CN102219848B - 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 purification process of albumen.Particularly, the invention discloses the new technology for purifying of recombinant human interferon beta-1a.The present invention has abandoned affects reversed phase chromatography and these steps of metal chelate chromatography that protein-active is larger, 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, according to composite factors such as the generation cell of Interferon, rabbit, acceptor and activity, be divided into 2 types: 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.The glycoprotein that interferon beta-1a (IFN β-1a) is comprised of 166 amino acid, the about 22.5KD of molecular weight, as removed sugar chain, molecular weight is about 18KD, and glycosylation site is positioned at Asn80.IFN β contains 3 halfcystine: Cys17, Cys31 and Cys141, wherein disulfide linkage in Cys31 and Cys141 ingredient.IFN β-1a mainly produces by the mammalian cell of induction human fibroblasts or process genetic modification.The Cys17 of reduced form is easy to cause albumen unstable, after being mutated into Ser, can improve protein stability, can with inclusion body form, be expressed by intestinal bacteria, by becoming renaturation, obtain the activated albumen of tool, but do not there is sugar chain, and at N-terminal, lack methionine(Met), therefore only have 165 amino acid, the Interferon, rabbit called after IFN β-1b obtaining thus.
IFN β relates to the non-specific humoral immunization of adjusting and antiviral immunity, and its clinical treatment effect is mainly manifested in following several respects:
1) multiple sclerosis (multiplesclerosis, MS).Multiple sclerosis is mainly with central nervous system (CNS) white matter demyelinating disease, to become the autoimmune disorder of feature, immune modulating treatment is main policies [the Kieseier BC for the treatment of 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.IFN β all belong to I type IFN the same as IFN α, IFN β has broad-spectrum disease resistance toxic action, and they can induce host cell to produce the links that various active enzyme comes viral interference to copy.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 good therapeutic action, can be applicable to prevention and the treatment of multiple DNA virus, picornavirus infection.
IFN β is mainly used in the treatment of multiple sclerosis at present, comprises IFN β-1a that cell cultures obtains and passes through the IFN β-1b of 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.Current Rebif
introduced China, Chinese commodity are called " Libiee "; Formulation comprises freeze-dried powder, and pre-filling injection liquid etc. there is no homemade goods listing.IFN β-the 1b of the Diichi of Japan and the development of Mochida company has ratified for 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.
Utilize the recombinant human IFN β that mammalian cell expression obtains to there is the disulfide linkage structure the same with naive IFN β, and more close glycosylation structure, so its biologic activity is almost equal to natural protein.And the IFN β obtaining by escherichia coli expression is inclusion body form, do not there is biologic activity, need to be through complicated sex change, the recovery that renaturation realizes its activity, the loss of protein is larger therebetween, and the protein obtaining is in this way without any glycosylation modified, its biologic activity is compared with native protein, only has the latter's 1/10th left and right.At present China does not also have successful exploit person IFN β listing, is escherichia coli expression, so adopt mammalian cell expression Restruction people IFN β to remain technological difficulties and the market vacancy and unique certain company in registration phase adopts.We have designed the carrier for expression of eukaryon of cance high-expression gene, have realized high efficient expression and the homogeneity of recombinant human IFN β-1a and have expressed, and obtained highly purified IFN β-1a, and its antiviral activity is studied.Now successfully build the eucaryotic cell strain (CHO-K1-9A3) of stably express recombinant human IFN β-1a, and can stable being expressed in vitro.
Natural human IFN beta molecule amount is about 22.5KD, molecular weight, but it has again the features such as high hydrophobicity, causes its purifying difficulty to be greater than other common protein.In document, majority is used dye affinity chromatographies or CPG controllable bore diameter glass fillers as the means of catching at present, later stage is passed through reversed phase chromatography, ion exchange chromatography, sieve chromatography, the 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.].What wherein, reversed phase chromatography or metal chelate chromatography were these purge processes must have approach.
Reversed phase chromatography needs with an organic solvent as the wash-out medium of protein, and protein is exposed to for a long time in organic solvent and will causes molecular structure change in various degree, and serious caused irreversible denaturation has a strong impact on the biologic activity of albumen.Metal chelate chromatography can cause high-concentration metallic ions to be introduced in protein solution, causes in various degree the reduction of protein-active, and has increased the difficulty that metal ion is removed.For this reason, developing a kind of IFN β purifying process that reduces protein-active forfeiture has important practical significance.
A patent of invention (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, but it is how to combine that 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 indices and meet the requirement as drug development, the present invention attempts the purifying process of a set of recombinant human IFN β-1a, 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 exchange chromatography, remove and compare the foreign protein with different electric charges from 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, comprises the following steps:
1) affinity chromatography: after cell conditioned medium liquid concentration, loading after balance affinity column, then use elutriant elution chromatography post, collect elution peak; Concentrated affinity chromatography is collected liquid.
2) reinforcing yin essence ion exchange chromatography: balance anion chromatography column; By the collection liquid loading of upper step, collect the stream that contains recombinant human IFN β-1a and wear component.
3) weak cation exchange chromatography: balance cation chromatography column; The positively charged ion chromatography post stream of collection is worn to component loading; With elutriant elution chromatography post, collect the component that contains recombinant human IFN β-1a.By the concentrated preservation of target protein.
Affinity chromatography process of the present invention, affinity column used 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 exchange chromatography column is cationic exchange coloum CM-sepharose.
The purification process that the invention also discloses another kind of recombinant human IFN β-1a, comprising:
1) affinity chromatography: after cell conditioned medium liquid concentration, loading after balance affinity column, then use elutriant elution chromatography post, collect elution peak; Concentrated affinity column is collected liquid.
2) weak cation exchange chromatography: balance cation chromatography column; By the collection liquid loading of upper step, elutriant elution chromatography post, collects the component that contains recombinant human IFN β-1a.
3) reinforcing yin essence ion exchange chromatography: balance anion chromatography column; By the collection liquid dilution loading of upper step, and collect the stream that contains recombinant human IFN β-1a and wear component.By the concentrated preservation of target protein.
Affinity chromatography process of the present invention, affinity column used 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 exchange chromatography column is cationic exchange coloum CM-sepharose.
First ion exchange chromatography process of the present invention can carry out anion-exchange chromatography with reinforcing yin essence ion chromatography post, then carries out cation-exchange chromatography with weak cation chromatography column; Also can first with weak cation chromatography column, carry out cation-exchange chromatography, then carry out anion-exchange chromatography with reinforcing yin essence ion chromatography post.
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, through the checking of recombinant human IFN β-1a purity is verified with active, and has shown good actual effect.The present invention prepares recombinant human IFN β-1a and commercially available IFN β-1a Rebif
(Libiee), through contrast, shown that the present invention prepares product and has higher biologic activity.
Accompanying drawing explanation
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 exchange 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 A280 ultraviolet absorption value, and X-coordinate is volume.
The RP-HPLC collection of illustrative plates of IFN β-1a after Fig. 3 Blue affinity chromatography-reinforcing yin essence ion exchange chromatography-weak cation exchange chromatography purification (embodiment 2).Wherein, ordinate zou mAU represents A214 ultraviolet absorption value, and X-coordinate is the time, and target protein goes out peak in 11.8min.
The SEC-HPLC collection of illustrative plates of IFN β-1a after Fig. 4 Blue affinity chromatography-reinforcing yin essence ion exchange chromatography-weak cation exchange chromatography purification (embodiment 2).Wherein, ordinate zou mAU represents A214 ultraviolet absorption value, and X-coordinate is the time, and target protein goes out peak in 14.5min.
The RP-HPLC collection of illustrative plates of IFN β-1a after Fig. 5 Blue affinity chromatography-weak cation exchange chromatography-reinforcing yin essence ion exchange chromatography purifying (embodiment 3).Wherein, ordinate zou mAU represents A214 ultraviolet absorption value, and X-coordinate is the time, and target protein goes out peak in 11.8min.
The SEC-HPLC collection of illustrative plates of IFN β-1a after Fig. 6 Blue affinity chromatography-weak cation exchange chromatography-reinforcing yin essence ion exchange chromatography purifying (embodiment 3).Wherein, ordinate zou mAU represents A214 ultraviolet absorption value, and X-coordinate is the time, and target protein goes out 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 in embodiment 2 after Blue affinity chromatography-reinforcing yin essence ion exchange chromatography-weak cation exchange chromatography purification;
Lane 2: the recombinant human IFN β-1a in embodiment 3 after IFN β antibody affinity chromatography-weak cation exchange 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 for passing through the recombinant human IFN β-1a after Blue dye affinity chromatography-reinforcing yin essence ion exchange chromatography-weak cation exchange chromatography purification.Figure B target protein is the recombinant human IFN β-1a after IFN β antibody affinity chromatography-weak cation exchange chromatography-reinforcing yin essence ion exchange chromatography purifying.Result shows that both have the biologic activity being equal to.Wherein zero represents Libiee, and represents minute other two batch sample.
Embodiment
1) structure of cell strain
We adopt the codon of Mammals bias, the gene order of people IFN β in Genbank is optimized, in the situation that guaranteeing that encoding amino acid sequence (Sequence No.1) is constant, synthesize by artificial means IFN beta gene sequence (Sequence No.2).The gene fragment of this synthetic is inserted to a kind of highly effective eukaryon expression plasmid vector pCG-IFN β that we build, transfection CHO-K1 cell, with first sulfonyl sulfuric acid amine, screened and set up the cell strain CHO-K1-9A3 of stable transfection, with immunoblotting and ELISA, detect the expression of albumen, obtained the cell strain that a strain can this albumen of high-level secretory expression.By serum-free culture, tame again, obtained the suspension growth cell strain that can be adapted to serum-free culture.
2) evaluation of recombinant human IFN β-1a cell strain
The IFN β albumen that all detects this cell strain secreting, expressing with immunoblotting and ELISA, its expression amount in Tissue Culture Flask reaches 0.5-1 μ g/10
6cell.With this cell strain, set up master cell bank, on master cell bank basis, set up master cell bank and work storehouse.To master cell bank, calibrating shows that it pollutes without exogenous factors such as bacterium, fungi, mycoplasma and viruses, and working cardial cell is surpassed to the cell strain calibrating more than 10 generations of production generation, shows without tumorigenicity.Synthetic primer Sequence No.3 and Sequence No.4, with PCR and real-time fluorescence quantitative PCR, cells more than 50 generations of continuous passage is carried out to evaluation and the gene copy number analysis of IFN gene, with immunoblotting and elisa assay recombinant human IFN β-1a protein expression level, result shows that this IFN stable gene is incorporated in Chinese hamster ovary celI karyomit(e), more than its copy number reaches 72 copies, expression level is stable.
In vitro tests shows, this albumen has and suppresses the activity that VSV virus causes 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
In 300L bioreactor scale, adopt import serum-free cell culture medium, coordinate the supplemented medium for this cell strain exploitation, realize cell density and be up to 8 * 10
6/ ml.Through cultivation in 12 days, in nutrient solution, target protein concentration reached 10～15mg/L.
We have successfully set up and can stablize the CHO-K1-9A3 cell strain that high level expression has recombinant human IFN β-1a albumen of high biologic activity, and the indices in master cell bank, master cell bank and the working cardial cell storehouse of setting up based on this cell strain all meets produces the requirement with zooblast for biological products in three of Pharmacopoeia of the People's Republic of China versions in 2005.This series work is set up the production technique of this albumen and carry out the researchs such as its pharmacology, pharmacodynamics and lay a good foundation.
The purification route A of embodiment 2 recombinant human IFN β-1a albumen
The present embodiment preferably be take the affine filler of dyestuff that hydroxylated polymethacrylic acid resin is matrix and is replaced and take the affine filler that dextran is matrix, and the former advantage is that filler hardness is high, and back-pressure is low, can bear larger process flow rate.Next selects the chromatography method of twice ion-exchange, first uses reinforcing yin essence ion exchange chromatography, and the rear weak cation exchange chromatography of using replaces the complex process such as reversed phase chromatography, easily causes the chromatography method of protein denaturation.Its method schematic 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 is concentrated
Clean ultrafiltration system, concentrated supernatant, 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
Clean AF-Blue chromatographic system, and the former processing of reducing phlegm and internal heat, carry out following operation: successively with (0.025mol/L SODIUM PHOSPHATE, MONOBASIC+0.025mol/L Sodium phosphate dibasic) damping fluid (pH7.2) balance chromatography column; After loading, use (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) 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.
Clean ultrafiltration system, and the former processing of reducing phlegm and internal heat, with after 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
Clean 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 (0.01mol/L SODIUM PHOSPHATE, MONOBASIC+0.01mol/L Sodium phosphate dibasic) damping fluid (pH7.0) balance chromatography column, recombinant human IFN β-1a albumen is not combined on anion-exchange column, and impurity is combined on anion-exchange column, collects stream and wear 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
Clean 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 collection is worn to protein solution, loading; 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, collects elution peak.
5) ultrafiltration and concentration and damping fluid displacement
By 0.01mol/L sodium-acetate buffer (pH3.5) dilution for the protein solution of the cation seperation column chromatography of collecting, use Millipore Pellicon ultrafiltration system thoroughly to replace damping fluid for 0.01mol/L sodium-acetate (pH3.5).
The purification route B of embodiment 3 recombinant human IFN β-1a purifying
The chromatography method that the present embodiment comprises twice ion-exchange, preferably first adopts weak cation exchange chromatography, the rear reinforcing yin essence ion exchange chromatography of using.Its method schematic 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 is concentrated
Clean ultrafiltration system, concentrated supernatant, with 0.05mol/L Tris-HCl damping fluid (pH7.2) displacement concentrated solution.
2) Blue dye affinity chromatography
Clean Capto-Blue chromatographic system, and the former processing of reducing phlegm and internal heat, carry out following operation: successively with 0.05mol/L Tris-HCl damping fluid (pH7.2) balance chromatography column; After loading, use 0.05mol/L Tris-HCl damping fluid (pH7.2) balance chromatography column; 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.Clean ultrafiltration system.Capto-Blue tomographic results as shown in Figure 2.
3) CM-sepharose cation-exchange chromatography
Clean CM-sepharose column chromatography system, and the former processing of reducing phlegm and internal heat, carry out: successively according to the following steps by 0.05mol/L Tris-HCl (pH6.7) balance chromatography column, loading; 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, collects elution peak.
4) Q-sepharose anion-exchange chromatography
Clean 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 is not combined on anion-exchange column, and impurity is combined on anion-exchange column, by the collection liquid loading of upper 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
By 0.01mol/L sodium-acetate buffer (pH3.5) dilution for the protein solution of the anion column chromatography of collecting, use Millipore Pellicon ultrafiltration system thoroughly to replace damping fluid for 0.01mol/L sodium-acetate (pH3.5).
The purification route C of embodiment 4 recombinant human IFN β-1a purifying
The present embodiment, preferably with after antibody affinity chromatography sample, selects the chromatography method of twice ion-exchange first to use reinforcing yin essence ion exchange chromatography, the rear weak cation exchange chromatography of using.Its method schematic 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 is concentrated
Clean ultrafiltration system, concentrated supernatant, with 0.03mol/L Tris-HCl damping fluid (pH7.2) displacement concentrated solution.
2) antibody affinity chromatography
Utilize recombinant human IFN β-1a as antigen immune mouse, build the hybridoma cell strain of the anti-recombinant human IFN β-1a monoclonal antibody of secretion, by this cell of vitro culture, or by this cell infection mouse results ascites, prepare anti-recombinant human IFN β-1a monoclonal antibody.This antibody and Sepharose 4B gel are prepared to the affine filler for specificity purification of recombinant human IFN β-1a by CNBr coupling.By the direct loading of cell cultures concentrated solution or after preliminary purification, loading is to affinity column, and target protein is incorporated on post, utilizes 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
After the present embodiment selection is affine with AF-Blue dyestuff, select the chromatography method of twice ion-exchange first to use strong cation exchange chromatography, the rear weak anionic displacement chromatography of using.
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
Clean 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; The protein solution loading that a upper purification step is collected; With phosphate buffered saline buffer balance chromatography column; Set chromatographic system gradient elution program parameter, elution chromatography post, collects elution peak.
4) DEAE-Sepharose anion-exchange chromatography
Clean DEAE-Sepharose chromatographic system, and the former processing of reducing phlegm and internal heat, by SP purified product loading after dilution reduces specific conductivity, collect stream and wear component.
5) ultrafiltration and concentration and damping fluid displacement (with embodiment 2)
The detection of embodiment 6 recombinant human IFN β-1a albumen
1) SDS-PAGE detects
The protein sample of separation and purification is carried out to 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 in, about 22.5kD.
2) ELISA detects
Use Human IFN b ELISA kit (Pestka Biomedical Laboratories company product), testing process by specification carries out.
3) RP-HPLC method
Use C4RP-HPLC pillar, 4.6mm * 25mm, NOBEL company product.Wherein, A phase: 0.1% phosphoric acid; B phase: 80% acetonitrile, 0.1% phosphoric acid.Detection wavelength is 214nm; Flow rate of mobile phase is 1ml/min, and the mobile composition using in each time period is mutually as shown in table 1 below.By embodiment 2 obtain target protein detected result as shown in Figure 3, by embodiment 3 obtain target protein detected result as shown in Figure 5, target protein goes out peak in 11.8min.
Table 1 RP-HPLC method is measured 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
Use material is G2000 pillar, 4.6mm * 25mm (TOSOH company product); Moving phase is 20mmol/L PBS, 0.2mol/L NaCl, pH6.8; Detection wavelength is 214nm; Flow rate of mobile phase: 0.5ml/min.By embodiment 2 obtain target protein detected result as shown in Figure 4, by embodiment 3 obtain target protein detected result as shown in Figure 6, target protein goes out peak in 14.5min.
5) protein active and intracellular toxin detect
Biological activity assay is with reference to three appendix XC cytopathic-effect inhibition assay of Pharmacopoeia of the People's Republic of China version in 2005.By embodiment 2 and embodiment 3 target protein that obtains activity detected result in Table 2 and Fig. 8.As follows with other quality examination result of the embodiment 3 target protein solution that obtains by embodiment 2: intracellular toxin < 1EU/22ug, the residual < 10pg/22ug of host DNA, the residual < 0.1% of host protein, is all better than the quality standard of pharmacopeia.
By ELISA test kit, detect target protein concentration 12mg/L in 300L culture supernatant.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: AF-Blue and the Capto-Blue chromatography target protein rate of recovery are all in 70% left and right, and after purifying, lipidated protein approximately 85%; Ultrafiltration and the damping fluid displacement target protein rate of recovery 80%; The Q-Sepharose chromatography target protein rate of recovery 90%, after purifying, lipidated protein approximately 95%; The CM-Sepharose chromatography target protein rate of recovery 80%, after purifying, lipidated protein approximately 98%; 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 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 obtain specific activity
Experiment shows, after affinity chromatography, the effect that adopts strong cation exchange chromatography and weak anionic displacement chromatography to be used in conjunction is poor, and its purifying target protein purity, between 70%～85%, can not meet needs of production far away.Result of the present invention shows: to recombinant human IFN β-1a albumen, adopt the purification effect of weak cation exchange chromatography and reinforcing yin essence ion exchange chromatography 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, through experimental verification, determined the clear and definite condition of ion exchange chromatography and affinity chromatography, after affinity chromatography, adopt one time weak cation exchange chromatography, a reinforcing yin essence ion exchange chromatography, can reach more than 98% high purity.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 is all 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 impact 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 residual in protein soln of metal ion.By our 300L cell culture system, by method of the present invention, carried out the large scale purification of recombinant human IFN β-1a, respond well.
In sum, this purifying process process of recombinant human IFN β-1a is convenient, and filler cost is low, and target protein activity is high, is applicable to being applied to commercial production scale.
Claims (6)
1. the purification process of a recombinant human interferon beta-1a, it is characterized in that, described purification process comprises affinity chromatography process and twice ion exchange chromatography process subsequently, wherein in twice ion exchange chromatography process, first carry out reinforcing yin essence ion exchange chromatography and then carry out weak cation exchange chromatography, comprising:
1) affinity chromatography: after cell conditioned medium liquid concentration, loading after balance affinity column, then use elutriant elution chromatography post, collect elution peak; Concentrated affinity chromatography is collected liquid and replaces damping fluid; Elutriant composition is the phosphate buffered saline buffer that contains propylene glycol and NaCl, and its composition is 0.025mol/L SODIUM PHOSPHATE, MONOBASIC, 0.025mol/L Sodium phosphate dibasic, 2mol/L NaCl, 50% propylene glycol, and pH is 7.2; Displacement damping fluid composition is 0.02mol/L phosphate buffered saline buffer, and pH is 7.0;
2) reinforcing yin essence ion exchange chromatography: balance anion chromatography column; By the collection liquid loading of upper step, collect the stream that contains recombinant human interferon beta-1a and wear component; Level pad composition is 0.01mol/L SODIUM PHOSPHATE, MONOBASIC, 0.01mol/L Sodium phosphate dibasic, and pH is 7.0; Elutriant composition is 0.01mol/L SODIUM PHOSPHATE, MONOBASIC, 0.01mol/L Sodium phosphate dibasic, 1.0mol/L NaCl, and pH is 7.0;
3) weak cation exchange chromatography: balance cation chromatography column; The positively charged ion chromatography post stream of collection is worn to component loading; With elutriant elution chromatography post, collect the component that contains recombinant human interferon beta-1a; Level pad composition is 0.01mol/L SODIUM PHOSPHATE, MONOBASIC, 0.01mol/L Sodium phosphate dibasic, and pH is 6.7;
In affinity chromatography process, affinity column used is selected from one of Blue dye affinity chromatography post and antibody affinity chromatography.
2. purification process according to claim 1, is characterized in that, in reinforcing yin essence ion exchange chromatography process, reinforcing yin essence ion exchange column used is anion-exchange column Q-sepharose.
3. purification process according to claim 1, is characterized in that, in cation-exchange chromatography process, weak cation exchange chromatography column used is cationic exchange coloum CM-sepharose.
4. the purification process of a recombinant human interferon beta-1a, it is characterized in that, described purification process comprises affinity chromatography process and twice ion exchange chromatography process subsequently, wherein in twice ion exchange chromatography process, first carry out weak cation exchange chromatography and then carry out reinforcing yin essence ion exchange chromatography, comprising:
1) affinity chromatography: after cell conditioned medium liquid concentration, loading after balance affinity column, then use elutriant elution chromatography post, collect elution peak; Concentrated affinity column is collected liquid and replaces damping fluid; Elutriant composition is 0.05mol/L Tris-HCl, 2mol/L NaCl, 50% propylene glycol, and pH is 7.2;
2) weak cation exchange chromatography: balance cation chromatography column; By the collection liquid loading of upper step, elutriant elution chromatography post, collects the component that contains recombinant human interferon beta-1a; Elutriant composition is 0.05mol/LTris-HCl, and pH is 6.7;
3) reinforcing yin essence ion exchange chromatography: balance anion chromatography column; By the collection liquid loading of upper step, and collect the stream that contains recombinant human interferon beta-1a and wear component; Purifying damping fluid composition is 0.03mol/L Tris-HCl, 1.0mol/L NaCl, and pH is 7.0;
In affinity chromatography process, affinity column used is selected from one of Blue dye affinity chromatography post and antibody affinity chromatography.
5. purification process according to claim 4, is characterized in that, in reinforcing yin essence ion exchange chromatography process, reinforcing yin essence ion exchange column used is anion-exchange column Q-sepharose.
6. purification process according to claim 4, is characterized in that, in weak cation exchange chromatography process, weak cation exchange chromatography column used is cationic exchange coloum CM-sepharose.
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| CN103965346A (en) * | 2013-10-29 | 2014-08-06 | 王明丽 | RPoIFN alpha1 (recombinant porcine interferon alpha 1) separation and purification method |
| CN105017418B (en) * | 2014-03-27 | 2021-02-23 | 上海药明康德新药开发有限公司 | Monoclonal antibody purification process |
| EA027609B9 (en) * | 2015-04-29 | 2017-11-30 | Тютор С.А.С.И.Ф.И.А. | Method of producing interferon beta 1-alfa and pharmaceutical composition comprising interferon beta 1-alfa |
| KR20210009982A (en) * | 2019-07-18 | 2021-01-27 | 에이비온 주식회사 | Method for purification of interferon-beta protein with 2 glycosylation sites |
| CN112625115B (en) * | 2020-12-25 | 2022-09-06 | 山东睿鹰制药集团有限公司 | Method and kit for purifying recombinant human interferon-kappa |
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