CN103864915A - Method for purifying recombinant human interferon alpha2b and kit - Google Patents
Method for purifying recombinant human interferon alpha2b and kit Download PDFInfo
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Abstract
The invention belongs to the biochemistry field, and concretely belongs to the protein purifying field. More specifically, the invention relates to a method for purifying recombinant human interferon alpha2b and a kit. The method comprises the following steps: 1)performing hydrophobic chromatography (HIC) on a broth supernatant containing recombinant human interferon alpha2b; 2)performing anion exchange on the products obtained in the step 1) by a tolerable and high flow velocity multitime usage mode; 3) performing cation exchange on the products obtained in the step 2) by a tolerable and high flow velocity multitime usage mode; and 4) performing anion exchange on the products obtained in the step 3). According to the invention, the interferon yield is increased by about 25%, and the method is suitable for large scale industrial production/purifying the recombinant human interferon alpha2b, so that purifying cost is saved, and the purifying efficiency is increased.
Description
Technical field
The invention belongs to biochemical field, particularly, belong to protein purification field.More specifically, the present invention relates to a kind of method and test kit of purification of Recombinant human interferon-alpha-2 b.
Background technology
Interferon, rabbit is to be subject to virus or other inductors by body cell to stimulate and a histone matter of secretion, has the multiple physiologically active such as antiviral, antiproliferative and immunomodulatory of wide spectrum.The antivirus action of Interferon, rabbit is to act on viral target cell on the one hand, and induction produces a series of active substances, by multiple biochemical route, the different links in virus replication cycle is disturbed, and suppresses viral intrusion and copies.Interferon, rabbit acts on body immune system on the other hand, strengthens immunity system inhibition or removes viral ability.In addition, Interferon, rabbit can also regulate immunologic function, suppresses cell fission, and the especially growth of inhibition tumor cell, therefore the clinical treatment for anti-virus infection and some malignant tumour.
According to antigenic characteristic and molecular structure, Interferon, rabbit can be divided into three types: α or LeIF, β or fiblaferon, γ or type II interferon conventionally.Its broad-spectrum disease resistance possible mechanism of its cytotoxicity is to produce plurality of enzymes by induction host cell to carry out the links of viral interference reproduction process, and a kind of Interferon, rabbit can suppress the propagation of multiple virus, in the antiviral that is medically a class wide spectrum.It can also activate NK cell, scavenger cell, CTL cell and kill and wound the target cell of virus infection.Applying in the market maximum is alpha-interferon.
α 2b Interferon, rabbit is a kind of antiviral, antitumor drug, and has immunoregulatory activity.Can be used for the treatment of Type B viral hepatitis, hepatitis C, viral meningitis, viral eye infections, kidney, capillary leukemia, malignant lymphoma of skin, osteogenic sarcoma, multiple sclerosis etc.
The single chain polypeptide that the recombinant human interferon alpha 2 b that pseudomonas is expressed is made up of 165 amino acid, molecular weight is 19.2 ± 10%KDa, and iso-electric point is between 5.7-6.7, and sugar based in molecule, has four alpha-helixs in molecule; CYS
1and CYS
98, CYS
29and CYS
138form two disulfide linkage, wherein the disulfide linkage between 29-138 is very important for interferon biological activity.The three-D space structure schematic diagram of the recombinant human interferon alpha 2 b that pseudomonas is expressed as shown in Figure 1.
The aminoacid sequence of recombinant human interferon alpha 2 b (pseudomonas expression) is as shown in SEQ IDNO:1 below:
165aa
Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg?Thr?LeuMET?Leu?Leu?Ala?Gln?MET?Arg?Arg?Ile?Ser?Leu?Phe?Ser?Cys?Leu?LysAsp?Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?PheGln?Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?MET?Ile?Gln?Gln?IlePhe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?ThrLeu?Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?LeuGlu?Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Thr?Glu?Thr?Pro?Leu?METLys?Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?ThrLeu?Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?ValArg?Ala?Glu?Ile?MET?Arg?Ser?Phe?Ser?Leu?Ser?Thr?Asn?Leu?Gln?GluSer?Leu?Arg?Ser?Lys?Glu（SEQ?ID?NO：1）
Early stage human interferon albumen mainly from blood purifying obtain, there is a large amount of shortcomings in this method, as: be difficult to obtain a large amount of blood, economy is low, is subject to carry in various blood viral ground contamination.Recombinant DNA technology Restruction human interferon albumen is applied to after the production of human interferon albumen, and the problems referred to above are resolved, but it is unsatisfactory from fermented liquid, to extract the yield of Purification of Human Interferon, rabbit.
In currently available technology, there is the method about recombinant human interferon alpha 2 b purifying, for example method below:
Ion-exchange chromatography: adopt DEAE Sepharose (FF) post, column type XK30/50.With after F liquid balance, by the renaturation solution sample loading after above-mentioned dialysis, flow velocity is 20ml/min, after end of the sample, with the F liquid wash-out containing different concns NaCl, collects interferon activity peak.Select Sephacryl00/50XK chromatography column, S2100 filler, column volume 1700ml, with after G liquid balance, the interferon activity peak sample that ion-exchange chromatography is collected is through gel-filtration, and applied sample amount is column volume 5%, regather interferon activity peak, merging sample carries out following calibrating, and (Liu's Peng is real, Jiang Wei, Sui Baozhen, Zhang Chunli, the extraction of recombinant human interferon alpha 2 b and purifying, microbiology immunology progress, the 32nd the 1st phase of volume in 2004, p13-16).
But the inventor finds under study for action, existing purification process is not very good to the yield of Interferon, rabbit, therefore, and this area a kind of purification process that improves Interferon, rabbit yield of still needing.
Summary of the invention
The inventor, through deep research and performing creative labour, through comprehensive application and optimization to multiple conditioned disjunction parameters such as reagent used, applied sample amount, flow velocity, pH, has obtained a kind of method or test kit of purification of Recombinant human interferon-alpha-2 b.And the inventor is surprised to find, method of the present invention or test kit be particularly human interferon-alpha-2 b (for example recombinant human interferon alpha 2 b) of purifying alpha-interferon effectively, and the yield of albumen and Interferon, rabbit is all higher, and the interferon activity obtaining is good.Following invention is provided thus:
One aspect of the present invention relates to a kind of method of purifying alpha-interferon, comprises the steps:
1) the fermented liquid supernatant liquid containing Interferon, rabbit is carried out to hydrophobic chromatography (HIC);
2) product obtaining in step 1) is carried out tolerating the nonexpondable anion chromatography of high flow rate;
3) by step 2) in the product that obtains carry out tolerating the nonexpondable positively charged ion chromatography of high flow rate; With
4) product obtaining in step 3) is carried out to anion chromatography.
The above-mentioned method according to the present invention, wherein, step 2) and step 4) described in anion chromatography be weak anionic chromatography.
For the purpose of the present invention, the obtaining of fermented liquid supernatant liquid of Interferon, rabbit is not particularly limited, and the method that for example can know by those skilled in the art obtains.
Can tolerate the nonexpondable anionic exchange medium of high flow rate and be by charge effect and hydrogen bond and/or hydrophobic interaction and can tolerate the nonexpondable anionic exchange medium of high flow rate and interact.
Be not limited to theoretical restriction, it is stationary phase that hydrophobic chromatography utilizes the hydrophobic adsorbent of the weak hydrophobic grouping (hydrophobicity aglucon) of surperficial coupling, is the chromatography of carrying out protein-based separation and purification of biological macromolecule according to the difference of the weak hydrophobic interaction between protein and hydrophobic adsorbent.A certain amount of hydrophobic group is all contained on hydrophilic protein matter surface, and the more protein hydrophobic group of hydrophobic amino acid (as tyrosine, phenylalanine etc.) content is many, and hydrophobicity is also strong.And in different media, how many differences to some extent also that hydrophobic grouping exposes.In the higher salts solution of ionic strength, the hydration layer at the hydrophobic position of protein surface is destroyed, and the structure of protein is also overturn, and exposes hydrophobic position and increases, and the hydrophobicity of protein strengthens; In the lower solution of ionic strength, the hydrophobicity of protein reduces.Loading under the condition of high conductivity, the target protein that hydrophobic group is more and aglucon effect absorption, the albumen that lacks hydrophobic grouping is rinsed down, then the specific conductivity that reduces elutriant reduces albumen hydrophobicity, is eluted from resin.
Be not limited to theoretical restriction, to be ion-exchanger be combined with the net charge of protein with self institute's oppositely charged ion exchange chromatography.The ion-exchanger of standard is formed by a large amount of charged side chains and insoluble resin-bonded.The resin anion(R.A) side-chain charges such as DEAE are being for just, can with electronegative protein binding.When ion exchange chromatography, first with ionic strength lower damping fluid loading and washing.Protein soln enters after ion exchange column, is rinsed without the albumen of binding ability with ion exchange resin, and remaining protein all can be combined on resin.Then in damping fluid, add strong electrolyte (as sodium-chlor), the binding ability of salt ion and ion-exchanger is better than the binding ability of albumen and ion-exchanger, and albumen will be eluted competitively.Because the avidity of different types of albumen and resin is different, wash-out is out one by one by it for the available method that increases gradually sodium chloride concentration in elutriant.Sodium chloride solution elution method has two kinds of modes: stepped start-stop system wash-out (in elutriant, sodium chloride concentration is interim improves) and linear gradient wash-out (in elutriant, sodium chloride concentration is linear raising).In general, when foreign protein and target protein binding ability differ larger, the suitable stepped start-stop system wash-out of using; Linear gradient elution is suitable for the separately close protein of binding ability.
Positively charged ion aglucon includes but not limited to: sulfonate radical (-SO
3 –/-SO
3– H), sulfate radical (-OSO
3 –/-OSO
3– H), carboxylate radical (-COO
–/-COOH), phosphate radical (-OPO
3 2 –/-OPO
3– H/-OPO
3h
2) and orthophosphite (-PO
3 2-/-PO
3– H/-PO
3h
2).Conventional weak cation exchange medium group is carboxylate radical (-COO
–/-COOH), phosphate radical (-OPO
3 2 –/-OPO
3– H/-OPO
3h
2) and orthophosphite (-PO
3 2 –/-PO
3– H/-PO
3h
2).
Less important conjugated group has a hydrogen bond action atom at least, and between this atom and cation exchange group, should have the distance of 1 to 7 atom.The hydrogen bond atomic time can participate in forming the atom of hydrogen bond.Hydrogen bond atom can be selected from contain heteroatomic group, as Sauerstoffatom, and nitrogen-atoms, sulphur atom; And sp and sp
2-hydridization carbon atom; Halogen family group, as F, Cl, Br, I, particularly F.The less important conjugated group of typical case contains uncharged atom or changes with pH can charged atom.
Be not limited to theoretical restriction, step 1) main purpose is to eliminate peptide class, prothetic group, and the materials such as immunoglobulin (Ig), therefore use hydrophobic chromatography resin (HIC).Owing to recombinant human interferon alpha 2 b fermented liquid supernatant liquid being carried out to ammonium sulfate precipitation processing before step 1), sample solution is high saline solution, and this step adopts HIC, can avoid the direct loading of desalting treatment, reduces complex process degree.HIC, by fermented supernatant fluid purifying and with small volume wash-out, reaches concentrated effect and has significantly reduced the salt concn in fermented supernatant fluid, for downstream purification provides desirable purification condition simultaneously.Hydrophobic chromatography matrix used is after sepharose is activated by BrCN, from different alpha-amino group alkane effects, produces the homologous series alkyl agarose Seph-Cn that carbon chain lengths successively changes, and wherein n=1-6, represents the carbonatoms in carbochain.
Being not limited to theoretical restriction, step 2) main purpose is to separate the charged biomolecules such as macromole polypeptide protein, nucleic acid, amino acid, therefore uses ion chromatography resin (IEC).On agarose molecule, be combined with diethylaminoethyl-(Dicthylaminoethyl, DEAE), contain positively charged agar, cationic sugar-O-C6 H14N
+h, its gegenion is that negatively charged ion is (as Cl
-deng), can exchange with electronegative protein negatively charged ion.Exchanger all has the ability of exchange adsorption to colloid ion (as protein) and inorganic ion (as NaCl), when both are present in a chromatography process simultaneously, and the exchange adsorption of competing property.Work as Cl
-concentration when large, protein is not easy to be adsorbed, and after absorption, is also easy to by wash-out, works as Cl
-concentration hour, protein is easily adsorbed, and after absorption, is also not easy by wash-out.Therefore, adopt the method for the ionic strength that increases elutriant to reach the object of isolated protein.
Be not limited to theoretical restriction, in step 3), main purpose is to separate the charged biomolecules such as macromole polypeptide protein, nucleic acid, amino acid, therefore uses ion chromatography resin (IEC).On agarose molecule, be combined with carboxymethyl (Carboxymethy, CM).Its gegenion is that positively charged ion is (as Na
+deng), can exchange with positively charged protein positively charged ion.
Be not limited to theoretical restriction, in step 4), main purpose is to separate the biological charged molecule of intracellular toxin, therefore uses ion chromatography resin (IEC).Endotoxic main chemical compositions is that the lipoid A composition in lipopolysaccharides is electronegative, therefore selects anion chromatography resin isolation.
A method for purifying alpha-interferon, comprises the steps:
1) be under 180 ± 100mS/cm, to utilize the agar carbohydrate chromatographic resin of Phenyl aglucon to carry out wash-out separation by Interferon, rabbit fermented liquid supernatant liquid in specific conductivity;
2) product obtaining in step 1) is regulated to pH to 7.5-9.0, utilize the agar carbohydrate chromatographic resin of DEAE aglucon to carry out wash-out separation;
3) by step 2) in the product that obtains regulate pH to 4.0-5.5, utilize the agar carbohydrate chromatographic resin of CM aglucon to separate wash-out; With
4) product obtaining in step 3) is regulated to pH to 7-9, utilize the agar carbohydrate chromatographic resin of DEAE aglucon to carry out wash-out separation.
Method according to the present invention described in any one, it comprises the steps:
1) recombinant human interferon alpha 2 b fermented liquid supernatant liquid is utilized under conductivity value 180 ± 100mS/cm the agar carbohydrate chromatographic resin of Phenyl aglucon carry out wash-out separation, flow velocity 10-30L/hr, an applied sample amount 5-9 column volume; Elutriant filters by microfiltration membrane;
2) product obtaining in step 1) is carried out to 3-5 with ultra-filtration membrane and doubly concentrate, concentrated rear pH regulator, to pH7.5-8.5, utilizes the agar carbohydrate chromatographic resin of DEAE aglucon to carry out wash-out separation; Flow velocity 10-30L/hr, an applied sample amount 3-7 column volume;
3) by step 2) in the product that obtains be adjusted to pH4.0-5.5, utilize the agar carbohydrate chromatographic resin of CM aglucon to separate wash-out, flow velocity 10-30L/hr; An applied sample amount 8-11 column volume; With
4) product obtaining in step 3) is adjusted to pH7-9, utilizes the agar carbohydrate chromatographic resin of DEAE aglucon to carry out wash-out separation, flow velocity 10-30L/hr, an applied sample amount 8-11 column volume; Elutriant is concentrated with ultra-filtration membrane, be concentrated into 1-3mg/ml.
The method that concentrated concentration can be known by those skilled in the art records, for example, retain liquid by monitoring and obtain in the ultraviolet absorption value at 280nm place.
Method according to the present invention described in any one, is characterized in that any one in following (1)-(17) or multinomial:
(1) flow velocity in step 1) is 20-40L/hr; Be preferably 25-35L/hr, for example 25,26,27,28,29,30,31,32,33,34 or 35L/hr;
(2) step 2) in flow velocity be 10-30L/hr; Be preferably 10-20L/hr, for example 10,11,12,13,14,15,16,17,18,19 or 20L/hr;
(3) flow velocity in step 3) is 10-30L/hr; Be preferably 10-20L/hr, for example 10,11,12,13,14,15,16,17,18,19 or 20L/hr;
(4) flow velocity in step 4) is 10-30L/hr; Be preferably 10-20L/hr, for example 10,11,12,13,14,15,16,17,18,19 or 20L/hr;
(5) applied sample amount in step 1) is 3-9 column volume; Be preferably 5-9 column volume (for example 5,6,7,8 or 9 column volumes);
(6) step 2) in applied sample amount be 1-7 column volume; Be preferably 3-7 column volume (for example 3,4,5,6 or 7 column volumes);
(7) applied sample amount in step 3) is 6-11 column volume; Be preferably 8-11 column volume (for example 8,9,10 or 11 column volumes);
(8) applied sample amount in step 4) is 6-11 column volume; Be preferably 8-11 column volume (for example 8,9,10 or 11 column volumes);
(9) step 2) in, pH to 8.0-9.0 regulated, for example 8.0,8.1,8.2,8.3,8.4,8.5,8.6,8.7,8.8,8.9 or 9.0; Be preferably 8.2-8.8;
(10), in step 3), regulate pH to 4.2-5.2; Be preferably 4.5-5.0, for example 4.5,4.6,4.7,4.8,4.9 or 5.0;
(11), in step 4), regulate pH to 7.5-8.5, for example 7.6,7.7,7.8,7.9,8.0,8.1,8.2,8.3,8.4 or 8.5; More preferably 7.7-8.2, for example 7.7,7.8,7.9,8.0,8.1 or 8.2;
(12) in step 1), also comprise and elutriant is filtered to (for example filtering with ultra-filtration membrane), obtain the step of filtrate;
(13) step 2) in, before being also included in adjusting pH, the product in step 1) is carried out to concentrated step (for example carrying out 3-5 with ultra-filtration membrane doubly concentrates);
(14), in step 4), also comprise that the elutriant in step 4) is carried out to concentrated step (is for example concentrated into 1-3mg/ml by elutriant ultra-filtration membrane; Preferably simmer down to 2-3mg/ml; More preferably simmer down to 2.5-3mg/ml, for example 2.5,2.6,2.7,2.8,2.9 or 3mg/ml.）。
(15) specific conductivity in step 1) is 80-280mS/cm; Preferably, be 100-190mS/cm; More preferably, be 120-140mS/cm;
(16), before carrying out step 1), just carry out cracking processing containing the fermented liquid supernatant liquid of recombinant human interferon alpha 2 b;
(17) step 2) in the half (calculating according to volume) of usage quantity of the amount of agar carbohydrate chromatographic resin of the DEAE aglucon that the uses agar carbohydrate chromatographic resin that is the Phenyl aglucon that uses in step 1).
Method according to the present invention described in any one, wherein, the specific conductivity in step 1) is 80-280mS/cm; Preferably, be 100-190mS/cm; More preferably, be 120-140mS/cm or 125-135mS/cm, for example 125,126,127,128,129,130,131,132,133,134 or 135mS/cm.
Method according to the present invention described in any one, wherein, before carrying out step 1), just carries out cracking processing containing the fermented liquid supernatant liquid of recombinant human interferon alpha 2 b.
The method that cracking can be known according to those skilled in the art is carried out, for example, use the method for high osmotic pressure that fermentation thalline is carried out to cracking processing.
Method according to the present invention described in any one, wherein step 2) in the half (calculating according to volume) of usage quantity of the hydrophobic medium that uses for the hydrophobic chromatography in step 1) of the amount of anionic exchange medium that anion chromatography uses.
The difference of weak anionic and reinforcing yin essence ion is to make the complete Ionized pH scope of exchang medium, and wider person is strong, and narrower person has nothing to do with bonding strength a little less than being.Weak anion exchange resin: this resinoid contains weakly alkaline group, as primary amine groups (also claiming one-level amido)-NH2, secondary amine (secondary amine)-NHR or tertiary amine groups (tertiary amine base)-NR2, they can dissociation go out OH-and be weakly alkaline in water.The positive electric group of this resin can be combined by the Anion-adsorption in solution, thereby produces anionresin effect.It can only work under neutrality or alkaline environment.And the applicable scope of reinforcing yin essence ionic is wider, under sour environment, also can.
Method according to the present invention described in any one, wherein, step 2) in elution flow rate be 50-120cm/h; Be preferably 70-110cm/h; More preferably 110cm/h.
Method according to the present invention described in any one, wherein, the elution flow rate in step 4) is 50-80cm/h; Be preferably 60-70cm/h; More preferably 70cm/h.
Method according to the present invention described in any one, wherein, described Interferon, rabbit is human interferon; Particularly, be human interferon-alpha-2 b; More specifically, be recombinant human interferon alpha 2 b; The recombinant human interferon alpha 2 b of expressing for pseudomonas especially particularly.
A kind of test kit of another aspect of the present invention, comprise: the agar carbohydrate chromatographic resin (in the present invention, the schematic diagram of three kinds of resins is respectively as shown in Fig. 2 A, Fig. 2 B, Fig. 2 C) of the agar carbohydrate chromatographic resin of Phenyl aglucon, the agar carbohydrate chromatographic resin of DEAE aglucon and CM aglucon.Particularly, described test kit comprises Phenyl hydrophobic resin, DEAE resin anion(R.A) and CMFF resin cation (R.C.).Described test kit of the present invention can be used in purifying alpha-interferon.
The purposes of test kit of the present invention in preparation or purifying alpha-interferon that relate in one aspect to again of the present invention; Particularly, described Interferon, rabbit is human interferon; More specifically, be human interferon-alpha-2 b; Especially particularly, be recombinant human interferon alpha 2 b; The recombinant human interferon alpha 2 b of expressing for pseudomonas further particularly.
In the present invention, term " Interferon, rabbit ", refers to human interferon particularly; More specifically, be human interferon-alpha-2 b; Especially particularly, be recombinant human interferon alpha 2 b; Further particularly, be the recombinant human interferon alpha 2 b that pseudomonas is expressed, for example recombinant human interferon alpha 2 b shown in SEQ ID NO:1.
The beneficial effect of the invention
The yield of the Interferon, rabbit of purification process of the present invention is more than 35% even more than 40% even more than 42%.The present invention has saved purifying cost, has improved purification efficiency, applicable to the large-scale industrial production/purifying of recombinant human interferon alpha 2 b.In addition, test kit of the present invention can stand higher pressure and larger elution flow rate, can repeatedly use.
Accompanying drawing explanation
Fig. 1: the space structure schematic diagram of pseudomonas recombinant human interferon alpha 2 b.
Fig. 2: Fig. 2 A, hydrophobic chromatography resin Phenyl schematic diagram, wherein the ball in left side represents agarose matrix; Fig. 2 B, anion chromatography resin DEAE schematic diagram, wherein the ball in left side represents Cross-linked Agar sugar substrate; Fig. 2 C, positively charged ion chromatography resin CM schematic diagram, wherein the ball in left side represents Cross-linked Agar sugar substrate.
The hydrophobic chromatography wash-out collection of illustrative plates of step 1) in Fig. 3: embodiment 1.
Step 2 in Fig. 4: embodiment 1) anion chromatography wash-out collection of illustrative plates.
The positively charged ion chromatography elution profile of step 3) spectrum in Fig. 5: embodiment 1.
The secondary anion chromatography wash-out collection of illustrative plates of step 4) in Fig. 6: embodiment 1.
Step 2 in Fig. 7: embodiment 1) the electrophoretic analysis of anion chromatography product.The sample of swimming lane 1-13 is respectively successively: molecular weight standard product, interferon standard substance, negatively charged ion sample solution, anion chromatography part are collected 1-9 and anion chromatography product.Described " part is collected " refers to when chromatography process enters into gradient elution and collects one by one according to fixed volume sub-bottle, until wash-out completes.
The cationic layer division thing electrophoretic analysis of step 3) in Fig. 8: embodiment 1.The sample of swimming lane 1-13 is respectively successively: molecular weight standard product, interferon standard substance, positively charged ion sample solution, positively charged ion chromatography part are collected 1-9 and cationic layer division thing.Described " part is collected " refers to when chromatography process enters into gradient elution and collects one by one according to fixed volume sub-bottle, until wash-out completes.
The anion chromatography product electrophoretic analysis of step 3) in Fig. 9: embodiment 1.The sample of swimming lane 1-13 is respectively successively: molecular weight standard product, interferon standard substance, negatively charged ion sample solution, anion chromatography part are collected 1-9 and anion chromatography product.Described " part is collected " refers to when chromatography process enters into gradient elution and collects one by one according to fixed volume sub-bottle, until wash-out completes.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example is only for the present invention is described, and should not be considered as limiting scope of the present invention.Unreceipted actual conditions person in embodiment, carries out according to the condition of normal condition or manufacturers's suggestion.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.
embodiment 1: the purifying of recombinant human interferon alpha 2 b
One, experiment material and laboratory apparatus:
Phenyl hydrophobic resin, GE Healthcare company, 17-0973-04; DEAE resin anion(R.A), GE Healthcare company, 17-0709-05; CM FF resin cation (R.C.), GEHealthcare company, 17-0719-05.
Damping fluid: phosphoric acid salt+sodium-chlor buffer system.
Full-automatic liquid phase chromatographic system, GE Healthcare company, AKTA Explorer100; Biological analyser, Agilent company, Agilent 2100 bioanalyzer; Electrophoresis system, GE Healthcare company, SE600 RUBY.
Two, experimental technique:
1) recombinant human interferon alpha 2 b fermented liquid supernatant liquid is utilized under conductivity value 130mS/cm the agar carbohydrate chromatographic resin of Phenyl aglucon carry out wash-out separation, elutriant filters with ultra-filtration membrane; Flow velocity 30L/hr, 6 column volumes of applied sample amount;
2) product obtaining in step 1) is carried out to 5 times with ultra-filtration membrane and concentrate, concentrated rear pH regulator pH to 8.5, utilizes the agar carbohydrate chromatographic resin of DEAE aglucon to carry out wash-out separation; Flow velocity 15L/hr, 3 column volumes of applied sample amount;
3) by step 2) in the product that obtains regulate pH to 4.5, utilize the agar carbohydrate chromatographic resin of CM aglucon to separate wash-out, flow velocity 12L/hr; 10 column volumes of applied sample amount; With
4) product obtaining in step 3) is regulated to pH to 7.8, utilize the agar carbohydrate chromatographic resin of DEAE aglucon to carry out wash-out separation, flow velocity 15L/hr, 9 column volumes of applied sample amount; Elutriant is concentrated with ultra-filtration membrane, be concentrated into 2.8mg/ml.
Three, experimental result
The hydrophobic chromatography wash-out collection of illustrative plates of step 1) as shown in Figure 3; Step 2) anion chromatography wash-out collection of illustrative plates as shown in Figure 4; The positively charged ion chromatography elution profile of step 3) is composed as shown in Figure 5; The secondary anion chromatography wash-out collection of illustrative plates of step 4) as shown in Figure 6.
From Fig. 3-6, the peak position point that goes out of α 2b albumen conforms to iso-electric point character, and the elution requirement of setting can reach predetermined resolving power, effectively purification of Recombinant human interferon-alpha-2 b albumen accurately.
comparative example: the purifying of recombinant human interferon alpha 2 b
According to operating as Publication about Document:
Liu's Peng is real, Jiang Wei, Sui Baozhen, Zhang Chunli, the extraction of recombinant human interferon alpha 2 b and purifying, microbiology immunology progress, the 32nd the 1st phase of volume in 2004, p13-16.
experimental example 1: gel electrophoresis analysis
One, laboratory sample
Step 2 in embodiment 1) to the product in step 4).
Two, method is as follows:
1. the preparation of gel
Each component is as shown in table 1 below and table 2.
Each component of table 1:15% lower floor separation gel
| Component | Volume (ml) |
| Ultrapure water | 9.4 |
| 30% acrylamide/bisacrylamide (29:1) | 20 |
| 1.5M?Tris-HCl，pH8.8 | 10 |
| 10%SDS | 0.4 |
| 10% ammonium persulphate | 0.2 |
| TEMED | 0.02 |
After mixing, pour in glass chamber, to stopping along 4cm place on short sheet glass, then get 1ml ultrapure water and carry out water seal.In the time that the different boundary of specific refractory power appears in gel and water seal interlayer, the ultrapure water of sealing of anhydrating inclines.
Each component of the concentrated glue in table 2:7.5% upper strata
| Component | Volume (ml) |
| Ultrapure water | 9.8 |
| 30% acrylamide/bisacrylamide (29:1) | 5.0 |
| 0.5M?Tris-HCl，pH6.8 | 5.0 |
| 10%SDS | 0.2 |
| 10% ammonium persulphate | 0.1 |
| TEMED | 0.02 |
After mixing, pour in glass chamber, until stop apart from short sheet glass upper limb 0.5cm place, loading comb is inserted in the glue of upper strata, room temperature is placed gel polymerisation after 20-30 minute.After polymerization, before gel loading, remove loading comb, gel glass plate is connected with upper strata electrophoresis chamber.
2. the processing of standard substance and sample
Interferon standard substance is done to suitable multiple dilution, and by mixed to 30 μ l interferon solutions and 10 μ l buffered soln, boiling water heating 5min, is cooled to room temperature.
Sample and buffered soln are mixed with 3:1 ratio, and boiling water heating 5min, is cooled to room temperature.
3. electrophoresis
The positive and negative electrode wire of electrophoresis chamber is connected with DC voltage-stabilizing electrophoresis apparatus, connects water coolant.Add 10 μ l molecular weight standard product solution to gel loading hole, add 10 μ l Interferon, rabbit standardized solution to loading hole, add 25 μ l sub-bottle sample solutions (comprising chromatography sample solution, i.e. BefDE, BefCM or BefDE II) to loading hole.
After application of sample, gel slab is put into electrophoresis chamber, build electrophoresis chamber lid, in electrophoresis chamber, add electrophoretic buffer, switch on power and start electrophoresis with water coolant.Deposition condition is constant current 20mA/gel.Run out of separation gel base 5-10 minute when tetrabromophenol sulfonphthalein forward position, cut off the electricity supply and water coolant.Open electrophoresis chamber lid, take out gel.
4. silver dyes
Gel soaks in stop bath, vibration 30min.Discard stop bath, add photosensitive solution, vibration 5min.Discard photosensitive solution, rinse gel 3 times, each 10min with ultrapure water.Add 0.2% silver nitrate solution, vibration 15min.Outwell silver nitrate solution, rinse gel 2 times, each 1min with ultrapure water.Add the vibration of gel nitrite ion, until interferon standard substance band is high-visible, stop immediately dyeing.Discard nitrite ion, add 5% acetic acid stop buffer, vibration 10min.Then clean gel 3-4 time with ultrapure water.
Three, experimental result
Step 2) anion chromatography electrophoretic analysis as shown in Figure 7; The positively charged ion chromatography electrophoretic analysis of step 3) as shown in Figure 8; The anion chromatography electrophoretic analysis of step 4) as shown in Figure 9.
From Fig. 7-9, by each step chromatography, the impurity in sample is effectively removed, and the purity of the product finally obtaining meets the requirements.
experimental example 2: Interferon, rabbit yield determination experiment
One, experiment reagent and laboratory apparatus
1. the required reagent of Interferon, rabbit assay:
Water for injection; DTT, Bole company; Protein 80 assay kit, Agilent company; Interferon standard substance CRS.
2. the required instrument of Interferon, rabbit assay and utensil
2100 biological analysers; Refrigerated centrifuge; Software control system; Water for injection system; Refrigerator; Computer, printer; Mini vibrator; Mini whizzer; Single track liquid getting device; Analytical balance; Beaker.
The sample of measuring is sample prepared by embodiment 1 and comparative example.
Two, experimental technique
1. the preparation of interferon alpha 2 b standardized solution:
Get interferon standard substance and inject dilute with water, make the Interferon, rabbit standardized solution of 300 μ g/ml.
Get 5 centrifuge tubes according to following table dilution interferon alpha 2 b standardized solution, and mix.
Table 3: the dilution of interferon alpha 2 b standardized solution
Step 3) sample solution, step 3) collect liquid and step 4) sample solution sample uses No.2-No.6 standardized solution; Step 1) sample solution, step 1) are collected liquid, step 2) sample solution, step 2) collect liquid, step 4) and collect liquid sample, comparative example sample solution, comparative example and finally collect liquid and use No.1-No.5 standardized solution.The effect of standardized solution is to generate typical curve, determines that according to the concentration of different samples sample need use many typical curves on a large scale.Can only use the standardized solution of 5 concentration to be determined by equipment self problem, the design requirements of chip can only be used the standardized solution of 5 concentration.
Draw respectively in Interferon, rabbit standardized solution to 5 centrifuge tube of 4 μ l different concns, then add successively 2 μ l reduction buffered soln, in boiling water, boil 5min.In every pipe, add 84 μ l waters for injection successively again.
2. the processing of sample
Draw respectively in the extremely corresponding centrifuge tube of sample that 4 μ l are different, every pipe adds 2 μ l reducing solutions successively, boils 5min in boiling water.In every pipe, add 84 μ l waters for injection successively again.
3. the preparation of molecular weight standard product
Get in 6 μ l molecular weight standard product to centrifuge tube.Boiling water heating 5min.Then in centrifuge tube, add 84 μ l waters for injection.
4. loading
Getting a chip of not sealing off adds 12 μ l gel-dyestuff mixed solution to be marked with to chip
hole in, be positioned on chip enable device chassis.Adjust the interior bar of syringe to 1.0ml scale, starter gear then closes.Press lower inner push-rod and fix with clamp, keeping 60s.Stir clamp inner push-rod is gone up, slowly promote inner push-rod to 1.0ml scale marks place.In other 3 holes that indicate G, add successively 12 μ l gel-dyestuff mixed solution.Add again 12 μ l decolouring glue to indicating in the hole of DS.Add respectively the interferon alpha 2 b standard substance of 6 μ l different concns in the hole that is 1-5 to label.Add in the hole that 6 μ l analytic samples are 6-10 to label.Finally add 6 μ l molecular weight standard product to indicating
hole in.
5. sample analysis operation
Open software " 2100expert ".The chip of loaded sample is placed on biological analyser, closes upper cover, select the Protein 80 in software approach, click " start " button, program brings into operation.
6. data processing
Typical curve correlation coefficient r >=0.97.
The slope range of typical curve is 1.8-4.5.
In software meeting establishing criteria curve automatic analysis of samples, (ng/ μ l) and by calculation result lists in " the Calib.Conc. μ g/ml " hurdle in form to the concentration of recombinant human interferon alpha 2 b.
7. result is calculated
In sample, concentration (C, the mg/ml) calculation formula of recombinant human interferon alpha 2 b is:
α
ithe concentration of the interferon alpha 2 b that-typical curve calculates, ng/ μ l;
P-diluted sample multiple;
I-replicated experimental units.
Three, experimental result
As shown in table 4.
Table 4: Interferon, rabbit assay and yield calculation result
| | Embodiment | 1 | Sample | Comparative example |
| Step 1) sample solution | 0.081mg/ml | Initial sample solution | 0.345mg/ml | |
| Step 4) is collected liquid | 0.016mg/ml | The final liquid of collecting | 0.048mg/ml | |
| Total recovery | 42.5% | Total recovery | 33.0% |
Result demonstration, the yield of the Interferon, rabbit of purification process of the present invention is more than 40%, apparently higher than comparative example.
Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all instructions, can carry out various modifications and replacement to those details, these change all within protection scope of the present invention.Four corner of the present invention is provided by claims and any equivalent thereof.
Claims (10)
1. a method for purifying alpha-interferon, comprises the steps:
1) the fermented liquid supernatant liquid containing Interferon, rabbit is carried out to hydrophobic chromatography;
2) product obtaining in step 1) is carried out tolerating the nonexpondable anion chromatography of high flow rate;
3) by step 2) in the product that obtains carry out tolerating the nonexpondable positively charged ion chromatography of high flow rate; With
4) product obtaining in step 3) is carried out to anion chromatography.
2. method according to claim 1, wherein, step 2) and step 4) described in anion chromatography be weak anionic chromatography.
3. a method for purifying alpha-interferon, comprises the steps:
1) be under 180 ± 100mS/cm, to utilize the agar carbohydrate chromatographic resin of Phenyl aglucon to carry out wash-out separation by Interferon, rabbit fermented liquid supernatant liquid in specific conductivity;
2) product obtaining in step 1) is regulated to pH to 7.5-9.0, utilize the agar carbohydrate chromatographic resin of DEAE aglucon to carry out wash-out separation;
3) by step 2) in the product that obtains regulate pH to 4.0-5.5, utilize the agar carbohydrate chromatographic resin of CM aglucon to separate wash-out; With
4) product obtaining in step 3) is regulated to pH to 7.0-9.0, utilize the agar carbohydrate chromatographic resin of DEAE aglucon to carry out wash-out separation.
4. method according to claim 3, it comprises the steps:
1) recombinant human interferon alpha 2 b fermented liquid supernatant liquid is utilized under conductivity value 180 ± 100mS/cm the agar carbohydrate chromatographic resin of Phenyl aglucon carry out wash-out separation, flow velocity 20-40L/hr, an applied sample amount 5-9 column volume; Elutriant filters by microfiltration membrane;
2) product obtaining in step 1) is carried out to 3-5 with ultra-filtration membrane and doubly concentrate, concentrated rear pH regulator, to pH7.5-9.0, utilizes the agar carbohydrate chromatographic resin of DEAE aglucon to carry out wash-out separation; Flow velocity 10-30L/hr, an applied sample amount 3-7 column volume;
3) by step 2) in the product that obtains be adjusted to pH4.0-5.5, utilize the agar carbohydrate chromatographic resin of CM aglucon to separate wash-out, flow velocity 10-30L/hr; An applied sample amount 8-11 column volume; With
4) product obtaining in step 3) is adjusted to pH7.0-9.0, utilizes the agar carbohydrate chromatographic resin of DEAE aglucon to carry out wash-out separation, flow velocity 10-30L/hr, an applied sample amount 8-11 column volume; Elutriant is concentrated with ultra-filtration membrane, be concentrated into 1-3mg/ml.
5. according to the method described in claim 3 or 4, it is characterized in that any one in following (1)-(17) or multinomial:
(1) flow velocity in step 1) is 20-40L/hr;
(2) step 2) in flow velocity be 10-30L/hr;
(3) flow velocity in step 3) is 10-30L/hr;
(4) flow velocity in step 4) is 10-30L/hr;
(5) applied sample amount in step 1) is 3-9 column volume; Be preferably 5-9 column volume;
(6) step 2) in applied sample amount be 1-7 column volume; Be preferably 3-7 column volume;
(7) applied sample amount in step 3) is 6-11 column volume; Be preferably 8-11 column volume;
(8) applied sample amount in step 4) is 6-11 column volume; Be preferably 8-11 column volume;
(9) step 2) in, pH to 8.0-9.0 regulated;
(10), in step 3), regulate pH to 4.2-5.2;
(11), in step 4), regulate pH to 7.5-8.5;
(12) in step 1), also comprise and elutriant is filtered to (for example filtering with ultra-filtration membrane), obtain the step of filtrate;
(13) step 2) in, before being also included in adjusting pH, the product in step 1) is carried out to concentrated step (for example carrying out 3-5 with ultra-filtration membrane doubly concentrates);
(14), in step 4), also comprise the elutriant in step 4) is carried out to concentrated step (for example elutriant ultra-filtration membrane being concentrated into 1-3mg/ml)
(15) specific conductivity in step 1) is 80-280mS/cm; Preferably, be 100-190mS/cm; More preferably, be 120-140mS/cm;
(16), before carrying out step 1), just carry out cracking processing containing the fermented liquid supernatant liquid of recombinant human interferon alpha 2 b;
(17) step 2) in the half of usage quantity of the amount of agar carbohydrate chromatographic resin of the DEAE aglucon that the uses agar carbohydrate chromatographic resin that is the Phenyl aglucon that uses in step 1).
6. according to the method described in claim 3 or 4, wherein, step 2) in elution flow rate be 5-50cm/h; Be preferably 10-30cm/h; More preferably 15cm/h.
7. according to the method described in claim 3 or 4, wherein, the elution flow rate in step 4) is 5-50cm/h; Be preferably 10-30cm/h; More preferably 15cm/h.
8. according to the method described in any one in claim 1 to 7, wherein, described Interferon, rabbit is human interferon; Particularly, be human interferon-alpha-2 b; More specifically, be recombinant human interferon alpha 2 b; The recombinant human interferon alpha 2 b of expressing for pseudomonas especially particularly.
9. a test kit, comprising: the agar carbohydrate chromatographic resin of the agar carbohydrate chromatographic resin of Phenyl aglucon, the agar carbohydrate chromatographic resin of DEAE aglucon and CM aglucon; Particularly, described test kit comprises Phenyl hydrophobic resin, DEAE resin anion(R.A) and CM FF resin cation (R.C.).
10. the purposes of test kit claimed in claim 9 in preparation or purifying alpha-interferon; Particularly, described Interferon, rabbit is human interferon; More specifically, be human interferon-alpha-2 b; Especially particularly, be recombinant human interferon alpha 2 b; The recombinant human interferon alpha 2 b of expressing for pseudomonas further particularly.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107033233A (en) * | 2008-01-18 | 2017-08-11 | 弗·哈夫曼-拉罗切有限公司 | Purification of not-glycosylated polypeptides |
| CN109206476A (en) * | 2018-11-20 | 2019-01-15 | 中国科学院青岛生物能源与过程研究所 | A kind of method of separation and purification of protein |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101033254A (en) * | 2007-02-09 | 2007-09-12 | 广州市恺泰生物科技有限公司 | Method of purifying and combining human interleukins 12 |
| CN102532254A (en) * | 2010-12-24 | 2012-07-04 | 武汉禾元生物科技有限公司 | Method for separating and purifying recombinant human serum albumin (rHSA) from rice seeds |
| US20120309057A1 (en) * | 2010-02-01 | 2012-12-06 | Digna Biotech, S.L. | Method for producing interferon alpha 5 |
-
2012
- 2012-12-07 CN CN201210526437.XA patent/CN103864915B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101033254A (en) * | 2007-02-09 | 2007-09-12 | 广州市恺泰生物科技有限公司 | Method of purifying and combining human interleukins 12 |
| US20120309057A1 (en) * | 2010-02-01 | 2012-12-06 | Digna Biotech, S.L. | Method for producing interferon alpha 5 |
| CN102532254A (en) * | 2010-12-24 | 2012-07-04 | 武汉禾元生物科技有限公司 | Method for separating and purifying recombinant human serum albumin (rHSA) from rice seeds |
Non-Patent Citations (1)
| Title |
|---|
| 李秋儒: "干扰素α-2a纯化工艺中液相层析的研究", 《中国优秀硕士论文数据库 基础科学辑》, 31 August 2009 (2009-08-31) * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107033233A (en) * | 2008-01-18 | 2017-08-11 | 弗·哈夫曼-拉罗切有限公司 | Purification of not-glycosylated polypeptides |
| CN109206476A (en) * | 2018-11-20 | 2019-01-15 | 中国科学院青岛生物能源与过程研究所 | A kind of method of separation and purification of protein |
| CN109206476B (en) * | 2018-11-20 | 2021-08-17 | 中国科学院青岛生物能源与过程研究所 | Method for separating and purifying protein |
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