CN103360461B - A kind of people's IL-12 affinity purification chromatographic column, its preparation method and the method utilizing its purification of recombinant human IL-12 - Google Patents
A kind of people's IL-12 affinity purification chromatographic column, its preparation method and the method utilizing its purification of recombinant human IL-12 Download PDFInfo
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Abstract
The present invention relates to a kind of prepare recombined human IL 12 affinity purification chromatographic column technique, the recombined human IL 12 affinity purification chromatographic column prepared by this technique and by the method for this affinity purification column chromatography recombined human IL 12.Specifically, the present invention relates to prepare anti-human IL 12 monoclonal antibody with the hybridoma cell strain of anti-human IL 12, and prepare recombined human IL 12 affinity purification chromatographic column with this antibody, then with the technological process of this affinity purification column chromatography recombined human IL 12.More specifically, the present invention relates to a kind of method using affinity column purification of recombinant human IL 12, it is characterized in that preparing antibody with the hybridoma cell strain of anti-human IL 12 monoclonal antibody, people IL 12 affinity purification chromatographic column, the method being then purified recombined human IL 12 based on this affinity purification chromatographic column is prepared with this antibody.
Description
Technical field
The present invention relates to a kind of prepare the technique of rHuIL-12 affinity purification chromatographic column, by this technique
The rHuIL-12 affinity purification chromatographic column for preparing and by this affinity purification column chromatography recombined human
The method of IL-12.Specifically, the present invention relates to prepare with the hybridoma cell strain of anti-human IL-12 anti-
People's IL-12 monoclonal antibody, and prepare rHuIL-12 affinity purification chromatographic column with this antibody, so
Afterwards with the technological process of this affinity purification column chromatography rHuIL-12.
Background technology
Recombinant human interleukin 12 (IL-12) is initially referred to as natural kill cell stimulating factor (natural
Killer cell stimulatory factor, NKSF) or cytotoxic lymphocytic maturation factor
(cytotoxic lymphocyte maturation factor, CLMF), mainly by the monokaryon activated
Cell and other kinds of cell (dendritic cell, B cell, neutrophilic granulocyte and horn cell)
Produce.Increase NK cell and the cytotoxic activity of activating T cell that IL-12 is possessed, induce
IFN-γ, the immunologic competence such as growth of regulation Th1 cell are IL-12 antitumor, antiviral effect
Theoretical basis, zoopery the most fully confirms its medical value.Based on this, rHuIL-12
(rhIL-12) the II/III phase clinic of antitumor, antiviral property disease is come into as a kind of medicine
Experimental stage.
People IL-12 is to be formed heterodimer by two subunits of p40 and p35 by disulfide bond is covalently bound
Glycoprotein, its molecular weight is about 70-75kDa, and isoelectric point, IP is 4.5-5.5, and wherein p35 subunit has
197 amino acid residues, containing 7 cysteine (cys) and 3 N glycosylation sites, p40 is sub-
306 amino acid residues of unit, have 10 cysteine, 4 N-glycosylation sites.P35 has 3
Individual intramolecular disulfide bond, p40 has 4 intramolecular disulfide bonds, has one simultaneously between p35 and p40
Intermolecular disulfide bond is existed.Individually p40 or p35 equal abiology activity, and free p40 can
To compete the binding site of IL-12p70 Yu IL-12 receptor, thus suppress the activity of IL-12.Due to
The particularity of the molecule of people IL-12 and complexity, express with prokaryotic expression system or Yeast system
Time, its product abiology activity, generally use insecticide or mammalian cell to express.This class
Topic group uses Chinese hamster ovary cell (CHO) to express people IL-12 (see patent ZL03131567.4).
The purifying process of rHuIL-12 is the industrial important step of people IL-12, the most
All there are Patents or document report in individual seminar or company, such as in the published patent of this seminar
Application 200410080518.7 (methods Tian Zhigang of purifying and recombining human iterleukin-12 etc. 2004
Year) in have employed ultrafiltration, anion, cation, five step process methods hydrophobic, molecular sieve carry out pure
Change.Patent CN 101033254 (the method Pu of a kind of purifying and recombining human iterleukin-12 at home
Diligent wait 2007) in have employed ultrafiltration, Anionic/Cationic, thiamine precipitation, anionic/cationic,
Six step process methods hydrophobic, molecular sieve are purified, and both approaches is relatively cumbersome, and step is more.
Although domestic patent ZL01133658.7 (hIL-12 expression in three-spotted plusia and
The method holt in the Meng of purification etc. 2004) the affine method Purification of Human IL-12 of middle employing, but this process
For single-step process of affinity chromatography purification, n.s pretreatment, easily pollute affinity column, and unmanned IL-12
Aggressiveness removal process.Additionally, due to the difficulty of rHuIL-12 purification, institute of current biotech firm
The business rHuIL-12 product provided, price is the highest, is highly detrimental to relate to people IL-12
Research and the carrying out of extensive medicinal production.
The hybridoma cell strain using anti-human IL-12 in the present invention prepares monoclonal antibody, and resists with this
Body prepares rHuIL-12 affinity purification chromatographic column, then recombinates with this affinity purification column chromatography
The technological process of people IL-12.This flow process only needs 4 steps i.e. to can get the purity rhIL-12 more than 95%
Sample, can be greatly shortened workflow and time, and improve the purity of the response rate and product.
Summary of the invention
In order to overcome the shortcoming of prior art, it is affine pure that the present invention provides one to prepare rHuIL-12
The technique changing chromatographic column, and the rHuIL-12 affinity purification chromatographic column prepared by this technique.Tool
Say body, the present invention relates to prepare with the hybridoma cell strain of anti-human IL-12 monoclonal antibody, and use
This antibody prepares rHuIL-12 affinity purification chromatographic column.The invention still further relates to utilize this affinity purification
The method of column chromatography rHuIL-12.
In one embodiment of the invention, the present invention relates to a kind of employing affinity chromatograph column purification weight
The method of group people IL-12, it is characterised in that with the hybridoma cell strain of anti-human IL-12 monoclonal antibody
Prepare antibody, prepare people's IL-12 affinity purification chromatographic column with this antibody, then with this affinity purification layer
The method being purified rHuIL-12 based on analysis post.
It is an object of the present invention to provide the people utilizing people's IL-12 affinity column purification of Recombinant
The method of IL-12.Specifically, described method includes ultrafiltration, anion-exchange chromatography, affinity chromatograph
With four purification steps of molecular sieve:
A. ultrafiltration: obtain the culture supernatant containing rHuIL-12 (rhIL-12), selects 10-30kD
Ultrafilter membrane, supernatant is concentrated by ultrafiltration about 10-15 times, 0.22-0.45 μm membrane filtration remove insoluble
Property microgranule；
B. anion-exchange chromatography: use anion-exchange chromatography post, 20mM Tris-HCl balance liquid
Balance, 40mM histidine buffering liquid eluting foreign protein, 0.25M NaCl 20mM Tris-HCl eluting
Destination protein；
C. affinity chromatograph: use people's IL-12 affinity column (this prepared according to the technique of the present invention
Constitute another aspect of the present invention, see below) purification rhIL-12, with 20mM Tris-HCl balance liquid
Balance, by 20mM Gly-HCl buffer solution elution, is adjusted to 7.0 with 1M Tris by the pH of eluted protein
Left and right；
D. molecular sieve: utilize molecular sieve to remove glycoprotein polyprotein precursor, obtain the rHuIL-12 monomer of purification.
It should be appreciated by those skilled in the art that wherein, it is thus achieved that containing rHuIL-12 (rhIL-12)
Culture supernatant can be expressed by cultivating by the cultural method disclosed in domestic patent ZL03131567.4
The Chinese hamster ovary celI of rHuIL-12 and obtain.In ZL03131567.4, inventor builds table respectively
The p40 subunit of intelligent IL-12 and the carrier for expression of eukaryon pcDNA3/p40 of p35 subunit and
PEF13/p35, cotransfection Chinese hamster ovary celI, utilize G418 and methionine sulfoxide screening to obtain high expressed
The engineering cell strain of rHuIL-12.Described method simply utilizes people's natural table of IL-12 P40 and P35
Reach unbalanced characteristic, use the carrier that two kinds of expression efficiencies are different, so that the expression of two chains is as far as possible
Balance.The content of this patent is completely combined in this application by quoting.Certainly, the present invention is to how
The culture supernatant that acquisition comprises rHuIL-12 (rhIL-12) does not has any restriction, if described training
Foster supernatant comprises activated rHuIL-12 and i.e. can be used for the purification process of the present invention.
It should be appreciated by those skilled in the art that described anion-exchange chromatography post includes, but not limited to
Sepharose, Capto, Sephadex, Sephacel etc. are skeleton, and Q, QAE, DEAE are for living
The gel of property group, such as: Q-Sepharose FF, Capto Q, Q-Sepharose HP, Capto
DEAE, QAE Sephadex, DEAE-Sepharose FF, DEAE Sephadex etc.；Can use
Molecular sieve includes, but not limited to Sephacryl, Superdex, Sephadex, Superose, Sepharose
Deng the gel for skeleton, such as: Sephacryl S-200 HR, Superdex 200, Sephadex G-100
Deng.
It addition, it should also be realized by those skilled in the art that what the present invention provided utilizes rHuIL-12
Ultrafiltration that the method for the people IL-12 of affinity column purification of Recombinant is comprised, anion-exchange chromatography, parent
With the order between chromatography and four purification steps of molecular sieve is not changeless, people in the art
Member can carry out suitable adjustment according to actual needs, for example, it is possible to after carrying out ultrafiltration step,
Carry out affinity chromatograph, carry out anion-exchange chromatography the most again, etc..
It is also another object of the present invention to provide that to utilize the monoclonal antibody of anti-human IL-12 to prepare affine pure
The method changing chromatographic column, described method comprises the steps:
A. activated gel pretreatment:
Take appropriate activated gel (such as, the agarose gel of CNBr or NHS activation, be purchased from
GE company), it is completely dissolved with 1mM HCl, and rinses colloid with 1mM HCl, the most again
Use 0.1M NaHCO3, rinse colloid；
The coupling of the most anti-rhIL-12 antibody and the preparation of affinity column:
The method being concentrated by ultrafiltration by that prepare by present invention method provided below or buy anti-
Anti-rhIL-12 antibody, to 4-5mg/ml, is then added in activated gel, puts by rhIL-12 Antibody Concentration
4 DEG C overnight, then uses 0.1M NaHCO3Rinse colloid, with 0.1M Tris-HCl buffer blind
Unconjugated active group, then delays with 0.1M Tris-HCl buffer and 0.1M Acetic acid-sodium acetate
Rush liquid and alternately rinse colloid, finally the gel of anti-for covalent bond rhIL-12 antibody is filled post, be prepared as
The affinity column of purification rhIL-12.
Wherein, the activated gel in step a can be CNBr (Bromine cyanide .), NHS (N-hydroxyl sulfur
For butanimide), the gel that Sepharose is skeleton of the activation such as Epoxy (epoxy resin),
Such as: CNBr-activated Sepharose 4FF, NHS-activated Sepharose 4FF, CNBr
The Sepharose 4B of activation, Epoxy-activated Sepharose 6B, Activated CH
Sepharose 4B、EAH Sepharose 4B、ECH Sepharose 4B、Thiopropyl Sepharose
6B etc..
A further object of the present invention is to provide and utilizes people's IL-12 hybridoma to prepare anti-human IL-12 Dan Ke
The method of grand antibody.
Generally, from produce anti-human IL-12 antibody hybridoma (such as, ATCC CRL-2382,
Clone number is 20C2, purchased from American type culture collection (ATCC)) collect the present invention's
During anti-human IL-12 monoclonal antibody, antibody can be obtained from Hybridoma Cell Culture supernatant, specifically
For, passage receives culture supernatant after cultivating 3-5 days, containing certain density monoclonal in supernatant
Antibody.Or hybridoma is expelled to the pretreated mouse peritoneal of incomplete Freund's adjuvant, logical
Cross acquisition monoclonal antibody in extraction mouse ascites.Generally, hybridoma all uses BALB/c mouse to make
Carrying out antibody producing for immune animal, Normal practice is paraffin or blood fat reducing alkane lumbar injection after 1 week, then
Lumbar injection 1-2 × 106Hybridoma, produces ascites in about 7-10 days.But clone number is 20C2
The conventional paraffin of hybridoma anticipate without ascites generation.Although cannoing be used up, full Freund adjuvant is in advance
Process can produce ascites at about 10-14 days, but its potency ratio nude mice is low at least 100 times (schemes
1).Therefore, the present invention provides and utilizes people's IL-12 hybridoma to prepare anti-human IL-12 monoclonal antibody
Method, the described method full Freund adjuvant that comprises the steps: to cannot be used up processes the nude mice of 8-10 week old,
Every mouse peritoneal injects 500 μ l incomplete Freund's adjuvants；Lumbar injection hybridoma after 3 days,
Every mice 1 × 106Cell；Within about 7-10 days, produce ascites, collect mouse ascites, centrifugal receipts supernatant
I.e. ascites antibody.The anti-human IL-12 antibody obtained by said method, can be with the affine layer of Protein G
Analysis method purification.
Beneficial effect: the invention provides the purification process of a kind of rhIL-12 rapidly and efficiently, pass through
Use the technological process of hIL-12 affinity purification column chromatography rHuIL-12, only need 4 steps
Obtain the purity rhIL-12 sample more than 95%, workflow and time can be greatly shortened, and carry
High-recovery and the purity of product, be particularly suitable for commercial production.
Accompanying drawing explanation
From detailed description below in conjunction with the accompanying drawings, features described above and the advantage of the present invention will be apparent from,
Wherein:
Fig. 1, BALB/c and nude mice the affinity of the anti-human IL-12 antibody generated compares.
Fig. 2, the Protein G purification collection of illustrative plates of anti-human IL-12 antibody.
Fig. 3, anti-human IL-12 antibody purity qualification result.
Fig. 4, the result of anion exchange column purification rhIL-12.
Fig. 5, utilizes the result of the people IL-12 affinity column purification rhIL-12 of the present invention.
Fig. 6, molecular sieve purification rhIL-12 result.
Detailed description of the invention
Following example will assist in those of ordinary skill in the art and are further appreciated by the present invention, but not
Limit the present invention in any form.Experimental technique in embodiment, if no special instructions, all uses this
Field routine techniques, experiment reagent is commercially available analytical pure grade product.
Embodiment 1: the preparation of monoclonal antibody
1. prepared by culture supernatant or ascites:
From the hybridoma producing anti-human IL-12, (ATCC CRL-2382 trains purchased from U.S. typical case
Support thing preservation center (ATCC)) collect the present invention anti-human IL-12 monoclonal antibody time, Ke Yicong
Obtaining antibody in Hybridoma Cell Culture supernatant, specifically, passage receives training after cultivating 2-3 days
Support supernatant, containing certain density monoclonal antibody in supernatant.Or hybridoma is expelled to not
The pretreated mouse peritoneal of complete Freund's adjuvant, by obtaining monoclonal anti in extraction mouse ascites
Body, specifically, the full Freund adjuvant that cannots be used up processes the nude mice of 8-10 week old, every mouse peritoneal note
Penetrate 500 μ l incomplete Freund's adjuvants.Lumbar injection hybridoma after one week, every mice 1-2
×106Cell, produces ascites in about 7-10 days, collects mouse ascites, centrifugal receipts supernatant, uses PBS
(pH7.0) with 0.45 μm membrane filtration after dilution 3-5 times.
The purification of the most anti-human IL-12 monoclonal antibody:
Culture supernatant/the ascites obtained by said method, can use Protein G affinity chromatograph method
Purification.Protein G affinity column is balanced, by the ascites processed or cultivation with PBS
Supernatant loading, then balance Protein G affinity column with PBS, use 0.1M citric acid
Eluting purpose antibody, and use 0.5M Na immediately2CO3The pH of regulation purpose antibody be about 7,
In order to avoid albuminous degeneration.Purity SDS-PAGE of antibody is identified.As in figure 2 it is shown, it is purified
The purity of monoclonal antibody reaches more than 95%, monoclonal antibody molecule amount heavy chain about 50kDa, light chain
About 26kDa.
Embodiment 2: the preparation of people's IL-12 affinity column
The Sepharose 4B gel pretreatment of 1.CNBr activation
Weigh the Sepharose 4B gel (purchased from GE company) that the addition of C NBr (Bromine cyanide .) activates,
It is slowly added in 1mM HCl and constantly shakes up, treating that gel is completely dissolved, standing to gel
Sink to the bottom, supernatant discarded, add appropriate 1mM HCl and again hanged gel, then stand 15min,
Supernatant discarded, repeatable operation 5 times.Use 0.1M NaHCO the most again3(pH=8.3), colloid is rinsed,
The colloid at least 5ml 0.1M NaHCO of every milliliter3(pH=8.3) rinse.
The coupling of the most anti-rhIL-12 antibody and the preparation of affinity column
The method being concentrated by ultrafiltration is by (article No. that is that prepare by embodiment 1 or that buy from BD company
Being 555065, clone number be 20C2) Antibody Concentration of anti-rhIL-12 to 4-5mg/ml, then general
RhIL-12 antibody adds in the gel that step 1 processes, and surveys supernatant protein concentration, puts 4 DEG C overnight.
Then 0.1M NaHCO is used3(pH=8.3) colloid is rinsed 5 times, with 0.1M Tris-HCl buffer
(pH=8.0) close unconjugated active group 3 times, and stand 2h.Then 0.1M Tris-HCl is used
Buffer (pH=8.0) and 0.1M Acetic acid-sodium acetate buffer (pH=5.0) alternately rinse colloid,
At least wash 5 circulations.The colloid combining rhIL-12 antibody is filled post, is prepared as purification rhIL-12 and resists
The affinity column of body.
Embodiment 3: the method utilizing people IL-12 affinity chromatograph column purification rhIL-12 prepared by embodiment 2
1. ultrafiltration: the culture supernatant containing rhIL-12, selects the ultrafilter membrane (purchased from Millipore) of 10-30kD,
Supernatant is concentrated by ultrafiltration to about 10-15 times, with 0.22-0.45 μm filter membrane (purchased from Millipore)
It is filtered to remove particulate matter.
2. anion-exchange chromatography: use Q-Sepharose Fast Flow post (purchased from GE company), 20mM
Tris-HCl balance liquid (pH=8.0) balances, 40mM histidine buffering liquid eluting foreign protein, 0.25M
NaCl 20mM Tris-HCl (pH=8.0) eluting destination protein.The rhIL-12 sample of anion column purification
The purity of product reaches more than 20% (result is shown in Fig. 4).
3. affinity chromatograph: use the people IL-12 affinity chromatograph column purification rhIL-12 of embodiment 2 preparation, use 20mM
PBS balance liquid (pH=7.2) balances, with 20mM Gly-HCl buffer (pH=2.5) eluting,
With 1M Tris (pH=9.0), the pH of eluted protein is adjusted to about 7.0.The rhIL-12 sample of purification
Purity reaches more than 80% (result is shown in Fig. 5).
4. molecular exclusion chromatography: utilize Sephacryl S-200HR (purchased from GE company) to remove glycoprotein polyprotein precursor,
Final rhIL-12 purity of protein is more than 95% (result is shown in Fig. 6).
It should also be realized by those skilled in the art that what the present invention provided utilizes the affine layer of rHuIL-12
Ultrafiltration that the method for people IL-12 of analysis column purification restructuring is comprised, anion-exchange chromatography, affine layer
Order between analysis and four purification steps of molecular sieve is not changeless, and those skilled in the art can
To carry out suitable adjustment according to actual needs, for example, it is possible to after carrying out ultrafiltration step, advanced
Row affinity chromatograph, carries out anion-exchange chromatography the most again, etc..
The determination of activity of embodiment 4:rhIL-12
With reference to domestic patent 201010134960.9 (a kind of detection rhIL-12(p70) albumen
The method of activity, Tian Zhigang etc.), use IFN-γ to induce method, use Bender Systems company
The content of the Human IFN-γ ELISA kit detection IFN-γ produced.Concrete detecting step is as follows:
1. standard substance and the dilution of measuring samples: IL-12 standard substance (purchased from NIBSC) are complete with 1640
25.6,12.8 culture fluid (purchased from GIBCO company) is diluted to following 11 dilution factors (ng/ml):,
6.4,3.2,1.6,0.8,0.4,0.2,0.1,0.05,0.025, measuring samples is also diluted to following
11 dilution factors (ng/ml): 25.6,12.8,6.4,3.2,1.6,0.8,0.4,0.2,0.1,0.05,
0.025, standby；
2.IFN-γ induces: collect 37 DEG C, 5%CO2The NKG cell being in exponential phase cultivated
(CGMCC No.2901, purchased from China Microbiological bacterial strain preservation committee's common micro-organisms center
(CGMCC)) in 50ml sterile centrifugation tube, 800rpm, 10min, centrifugal, abandon supernatant,
After PBS solution washs twice, being suspended from containing in 1640 complete mediums, adjusting cell concentration is 5 × 105
Individual/ml, adds 96 orifice plates by 100 μ l/ holes by cell.Add in the 96 orifice plate corresponding apertures added with cell
Enter the different dilution IL-12 standard substance of 100 μ l and supernatant to be measured (each dilution factor does duplicate hole).
37 DEG C, 5%CO2After cultivating 24 hours, every hole takes 50 μ l culture supernatant ELISA and measures IFN-γ
Content；
The mensuration of 3.IFN-γ: with reference to Bender Systems company Human IFN-γ ELISA kit
Description, makees standard curve by the concentration of standard substance and corresponding A value, inputs measuring samples A value
Directly obtain measuring samples IFN-γ content；
4.IL-12 activity calculates: with IFN-γ content, sample dilution is made curve, calculates each experiment sample
The ED50 of product (sample concentration when i.e. IFN-γ content is the half of Cmax), and count as the following formula
Calculation result: titer=Pr × ED50r/ED50s.Pr: standard substance titer, IU/mg；ED50r: standard
Product ED50；ED50s: measuring samples ED50.
Result shows, its specific activity of rhIL-12 of the affine method purification of the embodiment of the present invention 3 exceed
5.0×106IU/mg, therefore, according to said method the IL-12 of purification has higher activity.
Although it should be understood that with reference to its exemplary embodiment, the present invention being carried out specifically
It is shown and described, it should be understood by those skilled in the art that without departing substantially from by appended power
Under conditions of profit requires defined the spirit and scope of the present invention, various forms can be carried out wherein
With the change of details, the combination in any of various embodiment can be carried out.
Claims (7)
1. the monoclonal antibody utilizing anti-human IL-12 prepares people's IL-12 affinity purification chromatographic column
Method, described method comprises the steps:
A. activated gel pretreatment: take appropriate activated gel, is completely dissolved with 1mM HCl,
And rinse colloid with 1mM HCl, use 0.1M NaHCO the most again3Rinse colloid；
The coupling of the most anti-human IL-12 antibody and the preparation of affinity column: the method being concentrated by ultrafiltration
By anti-human IL-12 Antibody Concentration to 4-5mg/mL, then anti-human IL-12 antibody is added in step a
In the activated gel of pretreatment, put 4 DEG C overnight, then use 0.1M NaHCO3Rinse colloid, use
The unconjugated active group of 0.1M Tris-HCl buffer blind, then buffers with 0.1M Tris-HCl
Liquid and 0.1M Acetic acid-sodium acetate buffer alternately rinse colloid, finally will be in conjunction with anti-human IL-12 antibody
Colloid dress post, be prepared as the affinity column of Purification of Human IL-12；
Wherein the activated gel described in step a selected from CNBr activation Sepharose 4B gel,
The Sepharose 4FF of Sepharose 4FF or the NHS activation of CNBr activation, and
Wherein said anti-human IL-12 antibody is prepared by following step: the full Freund adjuvant that cannots be used up processes
The nude mice of 8-10 week old, every mouse peritoneal injects 500 μ L incomplete Freund's adjuvants；After 3 days, will
Preserving number is that the hybridoma of ATCC CRL-2382 is expelled in nude mice abdominal cavity, every nude mice
1×106Individual cell；Producing ascites after 7-10 days, collect nude mice ascites, purification prepares described anti-human IL-12
Antibody.
2. a method of the people IL-12 of purification of Recombinant, described method includes ultrafiltration, anion exchange
Chromatography, affinity chromatograph and four purification steps of molecular sieve, the feature of described step is as follows:
A. ultrafiltration: obtain the culture supernatant containing rHuIL-12, selects the ultrafilter membrane of 10-30kD,
Supernatant is concentrated by ultrafiltration 10-15 times, and 0.45 μm membrane filtration removes particulate matter；
B. anion-exchange chromatography: use anion-exchange chromatography post, 20mM Tris-HCl balance liquid
Balance, 40mM histidine buffering liquid eluting foreign protein, 0.25M NaCl 20mM Tris-HCl eluting
Destination protein；
C. affinity chromatograph: use the method preparation of claim 1 to come pure for the chromatographic column of affinity chromatograph
Change rHuIL-12, balance with 20mM Tris-HCl balance liquid, buffer with 20mM Gly-HCl
Liquid eluting, is adjusted to 7.0 with 1M Tris by the pH of eluted protein；
D. molecular sieve: utilize molecular sieve to remove glycoprotein polyprotein precursor, obtain the rHuIL-12 monomer of purification.
Method the most according to claim 2, the wherein said culture supernatant containing rHuIL-12
Obtained by cultivating the Chinese hamster ovary celI expressing rHuIL-12.
Method the most according to claim 2, wherein said anion-exchange chromatography is with being filled with
With Sepharose, Capto, Sephadex or Sephacel as skeleton and with Q, QAE, DEAE
Carry out for the anion-exchange chromatography post of the gel of active group.
Method the most according to claim 4, wherein said anion-exchange chromatography post is selected from
Q-Sepharose FF, Capto Q, Q-Sepharose HP, Capto DEAE, QAE Sephadex,
DEAE-Sepharose FF, or DEAE Sephadex.
Method the most according to claim 2, wherein said molecular sieve be with Sephacryl,
Superdex, Sephadex, Superose or Sepharose are the gel of skeleton.
Method the most according to claim 6, wherein said molecular sieve is selected from Sephacryl S-200
HR, Superdex 200 or Sephadex G-100.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1119018A (en) * | 1993-03-05 | 1996-03-20 | 先灵公司 | Purification of human interleukin-10 |
| CN1417340A (en) * | 2001-11-08 | 2003-05-14 | 武汉大学 | Expression of human interleukin-12 in craze noctuid and its purifying process |
| CN1467292A (en) * | 2003-05-27 | 2004-01-14 | 中国科学技术大学 | Highly effective expressing method of interleukin-12 |
| US20060073591A1 (en) * | 2004-01-09 | 2006-04-06 | Abitorabi M A | Cell culture media |
-
2012
- 2012-03-31 CN CN201210093656.3A patent/CN103360461B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1119018A (en) * | 1993-03-05 | 1996-03-20 | 先灵公司 | Purification of human interleukin-10 |
| CN1417340A (en) * | 2001-11-08 | 2003-05-14 | 武汉大学 | Expression of human interleukin-12 in craze noctuid and its purifying process |
| CN1467292A (en) * | 2003-05-27 | 2004-01-14 | 中国科学技术大学 | Highly effective expressing method of interleukin-12 |
| US20060073591A1 (en) * | 2004-01-09 | 2006-04-06 | Abitorabi M A | Cell culture media |
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