CN1563090A - High performance quick purifying method for preparing piecewise antibody - Google Patents
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- CN1563090A CN1563090A CN 200410026022 CN200410026022A CN1563090A CN 1563090 A CN1563090 A CN 1563090A CN 200410026022 CN200410026022 CN 200410026022 CN 200410026022 A CN200410026022 A CN 200410026022A CN 1563090 A CN1563090 A CN 1563090A
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Abstract
The invention discloses a method for high-effectively quickly purifying and preparing fragment antibody from animal cell scale culture. Said method adopts the processes of dilatant column bed adsorption, anion chromatography concentration and hydrophobic chromatography purification, and includes the following steps: using dilatant column bed cation chromatography to adsorb antibody IgG from large-scale cell culture, changing pH value of buffer system to make elution, making collected elution peak be directly andergone the process of anion chromatography, using pepsinum to directly enzyme-out the fully-concentrated and purified antibody IgG to obtain fragment antibody, making hydrophobic chromatography and purification so as to obtain final fragment antibody whose purity is up to above 97%, and its whole purification process can be completed within 12 hr. Said invented method can be substituted for traditional ammonium sulfate precipitation, concentration method, and has the characteristics of high efficiency, rapid speed, high purity and stable operation and result, etc.
Description
Technical field
The invention belongs to the purified technology of protein of technical field of bioengineering, relate generally to antibody class and comprise monoclonal antibody and small molecule segment antibody, the purifying preparation technology of humanization or people-mouse chimeric antibody and fragment antibody.Be particularly related to the method that a kind of efficient fast purifying prepares fragment antibody, be applicable to that treatment prepares with the antibody class medicine production.
Background technology
Antybody therapy has become the important means of current biotherapy tumour, popularity and infectious diseases.To in February, 2004,16 Antybody therapy medicine listings of U.S. FDA approved, the market sales revenue surpasses 2 * 10
9U.S. dollar.The antibody class medicine becomes a big class new therapeutic agent on the international drug market with specificity, validity and the security of its height.In 16 Antybody therapy medicines, 12 are humanization or human mouse chimeric antibody (U.S. FDA webpage http://www.fda.gov).
Radioimmunotherapy is (Radioimmuno-therapy, RIT) with antibody be carrier, the radionuclide that carries concentrated dense gathering by means of the guide effect of antibody, thereby improve the tumor cytotoxicity effect, reduce normal tissue injury, in 16 Antybody therapy medicines, Y is arranged
90And I
131(90Y-IbritumomabIDEC 2002 for the mouse originality antibody of two kinds of radioisotope labelings; Tositumomab and Iodine I
131Tositumomab Corixa 2003) is used for tumor targeting therapy.F (ab ') 2 fragment antibodies and Fab small molecular antibody, the relatively anti-full characteristics such as to have immunogenicity low, and penetration of tumours is stronger, and the tumor locus residence time is long of mouse source property.Therefore be more suitable for targeting vector as the tumour radiotherapy immunotherapy.
Animal cell large-scale is cultivated and is produced the main flow that the protein for treatment medicine has become current field of biological pharmacy.The treatment of clinical many antibody indications, antibody needs with higher dosage or delay long-term prescription.Therefore, Eco-power process amplification is the important indicator of technology assessment.Expansion column bed adsorption technology can be with the direct upper prop of recovery liquid of large scale culturing, and a step is finished absorption, concentrated and purifying, reduces processing step, saves fund input and increases product yield, is accepted extensively [6] by the biological-pharmacy field.It is the most normal for adopting operating method [7] that Protein A affinity chromatography expansion column bed is adsorbed in the Antibody Preparation technology, because Protein A affinity chromatography is optionally effectively in conjunction with mammiferous IgG molecule, antibody in one step high efficiente callback and the purifying cells product mixtures, the rate of recovery is greater than 99.5%, and affinity chromatography also can effectively be removed virus simultaneously; Because the affinity chromatography operation steps is simply quick, accomplishes quality control and consistency of product [8] in complicated manufacturing process easily.
The unfavorable factor of Protein A affinity chromatography mainly is the stability [9] of technology cost height and synthetic resins.The total cost of Protein A affinity chromatography is higher than 30% of ion-exchange chromatography in Antibody Preparation technology, adds stratographic regeneration and other Master Cost, and cost rises to 35%[10].In addition, the deformable under treatment condition extremely of chromatogram ligand, the Protein A molecule that comes off has immunogenicity and can cause physiological response human body.Genentech company technology the most commonly used comprises three step chromatographys, Protein A affinity chromatography-cation chromatography-anion chromatographic [13].
Relevant F (ab ')
2The enzyme of fragment antibody is cut with purifying and is reported in recent years seldom.Early stage method is to adopt ammonium sulfate precipitation and thermal treatment combination, and this method was both time-consuming, and cost is high again, can't be as production; Affinity chromatography Protein A and Protein G are because they, can not be used for F (ab ') in conjunction with the Fc of IgG end
2Purifying [11].Morimoto andInouye and Inouye and Morimoto hydrophobic chromatography adsorption and purification F (ab ')
2Fragment antibody filters two kinds of methods at SDS-PAGE and glue and shows F (ab ')
2Higher uniformity is arranged in single chromatographic peak; Koichi Morimoto and Kuniyo Inouye Tsk gel Phenyl-5PW hydrophobic chromatography adsorption and purification IgGl type F (ab ')
2Fragment antibody, its purity reaches 98%, but it all carries out in the small scale experiments stage, does not enter the pilot scale amplification technique as yet and produces [12].
In the purifying process of therapeutic antibodies, remove purity, the rate of recovery of considering product, also the more important that must consider is the quality control of technological process, effectively remove impurity and comprise albumen, DNA, antibody isomer, fragment, and small molecules and potential pollutent are as intracellular toxin, filtrable virus etc. from host cell.Therapeutic F (ab ')
2The purifying process of fragment antibody is whole antibody relatively, and is more complicated with regard to its process control, needs to consider that more process is connected product yield, and [14] such as pollutions of residual enzyme amount and small molecules fragment.
The applicant is at anti-human liver cancer F (ab ')
2In the Antibody Preparation technology, adopt three step chromatogram purifications, the first step STREAMLINESP cation-adsorption chromatography obtains the IgG in the Hybridoma Cell Culture liquid; The negatively charged ion absorption of second step concentrates, and the stomach en-enzyme is cut IgG then; The 3rd step Phenyl-5PW hydrophobic chromatography purifying F (ab ')
2Fragment antibody is removed bacterial endotoxin simultaneously.In the whole technological process, the applicant compares analysis with regard to the key parameter of technological process, as product recovery rate and condition, technology is connected with method to be selected, and finished product purity keeps with active, and the amplification of technological process, select the optimum parameter standard, with obtain efficiently, F (ab ') fast
2Antibody purification technology.
At present relevant F (ab ')
2Fragment antibody purifying preparation technology's patent has following several pieces:
Mi Li, Chinese invention patents such as Chen Zhinan: ZL 99115730.3[1], name is called: at monoclonal antibody F (ab ')
2With the segmental preparation method of Fab, being described as of its claim:
Monoclonal antibody ascites is slightly carried secondary with 50% saturated sulfuric acid amine, F (ab ')
2Preparation is 1: 100 with stomach en-and the IgG enzyme ratio of cutting, 37 ℃ of reaction 2-3h; The Fab papoid, the ratio of enzyme and IgG 1: 100,37 ℃ of reaction 1-2h; HPLC hydrophobic liquid phase chromatography purifying F (ab ')
2, the Fab fragment antibody, divide secondarily purified with Phenyl Sepharose HighPerformance dewatering filling (Pharmacia): one time purifying A liquid is 1～1.4M sulfuric acid amine 0.05MpH5.0 sodium-acetate buffer, and B liquid is 0.05M pH5.0 sodium-acetate buffer; Secondarily purified A liquid is with 0.7～1.0M sulfuric acid amine 0.05M pH5.0 sodium-acetate buffer, and B liquid is 0.05M pH5.0 sodium-acetate buffer, 30-40 minute linear gradient elution time; Freeze-drying immediately behind the purifying adds that 10% low molecule dextrose is intoxicated does the renaturation agent.
Chinese invention patent such as Mi Li, Li Ling (ZL 01131736.1) [2], name is called: the method for continuously perfused culture of hybrid tumor cell preparation antibody, the description that relevant antibody reclaims purifying in its claim:
Product reclaims purifying, adopts Streamline SP medium, and a step is reclaimed the antibody in the culture supernatant, the thermal source matter behind the DEAE-Sepharose FF anionite removal purifying in the antibody product, and the freeze-drying of antibody finished product is preserved.Antibody product culture supernatant is reconciled pH value 4-7, electric conductivity value 2-5mS/cm; Balance liquid (A liquid) pH4-7; Elutriant (B liquid) A liquid+1M NaCl.With the thermal source matter in the antibody product behind the DEAE-Sepharose FF anionite removal purifying.Under the condition of pH4～7, thermal source is adsorbed on the post, and antibody flows out in passing the peak; Remove the thermal source that is adsorbed on the post with 1M NaCl and 0.5M NaOH again, pillar is reusable.
Ngo; The relevant antibody purification technology of That T. (United States Patent, No:4,933,435) [3], its claim is mainly described with stationary phase proteinA, proteinG chromatogram in conjunction with the purifying process that adsorbs immunoglobulin (Ig), the pH6-10 of used buffering system comprises at least a many carboxylic acids, concentration 0.5-0.9M.
Sullivan; John B, Russell; In the relevant antibody purification technology of Findlay E. (United States Patent, No 4,849, the 352) patent [4], the F that the Fab fragment antibody cut with poly benzene alkene acyl ammonia gel affinity chromatography separation papoid and stomach en-cut (ab ') is described
2Antibody is not described specifically the condition and the method for purifying process.
Ngo; Fixedly Protein A affinity chromatography monoclonal antibody purification technology patent (United StatesPatent of That T., No 4,801,687) [5], the adsorption-buffering liquid pH7.5-10 of processing condition immunoglobulin (Ig), buffer concentration 0.6-1.75M is the anionic buffering system of monovalent positively charged ion or polybase base.The desorption buffer conditions pH3-6 of immunoglobulin (Ig).
According to the data-searching that the applicant carried out, retrieve have below with reference to document relevant with the present invention:
[1] Mi Li, Chen Zhinan etc.Monoclonal antibody F (ab ')
2With the segmental preparation method of Fab, 2004, Chinese invention patent ZL99115730.3.
[2] Mi Li, Li Ling etc.The method of continuously perfused culture of hybrid tumor cell preparation antibody, 2004, Chinese invention patent ZL01131736.1.
[3]Ngo；That?T.Antibody?purification?process，1990.United?StatesPatent，No：4,933,435。
[4]Sullivan；John?B.Russell；Findlay?E.Antibody?purification?process，1989，United?States?Patent，No：4,849,352。
[5]Ngo；That?T.Monoclonal?antibody?purification?process?using?protein?A，1989，United?States?Patent，No：4,801,687。
[6]J.Feuser?a，M.Halfar?a，D.Lutkemeyer?b，etal.Interaction?of?mammalian?cellculture?broth?with?adsorbents?in?expanded?bed?adsorption?of?monoclonalantibodies.Process?Biochemistry?34(1999)159-165。
[7]Jorg?Thommes*，Andrea?Bader，Markus?Halfar，etal.Isolation?of?monoclonalantibodies?from?cell?containing?hybridoma?broth?using?a?protein?A?coatedadsorbent?in?expanded?beds.Journal?of?Chromatography?A.752(1996)111-122。
[8]R.L.Fahrner，D.H.Whitney，M.Vanderlaan，G.S.Blank，Performance?comparisonof?protein?A?affinity-chromatography?sorbents?for?purifying?recombinantmonoclonal?antibodies.Biotechnol.Appl.Biochem.30(1999)121。
[9]G.Hale，A.Drumm，P.Harrison，J.Phillips，Repeated?cleaning?of?protein?Aaffinity?column?with?sodium?hydroxide.J.Immunol.Methods.171(1994)15-21.
[10]Deborah?K.Follman.，Robert?L.Fahrner.Factorial?screening?of?antibodypurification?processes?using?three?chromatography?steps?without?proteinA.Journal?of?Chromatography?A，1024(2004)79-85。
[11]Peter?Kumpalume，Nigel?K.H.Slater.Comparative?study?of?thiophilicfunctionalised?matrices?for?polyclonal?F(ab_)
2?purification.Journal?ofChromatography?A，1022(2004)41-50。
[12]Koichi?Morimoto，Kuniyo?Inouye.Single-step?purification?of?F(ab’)
2fragments?of?mouse?monoclonal?antibodies(IgGl)by?hydrophobic?interaction?highperformance?liquid?chromatography?using?TSKgel?Phent-5PW.J.BiochemistryBiophysics?Methods..1992，24：107-117。
[13]R.L.Fahrner，H.L.Knudsen，C.D.Basey，W.Galan，D.Feuerhelm，M.Vanderlaan，G.S.Blank.Industrial?purification?of?pharmaceutical?antibodies：development，operation.and?validation?of?chromatograpgy?processes.Biotechnol.Genet.Eng.Rev.18(2001)301。
[14]R.L.Fahrner，D.H.Whitney，M.Vanderlaan，G.S.Blank，Performance?comparisonof?protein?A?affinity-chromatography?sorbents?for?purifying?recombinantmonoclonal?antibodies.Biotechnol.Appl.Biochem.30(1999)121。
Summary of the invention
At defective or the deficiency that above-mentioned prior art exists, the purpose of this invention is to provide a kind of efficient, method that fast purifying prepares fragment antibody.
Realize that foregoing invention purpose technical solution is,---anion chromatography concentrates---hydrophobic chromatography purifying three goes on foot purifying to adopt the absorption of expansion column bed, separation and purification IgG antibody from cell scale culture, enzyme is cut the back purifying and is prepared fragment antibody, same buffering system is adopted in the linking of whole purifying process, carry out wash-out by the method that changes change pH values, reduced technological process buffering system metathetical step.
At first adopt expansion column cationic layer to analyse the IgG antibody of adsorbing in the mass cell culture, carry out wash-out, collect IgG antibody by changing buffering system pH value (pH7-9); Directly carry out anion chromatography again, carry out wash-out by changing buffering system pH value (pH3-6) equally, reach the purpose of concentrated and purified IgG antibody; F (ab ') is cut and obtained to the IgG antibody of collecting this moment by the stomach en-enzyme
2Fragment, the sample after enzyme is cut be through hydrophobic chromatography, the F that gets access to (ab ')
2Work in-process purity is not less than 98%, the complete conformance with standard of quality arbitration.
Technical characterstic of the present invention is: (1) purifying process process is reasonable in design, and the result shows that---negatively charged ion is concentrated and purified---enzyme is cut---, and several steps of sulphur desalt have reached good separating effect to the separation of process cation-adsorption; (2) same buffering system is adopted in the linking of purifying process, carries out wash-out by the method that changes change pH values, has saved buffering system and has changed step, and purge process is connected closely before and after making; (3) passing through positively charged ion---the anion chromatographic concentration method substitutes traditional ammonium sulphate precipitation concentration method.Whole purifying process process was finished in 12 hours, its fragment antibody purity is not less than 98%, reach efficient, fast, cost is low, purity is high, stable, batch between characteristics such as result difference is little, the activity that keeps fragment antibody effectively is suitable for purifying and preparation that monoclonal antibody, enzyme are cut back antibody and gene engineering expression small molecular antibody especially.
The present invention includes following steps:
1) in the novel process for preparing IgG antibody, adopt two-step approach promptly earlier with the IgG antibody in the Streamline SP positively charged ion chromatography collection large volume Hybridoma Cell Culture thing, concentrate with the anion chromatography purifying again.The positively charged ion chromatography adopts the pH condition that changes damping fluid to carry out the wash-out (promptly adopting buffering system B to carry out wash-out) of target protein, and this buffering system just in time is an anion chromatography adsorption-buffering system, and therefore the protein solution of collecting can carry out anion chromatography.
2) adopting the main purpose of anion chromatography is the elutriant that concentrates the positively charged ion chromatography, cuts for next step enzyme and prepares.Anion chromatography also adopts the pH condition that changes damping fluid to carry out the wash-out (promptly adopting buffering system A to carry out wash-out) of target protein, and this buffering system just in time to be enzyme cut buffering system, and therefore the protein solution of collecting can carry out enzyme and cuts and prepare F (ab ')
2Fragment.
In this two steps chromatography process, all adopt the pH condition that changes damping fluid to carry out the wash-out of target protein.Purpose is saved time in order to have saved the process of exchange buffering liquid, makes purge process be connected closelyr.
3) sample after enzyme is cut need not precipitate, dialyse, and only needs to add the 0.8-1.5M solid ammonium sulfate, can carry out hydrophobic chromatography (the adsorption-buffering system adds ammonium sulfate for buffering system A), and the desorption buffering system is buffering system A simultaneously.
4) per step purification step sample all need be handled, and concrete parameter is as follows:
A. the Hybridoma Cell Culture thing that promptly reclaims of positively charged ion chromatography sample need not be clarified and concentrate, and being diluted to specific conductivity with ultrapure water is 4-6ms/ml, regulates pH then and goes up sample to the back.
B. the anion chromatography sample is that positively charged ion chromatography elutriant pH is 7-9.
C. to cut sample be that anion chromatography elutriant pH is 3-6 to enzyme.
D. the hydrophobic chromatography sample is that enzyme cuts that to stop back liquid pH be 3-6.
5) other parameter is as follows:
I positively charged ion chromatography flow velocity 100-200ml/min.
II anion chromatography flow velocity 5-15ml/min.
The III enzyme is cut time 0.5-2.0h, 37 ℃ of temperature.
IV hydrophobic chromatography flow velocity 3-10ml/min.
Above-mentioned buffering system (A) is the carboxylic acid buffering system, pH=3-6, concentration 0.01-0.1M; Buffering system (B) is the negatively charged ion buffering system of monovalent positively charged ion or polybase base, pH=7-9, concentration 0.01-0.05M.
The present invention has adopted parameter control first, sets pH, and electricity is led, flow velocity, and temperature index respectively goes on foot purifying with this standard control, and first in conjunction with the software of AKTA chromatographic system configuration, is provided with the purification scheme of automatic operation, has saved the time greatly, manpower.The present invention is suitable for fragment antibody, the enzyme of zooblast scale cultivation secreting, expressing especially and cuts back antibody and express the bad proteinic fast purifying preparation of rear stability.Have efficient, fast, purity is high, cost is low, stable and controllable strong, batch between characteristics such as result difference is little.
Description of drawings
Fig. 1 is F (ab ')
2The process flow sheet that fragment antibody is produced;
Fig. 2 is a positively charged ion chromatography color atlas;
Fig. 3 is a positively charged ion chromatography SDS-PAGE electrophorogram
Fig. 4 is the anion chromatography color atlas;
Fig. 5 is an anion chromatography SDS-PAGE electrophorogram;
Fig. 6 is restriction enzyme digestion and electrophoresis figure;
Fig. 7 is the hydrophobic chromatography color atlas;
Fig. 8 is a hydrophobic chromatography SDS-PAGE electrophorogram;
Fig. 9 is F (ab ')
2Fragment antibody SEC-HPLC figure.
Embodiment
The present invention is described in further detail below in conjunction with specific embodiment that accompanying drawing and contriver provide.
According to technical scheme of the present invention, the applicant adopts hybridoma excretory monoclonal antibody to carry out F (ab ')
2The preparation of fragment antibody.This hybridoma stably excreting is expressed monoclonal antibody (China Committee for Culture Collection of Microorganisms's common micro-organisms preservation center preservation, the preserving number: CGMCC No 0426) of anti-human liver cancer related antigen.
Use the mechanical agitation type bio-reactor, large scale and high density suspension culture hybridoma; Adopted the IgG antibody in the expansion column bed cation chromatography absorption mass cell nutrient solution in early stage and concentrate with anion chromatography, two step chromatographies all adopt by the method that changes buffering system pH value and carry out wash-out.IgG antibody is obtained F (ab ') by pepsin
2Fragment at last by hydrophobic chromatography, is obtained complete standard compliant F (ab ')
2Work in-process, whole purge process only need 12 hours, can freeze-drying make fragment antibody work in-process (preparation F (ab ')
2The fragment antibody process flow sheet is referring to Fig. 1).
Technology of the present invention is obtained F (ab ') with the continuous purification process
2Fragment antibody, its technological process mainly divide following a few step:
1. positively charged ion chromatography: reclaim the monoclonal antibody IgG in the purifying nutrient solution.
2. anion chromatography: concentrate the positively charged ion chromatography and collect liquid.
3. enzyme is cut: obtain F (ab ')
2Fragment antibody.
4. hydrophobic chromatography: the F (ab ') that obtains high purity, aseptic, no thermal source, highly active meeting " preparation of human mouse monoclonal antibody and key Quality Control " [defending medicine Chinese character (90) No. 37] standard
2Fragment antibody.
5. product is identified: comprise experiments such as aseptic, no thermal source, purity check, active detection, rate of recovery calculating.
Below above-mentioned steps is further elaborated.
One, positively charged ion chromatography
(1) the Hybridoma Cell Culture thing of Hui Shouing need not be clarified and concentrate, and is diluted to specific conductivity 4-6ms/cm with ultrapure water, regulates pH to 3-6.
(2) expansion column Streamline-50 (Pharmacia Biotech Sweden), adsorption medium: Streamline SP; BT00-300M, BT00-100M type peristaltic pump each (Baoding Lange constant flow pump company limited), HD-21-88 type nucleic acid-protein detector (the special Analytical Instrument Co., Ltd of Shanghai fine jade) and YOKOGAWA 3057 potable recording instrument (Sichuan instrument four factories).
(3) adsorption-buffering system: buffering system A (citrate buffer solution pH3-6); Desorption buffering system: buffering system B (Tris-HCl pH7-9).
(4) earlier with the abundant balanced expansion post of buffering system A bed Streamline, last sample goes not conjugated protein with buffering system A flushing after finishing again; Use the direct wash-out of buffering system B then, collect elution peak.
Two, anion chromatography
(1) regulating positively charged ion chromatography elution peak pH is 7-9.
(2) purification system: AKTA purifier-100; Chromatographic column: XK16/20 (Pharmacia Biotech Sweden); Chromatography media: DEAE SepharoseTM Fast Flow (Pharmacia Biotech Sweden).
(3) sample is gone up in the absorption of buffering system B condition, (pH7-9); Buffering system A desorption (pH is 3-6).
(4) the abundant balance DEAE post of buffering system B, last sample goes not conjugated proteinly again with buffering system B flushing after finishing, use the direct wash-out of buffering system A then, collects elution peak.
Three, enzyme is cut
(1) stomach en-(crystallization product, Sigma company), stop buffer 3mol/l Tris.
(2) enzyme is cut buffering system: buffering system A (citrate buffer solution pH3-6).
(3) stomach en-mixes 1: 100 in proportion (g/g) with antibody, 37 ℃ of water-baths, and 0.5-2h adds 3mol/lTris and adjusts about pH value to 7.0 termination reaction.
Four, hydrophobic chromatography
(1) regulatory enzyme is cut stop buffer pH to 3-6; Add 0.8-1.5M ammonium sulfate.
(2) purification system: AKTA purifier-100 (Amersham Biosciences Sweden); Chromatographic column: XK16/20 (Amersham Biotech Sweden); Chromatography media: Phenyl Sepharose High Performance (PharmaciaBiotech Sweden).
F after (3) buffering system A adding 0.8-1.5M ammonium sulfate adsorptive enzyme is cut (ab ')
2(citrate buffer solution pH3-6); Buffering system A gradient elution.
(4) with the abundant balance drainage column of adsorption-buffering system, last sample goes not conjugated proteinly again with adsorption-buffering system flushing after finishing, then with desorption buffering system gradient elution, collect elution peak, is F (ab ')
2Fragment antibody.
Five, product is identified
A. sterility test is pressed 79 pages of " biological products preparation and vertification regulation " 1995 editions two appendix of Chinese Pharmacopoeia, and cell cultures sterility test result should be negative.
B. bacterial endotoxin inspection is undertaken by 2000 editions " Chinese biological goods rules " general rules " biological products bacterial endotoxin test rules "; Bacterial endotoxin should＜0.5 (Eu) as a result.
C. determination of recovery rates, ELISA method are measured target protein concentration before and after the chromatography.According to the following formula calculate recovery rate:
D. purity testing adopts SDS-PAGE, carries out electrophoresis under non-reduced condition, and electrophoresis result is after Xylene Brilliant Cyanine G R-250 dyeing, with Shanghai image analysis system scanning in multiple day, and with the purity of software analysis product.
The result
1. the result who prepares IgG antibody
A. know by Fig. 2,3, by a large amount of foreign proteins of positively charged ion chromatography in passing the peak, and do not have target protein loss, target protein (IgG antibody) all concentrates (can concentrate 20 times) in elution peak and by obvious purifying, wherein 1,2,3 represents stoste respectively among Fig. 3, passes liquid, elutriant;
B. know by Fig. 4,5 that in passing the peak, target protein (IgG antibody) is obviously concentrated by the anion chromatography foreign protein in elution peak, and by purifying (can concentrate 10 times) once more.Wherein 1 among Fig. 5 ', 2 ', 3 ' represents that respectively anion chromatography stoste, anion chromatography pass peak, anion chromatography elution peak;
C. be 89.31% by positively charged ion, two step of the anion chromatography IgG antibody rate of recovery.
2. the enzyme cutting is equipped with F (ab ')
2The result
Know by Fig. 6, can carry out enzyme after the elution peak of anion chromatography is regulated and cut, and enzyme is cut efficient and reached 97%." represent F (ab ') respectively 1 ", 2 ", 3 wherein
2Standard substance, enzyme are cut preceding sample, enzyme is cut the back sample.
3. hydrophobic chromatography purification result
A. know by Fig. 7,8, and process hydrophobic chromatography F (ab ') the effective purifying of 2 fragments quilt.Wherein the 1* among Fig. 8,2*, 3* represent that respectively hydrophobic chromatography stoste, hydrophobic chromatography pass liquid, hydrophobic chromatography elutriant.
The b.SDS-PAGE electrophoresis result is through software analysis, and the F behind the hydrophobic chromatography purifying (ab ') 2 fragment purity are 98.9%;
C. know by Fig. 9, F behind the hydrophobic chromatography purifying (ab ')
2Fragment purity is 99.63%.
4. sterility test result
No bacterial growth.
5. bacterial endotoxin inspection
Bacterial endotoxin＜0.5 (Eu).
Claims (4)
1. an efficient fast purifying prepares the method for fragment antibody,---anion chromatography concentrates---hydrophobic chromatography purifying three goes on foot purifying to adopt the absorption of expansion column bed, separation and purification IgG antibody from cell scale culture, enzyme is cut the back purifying and is prepared fragment antibody, same buffering system is adopted in the linking of whole purifying process, carries out wash-out by the method that changes change pH values, has reduced technological process buffering system metathetical step, it is characterized in that, may further comprise the steps:
(1) separation and purification IgG antibody in the zooblast scale culture earlier with the IgG antibody in the Streamline SP positively charged ion chromatography absorption zooblast scale culture, obtains the IgG antibody of preliminary purification, and its process parameter is:
A.Streamline SP positively charged ion chromatography, animal cell culture need not be clarified and concentrate, and being diluted to specific conductivity with ultrapure water is 4ms/ml～6ms/ml, and adsorption conditions is directly gone up sample with buffering system (A);
B. the desorption condition is with buffering system (B), and directly wash-out is collected elution peak;
(2) anion chromatography, IgG antibody is by fully concentrated and purified; The stomach en-enzyme is cut and is obtained F (ab ')
2Fragment; Its process parameter is:
A. adsorption conditions is with buffering system (B), and positively charged ion chromatography elution peak is directly gone up sample; The desorption condition is collected elution peak, further concentrated and purified IgG antibody with buffering system (A);
B. enzyme is cut with buffering system (A), and enzyme and antibody 1: 100 (g/g) mix, and 37 ℃ of water-bath 0.5h-2h adjust the pH=6-8 termination reaction;
(3) hydrophobic chromatography purifying fragment antibody, its process parameter is:
A. adsorption conditions adds 0.8-1.5M ammonium sulfate with buffering system (A), and the sample after enzyme is cut is handled the back equally and gone up sample,
B. the desorption condition is collected elution peak with buffering system (A) gradient elution.
2. the fast purifying described in claim 1 prepares the method for fragment antibody, it is characterized in that, IgG antibody in the zooblast scale culture, the monoclonal antibody IgG that comprises Hybridoma Cell Culture, or CHO, SP2/0, NSO cell cultures secretor type people-mouse chimeric antibody IgG or humanized antibody IgG.
3. the fast purifying described in the claim 1 prepares the method for fragment antibody, it is characterized in that, buffering system (A) is the carboxylic acid buffering system, pH=3-6, concentration 0.01-0.1M; Buffering system (B) is the negatively charged ion buffering system of monovalent positively charged ion or polybase base, pH=7-9, concentration 0.01-0.05M.
4. the fast purifying described in the claim 1 prepares the method for fragment antibody, it is characterized in that, the fragment antibody purity that described three step purifying methods obtain is not less than in 97%, and whole purge process can be finished in 12 hours.
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