiment is a matrix with hydroxylated polymethacrylic acid resin preferably replaces with the dextran, and the former advantage is a filler hardness height, and back-pressure is low, can bear bigger process flow rate.Next selects the chromatography method of twice ion-exchange, uses earlier the reinforcing yin essence ion exchange chromatography, and back weak cation displacement chromatography replaces complex process such as reversed phase chromatography, causes the chromatography method of protein denaturation easily.Its method synoptic diagram as shown in Figure 1.
Material and instrument
AF-Blue HC 650M filler (TOSOH company product), CM-sepharose FF filler (GE healthcare company product), Q-sepharose FF filler (GE healthcare company product).Pellicon 2 ultra-filtration membranes (molecular weight 10K dams), 0.5M
2(Millipore company product), Pellicon ultrafiltration system (Millipore company product), Amicon ultra-filtration centrifuge tube (Millipore company product), AKTA Purifier chromatographic system (GE healthcare company product).
Experimental technique
1) supernatant liquor concentrates
The cleaning ultrafiltration system, concentrated supernatant is with (0.025mol/L SODIUM PHOSPHATE, MONOBASIC+0.025mol/L Sodium phosphate dibasic) damping fluid (pH7.2) displacement concentrated solution.
2) Blue dye affinity chromatography
Cleaning AF-Blue chromatographic system, and the former processing of reducing phlegm and internal heat are carried out following operation: successively with (0.025mol/L SODIUM PHOSPHATE, MONOBASIC+0.025mol/L Sodium phosphate dibasic) damping fluid (pH7.2) balance chromatography column; Use (0.025mol/L SODIUM PHOSPHATE, MONOBASIC+0.025mol/L Sodium phosphate dibasic) damping fluid (pH7.2) balance chromatography column behind the last sample; With (0.025mol/L SODIUM PHOSPHATE, MONOBASIC+0.025mol/L Sodium phosphate dibasic+2mol/L NaCl) damping fluid (pH7.2) washing chromatography column; With (0.025mol/L SODIUM PHOSPHATE, MONOBASIC+0.025mol/L Sodium phosphate dibasic+2mol/L NaCl+15% propylene glycol) damping fluid (pH7.2) washing chromatography column; With (0.025mol/L SODIUM PHOSPHATE, MONOBASIC+0.025mol/L Sodium phosphate dibasic) damping fluid (pH7.2) balance chromatography column; With (0.025mol/L SODIUM PHOSPHATE, MONOBASIC+0.025mol/L Sodium phosphate dibasic+2mol/L NaCl+50% propylene glycol) damping fluid (pH7.2) elution chromatography post, collect elution peak.
Cleaning ultrafiltration system, and the former processing of reducing phlegm and internal heat, behind 0.02mol/L phosphate buffered saline buffer (pH7.0) wash cycles ultrafiltration system, ultrafiltration and concentration AF-Blue post is collected liquid, and fully replaces with 0.02mol/L phosphate buffered saline buffer (pH7.0).Tomographic results as shown in Figure 2.
3) Q-sepharose anion-exchange chromatography
Cleaning Q-sepharose column chromatography system, and the former processing of reducing phlegm and internal heat, carry out successively according to the following steps:
With A phase (0.01mol/L SODIUM PHOSPHATE, MONOBASIC+0.01mol/L Sodium phosphate dibasic) damping fluid (pH7.0) balance chromatography column, recombinant human IFN β-debond of 1a albumen is on anion-exchange column, and impurity is combined on the anion-exchange column, collects stream and wears component; With A phase (0.01mol/L SODIUM PHOSPHATE, MONOBASIC+0.01mol/L Sodium phosphate dibasic) damping fluid (pH7.0) balance chromatography column; With B phase (0.01mol/L SODIUM PHOSPHATE, MONOBASIC+0.01mol/L Sodium phosphate dibasic+1.0mol/L NaCl) damping fluid (pH7.0) elution chromatography post, collect elution peak.
4) CM-sepharose cation-exchange chromatography
Cleaning CM-sepharose column chromatography system, and the former processing of reducing phlegm and internal heat carry out: successively according to the following steps with 0.01mol/L SODIUM PHOSPHATE, MONOBASIC+0.01mol/L Sodium phosphate dibasic damping fluid (pH6.7) balance chromatography column; The Q chromatography column stream of collecting is worn protein solution, last sample; With (0.01mol/L SODIUM PHOSPHATE, MONOBASIC+0.01mol/L Sodium phosphate dibasic) damping fluid (pH6.7) balance chromatography column; Set chromatographic system gradient elution program parameter, 15-20 column volume elution chromatography post collected elution peak.
5) ultrafiltration and concentration and damping fluid displacement
The protein solution of the cation seperation column chromatography collected with 0.01mol/L sodium-acetate buffer (pH3.5) dilution, is used Millipore Pellicon ultrafiltration system thoroughly to replace damping fluid and is 0.01mol/L sodium-acetate (pH3.5).
The purification route B of embodiment 3 recombinant human IFN β-1a purifying
Present embodiment comprises the chromatography method of twice ion-exchange, the preferred weak cation displacement chromatography that adopts earlier, back reinforcing yin essence ion exchange chromatography.Its method synoptic diagram as shown in Figure 1.
Material and instrument
Capto-Blue (GE healthcare company product), CM-sepharose FF filler (GE healthcare company product), Q-sepharose FF filler (GE healthcare company product).Pellicon 2 ultra-filtration membranes (molecular weight 10K dams), 0.5M
2(Millipore company product), Pellicon ultrafiltration system (Millipore company product), Amicon ultra-filtration centrifuge tube (Millipore company product), AKTA Purifier chromatographic system (GE healthcare company product).
Experimental technique
1) supernatant liquor concentrates
The cleaning ultrafiltration system, concentrated supernatant is with 0.05mol/L Tris-HCl damping fluid (pH7.2) displacement concentrated solution.
2) Blue dye affinity chromatography
Cleaning Capto-Blue chromatographic system, and the former processing of reducing phlegm and internal heat are carried out following operation: successively with 0.05mol/L Tris-HCl damping fluid (pH7.2) balance chromatography column; Use 0.05mol/L Tris-HCl damping fluid (pH7.2) balance chromatography column behind the last sample; With (0.05mol/L Tris-HCl+2mol/L NaCl) damping fluid (pH7.2) washing chromatography column; With (0.05mol/L Tris-HCl+2mol/L NaCl+15% propylene glycol) damping fluid (pH7.2) washing chromatography column; With 0.05mol/L Tris-HCl damping fluid (pH7.2) balance chromatography column; With (0.05mol/L Tris-HCl+2mol/L NaCl+50% propylene glycol) damping fluid (pH7.2) elution chromatography post, collect elution peak.The cleaning ultrafiltration system.The Capto-Blue tomographic results as shown in Figure 2.
3) CM-sepharose cation-exchange chromatography
Cleaning CM-sepharose column chromatography system, and the former processing of reducing phlegm and internal heat carry out: successively according to the following steps with 0.05mol/L Tris-HCl (pH6.7) balance chromatography column, last sample; With 0.05mol/L Tris-HCl damping fluid (pH6.7) balance chromatography column; Set chromatographic system gradient elution program parameter, 15-20 column volume elution chromatography post collected elution peak.
4) Q-sepharose anion-exchange chromatography
Cleaning Q-sepharose column chromatography system, and the former processing of reducing phlegm and internal heat, carry out according to the following steps successively: with A phase 0.03mol/L Tris-HCl damping fluid (pH7.0) balance chromatography column, recombinant human IFN β-1a albumen debond is on anion-exchange column, and impurity is combined on the anion-exchange column, with sample on the collection liquid in last step, collect stream and wear component; With A phase 0.03mol/L Tris-HCl damping fluid (pH7.0) balance chromatography column; With B phase (0.03mol/L Tris-HCl+1.0mol/L NaCl) damping fluid (pH7.0) elution chromatography post, collect elution peak.
5) ultrafiltration and concentration and damping fluid displacement
The protein solution of the anion column chromatography collected with 0.01mol/L sodium-acetate buffer (pH3.5) dilution, is used Millipore Pellicon ultrafiltration system thoroughly to replace damping fluid and is 0.01mol/L sodium-acetate (pH3.5).
The purification route C of embodiment 4 recombinant human IFN β-1a purifying
Present embodiment selects the chromatography method of twice ion-exchange to use earlier the reinforcing yin essence ion exchange chromatography, back weak cation displacement chromatography preferably with after the antibody affinity chromatography sample.Its method synoptic diagram as shown in Figure 1.
Material and instrument
IFN β antibody affinity column (self-control), CM-sepharose FF filler (GE healthcare company product), Q-sepharose FF filler (GE healthcare company product).Pellicon 2 ultra-filtration membranes (molecular weight 10K dams), 0.5M
2(Millipore company product), Pellicon ultrafiltration system (Millipore company product), Amicon ultra-filtration centrifuge tube (Millipore company product), AKTA Purifier chromatographic system (GE healthcare company product).
Experimental technique
1) supernatant liquor concentrates
The cleaning ultrafiltration system, concentrated supernatant is with 0.03mol/L Tris-HCl damping fluid (pH7.2) displacement concentrated solution.
2) antibody affinity chromatography
Utilize recombinant human IFN β-1a as the antigen immune mouse, make up the hybridoma cell strain of secretion anti-recombinant human IFN β-1a monoclonal antibody, by this cell of vitro culture, perhaps, prepare anti-recombinant human IFN β-1a monoclonal antibody with this cell infection mouse results ascites.This antibody and Sepharose 4B gel are prepared the affine filler that is used for specificity purification of recombinant human IFN β-1a by the CNBr coupling.The cell cultures concentrated solution is directly gone up sample or through going up sample behind the preliminary purification to affinity column, target protein is incorporated on the post, utilize low pH buffer solution elution target protein.
3) CM-sepharose cation-exchange chromatography (with embodiment 3)
4) Q-sepharose anion-exchange chromatography (with embodiment 3)
5) ultrafiltration and concentration and damping fluid displacement (with embodiment 3)
The purification route D of embodiment 5 recombinant human IFN β-1a purifying
Present embodiment select with the AF-Blue dyestuff affine after, select the chromatography method of twice ion-exchange use earlier the strong cation exchange chromatography, then use the weak anionic displacement chromatography.
Material and instrument
AF-Blue HC 650M filler (TOSOH company product), SP-Sepharose (GE healthcare company product); DEAE-Sepharose (Tosoh company product).Pellicon 2 ultra-filtration membranes (molecular weight 10K dams), 0.5M
2(Millipore company product), Pellicon ultrafiltration system (Millipore company product), Amicon ultra-filtration centrifuge tube (Millipore company product), AKTA Purifier chromatographic system (GE healthcare company product).
Experimental technique
1) concentrated (with the embodiment 2) of supernatant liquor
2) AF-Blue dye affinity chromatography (with embodiment 2)
3) SP-Sepharose cation-exchange chromatography
Cleaning SP-Sepharose column chromatography system, and the former processing of reducing phlegm and internal heat carry out: successively according to the following steps with phosphate buffered saline buffer balance chromatography column; With sample on the protein solution of last purification step collection; With phosphate buffered saline buffer balance chromatography column; Set chromatographic system gradient elution program parameter, the elution chromatography post is collected elution peak.
4) DEAE-Sepharose anion-exchange chromatography
Cleaning DEAE-Sepharose chromatographic system, and the former processing of reducing phlegm and internal heat go up sample with the SP purified product after dilution reduces specific conductivity, collect stream and wear component.
5) ultrafiltration and concentration and damping fluid displacement (with embodiment 2)
The proteic detection of embodiment 6 recombinant human IFN β-1a
1) SDS-PAGE detects
The protein sample of separation and purification is carried out the SDS-PAGE analysis, detected result by embodiment 2 and embodiment 3 target protein that obtains, standard substance (Libiee) and protein Marker as shown in Figure 7, target protein has consistent molecular weight with standard substance, and this molecular weight meets document [Goodin DS.Treatment of Multiple Sclerosis with Human Beta Interferon.The International MS Journal.2005,12:96-108] described, about 22.5kD.
2) ELISA detects
Use Human IFN b ELISA kit (Pestka Biomedical Laboratories company product), the testing process by specification carries out.
3) RP-HPLC method
Use the C4RP-HPLC pillar, 4.6mm * 25mm, NOBEL company product.Wherein, A phase: 0.1% phosphoric acid; B phase: 80% acetonitrile, 0.1% phosphoric acid.The detection wavelength is 214nm; Flow rate of mobile phase is 1ml/min, and employed mobile composition is mutually as shown in table 1 below in each time period.By embodiment 2 obtain the target protein detected result as shown in Figure 3, by embodiment 3 obtain the target protein detected result as shown in Figure 5, target protein goes out the peak in 11.8min.
Table 1 RP-HPLC method is measured the IFN β-1a albumen of purifying
| Time (min) | A phase (%) | B phase (%) |
| 0 | 60 | 40 |
| 3 | 60 | 40 |
| 18 | 0 | 100 |
| 18.5 | 0 | 100 |
| 22.5 | 60 | 40 |
| 26.5 | 60 | 40 |
4) SEC-HPLC method
Materials used is the G2000 pillar, 4.6mm * 25mm (TOSOH company product); Moving phase is 20mmol/L PBS, 0.2mol/L NaCl, pH6.8; The detection wavelength is 214nm; Flow rate of mobile phase: 0.5ml/min.By embodiment 2 obtain the target protein detected result as shown in Figure 4, by embodiment 3 obtain the target protein detected result as shown in Figure 6, target protein goes out the peak in 14.5min.
5) protein active and intracellular toxin detect
Biological activity assay is with reference to three appendix XC of Pharmacopoeia of the People's Republic of China version in 2005 cytopathic-effect inhibition assay.See Table 2 and Fig. 8 by embodiment 2 and embodiment 3 target protein that obtains activity detected result.By embodiment 2 and embodiment 3 to obtain other quality examination of target protein solution result as follows: intracellular toxin＜1EU/22ug, host DNA is residual＜10pg/22ug, host protein is residual＜0.1%, all is better than the officinal quality standard.
Detect target protein concentration 12mg/L in the 300L culture supernatant by the ELISA test kit.Through ultrafiltration and concentration and damping fluid displacement, the target protein rate of recovery 95%.The concrete rate of recovery step by step and target protein purity are: all about 70%, lipidated protein about 85% behind the purifying for the AF-Blue and the Capto-Blue chromatography target protein rate of recovery; The ultrafiltration and the damping fluid displacement target protein rate of recovery 80%; The Q-Sepharose chromatography target protein rate of recovery 90%, lipidated protein about 95% behind the purifying; The CM-Sepharose chromatography target protein rate of recovery 80%, lipidated protein about 98% behind the purifying; Total yield 38.3%.Measuring active standard substance is the former medicine Libiee (REBIF) that grinds.The present invention obtains recombinant human IFN β-1a biological activity of albumen detected result and shows, the purifying gained protein-active of embodiment 2 is Libiee's 260%, and the purifying gained protein-active of embodiment 3 is Libiee 237% (table 2).
The recombinant human IFN β-1a of table 2 purifying of the present invention and formerly grind medicine Libiee and get specific activity
Experiment shows, after affinity chromatography, adopts the effect of strong cation exchange chromatography and the logotype of weak anionic displacement chromatography relatively poor, and its purifying target protein purity can not satisfy needs of production far away between 70%～85%.Result of the present invention shows: to recombinant human IFN β-1a albumen, the purification effect that adopts weak cation displacement chromatography and reinforcing yin essence ion exchange chromatography is apparently higher than the purification effect that adopts strong cation exchange chromatography and weak anionic displacement chromatography.
The chromatography method of the recombinant human IFN β that patent of invention (production method of recombinant human IFN β, application number 200510025595.7) is mentioned is fuzzy, does not have embodiment clearly to verify.The present invention is directed to recombinant human IFN β-1a protein purification,, determined the clear and definite condition of ion exchange chromatography and affinity chromatography through experimental verification, promptly after affinity chromatography, adopt one time the weak cation displacement chromatography, a reinforcing yin essence ion exchange chromatography can reach the high purity more than 98%.And take the ion exchange chromatography of other types, and comprising strong cation exchange chromatography and the coupling of weak anionic displacement chromatography, the purification effect of two kinds of cation-exchange chromatography couplings or two kinds of anion-exchange chromatography couplings all is relatively poor.
Affinity chromatography and ion exchange chromatography that this purifying process adopts, with respect to reversed phase chromatography, technological process is simple, to equipment require lowly, reduced the sex change influence that organic solvent produces target protein; With respect to sieve chromatography, the purifying time shortens greatly; With respect to metal chelate chromatography, avoided metal ion hanging column process, simplify purifying process, and avoided metal ion residual in protein soln.By our 300L cell culture system, carried out the large scale purification of recombinant human IFN β-1a with method of the present invention, respond well.
In sum, this purifying process process of recombinant human IFN β-1a is convenient, and the filler cost is low, and target protein is active high, is fit to be applied to commercial production scale.
Claims (10)
1. the purification process of a recombinant human interferon beta-1a, it is characterized in that, described purification process comprises affinity chromatography process and subsequently twice ion exchange chromatography process, wherein in twice ion exchange chromatography process, carry out the reinforcing yin essence ion exchange chromatography earlier and then carry out the weak cation displacement chromatography.
2. the purification process of recombinant human interferon beta-1a according to claim 1 comprises:
1) affinity chromatography: after the cell conditioned medium liquid concentration, go up sample behind the balance affinity column, use elutriant elution chromatography post again, collect elution peak; Concentrating affinity chromatography collects liquid and replaces damping fluid;
2) reinforcing yin essence ion exchange chromatography: balance anion chromatography column; With sample on the collection liquid in last step, collect the stream that contains recombinant human interferon beta-1a and wear component;
3) weak cation displacement chromatography: balance cation chromatography column; The positively charged ion chromatography post stream of collecting is worn sample on the component; With elutriant elution chromatography post, collect the component that contains recombinant human interferon beta-1a.
3. purification process according to claim 1 and 2 is characterized in that used affinity column is selected from one of Blue dye affinity chromatography post and antibody affinity chromatography in the affinity chromatography process.
4. purification process according to claim 1 and 2 is characterized in that, used reinforcing yin essence ion exchange column is anion-exchange column Q-sepharose in the reinforcing yin essence ion exchange chromatography process.
5. purification process according to claim 1 and 2 is characterized in that, weak cation displacement chromatography post used in the cation-exchange chromatography process is cationic exchange coloum CM-sepharose.
6. the purification process of a recombinant human interferon beta-1a, it is characterized in that, described purification process comprises affinity chromatography process and subsequently twice ion exchange chromatography process, wherein in twice ion exchange chromatography process, carry out the weak cation displacement chromatography earlier and then carry out the reinforcing yin essence ion exchange chromatography.
7. the purification process of recombinant human interferon beta-1a according to claim 6 comprises:
1) affinity chromatography: after the cell conditioned medium liquid concentration, go up sample behind the balance affinity column, use elutriant elution chromatography post again, collect elution peak; Concentrating affinity column collects liquid and replaces damping fluid;
2) weak cation displacement chromatography: balance cation chromatography column; With sample on the collection liquid in last step, elutriant elution chromatography post is collected the component that contains recombinant human interferon beta-1a;
3) reinforcing yin essence ion exchange chromatography: balance anion chromatography column; With sample on the collection liquid in last step, and collect the stream that contains recombinant human interferon beta-1a and wear component.
8. according to claim 6 or 7 described purification process, it is characterized in that used affinity column is selected from one of Blue dye affinity chromatography post and antibody affinity chromatography in the affinity chromatography process.
9. according to claim 6 or 7 described purification process, it is characterized in that used reinforcing yin essence ion exchange column is anion-exchange column Q-sepharose in the reinforcing yin essence ion exchange chromatography process.
10. according to claim 6 or 7 described purification process, it is characterized in that weak cation displacement chromatography post used in the weak cation displacement chromatography process is cationic exchange coloum CM-sepharose.
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