CN102952836A - Recombinant human long-acting interleukin-1 receptor antagonist fusion protein purification method - Google Patents

Recombinant human long-acting interleukin-1 receptor antagonist fusion protein purification method Download PDF

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CN102952836A
CN102952836A CN2012104336421A CN201210433642A CN102952836A CN 102952836 A CN102952836 A CN 102952836A CN 2012104336421 A CN2012104336421 A CN 2012104336421A CN 201210433642 A CN201210433642 A CN 201210433642A CN 102952836 A CN102952836 A CN 102952836A
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chromatography
target protein
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purification process
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徐岩
李征
王海彬
陈枢青
沈其
聂磊
白骅
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Zhejiang Hisun Pharmaceutical Co Ltd
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Zhejiang Hisun Pharmaceutical Co Ltd
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Abstract

The present invention provides a recombinant human long-acting interleukin-1 receptor antagonist fusion protein purification method, which comprises: carrying out fermentation expression to obtain a fermentation broth containing target protein, carrying out a pretreatment, carrying out strong anion exchange chromatography, carrying out blue gel affinity chromatography, and carrying out weak anion exchange chromatography. According to the present invention, the high purity target protein is obtained, such that the difficult problem that the target protein with sufficient purity can not be obtained in large-scale fermentation production is effectively solved.

Description

The purification process of the long-acting interleukin 1 receptor antagonist fusion rotein of recombinant human
Technical field
The present invention relates to the protein purification field, more specifically, the invention provides the purification process of the long-acting interleukin 1 receptor antagonist fusion rotein of a kind of recombinant human.
Background technology
As a main immune-regulating factor, il-1 (Interleukin-1, IL-1) plays important effect in the immunity of body and energy metabolism regulation.Research is found, IL-1 is at autoimmune disorder such as rheumatoid arthritis (Rheumatoid arthritis, RA), play very important effect in the pathogenic process of multiple scleroderma (Systemic sclerosis), type ii diabetes (Type2 diabetes), obesity (Obesity), nearest studies show that, the IL-1 also generation with the cardiovascular disorder of some kind is relevant.
Interleukin 1 receptor antagonist (Interleukin-1 receptor antagonist; IL-1Ra) be naturally occurring a kind of protein molecule in the human body; its in vivo can with the receptor-specific ground combination of IL-1; the corresponding signal transduction path of deactivation, thus the body injury that IL-1 causes there is provide protection.Can obtain the complete genome sequence of human interleukin-11 receptor antagonist by the GenBank inquiry, the gene order of IL-1Ra is M63099.1 corresponding GenBank number in this patent.U.S. food and drug administration (FDA) November calendar year 2001 ratify Amgen(pacify into) rhIL-1Ra (Kineret of company, Anakinra) listing, be mainly used in the intractable rheumatoid arthritis patients invalid to conventional medicine, this medicine also is used for the treatment of type ii diabetes at present, and has obtained good effect.Anakinra is the restructuring IL-1Ra protein of escherichia coli expression, needs drug administration by injection every day during the treatment, and medication has increased the weight of patient's health, psychology and economical load frequently, and therefore the research and development of long-acting IL-1Ra are significant.
Naturally occurring protein in target protein matter and the organism is carried out amalgamation and expression, is one of major way of a kind of prolong drug biology half life.Carrier protein commonly used has two kinds at present: human serum albumin (Human serum albumin, HSA), can obtain the complete genome sequence of human serum albumin by the GenBank inquiry, the HSA gene order is NM_000477 corresponding GenBank number in this patent.Obtain fusion rotein by the yeast cell secreting, expressing, the long-acting HSA-interferon fusion protein such as the development of U.S. HGS company has entered clinical use at present.Its average half-life in vivo is 157 hours, only needs per two weeks injection once can obtain satisfied clinical therapeutic efficacy; Another kind of carrier is the Fc fragment of immunoglobulin (Ig), usually select Chinese hamster ovary celI to express as host cell, as above the benefit of the strong pharmaceutcal corporation, Ltd of marine letter state production is matched the fusion rotein of general injection liquid behaviour II type Tumor Necrosis Factor Receptors-antibody, is used for clinically moderate and severe Active rheumatoid arthritis patient's treatment.
At present, Chen Shuqing etc. have made up the expression vector of four kinds of long-acting interleukin 1 receptor antagonist fusion roteins of recombinant human that merged by different modes by HSA and IL1-Ra, are respectively pPIC9-HSA-IL1-Ra plasmid, pPIC9-HSA-(G) n-IL1-Ra plasmid, pPIC9-IL1-Ra-HSA plasmid and pPIC9-IL1-Ra-(G) n-HSA plasmid (patent No.: ZL 200810060568.7), the present invention quotes this full patent texts, these four kinds of expression vectors transform by electric shock and are inserted among Pichi strain GS115 or the SMD1168, four kinds of fusion roteins that give expression to are respectively: HSA-IL-1Ra(is without connection peptides, and this paper is called for short the HI fusion rotein), HSA-(G) n-IL-1Ra(this paper is called for short H (G) nI fusion rotein), IL-1Ra-HSA(is without connection peptides, this paper is called for short the IH fusion rotein), IL-1-(G) n-HSA(this paper is called for short I (G) nH fusion rotein); Described (G) nSequence be [GlyGlyGlyGlySer] n, n is the integer of 1-4.Shen Qi etc. carry out purifying to the shake flask fermentation liquid supernatant of HSA-G-IL-1Ra in laboratory scale, first through affinity chromatography, at last with Q Sepharose XL chromatography obtain purified product (Shen its, purifying and the biological activity research of the blue or green .HSA/IL1ra fusion rotein of old pivot. journal of Zhejiang university, 2009, Vol 38, No 3:260-264).
We find when fusion rotein being carried out purifying research, four kinds of fusion roteins self contain the restriction enzyme site of some lytic enzymes, thereby can be fermented some lytic enzymes that produce in the process identifies and cuts off and produce some protein degradations, this phenomenon is more more obvious than shake flask fermentation in large scale fermentation, the some of them protein degradation is because character and fusion rotein are very approaching, therefore be difficult to remove, work has brought very large inconvenience to purifying.Simultaneously, in the large scale fermentation of yeast cell, because fermentation condition is different, impurity component in its fermented liquid supernatant than shake flask fermentation liquid supernatant complexity many, except some are different from the foreign protein of shake flask fermentation, also can produce a large amount of colors, differ larger just because of proteolytic degradation degree and impurity component, so laboratory scale shake flask fermentation purification process is too simple, can not fully enough remove the protein degradation and the some other impurity that produce when large scale fermentation is produced.In addition, in laboratory scale shake flask fermentation experiment, fermented liquid supernatant does not almost have other impurity except target protein and protein degradation, so the first step purifying adopts affinity chromatography can effectively catch target protein.And because the impurity component in large scale fermentation is produced is complicated, reduce its work-ing life thereby some of them impurity is easy to damage the affinity chromatography filler.Because affinity chromatography filler cost is higher, thus in large scale fermentation production affinity chromatography and be not suitable for the first step purifying.
Summary of the invention
The invention provides the purification process of the long-acting interleukin 1 receptor antagonist fusion rotein of a kind of recombinant human (referred to as fusion rotein), after having passed through a large amount of improvement and condition optimizing, obtained better operational path.This purification process can effectively be removed foreign protein and the protein degradation that produces when large scale fermentation is produced fusion rotein, both can save cost, can obtain highly purified target protein again.
Purification process provided by the present invention comprises:
(1) engineering strain that can express the long-acting interleukin 1 receptor antagonist fusion rotein of recombinant human carries out fermentation expression, acquisition contains the fermented liquid of target protein, make the concentrated solution that contains target protein through pre-treatment, described target protein is HSA-IL-1Ra, HSA-(G) n-IL-1Ra, IL-1Ra-HSA or IL-1Ra-(G) n-HSA, wherein (G) nBe peptide linker, the sequence of G is GlyGlyGlyGlySer, and n is the integer of 1-4;
(2) concentrated solution with gained of upper step carries out the reinforcing yin essence ion exchange chromatography, obtains to contain the elutriant of target protein, and the matrix that used chromatography media contains quaternary ammonium group or QAE aglucon and used medium is agarose or dextran;
(3) gained elutriant of upper step is carried out blue glue affinity chromatography, obtain to contain the elutriant of target protein, used chromatography media contains Cibacron Blue aglucon;
(4) gained elutriant of upper step is carried out the weak anionic displacement chromatography, acquisition contains the elutriant of target protein, wherein weak anionic displacement chromatography used medium contains diethylamino ethyl aglucon, and the matrix of described medium is agarose, dextran or polymethylmethacrylate; The damping fluid of used wash-out target protein contains 10 ~ 50mM NaH 2PO 4(be illustrated in the final concentration in the damping fluid, all continue to use in full this form of presentation) and 20 ~ 35mM NaCl, pH is 5.5 ~ 6.5.
Further, the long-acting interleukin 1 receptor antagonist fusion rotein of described recombinant human is IL-1Ra-G-HSA fusion rotein (this paper is called for short the IGH fusion rotein), and the sequence of G is GlyGlyGlyGlySer.
Further, in step 1 fermentation, the expression-form of target protein is secreting, expressing, and expression carrier used thereof is pPICZ α A, pPICZ α B, pPICZ α C, pPIC9 or pPIC9K: when carrier was pPICZ α A, pPICZ α B or pPICZ α C, used host cell was pichia pastoris phaff X33; When carrier was pPIC9 or pPIC9K, used host cell was pichia pastoris phaff GS115 or pichia pastoris phaff SMD1168.
Wherein recombination yeast uses this area ordinary method to make up, as utilizes my ZL 200810060568.7(CN101255197B of house journal) in four kinds of fusion protein expression vector pPIC9-HSA-IL1-Ra plasmids, pPIC9-HSA-(G) n-IL1-Ra plasmid, pPIC9-IL1-Ra-HSA plasmid and pPIC9-IL1-Ra-(G) n-HSA plasmid obtains four kinds of fusion protein gene fractions, and this gene fragment also can be inserted in the middle of other expression vectors by gene engineering method; The natural polymorphism that exists of human serum albumin, the human serum albumin part in the fusion rotein of the present invention also comprises these multiformities; Can carry out the 100L fermentation culture to fusion rotein according to the BSM basis salt culture medium and the cultural method that provide on the Invitrogen company pichia spp fermentation handbook, thereby obtain containing the fermented liquid of target protein.
Further, the used weak anionic displacement chromatography medium of step 4 is selected from DEAE Sepharose CL-6B, DEAE Sepharose F.F., DEAE Sephadex A-50, DEAE-650M, DEAE-650C and Macro-Prep DEAE, preferred DEAE Sepharose CL-6B.Because these media all contain diethylamino ethyl (DEAE) aglucon, through this step chromatography, finally can effectively some obstinate impurity components such as protein degradation of fusion rotein be removed, obtain highly purified fusion rotein.
Further, in the step 4: in advance with column equilibration, used level pad contains 10 ~ 50mM NaH before the chromatography 2PO 4With 20mM NaCl, pH is 5.5 ~ 6.5, and preferred level pad contains 20mM NaH 2PO 4With 20mM NaCl, pH is 5.5 or 6.5; The damping fluid of used wash-out target protein is for containing 20mM NaH 2PO 4With 35mM NaCl, pH be 5.5 damping fluid, or for containing 20mM NaH 2PO 4Damping fluid with 20mM NaCl, pH 6.5.In fact, in all damping fluids of this paper, amphoteric salt NaH 2PO 4All can be preferably 20mM, certainly, NaH in the damping fluid 2PO 4Be in other concentration conditions in 10 ~ 50mM scope, finally also can reach same purification effect.
Further, the described reinforcing yin essence ion-exchange chromatography media of step 2 is Q Sepharose Fast Flow, QAE Sephadex A-50 or QAE-550C, these several reinforcing yin essence ion exchange chromatography fillers are thick purification medium commonly used, the characteristics that the high carrying capacity of high flow rate is arranged, good thick pure effect is arranged, can when effectively catching target protein, remove most of impurity, preferred Q Sepharose Fast Flow.
Further, in the step 2: before the chromatography in advance with column equilibration, used level pad A 1Contain 10 ~ 50mM Tris and 10 ~ 50mM NaCl, pH is 6.5 ~ 7.5, preferred level pad A 1Contain 20mM Tris and 20mM NaCl, pH is 7.0; Used elution buffer B 1Contain 10 ~ 50mM Tris and 100 ~ 200mM NaCl, pH is 6.5 ~ 7.5, preferred elution buffer B 1Contain 20mM Tris and 160mM NaCl, pH is 7.0.Before the loading, the sample electricity can be led and adjust to 6mS/cm, pH adjusts to 7.0.
Further, the described affinity chromatography medium of step 3 is selected from AF-Blue HC-650M, Blue Sepharose 6 Fast Flow CL-6B, Capto Blue, Capto Blue (high sub) and Affi-Gel Blue, preferred AF-Blue HC-650M.
Further, in the step 3: before the chromatography in advance with column equilibration, used level pad A 2Contain Tris and the 20 ~ 200mM NaCl of 10 ~ 50mM, pH is 6.5 ~ 7.5, preferred level pad A 2Contain 20mM Tris and 0.1M NaCl, pH is 7.0; Used elution buffer B 2Contain 10 ~ 50mM Tris and 800 ~ 1300mM NaCl, pH is 6.5 ~ 7.5, preferred elution buffer B 2Contain 20mM Tris and 1000mM NaCl, pH is 7.0.Before the loading, the sample electricity can be led and adjust to 6mS/cm, pH adjusts to 7.0.
Further because that the electricity of the used elutriant of blue glue affinity chromatography is led is higher, so before the step 4 weak anionic displacement chromatography loading, turn down in advance the sample electricity and lead, in order to avoid in the loading process sample flow wear can't hanging column.Described inflation method comprises dilution, ultrafiltration displacement or gel desalination, preferred G25 gel desalination.
Further, described inflation method comprises G25 gel desalination process, and used desalination medium is selected from Sephadex G25 Fine, Sephadex G25 Medium, Sephadex G25 Coarse or Sephadex G25 superfine, preferred Sephadex G25 Fine; The desalination damping fluid that uses contains the Tris of 5 ~ 10mM, and the pH value is 6.5 ~ 7.5, and preferred desalination damping fluid contains 8mM Tris, and pH is 7.0.Before the loading, sample pH value can be adjusted to 7.0.When occurring, ultraviolet absorption peak begins to collect sample, when the ultraviolet peak drops to baseline, electricity is led and stopped to collect sample when the peak occurs.
Further, that described pre-treatment comprises is centrifugal, ultra filtering clarifying and ultrafiltration and concentration, can carry out in the steps below:
A) centrifugal fermented liquid is collected supernatant;
B) fermented liquid supernatant is clarified with the 500K ultra-filtration membrane;
C) clear liquor is carried out 10 times with the 30K ultra-filtration membrane and concentrate, make the sample concentration liquid that contains fusion rotein.
IGH fusion rotein with present method purifying detects through SDS-PAGE and SEC-HPLC, more than the purity to 98%, can reach the standard of pharmacodynamic study; Fusion rotein (being HI, H(G) by other three kinds of amalgamation modes formation nI, IH fusion rotein), with I(G) nH character approaches, and can prepare its same high-purity fusion rotein product with present method.
Advantage of the present invention is:
1) used the reinforcing yin essence ion exchange chromatography filler of the high carrying capacity character of high flow rate at the first step purifying of large scale fermentation product, wearing quality is better and the relative affinity chromatography filler of cost is lower, when effectively catching target protein, can remove foreign protein and colors that major part is different from shake flask fermentation, therefore reduce the loss of follow-up affine filler, thereby saved cost.
2) but the Blue medium specific adsorption that adopts contains the material of rHSA structure, can effectively remove the foreign protein that in the large scale fermentation of fusion rotein, produces, particularly the final step chromatography is used the medium that aglucon is the diethylamino ethyl instead, this chromatographic stuffing can be in protein degradation and some other obstinate impurity that active adsorption and target protein character approach, in hanging down under the electric sliver spare the target protein selective elution, thereby obtain the target protein of higher degree, efficiently solve the difficult problem that in large scale fermentation production, can't obtain the target protein of enough purity.
Description of drawings
M related in the following electrophoretogram all represents albumen Marker:
SDS-PAGE electrophoretogram before and after Fig. 1, the IGH fusion rotein Q Sepharose Fast Flow chromatography column purifying.1: be the electrophoresis result of embodiment 1 gained protein sample before the fusion rotein Q Sepharose Fast Flow chromatography column purifying; 2: be the electrophoresis result of embodiment 2 gained protein samples behind the fusion rotein Q Sepharose Fast Flow chromatography column purifying.
Sample SEC-HPLC detected collection of illustrative plates behind Fig. 2, the embodiment 2 IGH fusion rotein Q Sepharose Fast Flow chromatography column purifying, and target protein went out the peak in 9.2 minutes, and the protein peak area ratio is 45%.。
SDS-PAGE electrophoretogram before and after Fig. 3, the IGH fusion rotein AF-Blue HC-650M chromatography column purifying.1: be the electrophoresis result of embodiment 2 gained protein samples before the fusion rotein AF-Blue HC-650M chromatography column purifying; 2: be the electrophoresis result of embodiment 3 gained protein samples behind the fusion rotein AF-Blue HC-650M chromatography column purifying.
Sample SEC-HPLC collection of illustrative plates behind Fig. 4, the embodiment 3 IGH fusion rotein AF-Blue HC-650M chromatography column purifying, target protein went out the peak in 9.2 minutes, and protein degradation went out the peak in 9.6 minutes, and target protein peak area ratio is 65%.
SDS-PAGE electrophoretogram before and after Fig. 5, the IGH fusion rotein DEAE Sepharose CL-6B chromatography column purifying.1: be the electrophoresis result of embodiment 4 gained protein samples before the fusion rotein DEAE Sepharose CL-6B chromatography column purifying; 2: be the electrophoresis result of embodiment 5 gained protein samples behind the fusion rotein DEAE Sepharose CL-6B chromatography column purifying.
Sample SEC-HPLC collection of illustrative plates behind Fig. 6, the embodiment 5 IGH fusion rotein DEAE Sepharose CL-6B chromatography column purifying, target protein went out the peak in 9.2 minutes, and target protein peak area ratio is greater than 98%.
SDS-PAGE electrophoretogram before and after Fig. 7, the embodiment 6 IGH fusion rotein DEAE Sepharose CL-6B chromatography column purifying.1: be the electrophoresis result of embodiment 4 gained protein samples before the fusion rotein DEAE Sepharose CL-6B chromatography column purifying; 2: the embodiment 6 alone electrophoresis result that contain the buffer solution elution gained protein sample of 20mM NaCl; 3: the embodiment 6 alone electrophoresis result that contain the buffer solution elution gained protein sample of 35mM NaCl.
Fig. 8, the embodiment 6 alone SEC-HPLC collection of illustrative plates that contain the buffer solution elution gained sample of 35mM NaCl, target protein went out the peak in 9.2 minutes, and target protein peak area ratio is greater than 88%.
Fig. 9, the final purification result SDS-PAGE electrophoretogram of embodiment 5 IGH fusion roteins.M: albumen Marker; 1 ~ 6: the electrophoresis result of final purifying gained 6 duplicate samples of embodiment 5 IGH fusion roteins.
Figure 10, IGH fusion protein purification process SDS-PAGE electrophoresis cma staining collection of illustrative plates.1: embodiment 2 fusion rotein Q Sepharose Fast Flow chromatography column purifying rear electrophoresis cma staining results; 2: embodiment 3 fusion rotein AF-Blue HC-650M chromatography column purifying rear electrophoresis silver dye the result; 3: embodiment 5 fusion rotein DEAE Sepharose CL-6B chromatography column purifying rear electrophoresis silver dye the result.
Embodiment
The pre-treatment (take the IGH fusion rotein as example) of the long-acting interleukin 1 receptor antagonist fusion rotein of embodiment 1 recombinant human fermented liquid
One experiment material
500K and 30K ultra-filtration membrane bag and ultrafiltration anchor clamps are all available from Millipore Corp..
Two operation stepss
The bacterial strain that 1 fermentation is adopted is pPIC9-IGH/GS115, according to the BSM basis salt culture medium and the cultural method that provide on the Invitrogen company pichia spp fermentation handbook fusion rotein is carried out the 100L fermentation culture, thereby obtains containing the fermented liquid of target protein.
2 is centrifugal with fermented liquid 10000rpm, abandons thalline, collects supernatant liquor for subsequent use.
3 clarify fermented supernatant fluid after with the dilution of isopyknic purified water with 500K ultra-filtration membrane bag, inlet pressure＜20psi, and return pressure＜10psi abandons trapped fluid, and filtered solution is for subsequent use.
4 concentrate 500K ultra filtering clarifying liquid with 30K ultra-filtration membrane bag, inlet pressure＜20psi, and return pressure＜10psi abandons filtered solution, and trapped fluid is for subsequent use.
The long-acting interleukin 1 receptor antagonist fusion rotein of embodiment 2 recombinant human Q Sepharose Fast Flow chromatography column purifying (take the IGH fusion rotein as example)
One experiment material
The protein chromatographic post is available from GE company; Q Sepharose Fast Flow filler is available from GE company; The AKTA protein purification system is available from GE company.
Two operation stepss
1 solution preparation
Level pad A 1: 20mM Tris+20mM NaCl, the pH value is 7.0
Elution buffer B 1: 20mM Tris+160mM NaCl, the pH value is 7.0
2 balances
Use buffer A 12 of flushing post beds are more than the column volume, until it is identical with the damping fluid electric conductivity value and the pH that flow out chromatography column to flow into the damping fluid of chromatography column, in this moment ultraviolet return to zero.
3 loadings
1 that make with embodiment, as to contain the long-acting IL-1 receptor antagonist protein sample of recombinant human ultrafiltration and concentration liquid, electricity is led and is adjusted to 6mS/cm, after pH adjusts to 7.0, carries out loading.
4 wash-outs
Behind the end of the sample, continue to use buffer A 1Balance 1 column volume, then buffer B 1Liquid flushing chromatography column is collected the ultraviolet absorption peak sample with the wash-out target protein, finishes until go out the peak, and baseline is got back to the level before the loading.
Gained purification of samples SDS-PAGE detected result is referring to Fig. 1, and the HPLC detected result is referring to Fig. 2.
The long-acting interleukin 1 receptor antagonist fusion rotein of embodiment 3 recombinant human AF-Blue HC-650M chromatography column purifying (take the IGH fusion rotein as example)
One experiment material
The protein chromatographic post is available from GE company; AF-Blue HC-650M filler is available from TOSOH company; The AKTA protein purification system is available from GE company.
Two operation stepss
1 solution preparation
Level pad A 2: 20mM Tris+0.1M NaCl, the pH value is 7.0
Elution buffer B 2: 20mM Tris+1M NaCl, the pH value is 7.0
2 balances
Use buffer A 22 of flushing post beds are more than the column volume, until it is identical with the damping fluid electric conductivity value and the pH that flow out chromatography column to flow into the damping fluid of chromatography column, in this moment ultraviolet return to zero.
3 loadings
2 that make with embodiment, lead through the protein sample electricity of the first step reinforcing yin essence ion exchange chromatography wash-out and to adjust to 6mS/cm, after pH adjusts to 7.0, carry out loading.
4 wash-outs
Behind the end of the sample, continue to use buffer A 2Balance 1 column volume, then buffer B 2Wash-out is collected the ultraviolet absorption peak sample, finishes until go out the peak, and baseline is got back to the level before the loading.
Purification of samples SDS-PAGE detected result is referring to Fig. 3, and the HPLC detected result is referring to Fig. 4.
The long-acting interleukin 1 receptor antagonist fusion protein S of embodiment 4 recombinant human ephadex G25 Fine chromatography column desalination (take the IGH fusion rotein as example)
One experiment material
The protein chromatographic post is available from GE company; Sephadex G25 Fine filler is available from GE company; The AKTA protein purification system is available from GE company.
Two operation stepss
1 solution preparation
The desalination damping fluid: 8mM Tris, the pH value is 7.0
2 balances
With 2 of desalination damping fluid flushing post beds more than the column volume, until it is identical with the damping fluid electric conductivity value and the pH that flow out chromatography column to flow into the damping fluid of chromatography column, in this moment ultraviolet return to zero.
3 loadings
Embodiment 3 protein sample pH that make, process second step affinity chromatography wash-out are adjusted to 7.0, determine to carry out loading behind the loading volume according to the chromatography media column volume.
4 sample collections
Collect out the peak sample, finish until ultraviolet goes out the peak, this moment, electric lead curve began to rise, and stopped to collect sample.When electric lead curve drops to baseline, continue loading, repeatedly for several times, until till all samples desalination end.
The long-acting interleukin 1 receptor antagonist fusion rotein of embodiment 5 recombinant human DEAE Sepharose CL-6B chromatography column purifying (take the IGH fusion rotein as example)
One experiment material
The protein chromatographic post is available from GE company; DEAE Sepharose CL-6B filler is available from GE company; The AKTA protein purification system is available from GE company.
Two operation stepss
1 solution preparation
Level pad A 3: 20mM NaH 2PO 4+ 20mM NaCl, the pH value is 6.5
Elution buffer and level pad A 3Identical
2 balances
Use A 32 of liquid flushing post beds are more than the column volume, until it is identical with the damping fluid electric conductivity value and the pH that flow out chromatography column to flow into the damping fluid of chromatography column, in this moment ultraviolet return to zero.
3 loadings
The desalination sample that embodiment 4 is made carries out loading.
4 wash-outs
Behind the end of the sample, continue to use buffer A 3Have ultraviolet absorption peak about 1 column volume of balance and occur, collect out the peak sample, finish until ultraviolet goes out the peak, baseline is got back to the level before the loading, and this sample is final purpose albumen.
Purification of samples SDS-PAGE detected result is referring to Fig. 5, and the HPLC detected result is referring to Fig. 6.
The long-acting interleukin 1 receptor antagonist fusion rotein of embodiment 6 recombinant human DEAE Sepharose CL-6B chromatography column purifying (take the IGH fusion rotein as example)
One experiment material
The protein chromatographic post is available from GE company; DEAE Sepharose CL-6B filler is available from GE company; The AKTA protein purification system is available from GE company.
Two operation stepss
1 solution preparation
Level pad A 4: 20mM NaH 2PO 4+ 20mM NaCl, the pH value is 5.5
Elution buffer B 4: 20mM NaH 2PO 4+ 35mM NaCl, the pH value is 5.5
2 balances
Use A 42 of liquid flushing post beds are more than the column volume, until it is identical with the damping fluid electric conductivity value and the pH that flow out chromatography column to flow into the damping fluid of chromatography column, in this moment ultraviolet return to zero.
3 loadings
The desalination sample that embodiment 4 is made carries out loading.
4 wash-outs
Behind the end of the sample, continue to use buffer A 4Have ultraviolet absorption peak about 1 column volume of balance and occur, collect out the peak sample, finish until ultraviolet goes out the peak, baseline is got back to the level before the loading, then uses buffer B 4Wash-out is collected the ultraviolet absorption peak sample, finishes until go out the peak, and baseline is got back to the level before the loading.
Purification of samples SDS-PAGE detected result is referring to Fig. 7, and the HPLC detected result is referring to Fig. 8.
The purity detecting of the long-acting interleukin 1 receptor antagonist fusion rotein of embodiment 7 recombinant human (take the IGH fusion rotein as example)
One experiment material
TSKgel G2000SWXL liquid phase chromatography column is available from TOSOH company.
Two operation stepss
1, protein electrophoresis detects
Above-described embodiment gained purification of samples is 15% through non-reduced electrophoresis SDS-PAGE(resolving gel concentration), Coomassie brilliant blue dyeing, to the sample of embodiment 5 gained, except target protein, occur without obvious foreign protein, the result is referring to Fig. 9; It is 15% through non-reduced electrophoresis SDS-PAGE(resolving gel concentration that the IGH fusion rotein is respectively gone on foot purification of samples) the cma staining discovery, final purification of samples occurs without obvious foreign protein except target protein, and the result is referring to Figure 10.
2, HPLC detects
1) solution preparation
Moving phase is 50mM NaH 2PO 4+ 0.3%NaCl, pH6.5
2) experimental technique
Above-described embodiment gained purification of samples liquid phase detection method is SEC-HPLC, and the sample feeding amount is 50 μ l, and flow velocity is 0.7ml/min, and target protein goes out the peak at 9.2min.To embodiment 5 gained samples, referring to Fig. 6, the result judges the target protein peak area greater than 98%, and namely purity is greater than 98%.

Claims (12)

1. the purification process of the long-acting interleukin 1 receptor antagonist fusion rotein of recombinant human, the method comprises:
(1) engineering strain that can express the long-acting interleukin 1 receptor antagonist fusion rotein of recombinant human carries out fermentation expression, acquisition contains the fermented liquid of target protein, make the concentrated solution that contains target protein through pre-treatment, described target protein is HSA-IL-1Ra, HSA-(G) n-IL-1Ra, IL-1Ra-HSA or IL-1Ra-(G) n-HSA, wherein (G) nBe peptide linker, the sequence of G is GlyGlyGlyGlySer, and n is the integer of 1-4;
(2) concentrated solution with gained of upper step carries out the reinforcing yin essence ion exchange chromatography, obtains to contain the elutriant of target protein, and the matrix that used chromatography media contains quaternary ammonium group or QAE aglucon and used medium is agarose or dextran;
(3) gained elutriant of upper step is carried out blue glue affinity chromatography, obtain to contain the elutriant of target protein, used chromatography media contains Cibacron Blue aglucon;
(4) gained elutriant of upper step is carried out the weak anionic displacement chromatography, acquisition contains the elutriant of target protein, wherein weak anionic displacement chromatography used medium contains diethylamino ethyl aglucon, and the matrix of described medium is agarose, dextran or polymethylmethacrylate; The damping fluid of used wash-out target protein contains 10 ~ 50mM NaH 2PO 4With 20 ~ 35mM NaCl, pH is 5.5 ~ 6.5.
2. purification process as claimed in claim 1 is characterized in that, the long-acting interleukin 1 receptor antagonist fusion rotein of described recombinant human is the IL-1Ra-G-HSA fusion rotein, and the sequence of G is GlyGlyGlyGlySer.
3. purification process as claimed in claim 1, it is characterized in that, in step 1 fermentation, the expression-form of target protein is secreting, expressing, expression carrier used thereof is pPICZ α A, pPICZ α B, pPICZ α C, pPIC9 or pPIC9K: when carrier was pPICZ α A, pPICZ α B or pPICZ α C, used host cell was pichia pastoris phaff X33; When carrier was pPIC9 or pPIC9K, used host cell was pichia pastoris phaff GS115 or pichia pastoris phaff SMD1168.
4. such as each described purification process of claim 1-3, it is characterized in that, the used weak anionic displacement chromatography medium of step 4 is selected from DEAE Sepharose CL-6B, DEAE Sepharose F.F., DEAE Sephadex A-50, DEAE-650M, DEAE-650C and Macro-Prep DEAE, preferred DEAE Sepharose CL-6B.
5. purification process as claimed in claim 4 is characterized in that, in the step 4: in advance with column equilibration, used level pad contains 10 ~ 50mM NaH before the chromatography 2PO 4With 20mM NaCl, pH is 5.5 ~ 6.5, and preferred level pad contains 20mM NaH 2PO 4With 20mM NaCl, pH is 5.5 or 6.5; The damping fluid of used wash-out target protein is for containing 20mM NaH 2PO 4With 35mM NaCl, pH be 5.5 damping fluid, or for containing 20mM NaH 2PO 4Damping fluid with 20mM NaCl, pH 6.5.
6. such as each described purification process of claim 1-3, it is characterized in that, the described reinforcing yin essence ion-exchange chromatography media of step 2 is Q Sepharose Fast Flow, QAE Sephadex A-50 or QAE-550C, preferred Q Sepharose Fast Flow.
7. purification process as claimed in claim 6 is characterized in that, in the step 2: before the chromatography in advance with column equilibration, used level pad A 1Contain 10 ~ 50mM Tris and 10 ~ 50mM NaCl, pH is 6.5 ~ 7.5, preferred level pad A 1Contain 20mM Tris and 20mM NaCl, pH is 7.0; Used elution buffer B 1Contain 10 ~ 50mM Tris and 100 ~ 200mM NaCl, pH is 6.5 ~ 7.5, preferred elution buffer B 1Contain 20mM Tris and 160mM NaCl, pH is 7.0.
8. such as each described purification process of claim 1-3, it is characterized in that, the described affinity chromatography medium of step 3 is selected from AF-Blue HC-650M, Blue Sepharose 6 Fast Flow CL-6B, Capto Blue, Capto Blue (high sub) and Affi-Gel Blue, preferred AF-Blue HC-650M.
9. purification process as claimed in claim 8 is characterized in that, in the step 3: before the chromatography in advance with column equilibration, used level pad A 2Contain Tris and the 20 ~ 200mM NaCl of 10 ~ 50mM, pH is 6.5 ~ 7.5, preferred level pad A 2Contain 20mM Tris and 0.1M NaCl, pH is 7.0; Used elution buffer B 2Contain 10 ~ 50mM Tris and 800 ~ 1300mM NaCl, pH is 6.5 ~ 7.5, preferred elution buffer B 2Contain 20mM Tris and 1000mM NaCl, pH is 7.0.
10. such as each described purification process of claim 1-3, it is characterized in that, before the step 4 weak anionic displacement chromatography loading, turn down in advance the sample electricity and lead, described inflation method comprises dilution, ultrafiltration displacement or gel desalination, preferred G25 gel desalination.
11. purification process as claimed in claim 10, it is characterized in that, described inflation method comprises G25 gel desalination process, used desalination medium is selected from Sephadex G25 Fine, Sephadex G25 Medium, Sephadex G25 Coarse or Sephadex G25 superfine, preferred Sephadex G25 Fine; The desalination damping fluid that uses contains the Tris of 5 ~ 10mM, and the pH value is 6.5 ~ 7.5, and preferred desalination damping fluid contains 8mM Tris, and pH is 7.0.
12. such as each described purification process of claim 1-3, it is characterized in that, that described pre-treatment comprises is centrifugal, ultra filtering clarifying and ultrafiltration and concentration.
CN2012104336421A 2012-11-02 2012-11-02 Recombinant human long-acting interleukin-1 receptor antagonist fusion protein purification method Pending CN102952836A (en)

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Publication number Priority date Publication date Assignee Title
CN113929733A (en) * 2021-10-25 2022-01-14 江苏帆博生物制品有限公司 Monoclonal antibody purification method of mixed ion exchange filler

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Publication number Priority date Publication date Assignee Title
CN1339065A (en) * 1999-01-30 2002-03-06 达尔塔生物技术有限公司 Method for preparing highly pure albumin solution
CN101255197A (en) * 2008-03-28 2008-09-03 浙江大学 Fusion protein for serum albumin and interleukin 1 receptor antagonist and uses thereof

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
CN1339065A (en) * 1999-01-30 2002-03-06 达尔塔生物技术有限公司 Method for preparing highly pure albumin solution
CN101255197A (en) * 2008-03-28 2008-09-03 浙江大学 Fusion protein for serum albumin and interleukin 1 receptor antagonist and uses thereof

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113929733A (en) * 2021-10-25 2022-01-14 江苏帆博生物制品有限公司 Monoclonal antibody purification method of mixed ion exchange filler

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