CN103864915B - The method of purification of Recombinant human interferon-alpha-2 b and test kit - Google Patents

Abstract

The invention belongs to biochemical field, specifically, belong to protein purification art. More particularly it relates to an the method for purification of Recombinant human interferon-alpha-2 b and test kit. Specifically, described method includes: 1) the fermented liquid supernatant liquid containing recombinant human interferon alpha 2 b is carried out hydrophobic chromatography (HIC); 2) undertaken the product obtained in step 1) can tolerate the nonexpondable anion exchange of high flow rate; 3) by step 2) in the product that obtains carry out can tolerate the nonexpondable cation exchange of high flow rate; With 4) product obtained in step 3) is carried out anion exchange. The invention enables interferon yield to improve about 25%, be applicable to the large-scale industrial production/purification of recombinant human interferon alpha 2 b, save purification cost, improve purification efficiency.

Description

The method of purification of Recombinant human interferon-alpha-2 b and test kit

Technical field

The invention belongs to biochemical field, specifically, belong to protein purification art. More particularly it relates to an the method for purification of Recombinant human interferon-alpha-2 b and test kit.

Background technology

Interferon is the histone matter being subject to virus or the stimulation of other derivants by body cell and secreting, and has the multiple physiologically actives such as the antiviral of wide spectrum, antiproliferative and immunomodulating. The antivirus action of interferon is act on virus target cell on the one hand, and induction produces a series of active substances, by multiple biochemical route, the different links of virus replicative cycle is disturbed, it is suppressed that the intrusion of virus and duplication. Interferon acts on body immune system on the other hand, strengthens the ability that immune system suppresses or removes virus. Additionally, interferon can also regulate immunologic function, it is suppressed that cell division, especially suppress the growth of tumor cell, therefore be clinically used for the treatment of viral infection resisting and some malignant tumor.

According to antigenic characteristic and molecular structure, interferon is commonly divided into three types: �� or LeIF, �� or fiblaferon, �� or immune interferon. Its broad-spectrum disease resistance possible mechanism of its cytotoxicity is to produce multiple enzyme by inducing host cell to carry out the links of viral interference reproduction process, and a kind of interferon can suppress the propagation of multiple virus, is medically the antiviral drugs of a class wide spectrum. It can also activate NK cell, macrophage, CTL cell to kill the target cell of viral infection. Applying maximum in the market is alpha-interferon.

�� 2b interferon is a kind of antiviral, antitumor drug, and has immunoregulatory activity. May be used for treatment Type B viral hepatitis, hepatitis C, viral meningitis, viral eye infections, renal carcinoma, capillary leukemia, malignant lymphoma of skin, osteogenic sarcoma, multiple sclerosis etc.

The single chain polypeptide that is made up of 165 aminoacid of recombinant human interferon alpha 2 b that pseudomonas is expressed, molecular weight is 19.2 �� 10%KDa, isoelectric point, IP between 5.7-6.7, sugar based in molecule, molecule has four alpha-helixs; CYS¹And CYS⁹⁸��CYS²⁹And CYS¹³⁸Forming two disulfide bond, wherein the disulfide bond between 29-138 is particularly significant for interferon biological activity. The three-D space structure schematic diagram of the recombinant human interferon alpha 2 b that pseudomonas is expressed is as shown in Figure 1.

Shown in the SEQIDNO:1 that the aminoacid sequence of recombinant human interferon alpha 2 b (pseudomonas expression) is such as following:

165aa

CysAspLeuProGlnThrHisSerLeuGlySerArgArgThrLeuMETLeuLeuAlaGlnMETArgArgIleSerLeuPheSerCysLeuLysAspArgHisAspPheGlyPheProGlnGluGluPheGlyAsnGlnPheGlnLysAlaGluThrIleProValLeuHisGluMETIleGlnGlnIlePheAsnLeuPheSerThrLysAspSerSerAlaAlaTrpAspGluThrLeuLeuAspLysPheTyrThrGluLeuTyrGlnGlnLeuAsnAspLeuGluAlaCysValIleGlnGlyValGlyValThrGluThrProLeuMETLysGluAspSerIleLeuAlaValArgLysTyrPheGlnArgIleThrLeuTyrLeuLysGluLysLysTyrSerProCysAlaTrpGluValValArgAlaGluIleMETArgSerPheSerLeuSerThrAsnLeuGlnGluSerLeuArgSerLysGlu��SEQIDNO��1��

The human interferon albumen of early stage mainly from blood purification obtain, there is substantial amounts of shortcoming in this method, as: being difficult to obtain a large amount of blood, economy is low, is subject in various blood to carry virus ground contamination. After recombinant DNA technology produces the production that recombinant human interferon alpha 2 albumen is applied to human interferon albumen, the problems referred to above are resolved, but the yield of extraction purification human interferon is unsatisfactory from fermentation liquid.

The method being related to recombinant human interferon alpha 2 b purification in currently available technology, for instance method below:

Ion-exchange chromatography: adopt DEAESepharose (FF) post, column type XK30/50. After balancing with F liquid, by the renaturation solution sample loading after above-mentioned dialysis, flow velocity is 20ml/min, after end of the sample, with the F liquid eluting containing variable concentrations NaCl, collects interferon activity peak. Select Sephacryl00/50XK chromatographic column, S2100 filler, column volume 1700ml, after balancing with G liquid, the interferon activity peak sample collected by ion-exchange chromatography is through gel filtration, and applied sample amount is the 5% of bed volume, regathering interferon activity peak, merging sample carries out following calibrating, and (Liu is real, Jiang Wei, Sui Baozhen, Zhang Chunli, the extraction of recombinant human interferon alpha 2 b and purification, microbiology immunology is in progress, 32nd volume the 1st phase in 2004, p13-16).

But, the present inventor finds under study for action, and existing purification process is not very good to the yield of interferon, and therefore, still need a kind of purification process improving interferon yield in this area.

Summary of the invention

The present inventor, through deep research and performing creative labour, through multiple conditions such as reagent used, applied sample amount, flow velocity, pH or the comprehensive of parameter are applied and optimized, obtains method or the test kit of a kind of purification of Recombinant human interferon-alpha-2 b. And surprisingly, it was found that the method for the present invention or test kit can purifying alpha-interferon particularly human interferon-alpha-2 b (such as recombinant human interferon alpha 2 b) effectively, and the yield of albumen and interferon is all higher, and the interferon activity obtained is good.Thus provide following invention:

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 is carried out hydrophobic chromatography (HIC);

2) undertaken the product obtained in step 1) can tolerate the nonexpondable anion chromatography of high flow rate;

3) by step 2) in the product that obtains carry out can tolerate the nonexpondable cation chromatography of high flow rate; With

4) product obtained in step 3) is carried out anion chromatography.

According to the method that the present invention is above-mentioned, wherein, step 2) and step 4) described in anion chromatography be weak anionic chromatography.

For the purpose of the present invention, the acquisition of the fermented liquid supernatant liquid of interferon is not particularly limited, for instance can be obtained by the method that those skilled in the art know.

Can tolerate the nonexpondable anionic exchange medium of high flow rate is interacted with can tolerate the nonexpondable anionic exchange medium of high flow rate by charge effect and hydrogen bond and/or hydrophobic interaction.

It is not limited to the restriction of theory, hydrophobic chromatography utilizes the hydrophobic adsorbent of the weak hydrophobic group (hydrophobic ligands) of surface coupling for fixing phase, is the chromatography that carries out protein-based separation and purification of biological macromolecule of the difference according to 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 (such as tyrosine, phenylalanine etc.) content is many, and hydrophobicity is also strong. And in different media, how many also differences to some extent that hydrophobic group exposes. In the saline solution that ionic strength is higher, the hydrated sheath at the hydrophobic position of protein surface is destroyed, and the structure of protein also overturns, and exposes hydrophobic position and increases, and the hydrophobicity of protein strengthens; In the solution that ionic strength is relatively low, the hydrophobicity of protein reduces. The loading when high conductivity, target protein and aglucon effect that hydrophobic group is more are adsorbed, and the albumen lacking hydrophobic group is rinsed down, then the electrical conductivity reducing eluent makes albumen hydrophobicity reduce, and is eluted from resin.

Being not limited to the restriction of theory, ion-exchange chromatography is that ion-exchanger is combined with the net charge of protein with self institute's oppositely charged. 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 just, it is possible to electronegative protein binding. During ion-exchange chromatography, first with the relatively low buffer loading of ionic strength and washing. Protein solution is rinsed without the albumen of binding ability with ion exchange resin after entering ion exchange column, and remaining protein all may be incorporated on resin. Then the binding ability adding strong electrolyte (such as sodium chloride), salt ion and ion-exchanger in buffer is better than the binding ability of albumen and ion-exchanger, and albumen will be eluted competitively. Owing to the affinity of different types of albumen Yu resin is different, it is eluted out by the available method being gradually increased in eluent sodium chloride concentration one by one. Sodium chloride solution elution method has two ways: step-wise elution (in eluent, sodium chloride concentration stage improves) and linear gradient eluting (in eluent, sodium chloride concentration is linear raising). It is, in general, that when foreign protein differs bigger with destination protein binding ability, be suitable for step-wise elution; Linear gradient elution is suitable to the protein that separately binding ability is close.

Cation aglucon includes but not limited to: sulfonate radical (SO₃ ^�C/��SO₃H), sulfate radical (OSO₃ ^�C/��OSO₃H), carboxylate radical (COO^�C/ COOH), phosphate radical (OPO₃ ^2�C/��OPO₃�CH/��OPO₃H₂) and orthophosphite (PO₃ ^2-/��PO₃�CH/��PO₃H₂).Conventional weak cation exchange medium group is carboxylate radical (COO^�C/ COOH), phosphate radical (OPO₃ ^2�C/��OPO₃�CH/��OPO₃H₂) and orthophosphite (PO₃ ^2�C/��PO₃�CH/��PO₃H₂).

Secondary conjugated group has at least a hydrogen bond action atom, and should have the distance of 1 to 7 atom between this atom and cation exchange group. The hydrogen bond atomic time can participate in being formed the atom of hydrogen bond. Hydrogen bond atom can select from containing heteroatomic group, such as oxygen atom, nitrogen-atoms, sulphur atom; And sp and sp^2-Hydbridized carbon atoms; Halide groups, such as F, Cl, Br, I, particularly F. Typical case secondary conjugated group contain uncharged atom or with pH change can charged atom.

Being not limited to the restriction of theory, step 1) main purpose is to eliminate the materials such as peptides, prothetic group, immunoglobulin, therefore uses hydrophobic chromatography resin (HIC). Owing to recombinant human interferon alpha 2 b fermented liquid supernatant liquid being carried out ammonium sulfate precipitation process before step 1), sample solution is high saline solution, and this step adopts HIC, it is possible to avoid the direct loading of desalting processing, reduces complex process degree. HIC, by fermented supernatant fluid purification and with small size eluting, reaches concentrated effect and significantly reduces the salinity in fermented supernatant fluid simultaneously, provides desirable purification condition for downstream purification. Substrate used by hydrophobic chromatography is after being activated by BrCN by agarose gel, from different alpha-amido alkane effects, homologous series alkyl agarose Seph-Cn, the wherein n=1-6 that generation carbon chain lengths gradually changes, the carbon number in expression carbochain.

It being not limited to the restriction of theory, step 2) main purpose is to separate the charged biomolecules such as macromolecular polypeptides albumen, nucleic acid, aminoacid, therefore uses ion chromatography resin (IEC). Agarose molecules is combined with diethyl amino ethyl group (Dicthylaminoethyl, DEAE), containing positively charged agar, cationic sugar O C6H14N⁺H, its counter ion is that anion is (such as Cl^-Deng), can swap with electronegative protein anions. Colloid ion (such as protein) and inorganic ion (such as NaCl) are all had the ability of exchange adsorption by exchanger, when both are concurrently present in a chromatography process, then produce emulative exchange adsorption. Work as Cl^-Concentration big time, protein is not easy to be adsorbed, and is also easy to be eluted, works as Cl after absorption^-Concentration hour, protein is easily adsorbed, and is also not easy to be eluted after absorption. Therefore, the method for the ionic strength increasing eluent is adopted to reach the purpose of isolated protein.

Being not limited to the restriction of theory, in step 3), main purpose is to separate the charged biomolecules such as macromolecular polypeptides albumen, nucleic acid, aminoacid, therefore uses ion chromatography resin (IEC). Agarose molecules is combined with carboxymethyl (Carboxymethy, CM). Its counter ion is that cation is (such as Na⁺Deng), can swap with positively charged protein cation.

Being not limited to the restriction of theory, in step 4), main purpose is to separate endotoxin biology charged molecule, therefore uses ion chromatography resin (IEC). The lipoid A composition that endotoxic main chemical compositions is in lipopolysaccharide is electronegative, therefore selects anion chromatography resin to separate.

A kind of method of purifying alpha-interferon, comprises the steps:

1) it is that the agar saccharide chromatographic resin utilizing Phenyl aglucon under 180 �� 100mS/cm carries out eluting separation by interferon fermented liquid supernatant liquid in electrical conductivity;

2) product obtained in step 1) is regulated pH to 7.5-9.0, utilize the agar saccharide chromatographic resin of DEAE aglucon to carry out eluting separation;

3) by step 2) in the product that obtains regulate pH to 4.0-5.5, utilize the agar saccharide chromatographic resin of CM aglucon to be easily separated eluting; With

4) product obtained in step 3) is regulated pH to 7-9, utilize the agar saccharide chromatographic resin of DEAE aglucon to carry out eluting separation.

According to the method described in any one of the present invention, it comprises the steps:

1) recombinant human interferon alpha 2 b fermented liquid supernatant liquid utilize the agar saccharide chromatographic resin of Phenyl aglucon carry out eluting separation, flow velocity 10-30L/hr, 5-9 column volume of applied sample amount under conductivity value 180 �� 100mS/cm; Eluent micro-filtration membrane is filtered;

2) the product ultrafilter membrane obtained in step 1) being carried out 3-5 times to concentrate, after concentration, pH regulator is to pH7.5-8.5, utilizes the agar saccharide chromatographic resin of DEAE aglucon to carry out eluting separation; Flow velocity 10-30L/hr, 3-7 column volume of applied sample amount;

3) by step 2) in the product that obtains regulate to pH4.0-5.5, utilize the agar saccharide chromatographic resin of CM aglucon to be easily separated eluting, flow velocity 10-30L/hr; 8-11 column volume of applied sample amount; With

4) product obtained in step 3) is regulated to pH7-9, utilize the agar saccharide chromatographic resin of DEAE aglucon to carry out eluting separation, flow velocity 10-30L/hr, 8-11 column volume of applied sample amount; Eluent ultrafilter membrane is concentrated, is concentrated into 1-3mg/ml.

The concentration of concentration can be recorded by the method that those skilled in the art know, for instance obtains by monitoring the reservation liquid ultraviolet absorption value in 280nm place.

According to the method described in any one of the present invention, it is characterised in that any one in following (1)-(17) item or multinomial:

(1) flow velocity in step 1) is 20-40L/hr; It is preferably 25-35L/hr, for instance 25,26,27,28,29,30,31,32,33,34 or 35L/hr;

(2) step 2) in flow velocity be 10-30L/hr; It is preferably 10-20L/hr, for instance 10,11,12,13,14,15,16,17,18,19 or 20L/hr;

(3) flow velocity in step 3) is 10-30L/hr; It is preferably 10-20L/hr, for instance 10,11,12,13,14,15,16,17,18,19 or 20L/hr;

(4) flow velocity in step 4) is 10-30L/hr; It is preferably 10-20L/hr, for instance 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; It is preferably 5-9 column volume (such as 5,6,7,8 or 9 column volumes);

(6) step 2) in applied sample amount be 1-7 column volume; It is preferably 3-7 column volume (such as 3,4,5,6 or 7 column volumes);

(7) applied sample amount in step 3) is 6-11 column volume; It is preferably 8-11 column volume (such as 8,9,10 or 11 column volumes);

(8) applied sample amount in step 4) is 6-11 column volume; It is preferably 8-11 column volume (such as 8,9,10 or 11 column volumes);

(9) step 2) in, regulate pH to 8.0-9.0, for instance 8.0,8.1,8.2,8.3,8.4,8.5,8.6,8.7,8.8,8.9 or 9.0; It is preferably 8.2-8.8;

(10), in step 3), pH to 4.2-5.2 is regulated; It is preferably 4.5-5.0, for instance 4.5,4.6,4.7,4.8,4.9 or 5.0;

(11), in step 4), pH to 7.5-8.5 is regulated, for instance 7.6,7.7,7.8,7.9,8.0,8.1,8.2,8.3,8.4 or 8.5;It is more preferably 7.7-8.2, for instance 7.7,7.8,7.9,8.0,8.1 or 8.2;

(12), in step 1), also include eluent is filtered (being such as filtered with ultrafilter membrane), obtain the step of filtrate;

(13) step 2) in, before being additionally included in adjustment pH, the product in step 1) is carried out the step (such as carrying out 3-5 times with ultrafilter membrane to concentrate) concentrated;

(14), in step 4), eluent ultrafilter membrane (is such as concentrated into 1-3mg/ml by the step also including being undertaken concentrating by the eluent in step 4); Preferably concentration is 2-3mg/ml; More preferably concentration is for 2.5-3mg/ml, for instance 2.5,2.6,2.7,2.8,2.9 or 3mg/ml. ).

(15) electrical conductivity in step 1) is 80-280mS/cm; Preferably, for 100-190mS/cm; It is highly preferred that be 120-140mS/cm;

(16) before carrying out step 1), just the fermented liquid supernatant liquid containing recombinant human interferon alpha 2 b carries out cracking process;

(17) step 2) in the half (according to volume computing) making consumption of the amount of the agar saccharide chromatographic resin of DEAE aglucon that the uses agar saccharide chromatographic resin of Phenyl aglucon for using in step 1).

According to the method described in any one of the present invention, wherein, the electrical conductivity in step 1) is 80-280mS/cm; Preferably, for 100-190mS/cm; It is highly preferred that be 120-140mS/cm or 125-135mS/cm, for instance 125,126,127,128,129,130,131,132,133,134 or 135mS/cm.

According to the method described in any one of the present invention, wherein, before carrying out step 1), just the fermented liquid supernatant liquid containing recombinant human interferon alpha 2 b carries out cracking process.

Cracking can carry out according to the method that those skilled in the art know, for instance uses the method for hyperosmosis that fermentation thalli is carried out cracking process.

According to the method described in any one of the present invention, wherein step 2) in the half (according to volume computing) making consumption of hydrophobic medium that uses for the hydrophobic chromatography in step 1) of the amount of the used anionic exchange medium of anion chromatography.

Weak anionic and strong anion are distinctive in that the pH scope making the full ionizing of exchange media, and wider person is strong, and narrower person is weak, unrelated with bond strength. Weak anion exchange resin: this resinoid contains alkalescence group, such as primary amine groups (also known as one-level amido)-NH2, secondary amine (secondary amine)-NHR or tertiary amine groups (tertiary amine base)-NR2, they can dissociate out OH-in water and be alkalescence. The positive charged group of this resin can be combined with the Anion-adsorption in solution, thus producing anion exchange effect. It can only work under neutrality or alkaline environment. And the scope that strong anion type is suitable for is wider, can also under sour environment.

According to the method described in any one of the present invention, wherein, step 2) in elution flow rate be 50-120cm/h; It is preferably 70-110cm/h; It is more preferably 110cm/h.

According to the method described in any one of the present invention, wherein, the elution flow rate in step 4) is 50-80cm/h; It is preferably 60-70cm/h; It is more preferably 70cm/h.

According to the method described in any one of the present invention, wherein, described interferon is human interferon; Specifically, for human interferon-alpha-2 b; More specifically, be recombinant human interferon alpha 2 b; Especially specifically, the recombinant human interferon alpha 2 b expressed for pseudomonas.

Another aspect of the present invention one test kit, agar saccharide 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) including: the agar saccharide chromatographic resin of Phenyl aglucon, the agar saccharide chromatographic resin of DEAE aglucon and CM aglucon. Specifically, described test kit comprises Phenyl hydrophobic resin, DEAE resin anion (R.A.) and CMFF cationic resin. The described test kit of the present invention can be used in purifying alpha-interferon.

Another aspect of the invention relates to the test kit of present invention purposes in preparation or purifying alpha-interferon; Specifically, described interferon is human interferon; More specifically, be human interferon-alpha-2 b; Especially specifically, for recombinant human interferon alpha 2 b; Further specifically, the recombinant human interferon alpha 2 b expressed for pseudomonas.

In the present invention, term " interferon ", specifically refer to human interferon; More specifically, be human interferon-alpha-2 b; Especially specifically, for recombinant human interferon alpha 2 b; Further specifically, the recombinant human interferon alpha 2 b expressed for pseudomonas, for instance the recombinant human interferon alpha 2 b shown in SEQIDNO:1.

The beneficial effect of the invention

The yield of the interferon of the purification process of the present invention is more than 35% even more than 40% even more than 42%. Present invention saves purification cost, improve purification efficiency, be applicable to the large-scale industrial production/purification of recombinant human interferon alpha 2 b. It addition, the test kit of the present invention can stand higher pressure and bigger elution flow rate, repeatable 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 saccharide matrix; Fig. 2 C, cation chromatographic resin CM schematic diagram, wherein the ball in left side represents Cross-linked Agar saccharide matrix.

Fig. 3: the hydrophobic chromatography eluting collection of illustrative plates of step 1) in embodiment 1.

Fig. 4: step 2 in embodiment 1) anion chromatography eluting collection of illustrative plates.

Fig. 5: the cation chromatography elution profile spectrum of step 3) in embodiment 1.

Fig. 6: the secondary anion chromatography eluting collection of illustrative plates of step 4) in embodiment 1.

Fig. 7: step 2 in embodiment 1) anion chromatography product electrophoretic analysis. The sample of swimming lane 1-13 is respectively successively: molecular weight standards, interferon standard substance, anion sample solution, anion chromatography portion collection 1-9 and anion chromatography product. Described " portion collection " refers to and collects one by one according to fixed volume sub-bottle when chromatography process enters into gradient elution, until eluting completes.

Fig. 8: the cation chromatographed product electrophoretic analysis of step 3) in embodiment 1. The sample of swimming lane 1-13 is respectively successively: molecular weight standards, interferon standard substance, cation sample solution, cation chromatography portion collection 1-9 and cation chromatographed product. Described " portion collection " refers to and collects one by one according to fixed volume sub-bottle when chromatography process enters into gradient elution, until eluting completes.

Fig. 9: the anion chromatography product electrophoretic analysis of step 3) in embodiment 1. The sample of swimming lane 1-13 is respectively successively: molecular weight standards, interferon standard substance, anion sample solution, anion chromatography portion collection 1-9 and anion chromatography product. Described " portion collection " refers to and collects one by one according to fixed volume sub-bottle when chromatography process enters into gradient elution, until eluting completes.

Detailed description of the invention

Below in conjunction with embodiment, embodiment of the present invention are described in detail, it will be appreciated by those skilled in the art that the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Unreceipted actual conditions person in embodiment, conventionally the condition of condition or manufacturer's suggestion carries out. Agents useful for same or the unreceipted production firm person of instrument, be can pass through city available from conventional products.

Embodiment 1: the purification of recombinant human interferon alpha 2 b

One, experiment material and experimental apparatus:

Phenyl hydrophobic resin, GEHealthcare company, 17-0973-04; DEAE resin anion (R.A.), GEHealthcare company, 17-0709-05; CMFF cationic resin, GEHealthcare company, 17-0719-05.

Buffer: phosphate+sodium chloride buffer system.

Full-automatic liquid chromatographic system, GEHealthcare company, AKTAExplorer100; Biological analyser, Agilent company, Agilent2100bioanalyzer; Electrophoresis system, GEHealthcare company, SE600RUBY.

Two, experimental technique:

1) recombinant human interferon alpha 2 b fermented liquid supernatant liquid utilizing the agar saccharide chromatographic resin of Phenyl aglucon carry out eluting separation under conductivity value 130mS/cm, eluent ultrafilter membrane is filtered; Flow velocity 30L/hr, 6 column volumes of applied sample amount;

2) the product ultrafilter membrane obtained in step 1) is carried out 5 times of concentrations, pH regulator pH to 8.5 after concentration, utilize the agar saccharide chromatographic resin of DEAE aglucon to carry out eluting 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 saccharide chromatographic resin of CM aglucon to be easily separated eluting, flow velocity 12L/hr; 10 column volumes of applied sample amount; With

4) product obtained in step 3) is regulated pH to 7.8, utilize the agar saccharide chromatographic resin of DEAE aglucon to carry out eluting separation, flow velocity 15L/hr, 9 column volumes of applied sample amount; Eluent ultrafilter membrane is concentrated, is concentrated into 2.8mg/ml.

Three, experimental result

The hydrophobic chromatography eluting collection of illustrative plates of step 1) is as shown in Figure 3; Step 2) anion chromatography eluting collection of illustrative plates as shown in Figure 4; The cation chromatography elution profile of step 3) is composed as shown in Figure 5; The secondary anion chromatography eluting collection of illustrative plates of step 4) is as shown in Figure 6.

From Fig. 3-6, the peak position point that goes out of �� 2b albumen is consistent with isoelectric point, IP character, and the elution requirement of setting can reach predetermined resolution accurately, can purification of Recombinant human interferon-alpha-2 b albumen effectively.

Comparative example: the purification of recombinant human interferon alpha 2 b

It is operated according to such as Publication about Document:

Liu Shi, Jiang Wei, Sui Baozhen, Zhang Chunli, the extraction of recombinant human interferon alpha 2 b and purification, microbiology immunology is in progress, the 32nd volume the 1st phase 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

Shown in each component such as table 1 below and table 2.

Each component of table 1:15% lower floor separation gel

Component	Volume (ml)
		Ultra-pure water	9.4
30% acrylamide/bisacrylamide (29:1)	20
		1.5M Tris-HCl, pH8.8	10
10%SDS	0.4
		10% Ammonium persulfate.	0.2
TEMED	0.02

Pour into after mixing in glass chamber, stop along 4cm place to from short glass plate, then take 1ml ultra-pure water and carry out water seal. When the different boundary of refractive index occurs from water seal interlayer in gel, incline the ultra-pure water of sealing of anhydrating.

Each component of table 2:7.5% upper strata concentration glue

Component	Volume (ml)
		Ultra-pure water	9.8
30% acrylamide/bisacrylamide (29:1)	5.0

0.5M Tris-HCl, pH6.8	5.0
		10%SDS	0.2
10% Ammonium persulfate.	0.1
		TEMED	0.02

Pouring in glass chamber after mixing, until stopping from short glass plate upper limb 0.5cm place, loading comb being inserted in upper strata glue, room temperature place 20-30 minute after gel polymerisation.After polymerization, before gel loading, remove loading comb, gel glass plate is connected with upper strata electrophoresis tank.

2. the process of standard substance and sample

Interferon standard substance is done suitable multiple dilution, 30 �� l interferon solutions and 10 �� l buffer solution are mixed, boiling water heating 5min, it is cooled to room temperature.

Sample mixes with 3:1 ratio with buffer solution, boiling water heating 5min, is cooled to room temperature.

3. electrophoresis

The both positive and negative polarity wire of electrophoresis tank is connected with DC voltage-stabilizing electrophresis apparatus, connects cooling water. Add 10 �� l molecular weight standards solution to gel loading hole, add 10 �� l interferon standard solution to loading hole, add 25 �� l sub-bottle sample solution (including chromatography sample solution, i.e. BefDE, BefCM or BefDE II) to loading hole.

After application of sample, being put into by gel slab in electrophoresis tank, build electrophoresis tank lid, add electrophoretic buffer in electrophoresis tank, switching on power and cooling down water starts electrophoresis. Deposition condition is constant current 20mA/gel. When separation gel base is run out of 5-10 minute in bromophenol blue forward position, cut off the electricity supply and cool down water. Open electrophoresis tank lid, take out gel.

4. silver dye

Gel soaks in fixative solution, and vibrate 30min. Discarding fixative solution, add photosensitive solution, vibrate 5min. Discard photosensitive solution, with ultrapure water gel 3 times, each 10min. Adding the silver nitrate solution of 0.2%, vibrate 15min. Outwell silver nitrate solution, with ultrapure water gel 2 times, each 1min. Add the vibration of gel nitrite ion, until interferon standard substance band is high-visible, stop dyeing immediately. Discarding nitrite ion, add 5% acetic acid stop buffer, vibrate 10min. Then gel is cleaned 3-4 time with ultra-pure water.

Three, experimental result

Step 2) anion chromatography electrophoretic analysis as shown in Figure 7; The cation chromatography electrophoretic analysis of step 3) is as shown in Figure 8; The anion chromatography electrophoretic analysis of step 4) is 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 given meets the requirements.

Experimental example 2: interferon yield determination experiment

One, experiment reagent and experimental apparatus

1. reagent needed for interferon assay:

Water for injection; DTT, Bole company; Protein80assaykit, Agilent company; Interferon standard substance CRS.

2. instrument and utensil needed for interferon assay

2100 biological analysers; Refrigerated centrifuger; Software control system; Water for injection system; Refrigerator; Computer, printer; Mini agitator; Mini centrifuge; Single track liquid getting device; Analytical balance; Beaker.

The sample that the sample measured is embodiment 1 and prepared by comparative example.

Two, experimental technique

1. the preparation of interferon alpha 2 b standard solution:

Take interferon standard substance and inject dilute with water, prepare the interferon standard solution of 300 �� g/ml.

Take 5 centrifuge tubes and dilute interferon alpha 2 b standard solution mix homogeneously according to following table.

Table 3: the dilution of interferon alpha 2 b standard solution

Step 3) sample solution, step 3) collect liquid and step 4) sample solution sample uses No.2-No.6 standard solution; Step 1) sample solution, step 1) collect liquid, step 2) sample solution, step 2) collect liquid, step 4) collects liquid sample, comparative example sample solution, comparative example are finally collected liquid and used No.1-No.5 standard solution. The effect of standard solution is to generate standard curve, determines that sample need to use many standard curves on a large scale according to the concentration of different samples.The standard solution that can only use 5 concentration is determined by equipment self problem, and the designing requirement of chip can only use the standard solution of 5 concentration.

Draw the interferon standard solution of 4 �� l variable concentrations respectively to 5 centrifuge tubes, sequentially add 2 �� l reduction buffer solution, boiling water boils 5min. 84 �� l waters for injection are added again successively in often pipe.

2. the process of sample

Drawing 4 different for �� l samples respectively to corresponding centrifuge tube, often pipe is sequentially added into 2 �� l reducing solutions, boils 5min in boiling water. 84 �� l waters for injection are added again successively in often pipe.

3. the preparation of molecular weight standards

Take 6 �� l molecular weight standards to a centrifuge tube. Boiling water heating 5min. Then in centrifuge tube, add 84 �� l waters for injection.

4. loading

Take one piece of chip do not sealed off to add 12 �� l gels-dyestuff mixed solution and be marked with to chipHole in, be positioned over chip and start on device chassis. In adjustment syringe, bar is to 1.0ml scale, and then close startup device. Press lower inner push-rod and fix with clamp, keeping 60s. Stirring clamp makes inner push-rod go up, and slowly promotes inner push-rod to 1.0ml graduation mark place. It is sequentially added into 12 �� l gels-dyestuff mixed solution in other 3 holes indicating G. Add 12 �� l and decolour glue to the hole indicating DS. It is separately added into the interferon alpha 2 b standard substance of 6 �� l variable concentrations in the hole being numbered 1-5. Add 6 �� l and analyze sample in the hole being numbered 6-10. It is eventually adding 6 �� l molecular weight standards to indicatingHole in.

5. sample analysis runs

Open software " 2100expert ". Being placed on biological analyser by the chip of loaded sample, close upper cover, select the Protein80 in software approach, click " start " button, bring into operation program.

6. data process

Standard curve correlation coefficient r >=0.97.

The slope range of standard curve is 1.8-4.5.

Software is understood the concentration (ng/ �� l) of recombinant human interferon alpha 2 b in establishing criteria curve automatic analysis of samples and result of calculation is listed in " the Calib.Conc. �� g/ml " hurdle in form.

7. result calculates

In sample, concentration (C, the mg/ml) computing formula of recombinant human interferon alpha 2 b is:

$C=\frac{\mathrm{\Σ\αi}\×P}{i\×1000}$

��_iThe concentration of the calculated interferon alpha 2 b of-standard curve, ng/ �� l;

P-diluted sample multiple;

I-replicated experimental units.

Three, experimental result

As shown in table 4.

Table 4: interferon assay and yield result of calculation

Sample	Embodiment 1	Sample	Comparative example
				Step 1) sample solution	0.081mg/ml	Initial sample solution	0.345mg/ml
Step 4) collects liquid	0.016mg/ml	Finally collect liquid	0.048mg/ml
				Total recovery	42.5%	Total recovery	33.0%

Result shows, the yield of the interferon of the purification process of the present invention is more than 40%, hence it is evident that 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, it is possible to those details carry out various amendment and replacement, and these change all within protection scope of the present invention. The four corner of the present invention is provided by claims and any equivalent thereof.

Claims (24)

1. a method for purifying alpha-interferon, comprises the steps:

1) recombinant human interferon alpha 2 b fermented liquid supernatant liquid utilize the agar saccharide chromatographic resin of Phenyl aglucon carry out eluting separation, flow velocity 20-40L/hr, 5-9 column volume of applied sample amount under conductivity value 180 �� 100mS/cm; Eluent micro-filtration membrane is filtered;

2) by step 1) in the product ultrafilter membrane that obtains carry out 3-5 times and concentrate, after concentration, pH regulator is to pH7.5-9.0, utilizes the agar saccharide chromatographic resin of DEAE aglucon to carry out eluting separation; Flow velocity 10-30L/hr, 3-7 column volume of applied sample amount;

3) by step 2) in the product that obtains regulate to pH4.0-5.5, utilize the agar saccharide chromatographic resin of CM aglucon to be easily separated eluting, flow velocity 10-30L/hr; 8-11 column volume of applied sample amount; With

4) by step 3) in the product that obtains regulate to pH7.0-9.0, utilize the agar saccharide chromatographic resin of DEAE aglucon to carry out eluting separation, flow velocity 10-30L/hr, 8-11 column volume of applied sample amount; Eluent ultrafilter membrane is concentrated, is concentrated into 1-3mg/ml.

2. method according to claim 1, it is characterised in that any one in following (1)-(9) item or multinomial:

(1) step 2) in, regulate pH to 8.0-9.0;

(2) step 3) in, regulate pH to 4.2-5.2;

(3) step 4) in, regulate pH to 7.5-8.5;

(4) step 1) in, also include being filtered eluent, obtain the step of filtrate;

(5) step 2) in, before being additionally included in adjustment pH, by step 1) in product carry out the step that concentrates;

(6) step 4) in, also include step 4) in eluent carry out the step that concentrates;

(7) step 1) in electrical conductivity be 80-280mS/cm;

(8) step 1 is being carried out) before, just the fermented liquid supernatant liquid containing recombinant human interferon alpha 2 b carries out cracking process;

(9) step 2) in the amount of agar saccharide chromatographic resin of DEAE aglucon that uses be step 1) in the half making consumption of the agar saccharide chromatographic resin of Phenyl aglucon that uses.

3. method according to claim 2, wherein, is filtered into described in (4th) item and is filtered with ultrafilter membrane.

4. method according to claim 2, wherein, concentrates described in (5th) item and concentrates for carrying out 3-5 times with ultrafilter membrane.

5. method according to claim 2, wherein, described in (6th) item, concentration is for be concentrated into 1-3mg/ml by eluent ultrafilter membrane.

6. method according to claim 1, wherein, step 1) described in electrical conductivity be 100-190mS/cm.

7. method according to claim 1, wherein, step 1) described in electrical conductivity be 120-140mS/cm.

8. method according to claim 1, wherein, step 2) in elution flow rate be 5-50cm/h.

9. method according to claim 1, wherein, step 2) in elution flow rate be 10-30cm/h.

10. method according to claim 1, wherein, step 2) in elution flow rate be 15cm/h.

11. method according to claim 1, wherein, step 4) in elution flow rate be 5-50cm/h.

12. method according to claim 1, wherein, step 4) in elution flow rate be 10-30cm/h.

13. method according to claim 1, wherein, step 4) in elution flow rate be 15cm/h.

14. method according to claim 1, wherein, described interferon is human interferon.

15. method according to claim 14, wherein, described interferon is human interferon-alpha-2 b.

16. method according to claim 14, wherein, described interferon is recombinant human interferon alpha 2 b.

17. method according to claim 14, wherein, described interferon is the recombinant human interferon alpha 2 b that pseudomonas is expressed.

18. a test kit, including the agar saccharide chromatographic resin of: the agar saccharide chromatographic resin of Phenyl aglucon, the agar saccharide chromatographic resin of DEAE aglucon and CM aglucon.

19. test kit according to claim 18, wherein, described test kit comprises Phenyl hydrophobic resin, DEAE resin anion (R.A.) and CMFF cationic resin.

20. the purposes that the test kit described in claim 18 or 19 is in preparation or purifying alpha-interferon.

21. purposes according to claim 20, wherein, described interferon is human interferon.

22. purposes according to claim 20, wherein, described interferon is human interferon-alpha-2 b.

23. purposes according to claim 20, wherein, described interferon is recombinant human interferon alpha 2 b.

24. purposes according to claim 20, wherein, described interferon is the recombinant human interferon alpha 2 b that pseudomonas is expressed.