CN103505908A - Method for continuously separating and purifying antibody with mixed-mode chromatography medium - Google Patents
Method for continuously separating and purifying antibody with mixed-mode chromatography medium Download PDFInfo
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- CN103505908A CN103505908A CN201310415434.3A CN201310415434A CN103505908A CN 103505908 A CN103505908 A CN 103505908A CN 201310415434 A CN201310415434 A CN 201310415434A CN 103505908 A CN103505908 A CN 103505908A
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Abstract
The invention belongs to the technical field of antibody purification, and relates to a method for continuously adsorbing, separating and purifying an antibody with a mixed-mode chromatography medium, in particular to a method for separating and purifying an antibody with a multi-chromatographic column cascade continuous adsorption device. The method for continuously adsorbing, separating and purifying the antibody effectively solves the problems that a chromatographic column is limited in treatment capacity, the product purity and yield are low, and the like in the existing antibody purification process by using a mixed-mode chromatography technology. The antibody carrying capacity, and antibody yield and purity of the chromatographic column are increased and improved based on continuous separation and purification of the antibody. The mixed-mode chromatography medium is low in price, aglucon is not easy to fall, and the problems such as product contamination, reduction of dynamic adsorption capacity and the like due to fall of the protein A chromatography medium aglucon are effectively avoided. A preparation method of the chromatography medium by taking 4-(1-imidazole-group) phenylamine as the aglucon is simple to operate and low in cost, and can increase single column productive capacity of the antibody by above 2.5 times, and the antibody purity is higher than 90%.
Description
Technical field
The present invention relates to a kind of method of utilizing mixed mode chromatography media continuous adsorption technology antibody purification, specifically a kind of by the method for many chromatography column in series continuous adsorption device separation and purification antibody, belong to antibody purification technical field.
Background technology
Antibody is the special protein molecule of a class, is widely used in, in the Clinics and Practices of the diseases such as tumour and along with continuous development, having formed the three generations's antibody that comprises polyclonal antibody, monoclonal antibody and genetic engineering antibody.By the end of the year 2012,30 kinds of monoclonal antibody and genetic engineering antibody medicines that are used for the treatment of cancer, infection and immune deficiency disorder have been ratified to surpass in countries in the world; 2011, antibody drug occupied four seats in global sales amount of medicine top ten then, and sales volume is over 25,000,000,000 dollars.
Antibody product is mainly derived from the biological raw materials such as the plasma fractions of donor and all kinds of animals or animal cell culture supernatant.At present, antibody purification process is roughly divided into the precipitation method and chromatography.Precipitated separation technology is Main Means prepared by early antibody goods, the current main technique of extracting antibody from the body fluid such as blood plasma that is still of Cohn cold ethanol sedimentation mainly representing as it.Though it is simple, safe that the precipitation method have advantages of technique, working condition is harsh, the yield of product and purity is lower, automatization level is low is also the intrinsic persistent ailment of the precipitation method.In addition recovery, discharge and the pollution problem that, precipitation reagent causes is also to merit attention most in antibody preparation.By comparison, chromatography because possessing process conditions gentleness, flux is large, automaticity is high, sterilization easy to clean, meet the existing advantages such as GMP, is widely used in the separation and purification of antibody.Current for antibody purification mainly contain gel permeation chromatography, ion-exchange chromatography, hydrophobic chromatography, reversed phase chromatography and protein A affinity chromatography, wherein protein A affinity chromatography, anion-exchange chromatography, cation-exchange chromatography coupling antibody purification have become " golden standard " technique prepared by monoclonal antibody.But as the protein A affinity chromatography technology of above-mentioned technique core technology also exist that chromatography media is with high costs, wash-out and the defect such as regeneration condition is harsh, the ph stability of albumin A aglucon is poor and easily come off; the simultaneously common not enough 30mg/g medium of antibody carrying capacity of albumin A chromatography media coming off or enzymolysis and worsening along with albumin A aglucon; cause large-scale production process medium demand huge, raise production cost.
Above-mentioned defect is restricted albumin A chromatography media in actual applications, and novel chromatography medias some alternative albumin A chromatography medias, that the micromolecular compounds such as pigment derivative, heterocyclic compound of take are aglucon arise at the historic moment thus.The U.S. quite MEPHyperCel medium of your (Pall) company is exactly representative wherein.MEP HyperCel medium be a kind ofly in antibody separation, adopted, with 4-mercapto-ethyl-pyridine (4-Mercapto-Ethyl-Pyridine, MEP) be commercialization mixed mode chromatography (mixed-mode chromatography, the MMC) medium of aglucon.The pKa of described MEP aglucon is 4.8; When pH is greater than 4.8 (be generally 6～9 scope in), aglucon is combined with antibody by hydrophobic interaction; When pH is less than 4.8, (be generally 4.0 left and right), aglucon and antibody is all with positive charge, antibody because of and MEP aglucon between Coulomb repulsion wash-out, thereby realize the separation and purification of antibody.US Patent No. 6,498,236B1(UpFront Chromatography A/S company) chromatography media of the carboxylic aromatic compound of another kind of solid phase material surface coupling is disclosed for the separation of antibody.Chinese patent CN101185881A discloses a kind of chromatographic media for immunoglobulin class protein separation and preparation method thereof.Described chromatographic media be the coupling of spacerarm end contain imidazole group and phenyl ring dicyclic compound molecule as chromatographic media aglucon; The chromatography media antagonist preparing has selectively, can realize direct separation and purification antibody from blood plasma, ascites, cell culture supernatant.Bak and Thomas by the multiple MMC chromatography media that comprises MEP HyperCel performance and the albumin A chromatography media in antibody separation and purification carried out systematically comparing (J Chromatogr B, 2007,848:116-130).Result shows, antibody carrying capacity and the product purity of MMC chromatography media are all starkly lower than albumin A chromatography media.Therefore, MMC chromatography media replaces albumin A chromatography media and commercial road is still very long.
The key addressing the above problem is how to guarantee under the prerequisite of product yield, improves disposal ability and the product purity of MMC chromatography.For an existing MMC chromatographic column, between the disposal ability of chromatographic column and the yield of product and purity, normally mutually restrict.The lifting of chromatographic column disposal ability (applied sample amount) normally take that to sacrifice the yield of product be cost.In order to promote the disposal ability of albumin A chromatographic column, the people such as Mahajan proposed a kind of " semicontinuous " chromatography antibody purification technique (J Chromatogr A, 2012,1227:154-162).This technique is characterised in that, when albumin A chromatographic column (I post) exit AC reach concentration of raw material 1% after, a series connection access new albumin A chromatographic column (II post) after this chromatographic column, by that analogy; When I column outlet place AC reaches feed concentration 70%, adopt successively cleaning buffer solution and elution buffer to rinse I post wash-out antibody product then.This " semicontinuous " chromatography method is when improving albumin A chromatographic column disposal ability, and host protein in product (host cell protein, HCP) residual concentration also increases along with the rising of the antibody carrying capacity of albumin A chromatography media.Meanwhile, the automation of the complex operations of above-mentioned " semicontinuous " chromatography method is controlled more difficult.
Summary of the invention
Technical problem to be solved by this invention is in existing mixed mode chromatographic technique antibody purification technique that chromatographic column disposal ability is limited, product purity and the problem such as yield is low, thereby provide a kind of by many chromatography column in series continuous adsorption device, and the method for utilizing described mixed mode chromatography media continuous adsorption separation and purification antibody, on the basis producing in antibody separation and purification serialization thus, realize the raising of chromatographic column antibody carrying capacity and antibody yield and purity simultaneously.
For solving the problems of the technologies described above, the present invention is achieved by the following technical solutions:
A kind of mixed mode chromatography column in series adsorbent equipment, the chromatographic column of the mixed mode chromatography media that comprising that series connection arranges n is filled with MEP HyperCel chromatography media or ligand density to be 10-120mmol/g take 4(imidazoles-1-yl) aniline is aglucon, for carrying out separation and purification antibody from the biological raw material continuous adsorption antibody that comprises antibody and at least one foreign protein;
Wherein, the described 4(of take imidazoles-1-yl) the mixed mode chromatography media that aniline is aglucon is that to take Ago-Gel, cellulose or the hydroxylating methacrylic acid hydrophilic polymer microballoon that average grain diameter is 20～270 μ m be matrix, at described stromal surface coupling pi-allyl, then the chromatography media that the activation end group coupling 4(imidazoles-1-yl obtaining by pi-allyl and N-bromosuccinimide bromo-reaction) aniline is aglucon, its structural formula is M-L-AN; M represents matrix, and L represents the short-chain alkyl of being introduced by pi-allyl activation, and AN represents 4(imidazoles-1-yl) aniline.
Describedly take 4(imidazoles-1-yl) ligand density of the aniline mixed mode chromatography media that is aglucon is 21mmol/g-70mmol/g.Be preferably further 29-65mmol/g.
Described ligand density be 10-120mmol/g take 4(imidazoles-1-yl) preparation method of the aniline mixed mode chromatography media that is aglucon as:
The matrix M that repeatedly rinses and drain after moisture through deionized water mixes with dimethyl sulfoxide (DMSO), sodium hydroxide solution and allyl bromide, bromoallylene, in mixed liquor, the volume content of dimethyl sulfoxide (DMSO) (v/v) is 3-60%, the molal volume concentration of NaOH is 0.01-4mol/L, the mass volume ratio of matrix M and allyl bromide, bromoallylene (w/v) is 0.1-20, and mixed liquor reacts 1-64h and obtains pi-allyl activated media M-L in the shaking bath of 25 ℃ and 170rpm; Secondly, after activated media M-L cyclic washing, mix with water, dimethyl sulfoxide (DMSO) and N-bromosuccinimide, in above-mentioned mixed liquor, the volume ratio of water and dimethyl sulfoxide (DMSO) is 0-10, the volume content of dimethyl sulfoxide (DMSO) (v/v) is 3-70%, the mass ratio of activated media M-L and N-bromosuccinimide (w/w) is 0.5-7, and mixed liquor reacts 0.5-5h in the shaking bath of 25 ℃ and 170rpm; Finally, reacted activated media after deionized water is washed and is drained moisture with 4(imidazoles-1-yl) aniline, sodium hydroxide solution and dimethyl sulphoxide solution mix, activated media M-L and 4(imidazoles-1-yl) mass ratio (w/w) of aniline is 0.008-0.64, dimethyl sulfoxide (DMSO) is 0.2-5.0 times with the volume mass of activated media than (v/w), the molal volume concentration of NaOH is 0.01-1.0mol/L, above-mentioned mixed liquor reacts 1-72h in the shaking bath of 50 ℃ and 170rpm, obtains.
In described mixed mode chromatography column in series adsorbent equipment, n=2-5.
Utilize mixed mode chromatography column in series adsorbent equipment continuous separate from a method for antibody purification, comprise the steps:
(1) biological raw material sample chromatography, that contain target antibody product is treated in preparation;
(2) described biological sample is diluted with suitable level pad, the biological sample of dilution is input to described mixed mode chromatography column in series continuous adsorption device;
(3) input of dilution biological sample reaches after certain volume, leaves adsorption step enter into rinsing step in series connection continuous adsorption device chromatographic column foremost; Meanwhile, a new chromatographic column after regeneration is processed is linked into series connection continuous adsorption device least significant end; Dilution biological sample continues to be input to continuously in new series connection continuous adsorption device;
(4) to the chromatographic column that enters into the absorption antibody of rinsing step, inputting described level pad rinses;
(5) with applicable elution buffer by antibody wash-out from the chromatographic column after step (4) is rinsed, collect eluting peak, obtain antibody-solutions;
(6) input successively the chromatographic column after wash-out in described regeneration buffer solution and level pad regeneration step (5).
In described step (6), also comprise the chromatographic column after regeneration is linked into series connection continuous adsorption device least significant end, continue on for the continuous adsorption of antibody and the step of separation and purification.
In described step (4), also comprise the step that the chromatographic column effluent thing of described flushing is collected, for the dilution biological sample for chromatography absorption described in step (2).
In described step (2), also comprise the centrifugal step of the biological sample of described dilution, for removing the insoluble matter of biological sample and the impurity component of precipitation.
Described level pad comprises the 20mmol/L PB buffer solution of the 50mmol/L Tris-HCl buffer solution of the level pad that is applicable to described MEP HyperCel chromatography media: pH8.0-8.5 or the 10mmol/L PBS buffer solution of pH7.4 or pH6.0-8.0; And be applicable to described 4(imidazoles-1-yl of take) level pad of the aniline mixed mode chromatography media that is aglucon: the 50mmol/L Tris-HCl buffer solution containing 0.15-0.5mol/L NaCl of pH8.0-8.5 or the 10-50mmol/L PB buffer solution containing 0.15-0.5mol/L NaCl of pH6.0-8.0;
The concentration of described NaCl is 0.3-0.5mol/L.
Described dcq buffer liquid comprises the dcq buffer liquid that is applicable to described MEP HyperCel chromatography media: described in be applicable to the level pad of described MEP HyperCel chromatography media or be applicable to the level pad of described MEP HyperCel chromatography media containing described in 25mmol/L Sodium Caprylate; And be applicable to described 4(imidazoles-1-yl of take) the dcq buffer liquid of the aniline mixed mode chromatography media that is aglucon: described in be applicable to described 4(imidazoles-1-yl of take) level pad of the aniline mixed mode chromatography media that is aglucon;
Described elution buffer comprises the elution buffer that is applicable to described MEP HyperCel chromatography media: the citrate buffer of the acetate buffer of the 50mmol/L of pH4.0 or phosphoric acid-citrate buffer of the 20-100mmol/L of pH3.0-4.0 or the 50mmol/L of pH3.0-4.0; And be applicable to described 4(imidazoles-1-yl of take) elution buffer of the aniline mixed mode chromatography media that is aglucon: the 50mmol/L acetate buffer of pH4.0-5.0 or the 20mmol/L citrate buffer of pH3.5-4.0;
The described chromatographic column buffer solution used of regenerating comprises the NaOH solution of the regeneration buffer solution that is applicable to described MEP HyperCel chromatography media: 0.5-1.0mol/L or the 0.1mol/L citrate buffer of pH2.5 or the acetum of 0.1mol/L; And be applicable to described 4(imidazoles-1-yl of take) the regeneration buffer solution of the aniline mixed mode chromatography media that is aglucon: the NaOH solution of 0.5-1.0mol/L or the 0.1mol/L citrate buffer of pH2.5 or the acetum of 0.1mol/L.
In described step (1), described biological raw material behaviour source, the zoogenous cell conditioned medium liquid that contains antibody that contains antibody plasma or obtain through animal cell culture.
Mixed mode chromatography (mixed-mode chromatography) is a kind of finger based on two kinds and two or more fixedly phase aglucon and the interactional chromatographic technique between solute.The basis of mixed mode chromatography is that chromatography is fixedly on good terms multi-acting force is provided simultaneously, as fixing similar alkyl chain and the charge-site of comprising simultaneously, can provide hydrophobic effect power and electrostatic force, realizes reversed phase ion exchange mixed mode chromatography.Due to the existence of multi-acting force, mixed mode chromatography can improve separation selectivity significantly.Until nearly ten years, researchers just recognize that this fixing to have the chromatography pattern of several functions group on mutually may be a kind of important supplement to existing LC technology gradually.
Technique scheme of the present invention has the following advantages compared to existing technology:
(1) mixed mode chromatography column in series continuous adsorption device of the present invention, utilize a plurality of chromatography column in series to form, separation antibody from the biological raw material that comprises antibody and at least one foreign protein, has effectively avoided the problems such as antibody treatment amount is low in existing single-column chromatographic purifying antibody technique; Realize the serialization of antibody and produced, and can when improving antibody treatment ability, obtain high yield and highly purified antibody product;
(2) between described mixed mode chromatography media and antibody, there is the compatibility higher than impurity, thereby can realize accordingly antibody, with the separated of impurity and by antibody, the metathesis of impurity is further improved to the selective of chromatography;
(3), in the method for separation and purification antibody of the present invention, described take 4(imidazoles-1-yl) aniline is aglucon mixed mode chromatography media preparation method is simple to operate, with low cost, soda acid better tolerance;
(4) adsorbent equipment of the present invention, the chromatographic column in described chromatography column in series adsorbent equipment can recycle, and saves medium, thereby reduces costs;
(5) method of separation and purification antibody of the present invention, utilize the continuous adsorption device of described series connection, not only further promoted the efficiency of described separation, and in described separation process, can directly realize and use the wash-out of chromatographic column and regeneration and the process of chromatography again, realize the continuous lock out operation to described antibody.
Accompanying drawing explanation
For content of the present invention is more likely to be clearly understood, below in conjunction with accompanying drawing, the present invention is further detailed explanation, wherein,
Fig. 1 is the structural representation of series connection continuous adsorption device separation and purification antibody flow process of the present invention;
Fig. 2 is the chromatographic apparatus breakthrough curve figure of two MEP HyperCel chromatography column in series continuous adsorption separation and purification rabbit against human T cell blood plasma described in the embodiment of the present invention 4;
Fig. 3 is the electrophoresis result figure of two MEP HyperCel chromatography column in series continuous adsorption separation and purification rabbit against human T cell blood plasma described in the embodiment of the present invention 4;
Fig. 4 is the chromatographic apparatus breakthrough curve figure of three AN21SepFF chromatography column in series continuous adsorption separation and purification rabbit against human T cell blood plasma described in the embodiment of the present invention 5;
Fig. 5 is the electrophoresis result figure of three AN21SepFF chromatography column in series continuous adsorption separation and purification rabbit against human T cell blood plasma described in the embodiment of the present invention 5;
Fig. 6 is the chromatographic apparatus breakthrough curve figure of three AN55SepFF chromatography column in series continuous adsorption separation and purification rabbit against human T cell blood plasma described in the embodiment of the present invention 7;
Fig. 7 is the electrophoresis result figure of three AN55SepFF chromatography column in series continuous adsorption separation and purification rabbit against human T cell blood plasma described in the embodiment of the present invention 7.
The specific embodiment
Series connection continuous adsorption device separation and purification antibody flow process of the present invention comprises that continuous adsorption, flushing, wash-out and regeneration Si Ge operating space form, and continuous adsorption district is comprised of 2-5 chromatography column in series that is filled with mixed mode chromatography media.It is example that the following description be take three posts series connection continuous adsorption devices, as shown in Figure 1.Biological sample is inputted continuously from being positioned at series connection continuous adsorption device # 1 chromatographic column entrance foremost, and effluent flows out from being positioned at the #3 chromatographic column outlet of series connection continuous adsorption device end; When the input of biological sample reaches after certain volume, in continuous adsorption device # 1 chromatographic column foremost, enter rinsing area, the #4 chromatographic column after regeneration is processed is linked into the end (as shown in Figure 1-2) of continuous adsorption device simultaneously; Adopt suitable dcq buffer liquid to clean #1 chromatographic column, effluent is back to the biological sample injection port that is positioned at continuous adsorption district shown in figure for loading; #1 chromatographic column after flushing enters elution zone, is positioned at continuous adsorption device # 2 chromatographic column foremost and enters rinsing area, and the #5 chromatographic column after regeneration is processed is linked into the end (as Figure 1-3) of continuous adsorption device simultaneously; Adopt the antibody in suitable elution buffer wash-out #1 chromatographic column and collect product; #1 chromatographic column after wash-out enters renewing zone, be positioned at continuous adsorption device # 3 chromatographic column foremost and enter rinsing area, #2 chromatographic column after flushing enters elution zone, and the #6 chromatographic column after regeneration is processed is linked into the end (as Figure 1-4) of continuous adsorption device; Adopt successively suitable regeneration buffer solution and level pad to process #1 chromatographic column, the end that the chromatographic column after regeneration is processed accesses continuous adsorption device again recycles.In said process, elder is benchmark the switching of the chromatographic column access Yi Ge district operating time, can realize on this basis the continuous operation of mixed mode chromatography antibody.
It is that 21mmol/g be take 4(imidazoles-1-yl that embodiment 1 prepares ligand density) the aniline mixed mode chromatography media AN21SepFF that is aglucon.Preparation process is as follows: after being stored in Sepharose Fast Flow in 20% ethanol water and by deionized water, it being rinsed well repeatedly, through vacuum pump, drain 10 minutes; Take the Sepharose Fast Flow medium that 1g drains and insert in conical flask, add successively dimethyl sulfoxide (DMSO) 1.8mL, 2mol/L NaOH solution 0.5mL and allyl bromide, bromoallylene 0.3mL; Above-mentioned suspension mixes and with after polytetrafluoroethylraw raw material band sealing, the water bath with thermostatic control shaking table that is placed in 25 ℃ reacts 48h under 170rpm rotating speed; The activated media obtaining is placed in G3 glass funnel, uses successively deionized water, 0.1mol/LNaOH, 25%(v/v) ethanol water and 0.5mol/L NaCl solution clean until to the liquor potassic permanganate nondiscolouring dripping in cleaning fluid; The activation rate of medium is 130mmol/L.Activated media is placed in G3 glass funnel and drains after moisture, transfers in conical flask and adds successively deionized water 1mL, N-bromosuccinimide 0.3g and dimethyl sulfoxide (DMSO) 1mL; Above-mentioned suspension mixes and with after polytetrafluoroethylraw raw material band sealing, the water bath with thermostatic control shaking table that is placed in 25 ℃ reacts 2h under 170rpm rotating speed.After reaction finishes, medium is placed on G3 glass funnel again, by deionized water, repeatedly rinses and drain.1g drains medium and proceeds in conical flask, adds successively and contains 4(imidazoles-1-yl) dimethyl sulphoxide solution 1.2mL and the 3.0mol/L NaOH solution 1.0mL of aniline 14.0mg; After mixing, above-mentioned suspension reacts 4h in the shaking bath of 50 ℃ and 170rpm, obtains.
It is that 26mmol/g be take 4(imidazoles-1-yl that embodiment 2 prepares ligand density) the aniline mixed mode chromatography media AN26SepFF that is aglucon.Preparation process is as follows: after being stored in Sepharose Fast Flow in 20% ethanol water and by deionized water, it being rinsed well repeatedly, through vacuum pump, drain 10 minutes; Take the Sepharose Fast Flow medium that 1g drains and insert in conical flask, add successively dimethyl sulfoxide (DMSO) 0.2mL, 4mol/L NaOH solution 0.5mL and allyl bromide, bromoallylene 0.3mL; Above-mentioned suspension mixes and with after polytetrafluoroethylraw raw material band sealing, the water bath with thermostatic control shaking table that is placed in 25 ℃ reacts 52h under 170rpm rotating speed; The activated media obtaining is placed in G3 glass funnel, uses successively deionized water, 0.1mol/LNaOH, 25%(v/v) ethanol water and 0.5mol/L NaCl solution clean until to the liquor potassic permanganate nondiscolouring dripping in cleaning fluid; The activation rate of medium is 243mmol/L.Activated media is placed in G3 glass funnel and drains after moisture, transfers in conical flask and adds successively deionized water 3mL, N-bromosuccinimide 1.0g and dimethyl sulfoxide (DMSO) 1mL; Above-mentioned suspension mixes and with after polytetrafluoroethylraw raw material band sealing, the water bath with thermostatic control shaking table that is placed in 25 ℃ reacts 2h under 170rpm rotating speed.After reaction finishes, medium is placed on G3 glass funnel again, by deionized water, repeatedly rinses and drain.1g drains medium and proceeds in conical flask, adds successively and contains 4(imidazoles-1-yl) dimethyl sulphoxide solution 1.2mL and the 1.0mol/L NaOH solution 1.5mL of aniline 46.0mg; After mixing, above-mentioned suspension reacts 4h in the shaking bath of 50 ℃ and 170rpm, obtains.
The method of two MEP HyperCel chromatography column in series continuous adsorption separation and purification rabbit against human T cell blood plasma is as follows: MEP HyperCel chromatography media is filled in respectively after 5 Tricorn5/50 chromatographic columns, connect two chromatographic columns with the chromatographic column of 50mmol/L Tris-HCl buffer solution (pH8.0) the balance series connection of at least 10 times of column volumes (20mL), respectively hang oneself 50mmol/L Tris-HCl buffer solution (pH8.0) balance of 10mL of its excess-three root chromatographic column; Rabbit against human T cell blood plasma is as the biological sample for Image processing after ten times of dilutions of 50mmol/L Tris-HCl buffer solution (pH8.0), and in sample, the concentration of antibody is 0.876mg/mL; Above-mentioned biological sample is with in the MEP HyperCel chromatographic column of two series connection of the continuous input of flow velocity of 0.2mL/min (chromatography adsorption breakthrough curve as shown in Figure 2); Volume until biological sample reaches after 18mL, removes and to be positioned at the chromatographic column (being called A post herein) of series connection adsorbent equipment front end and to access the new chromatographic column after a balance at the end of series connection adsorbent equipment, and biological sample continues the series connection adsorbent equipment of the new assembling of input; A post enters into rinsing area, 50mmol/L Tris-HCl buffer solution (pH8.0) the cleaning A post that each is used 50mmol/L Tris-HCl buffer solution (pH8.0) of at least 5 column volumes and contains 25mmol/L Sodium Caprylate; Described 20mmol/L phosphoric acid-citrate buffer (pH4.0) wash-out antibody product for chromatographic column after cleaning; In chromatography process, the SDS-PAGE electrophoresis result of absorption, flushing and eluent as shown in Figure 3; The purity of product is that the antibody carrying capacity of 97%, MEP HyperCel medium is 8.5mg/g medium, and the yield of product approaches 100%; By comparison, the purity that single MEP HyperCel chromatographic column extracts antibody from rabbit against human T cell blood plasma is 87%, and antibody carrying capacity is only 0.97mg/g medium, and yield is 25%.
The 0.5mol/L NaOH solution of 5 column volumes and the regeneration of the 50mmol/L Tris-HCl buffer solution (pH8.0) of at least 10 column volumes are used in the regeneration of purifying chromatographic column successively; Chromatographic column after regeneration accesses series connection continuous adsorption device again, recycles.
The method of three AN21SepFF chromatography column in series continuous adsorption separation and purification rabbit against human T cell blood plasma is as follows: AN21SepFF chromatography media is filled in respectively after 6 Tricorn5/50 chromatographic columns, connect three chromatographic columns with the chromatographic column of 20mmol/L phosphate buffer (pH8.0) the balance series connection containing 0.5mol/LNaCl of at least 10 times of column volumes (30mL), respectively hang oneself 20mmol/L phosphate buffer (pH8.0) balance containing 0.5mol/L NaCl of 10mL of its excess-three root chromatographic column; Rabbit against human T cell blood plasma is through being used for the biological sample of Image processing containing conduct after ten times of dilutions of 20mmol/L phosphate buffer (pH8.0) of 0.5mol/L NaCl, in sample, the concentration of antibody is 0.876mg/mL; Above-mentioned biological sample is with in the AN21SepFF chromatographic column of two series connection of the continuous input of flow velocity of 0.2mL/min (chromatography adsorption breakthrough curve as shown in Figure 4); Volume until biological sample reaches after 9.1mL, dismounting is positioned at the chromatographic column (being called A post herein) of series connection adsorbent equipment front end and accesses the AN21SepFF chromatographic column after a balance at the end of series connection adsorbent equipment, and biological sample continues the series connection adsorbent equipment of the new assembling of input; A post enters into rinsing area, and each 20mmol/L phosphate buffer (pH8.0) containing 0.5mol/L NaCl with at least 5 column volumes cleans A post; Described 20mmol/L phosphoric acid-citrate buffer (pH4.0) wash-out antibody product for chromatographic column after cleaning; In chromatography process, the SDS-PAGE electrophoresis result of absorption, flushing and eluent as shown in Figure 5; The purity of product is that the antibody carrying capacity of 87.9%, AN21SepFF chromatography media is 1.54mg/g medium, and the yield of product approaches 58%; By comparison, single AN21SepFF chromatographic column extracts the purity of antibody and crosses rare cannot assessment because of solution from rabbit against human T cell blood plasma, and the total protein of AN21SepFF chromatography media (containing antibody and foreign protein) carrying capacity is 0.67mg/g medium, and yield is 36%.
The regeneration of purifying chromatographic column is successively with the 0.5mol/L NaOH solution of 5 column volumes and the regeneration of the 20mmol/L phosphate buffer (pH8.0) containing 0.5mol/L NaCl of at least 10 column volumes; Chromatographic column after regeneration accesses series connection continuous adsorption device again, recycles.
The method of three AN26SepFF chromatography column in series continuous adsorption separation and purification rabbit against human T cell blood plasma is as follows: AN26SepFF chromatography media is filled in respectively after 6 Tricorn5/50 chromatographic columns, connect three chromatographic columns with the chromatographic column of 20mmol/L phosphate buffer (pH8.0) the balance series connection containing 0.5mol/LNaCl of at least 10 times of column volumes (30mL), respectively hang oneself 20mmol/L phosphate buffer (pH8.0) balance containing 0.5mol/L NaCl of 10mL of its excess-three root chromatographic column; Rabbit against human T cell blood plasma is through being used for the biological sample of Image processing containing conduct after ten times of dilutions of 20mmol/L phosphate buffer (pH8.0) of 0.5mol/L NaCl, in sample, the concentration of antibody is 0.876mg/mL; Above-mentioned biological sample is inputted in the AN26SepFF chromatographic column of two series connection with the flow velocity of 0.2mL/min continuously; Volume until biological sample reaches after 12.7mL, dismounting is positioned at the chromatographic column (being called A post herein) of series connection adsorbent equipment front end and accesses the AN26SepFF chromatographic column after a balance at the end of series connection adsorbent equipment, and biological sample continues the series connection adsorbent equipment of the new assembling of input; A post enters into rinsing area, and each 20mmol/L phosphate buffer (pH8.0) containing 0.5mol/L NaCl with at least 5 column volumes cleans A post; Described 20mmol/L phosphoric acid-citrate buffer (pH4.0) wash-out antibody product for chromatographic column after cleaning; Purification result and single-column result more as shown in table 1.
The continuous adsorption chromatographic apparatus antibody purification result comparison of connecting with three posts of table 1AN26SepFF single-column
The regeneration of purifying chromatographic column is successively with the 0.5mol/L NaOH solution of 5 column volumes and the regeneration of the 20mmol/L phosphate buffer (pH8.0) containing 0.5mol/L NaCl of at least 10 column volumes; Chromatographic column after regeneration accesses series connection continuous adsorption device again, recycles.
Embodiment 7
The method of three AN55SepFF chromatography column in series continuous adsorption separation and purification rabbit against human T cell blood plasma is as follows: AN55SepFF chromatography media is filled in respectively after 6 Tricorn5/50 chromatographic columns, connect three chromatographic columns with the chromatographic column of 20mmol/L phosphate buffer (pH8.0) the balance series connection containing 0.5mol/LNaCl of at least 10 times of column volumes (30mL), respectively hang oneself 20mmol/L phosphate buffer (pH8.0) balance containing 0.5mol/L NaCl of 10mL of its excess-three root chromatographic column; Rabbit against human T cell blood plasma is through being used for the biological sample of Image processing containing conduct after ten times of dilutions of 20mmol/L phosphate buffer (pH8.0) of 0.5mol/L NaCl, in sample, the concentration of antibody is 0.876mg/mL; Above-mentioned biological sample is with in the AN55SepFF chromatographic column of two series connection of the continuous input of flow velocity of 0.2mL/min (chromatography adsorption breakthrough curve as shown in Figure 6); Volume until biological sample reaches after 25.0mL, dismounting is positioned at the chromatographic column (being called A post herein) of series connection adsorbent equipment front end and accesses the AN55SepFF chromatographic column after a balance at the end of series connection adsorbent equipment, and biological sample continues the series connection adsorbent equipment of the new assembling of input; A post enters into rinsing area, and each 20mmol/L phosphate buffer (pH8.0) containing 0.5mol/L NaCl with at least 5 column volumes cleans A post; Described 20mmol/L phosphoric acid-citrate buffer (pH4.0) wash-out antibody product for chromatographic column after cleaning; In chromatography process, the SDS-PAGE electrophoresis result of absorption, flushing and eluent as shown in Figure 7; The purity of product is that the antibody carrying capacity of 91.6%, AN55SepFF chromatography media is 11.0mg/g medium, and the yield of product is 97.4%; By comparison, the purity that single AN55SepFF chromatographic column extracts antibody from rabbit against human T cell blood plasma is that the antibody carrying capacity of 43.7%, AN55SepFF chromatography media is 4.28mg/g medium, and yield is 81%.
The regeneration of purifying chromatographic column is successively with the 0.5mol/L NaOH solution of 5 column volumes and the regeneration of the 20mmol/L phosphate buffer (pH8.0) containing 0.5mol/L NaCl of at least 10 column volumes; Chromatographic column after regeneration accesses series connection continuous adsorption device again, recycles.
Adopt the structure continuous adsorption of three chromatography column in series described in embodiment 7 from rabbit against human T cell blood plasma antibody purification, its difference is only that it is that 15mmol/L be take 4(imidazoles-1-yl that described chromatography media adopts ligand density) the aniline AN15SepFF chromatography media that is aglucon, when the volume of biological sample reaches after 4.22mL, penetrate and in night, have a large amount of antibody.The purity of product is crossed rare cannot assessment because of solution, and the total protein of single-column (containing antibody and foreign protein) carrying capacity is 0.26mg/g medium, and the yield of product is 12.5%; By comparison, single AN15SepFF chromatographic column extracts the purity of antibody and cannot assess equally from rabbit against human T cell blood plasma, and the total protein of single-column (containing antibody and foreign protein) carrying capacity is 0.24mg/g medium, and yield is 13.6%.
Adopt the structure continuous adsorption of three chromatography column in series described in embodiment 7 from rabbit against human T cell blood plasma antibody purification process, its difference is only that it is that 94mmol/L be take 4(imidazoles-1-yl that described chromatography media adopts ligand density) the aniline AN94SepFF chromatography media that is aglucon, when the volume of biological sample reaches after 10mL, penetrate and in night, occur antibody.The purity of product is 80%, and the carrying capacity of antibody is 4.82mg/g medium, and the yield of product is 55.2%.
Method with five MEP HyperCel chromatography column in series continuous adsorption purifying rabbit against human T cell blood plasma is as follows: MEP HyperCel chromatography media is filled in respectively after 8 Tricorn5/50 chromatographic columns, connect five chromatographic columns with the chromatographic column of 20mmol/L phosphate buffer (pH7.4) the balance series connection of at least 10 times of column volumes (50mL), respectively hang oneself 20mmol/L phosphate buffer (pH7.4) balance of 10mL of its excess-three root chromatographic column; Rabbit against human T cell blood plasma is as the biological sample for Image processing after 20mmol/L phosphate buffer (pH7.4) dilution, and in sample, the concentration of antibody is 0.96mg/mL; Above-mentioned biological sample is inputted in the MEP HyperCel chromatographic column of five series connection with the flow velocity of 0.2mL/min continuously; Volume until biological sample reaches after 27.7mL, dismounting is positioned at chromatographic column (being called A1 post herein) foremost of series connection adsorbent equipment and accesses the new chromatographic column after a balance at the end of series connection adsorbent equipment, and biological sample continues the series connection adsorbent equipment of the new assembling of input; A1 post enters into rinsing area, 20mmol/L phosphate buffer (pH7.4) the cleaning A1 post that each is used 20mmol/L phosphate buffer (pH7.4) of at least 5 column volumes and contains 25mmol/L Sodium Caprylate; Described 50mmol/L citrate buffer (pH3.0) wash-out antibody product for chromatographic column after cleaning; Result is as shown in table 2.
Table 2MEP HyperCel single-column, three posts and the comparison of five posts series connection continuous adsorption chromatographic apparatus antibody purification result
The 0.1mol/L citrate buffer (pH2.5) of 5 column volumes and the regeneration of the 20mmol/L phosphate buffer (pH7.4) of at least 10 column volumes are used in the regeneration of purifying chromatographic column successively; Chromatographic column after regeneration accesses series connection continuous adsorption device again, recycles.
Obviously, above-described embodiment is only for example is clearly described, and the not restriction to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without also giving all embodiments.And the apparent variation of being extended out thus or change are still among the protection domain in the invention.
Claims (10)
1. a mixed mode chromatography column in series continuous adsorption device, it is characterized in that, the chromatographic column of the mixed mode chromatography media that comprising that series connection arranges n is filled with MEP HyperCel chromatography media or ligand density to be 10-120mmol/g take 4(imidazoles-1-yl) aniline is aglucon, for carrying out separation and purification antibody from the biological raw material continuous adsorption antibody that comprises antibody and at least one foreign protein;
Wherein, the described 4(of take imidazoles-1-yl) the mixed mode chromatography media that aniline is aglucon is that to take Ago-Gel, cellulose or the hydroxylating methacrylic acid hydrophilic polymer microballoon that average grain diameter is 20-270 μ m be matrix, at described stromal surface coupling pi-allyl, then the chromatography media that the activation end group coupling 4(imidazoles-1-yl obtaining by pi-allyl and N-bromosuccinimide bromo-reaction) aniline is aglucon, its structural formula is M-L-AN; M represents matrix, and L represents the short-chain alkyl of being introduced by pi-allyl activation, and AN represents 4(imidazoles-1-yl) aniline.
2. a kind of mixed mode chromatography column in series continuous adsorption device according to claim 1, is characterized in that, describedly take 4(imidazoles-1-yl) ligand density of the aniline mixed mode chromatography media that is aglucon is 21-70mmol/g.
3. a kind of mixed mode chromatography column in series continuous adsorption device according to claim 1, is characterized in that, described ligand density is 10-120mmol/g take 4(imidazoles-1-yl) preparation method of the aniline mixed mode chromatography media that is aglucon as:
The matrix M that repeatedly rinses and drain after moisture through deionized water mixes with dimethyl sulfoxide (DMSO), sodium hydroxide solution and allyl bromide, bromoallylene, in mixed liquor, the volume content of dimethyl sulfoxide (DMSO) is 3-60%, the molal volume concentration of NaOH is 0.01-4mol/L, the mass volume ratio of matrix M and allyl bromide, bromoallylene is 0.1-20:1, and mixed liquor reacts 1-64h and obtains pi-allyl activated media M-L in the shaking bath of 25 ℃ and 170rpm; Secondly, after activated media M-L cyclic washing, mix with water, dimethyl sulfoxide (DMSO) and N-bromosuccinimide, in above-mentioned mixed liquor, the volume ratio of water and dimethyl sulfoxide (DMSO) is 0-10, the volume content of dimethyl sulfoxide (DMSO) is 3-70%, the mass ratio of activated media M-L and N-bromosuccinimide is 0.5-7:1, and mixed liquor reacts 0.5-5h in the shaking bath of 25 ℃ and 170rpm; Finally, reacted activated media after deionized water is washed and is drained moisture with 4(imidazoles-1-yl) aniline, sodium hydroxide solution and dimethyl sulphoxide solution mix, activated media M-L and 4(imidazoles-1-yl) mass ratio of aniline is 0.008-0.64:1, dimethyl sulfoxide (DMSO) is 0.2-5.0:1 with the volume mass ratio of activated media, the molal volume concentration of NaOH is 0.01-1.0mol/L, above-mentioned mixed liquor reacts 1-72h in the shaking bath of 50 ℃ and 170rpm, obtains.
4. according to the arbitrary described mixed mode chromatography column in series adsorbent equipment of claim 1-3, it is characterized in that n=2-5.
5. utilize the arbitrary described mixed mode chromatography column in series adsorbent equipment continuous separate of claim 1-4 from a method for antibody purification, it is characterized in that, comprise the steps:
(1) biological raw material sample chromatography, that contain target antibody product is treated in preparation;
(2) described biological sample is diluted with suitable level pad, the biological sample of dilution is input to described mixed mode chromatography column in series continuous adsorption device;
(3) input of dilution biological sample reaches after certain volume, leaves adsorption step enter into rinsing step in series connection continuous adsorption device chromatographic column foremost; Meanwhile, a new chromatographic column after regeneration is processed is linked into series connection continuous adsorption device least significant end; Dilution biological sample continues to be input to continuously in new series connection continuous adsorption device;
(4) to the chromatographic column that enters into the absorption antibody of rinsing step, inputting described level pad rinses;
(5) with applicable elution buffer by antibody wash-out from the chromatographic column after step (4) is rinsed, collect eluting peak, obtain antibody-solutions;
(6) input successively described regeneration buffer solution and level pad with the chromatographic column after wash-out in regeneration step (5).
6. continuous separate according to claim 5 is from the method for antibody purification, it is characterized in that, in described step (6), also comprise the chromatographic column after regeneration is linked into series connection continuous adsorption device least significant end, continue on for the continuous adsorption of antibody and the step of separation and purification.
7. the method from antibody purification according to the arbitrary described continuous separate of claim 5-6, it is characterized in that, in described step (4), also comprise the step that the chromatographic column effluent thing of described flushing is collected, for the dilution biological sample for chromatography absorption described in step (2).
8. the method from antibody purification according to the arbitrary described continuous separate of claim 5-7, is characterized in that, in described step (2), also comprises the centrifugal step of the biological sample of described dilution, for removing the insoluble matter of biological sample and the impurity component of precipitation.
9. the method from antibody purification according to the arbitrary described continuous separate of claim 5-8, is characterized in that,
Described level pad comprises the 20mmol/L PB buffer solution of the 50mmol/L Tris-HCl buffer solution of the level pad that is applicable to described MEP HyperCel chromatography media: pH8.0-8.5 or the 10mmol/L PBS buffer solution of pH7.4 or pH6.0-8.0; And be applicable to described 4(imidazoles-1-yl of take) level pad of the aniline mixed mode chromatography media that is aglucon: the 50mmol/L Tris-HCl buffer solution containing 0.15-0.5mol/L NaCl of pH8.0-8.5 or the 10-50mmol/L PB buffer solution containing 0.15-0.5mol/L NaCl of pH6.0-8.0;
Described dcq buffer liquid comprises the dcq buffer liquid that is applicable to described MEP HyperCel chromatography media: described in be applicable to the level pad of described MEP HyperCel chromatography media or be applicable to the level pad of described MEP HyperCel chromatography media containing described in 25mmol/L Sodium Caprylate; And be applicable to described 4(imidazoles-1-yl of take) the dcq buffer liquid of the aniline mixed mode chromatography media that is aglucon: described in be applicable to described 4(imidazoles-1-yl of take) level pad of the aniline mixed mode chromatography media that is aglucon;
Described elution buffer comprises the elution buffer that is applicable to described MEP HyperCel chromatography media: the citrate buffer of the acetate buffer of the 50mmol/L of pH4.0 or phosphoric acid-citrate buffer of the 20-100mmol/L of pH3.0-4.0 or the 50mmol/L of pH3.0-4.0; And be applicable to described 4(imidazoles-1-yl of take) elution buffer of the aniline mixed mode chromatography media that is aglucon: the 50mmol/L acetate buffer of pH4.0-5.0 or the 20mmol/L citrate buffer of pH3.5-4.0;
The described chromatographic column buffer solution used of regenerating comprises the NaOH solution of the regeneration buffer solution that is applicable to described MEP HyperCel chromatography media: 0.5-1.0mol/L or the 0.1mol/L citrate buffer of pH2.5 or the acetum of 0.1mol/L; And be applicable to described 4(imidazoles-1-yl of take) the regeneration buffer solution of the aniline mixed mode chromatography media that is aglucon: the NaOH solution of 0.5-1.0mol/L or the 0.1mol/L citrate buffer of pH2.5 or the acetum of 0.1mol/L.
10. the method from antibody purification according to the arbitrary described continuous separate of claim 6-9, it is characterized in that, in described step (1), described biological raw material behaviour source, the zoogenous cell conditioned medium liquid that contains antibody that contains antibody plasma or obtain through animal cell culture.
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