CN101087637A - Purification of immunoglobulins - Google Patents

Abstract

The present invention relates to a separation matrix comprised of porous particles to which antibody-binding protein ligands have been immobilised, wherein the ligand density is in the range of 5.0-10 mg/ml; the gel phase distribution coefficient of the particles expressed as Kav for a dextran of size 110 kDa is above 0.65 and the median particle di-ameter is between 65-84 mum. The carbohydrate material is preferably highly cross-linked agarose.

Description

The purifying of immunoglobulin (Ig)

Technical field

The present invention relates to the Antibody Preparation field, especially relate to the isolation medium that antagonist is emanated and/or separated.The present invention also comprises the chromatographic column that contains isolation medium of the present invention, the method that adopts described isolation medium separation antibody and the rapid technology of multistep of carrying out large scale purification antibody from raw material.

Background

Immune system is made up of a lot of complementary cell types, and described cell protects body to avoid the infection of bacterium, parasite, fungi, virus jointly, and the growth that prevents tumour cell.Immune bodyguard is the macrophage of constantly roaming in host's blood.When being infected or during immune activation, the huge system of biting produces response by engulfing the invador who is marked with the foreign molecules that is called as antigen.This phenomenon by the t helper cell mediation causes replying of a succession of complexity, and it causes the activation of B cell.And these B cells produce the albumen that is called antibody, and described albumen combines with the exotic invasive person.Combination between antibody and the antigen indicates that the person that causes the exotic invasive of the activation by phagocytosis or complement system is by broken ring.Have five kinds of dissimilar antibody or immunoglobulin (Ig): IgA, IgD, IgE, IgG and IgM.Not only physiological action is different but also their structure is also different for they.From the structure angle, IgG antibody is the special immunoglobulin (Ig) of a class that is widely studied, and it may be because they play remarkable effect in maturation immunity is replied.

In a series of different people and animal doctor's diagnosis, health care and treatment application, the biologically active of immunoglobulin (Ig) is studied at present.In fact, in nearest several years, monoclonal antibody and recombinant antibodies structure have become maximum one albuminoid of studying in the present clinical testing, and FDA ratifies it and is used for the treatment of and diagnoses.As to the replenishing of expression system and production strategy, the design purification scheme is with simply, mode obtains highly purified antibody cheaply.

The conventional method of separating immune globulin is stayed other protein component in the solution simultaneously based on the selectivity reversible precipitation to the protein component that comprises immunoglobulin (Ig).Typical precipitating reagent is promptly anti-unordered salt, for example ammonium sulfate and the potassium phosphate and sad of ethanol, polyethylene glycol, Yi Rong.Typically, these intermediate processings obtain very impure product, simultaneously consuming time, consumption power.And, in raw material, add feasible being difficult to of precipitating reagent supernatant is used for other purposes, thereby produced the problem of handling, with regard to the large scale purification immunoglobulin (Ig), this problem is particularly important.

Ion-exchange chromatography is a kind of known albumen stage division, is usually used in separating immune globulin.Yet because charged ion exchange ligands can be reacted with all compounds that has opposite charges, therefore, the selectivity of ion-exchange chromatography may be lower than other chromatographic isolation.

Hydrophobic interaction chromatography (HIC) is the method that another kind is used for separating immune globulin.But hydrophobic matrix requires to add lyotropic salt in raw material, thus effective binding domain-immunoglobulin.With continuously or gradient progressively reduce the concentration of lyotropic salt, the antibody of institute's combination discharges from matrix.If require highly purified product, recommend to adopt this hydrophobic interaction chromatography and in conjunction with further step.Thus, a shortcoming of this method is to add lyotropic salt in raw material, has proposed a problem and has increased cost to large-scale consumer thus.For the raw material except cell culture supernatant, for example whey, blood plasma and yolk, adding lyotropic salt in raw material is unallowed under many circumstances in large-scale application, its reason is that described salt may stop the raw-material any economically feasible application that does not contain immunoglobulin (Ig).An additional problem of large-scale application is a few kilolitre treatments of wastes produced.

Having a liking for the sulphur adsorption charomatography is at (J.Porath etc. in 1985 by J.Porath; FBBSLetters, the 185th volume, the 306th page, 1985) a kind of new chromatographic absorption method that is used for separating immune globulin of introducing.In this piece paper, described the 0.5M potassium sulfate be lyotropic salt in the presence of the agarose of divinyl sulfone activation how to show with the specificity of immunoglobulin (Ig) and combine that described agarose coupling has the different ligands that comprises free sulfhydryl groups.Although the described matrix that is used to have a liking for the sulphur adsorption charomatography has shown good performance usually,, can produce shortcoming mentioned above owing in raw material, add lyotropic salt to guarantee effective combination of immunoglobulin (Ig).

Affinity chromatography has uniqueness, effective function in isolation technics, thereby because this is the unique a kind of technology that can carry out purifying based on biological function or chemical constitution separately to biomolecule.High selectivity and high-effect this technology that makes are ideally suited in predetermined substance is separated from the biological mixture of complexity.In affinity chromatography, molecule to be purified is by covalently bound ligand specificity ground, reversibly combination to insoluble holder, and described part comprises complementary in conjunction with material.Help its with condition that described fixed ligands specificity combines under add sample.Unconjugated material is washed off, by experimental condition being changed under the situation that helps its desorb, can reclaim interested material.Affinity chromatography has concentrated effect, and it can be handled very easily to the sample of large volume.Albumin A and Protein G affinity chromatography are to separate and purifying immunoglobulin (Ig) the common and method of extensive employings is especially separated monoclonal antibody, and its main cause is to use and makes things convenient for and the high-purity of acquisition.

Nineteen eighty-two, Colbert etc. have described a kind of gene code of albumin A class material.In US5151350, successful clone and expression to described gene have been described for the first time.The clone of this gene and the sign of nucleotide sequence thereof make those skilled in the art obtain a certain amount of albumin A class fret oligonucleotide sequence, thereby clone in different hosts-vector system.The albumin A class material of described recombinant production and subfragrnent thereof have Fc zone and the activation polyclonal antibody synthetic characteristic of albumin A in conjunction with IgG.Therefore, these materials are used for chromatography in the mode identical with albumin A.In pharmaceuticals industry, the chromatographic clear superiority of recombinant protein A is to have eliminated the risk that contains the mammal residue in the isolation medium, and the risk that contains the mammal residue in the drug products thus.

US6399750 discloses a kind of medium in conjunction with IgG, especially has the separating medium of basic matrix and matrix conjugated group, and described group is the recombinant protein A (rProtein A) that contains cysteine.The formula of this group is :-B-X-rProtein A, and wherein B is for connecting the bridge of basic matrix, and X comprises hetero atom N or the S from rProtein A.In a preferred embodiment, X is for constituting the thioether in sulphur source, and C end residue is the cysteine of rProtein A.

Have various albumin A chromatogram products on the market.For example, Millipore (Billerca, Mass., USA) provide and used the natural Prosep-A High Capacity that makes from the albumin A of staphylococcus aureus, and the PROSEP-rPA High Capacity that adopts the recombinant protein A of in Escherichia coli Escherichiacoli, expressing to make.PROSEP matrix is made of the glass particle with interconnection aperture.

(Journal of Chromatography A such as McCue, 989 (2003) 139-153: " Evaluation of protein A chromatography media ") studied two kinds of albumin A media with different pore sizes, every kind all has the cellular glass skeleton.The material of smaller aperture due finds to have bigger static capacity, and it points out its reason is the higher ligand concentration that has bigger specific area and follow.The material of smaller aperture due also finds to have bigger dynamic binding ability.

MabSelect ^TMChromatogram product for the albumin A of Amersham Biosciences (Uppsala, Sweden) is particularly useful for catching monoclonal antibody from the raw material of large volume.Described part comprises by the C terminal cysteine and is coupled to recombinant protein A on the Sepharose holder.MabSelect ^TMParticle median diameter be 85um.

WO2004/074211 (Amershanm Biosciences AB) relates to a kind of method for preparing inorganic particle, and described particle has the pore structure of classification.Therefore, this method is not produced organic granular, for example carbohydrate particles.

US5672276 (BioSepra Inc) relates to the preparation method and the application of improved porosu solid supporter and this supporter.Especially, disclose the porous polymer supporter of passivation, it is characterized in that reversible high absorbent capacity, and do not had the non-specific adsorption or the interaction of biomolecule substantially.Miscellaneous non-passivation porous solid matrix can carry out passivation described in US5672276.These porous matrixes comprise (i) inorganic oxide supporter; (ii) " stabilisation " inorganic oxide supporter; thereby preventing by the thin protective layer at the surface-coated hydrophobic polymer that chemistry from invading analyses; and the porous matrix that (iii) constitutes separately, especially hydrophobic polymer by organic/polymeric material.Described among the US5672276 by polysaccharide derivates for example the organic ion exchanger made of derivatives such as agarose, glucan and cellulose have a lot of deficiencies.For example, the ion-exchanger that polysaccharide is derived is not very stable on mechanical strength, can not resist strong acid.Therefore, be described according to the porous media through passivation of this list of references that for example carbohydrate materials is different with polysaccharide.

At last, Meyer etc. have described inorganic particle in " Templating of porous polymeric beads toform porous silica and titania spheres " (Adv.Mater 2002 (14), the 23rd phase, 1768-72 page or leaf).Therefore, in this list of references, there is not to disclose the carbohydrate particles that coupling on it has the protein ligands of binding antibody.

Yet no matter prior art how, still needs another kind of isolation medium with separation antibody or antibody structure, it meets purity, security, usefulness and cost-benefit requirement.

Brief description of the drawings

Accompanying drawing 1 is a schematic diagram, shows that wherein dynamic binding ability (DBC) is the function according to the retention time of separating medium of the present invention.For relatively, shown the dynamic binding ability of a prior art products simultaneously.

Concise and to the point description of the present invention

An aspect of of the present present invention is a kind of new isolation medium that separates polyclone or monoclonal antibody.It can pass through the described realization of claim.

Another aspect of the invention is and allow antagonist to carry out isolation medium quicker than prior art, more effective purifying.It can realize by new by the appended claims isolation medium that when it was used for chromatography, its binding ability significantly improved.

Another aspect of the invention is aforesaid isolation medium, it is suitable for large-scale operation.More special aspect is described isolation medium, and it has the binding ability that significantly improves to raw material.

Another aspect of the present invention is the chromatographic column that is equipped with or is filled with according to isolation medium of the present invention.

Be kit on the other hand, it comprises according to chromatographic column of the present invention, the buffer solution that is suitable for antibody purification and written explanation.

Be the method that adopts isolation medium according to the present invention that antibody is separated from liquid on the other hand.This isolation medium can be arranged in according to splitter of the present invention.The method according to this invention can be used to obtain pure substantially specific antibodies material, perhaps obtains the liquid of one or more undesirable antibody wherein having been removed.

By claims and following description, further embodiment of the present invention and advantage will be conspicuous.

Definition

Herein, term " antibody " or " immunoglobulin (Ig) " exchange and use.

Herein, term " part " be meant can with target compound for example interactive molecule of antibody or compound.

Herein, term " spacerarm " or " bridge " are meant the isolated element of the supporter of part and isolation medium.The supporter of isolation medium also is called " basic matrix ".Term " isolation medium " also is called separating medium herein.

Herein, term " antibody binding proteins " is no matter be meant the albumen that binding mechanism can binding antibody.

Term " Fc-is in conjunction with albumen " is meant the albumen of crystallizable part (Fc) that can binding antibody, comprises for example albumin A and Protein G, or fragment or the hereditary derivative or the fusion of the described binding characteristic of its any maintenance.

Term " eluent " is the conventional sense in this field, the buffer solution that promptly has suitable PH and/or ionic strength, one or more chemicals are separated from isolation medium.

Term " K _Av" being meant the gel phase breadth coefficient, it is from wash-out or retention volume V for the molecule of giving sizing according to following formula _R(also be V _e) and the interstitial void volume V of post ₀Geometric volume (V with post _c) independent variable of the post that calculates:

K _av＝(V _R-V ₀)/(V _c-V ₀)

(referring to for example " Handbook of process chromatography, A Guide toOptimization, Scale-Up and Validation " (1997) Academic Press, San Siego.Gail Sofer﹠amp; Lars Hagel edits, ISBN0-12-654266-X, 368 pages)

Detailed description of the present invention

First aspect the present invention relates to the isolation medium be made up of porous particle, is fixed with the antibody binding proteins part on this porous particle, is the glucan of 110kDa for size wherein, and it is expressed as K _AvThe gel phase breadth coefficient of basic matrix greater than 0.65, median particle diameter is 65 to 84um.In one embodiment, the present invention relates to comprise the isolation medium of porous particle, be fixed with the antibody binding proteins part on this porous particle, wherein ligand density is 5.0-10, and median particle diameter is 65 to 84um.In another embodiment, the present invention relates to the isolation medium be made up of porous particle, be fixed with the antibody binding proteins part on this porous particle, wherein ligand density is 5.0-10mg/ml; For size is the glucan of 110kDa, and it is expressed as K _AvThe gel phase breadth coefficient of basic matrix greater than 0.65, median particle diameter is 65 to 84um.

In one embodiment, the part of isolation medium of the present invention comprises the albumen of binding antibody, albumin A for example, G and/or L.In one embodiment, described part comprises that Fc is in conjunction with albumen.In best embodiment, described Fc is an albumin A in conjunction with albumen.In preferred forms, described part comprises the recombinant protein A in nonmammalian source.In this article, term " comprises " that albumin A should be interpreted as comprising albumin A or its function equivalent, and it has kept the characteristic of albumin A in conjunction with IgG.Described recombinant protein A part can be coupled to particle by single or multiple attachments, preferably passes through cysteine.Replace in the embodiment one, described part comprises κ-in conjunction with albumen, for example albumen L.

In a specific implementations, the part of isolation medium of the present invention comprises monomer, dimer or the polymer of albumin A functional domain.Thus, described part can comprise one or more territory A, B, C, D and E, preference field B and/or territory C.In a specific implementations, described dimer or polymer comprise albumen Z, and it is the mutant form of territory B, referring to for example US5143844 (Abrahamsen etc.).In a favourable embodiment, it helps fixation and cleans (CIP) when keeping the good combination ability that is obtained according to the present invention, and described part comprises the albumin A functional domain that one or more alkali are stable.Thus, in this embodiment, described part comprises the albumen of sudden change, undergos mutation in wherein one or more described albumin A structure function territories, referring to for example WO03/080655 (Amersham Biosciences), this patent mode by reference is bonded to this paper.Replace in the embodiment one, part comprises the territory C of one or more albumin As.This comprises that the stable list of alkali isolation medium poly-or the poly part is easy to be prepared by those skilled in the art, for example described in the WO03/080655 (AmershamBiosciences).

Replace in the embodiment at one, described part is the antibody binding peptide.Therefore, comprising the porous particle that is fixed with the antibody binding proteins part according to the isolation medium of this embodiment, is the glucan of 110kDa for size wherein, and it is expressed as K _AvThe gel phase breadth coefficient of basic matrix greater than 0.65, median particle diameter is 65 to 84um.

Described basic matrix can comprise by the gel phase breadth coefficient be positioned at designated value with the porous particle that constitutes of any materials, as described herein the significantly improving of its dynamic binding ability (DBC).In another favourable embodiment of the present invention, particle is that the supporter of isolation medium is made by crosslinked carbohydrate materials, for example agarose, agar, cellulose, glucan, shitosan, konjac, poligeenan, gellan, alginates etc., it is easy to prepare by standard method, the for example anti-gelation that suspends (S Hjerten:Biochim Biophys Acta79 (2), 393-398 (1964)).In one embodiment, described carbohydrate materials is highly cross-linked agarose, for example Sepharose ^TM(AmershamBiosciences, Uppsala, Sweden).

In preferred forms of the present invention, above support is cross linked porous agarose material, and it shows favorable mechanical performance, allows flow at high speed thus and can not produce too high pressure.In this embodiment, before gelation, described gelose polymer is by allylation.Can be according to US6602990 (Amersham Biosciences, Uppsala, Sweden), SE0402322 (PCS/SE2005/001408) (AmershamBiosciences, Uppsala, Sweden) or SE0501610 (AmershamBiosciences, Uppsala, Sweden) the described agarose particle of preparation.In a favourable embodiment, described in the preparation such as US6602990 of particle, this patent mode by reference is bonded to this paper.In brief, before forming gel, the bi-functional cross-linking agent that will have an activation site and a disactivation site is introduced in the agarose solution, and allows the hydroxyl reaction of itself and agarose, and its chemical bond is on agarose thus.In the first step of this method, form the solution or the dispersion liquid of described polysaccharide.Form polysaccharide gel in the organic solvent by its aqueous solution is emulsifiable in then, activate the disactivation site of described crosslinking agent afterwards, and with the hydroxyl reaction of polysaccharide.Described initial crosslinked after, can be undertaken further crosslinked by conventional method.

The particle of isolation medium of the present invention is polydisperse, can be the particle diameter qualification of 30-140um by scope, preferred 43-128um, more preferably 70-84um.The mode of another defined particle size commonly used of this area is the median particle diameter (d that cumulative volume distributes _50v), for this separating medium, its scope is 65-84um, for example about 75um.In another embodiment, described particle is monodispersed, is restricted to particle diameter 74-76um, for example 75um.As known in the art, be easy to control granular size during the course, extracting sample from emulsion is estimated particle diameter at microscopically, next adjusts and stirs to reduce granular size.

According to the present invention, the method that can adopt any known for example epoxy coupling is fixed to particle with part.In a specific implementations, ligand density is 5-10mg/ml.Those skilled in the art is easy to control density, be the concentration of fixed ligands, referring to for example Hermanson, GregT., Mallia, A.Krishna, Smith, Paul K.Immobilized AffinityLigand Techniques, the 118th page, Academic Press.ISBN 0-12-342330-9. has also described for example fixing ad hoc approach of recombinant protein A of albumin A in the document.

Therefore, in an embodiment of isolation medium of the present invention, be the glucan of 110kDa for size, it is expressed as K _AvThe gel phase breadth coefficient of particle greater than 0.60, be preferably greater than 0.65.Therefore, in one embodiment, be the glucan of 110kDa for size, it is expressed as K _AvThe gel phase breadth coefficient of particle be 0.60-0.90, preferred 0.65-0.85, more preferably 0.65-0.75.Those skilled in the art is by adjusting the gel phase breadth coefficient that solids content is easy to control above support.Adopt K _AvValue and do not adopt the reason of the exact value etc. in aperture to be, for the agarose of hydrosol sample, it is very difficult accurately to measure the aperture under hygrometric state, and the hole size of estimating after hydrosol drying can not reflect described wet state truly.

The dynamic binding ability of isolation medium is the good indication that whether separating medium is fit to large-scale operation, and the increase of adhesion has improved the economy of technology greatly.In one embodiment, the present invention is a kind of isolation medium, and under 2.4 minutes retention time, its dynamic binding ability is higher than 40mg antibody/ml isolation medium.In a specific implementations, described isolation medium provides the dynamic binding ability that is higher than the 35mg/ml isolation medium, for example in the 35-50 scope, as about 39-40mg/ml isolation medium.

Therefore, if for example above-mentioned MabSelect of this isolation medium and prior art ^TMProduct is compared, and gel phase breadth coefficient of the present invention increases, and granular size reduces.Even be not directly related, the increase of gel phase breadth coefficient, the increase of the particle volume that can utilize will be accompanied by the increase in aperture usually.Yet, as mentioned above, McCue etc. (Journal ofChromatography A, 989 (2003) 139-153: " Evaluation of protein Achromatography media ") propose, in the research of being reported, the aperture reduce to have increased dynamic binding ability.Therefore, significantly increasing of the dynamic binding ability of isolation medium of the present invention is opposite with the instruction of prior art, is very beat all therefore.

Even the most preferred form of isolation medium of the present invention is a particle, but the present invention also comprises other form, for example material all in one piece, filter disc or film; Chip, surface, capillary etc.

Second aspect the present invention relates to a chromatographic column that comprises above-mentioned isolation medium.Contain the matrix that with good grounds conventional fill method is filled in this post, or the matrix with the formal operations of expansion bed is housed.For the operation of extended mode, described particle preferably provides highdensity filler, as known in the art.In a favourable embodiment, described post by any conventional material for example biocompatible plastic constitute for example polypropylene, stainless steel or glass.Described post can have the size that is suitable for laboratory or large scale purification and/or antibody detection.In a specific implementations, post according to the present invention has luer joint, pipe connections and domed nut.

The present invention also comprises the kit that is used for antibody purification, and described kit comprises the chromatographic column that is filled with above-mentioned isolation medium, one or more buffer solution that is arranged in different chamber and the written explanation that carries out extensive capture antibody from raw material.In a specific implementations, this kit also comprises luer joint, pipe connections and domed nut.

The third aspect, the present invention relates to method by the affinity chromatography antibody purification, this technology comprises raw materials technology is contacted with absorbing antigen with isolation medium according to the present invention, the selectivity step that the antibody that is adsorbed on the isolation medium is washed, thereby add eluent antibody is discharged from separating medium, from eluent, reclaim antibody.Adopt this method, can obtain, for example greater than the dynamic binding ability of 40mg antibody/ml isolation medium greater than 35mg antibody/ml isolation medium.Thus, when implementing this method, dynamically binding ability can be 35-50mg antibody/ml isolation medium, for example 39-40mg antibody/ml isolation medium.

This method can be used for from cultivate liquid and separation antibody in the supernatant.In a favourable embodiment, raw materials technology comprises zymotic fluid.In this embodiment, from host cell proteins, DNA, virus, endotoxin, nutrient, for example anti-blowing agent of cell culture medium component and antibiotic and the impurity relevant antibody purification misfolded sample and the polymer for example with product.In a specific implementations, described raw material is with before isolation medium contacts, and through mechanical filter, therefore, flowing mutually is the cell culture fluid of clarification.The suitable condition of absorption is well known to a person skilled in the art.

This method can be used for the monoclonal or the polyclonal antibody of purifying any kind of, for example derives from the antibody of mammalian hosts, and for example mouse, rodent, primate and the mankind perhaps derive from the antibody of cultured cell, for example hybridoma.In one embodiment, the antibody of recovery is behaved or humanized antibody.In another embodiment, described antibody is selected from the antibody from mouse, rat, rabbit, hamster, cavy, ox, sheep, goat, pig and chicken.Described antibody can be any kind, promptly is selected from IgA, IgD, IgE, IgG and IgM.In one embodiment, the antibody of recovery is immunoglobulin G (IgG).In a particular embodiment, described IgG is selected from human IgG1, human IgG2, human IgG 4, human IgG A, human IgG D, human IgG E, human IgG M, mouse IgG1, mouse IgG2a, mouse IgG2b, mouse IgG3, rabbit Ig, hamster Ig, cavy Ig, ox Ig and pig Ig, preferred human IgG1, human IgG2, human IgG 4, mouse IgG2a, rabbit Ig and cavy Ig.Therefore, in one embodiment, antibody is monoclonal antibody.As everyone knows, monoclonal antibody technique relates to the fusion of immortality cell with continuous replication capacity and mammalian cell to produce antibody.The Fusion of Cells that is produced or " hybridoma " next are arranged in generation the monoclonal antibody of cell culture.In the case, should be appreciated that term " antibody " also comprises antibody fragment and any fusion that comprises antibody or antibody fragment.Therefore, this method can be used for the separating immune globulin quasi-molecule, and it has the performance of albumin A and/or Protein G and/or albumen L binding domain-immunoglobulin.

This method can be used as conventional liquid chromatography, wherein the flow communication effect of crossing gravity and/or the pump isolation medium of flowing through.Therefore, in one embodiment, isolation medium is present in the chromatographic column, and described raw materials technology and eluent stream are through chromatographic column.

Replace in the embodiment at one, this aspect of the present invention relates to by affinity chromatography removes method with purification of liquid with one or more antibody, and this technology comprises and contacts liquid with absorption antibody with isolation medium according to the present invention and reclaim purified liquid.This embodiment for example can be used for the situation that liquid is blood or blood plasma, thereby wishes to remove from blood or blood plasma the blood product that one or more antibody obtain safety.In one embodiment, antibody is discharged from isolation medium by adding eluent, thus the isolation medium that preparation reuses.

Absorption and wash-out according to antibody of the present invention are easy to discharge by standard conditions, and for example similar commercial product is recommended, referring to for example MabSelect ^TMOperational manual (AmershamBiosciences, Uppsala, Sweden).Therefore, can carry out for example gradient elution of wash-out by the eluent that adds the PH that changes isolation medium.

Last aspect the present invention relates to the rapid technology of multistep that antagonist carries out purifying, and this technology comprises the aforesaid step of catching, and has one or more steps to carry out intermediate purification and/or refining with antagonist afterwards.In one embodiment, catch step and be hydrophobic reciprocation and/or ion-exchange chromatography afterwards.In a step of replacing, catch step and be multiple anion or cation-exchange chromatography afterwards.In the preferred forms of this technology, catch step at MabSelect ^TMCarry out on the Xtra (Amersham Biosciences, Uppsala, Sweden).

The detailed description of accompanying drawing

In the schematic diagram of accompanying drawing 1, for isolation medium according to the present invention (line on top) with for prior art products (dotted line of bottom), Y-axis is dynamic binding ability (DBC) (a mg antibody/ml isolation medium), its be on the X-axis retention time (minute) function.It clearly illustrates how to provide much higher dynamic binding ability according to isolation medium of the present invention, in fact, and high about 30%.

Experimental section

These examples not should be understood to only for illustration purposes to the restriction by claims institute restricted portion of the present invention.Hereinafter and whole lists of references mode by reference of providing of other places of this specification be combined in this paper.

Embodiment 1: isolation medium

Prepare the agarose particle by the disclosed suspension gel method of US6602990 (Amersham Biosciences).Especially, suitably adjust solids content, preparation K by principle well known in the art _AvIt is 0.69 particle.Further, by adjusting the speed and the time of stirring, median particle diameter is controlled to be 80um.

Above-mentioned particle is carried out the epoxy activation, according to known method, Hermanson for example, GregT., Mallia, A.Krishna, Smith, Paul K.Immobilizied AffinityLigand Techniques, the 118th page, described in the Academic Press.ISBN 0-12-342330-9, recombinant protein A (rProtein A) is coupled on the particle by the C end.The ligand density of rProteinA ligand coupling is 7.3mg/ml.

The isolation medium that is used for comparison is the MabSelect of Amersham Biosciences ^TM, according to the description of product, the median particle diameter of its accumulative total volume distributed median is 85um.

Embodiment 2: dynamic binding ability

According to the dynamic binding ability (DBC) of following measurement: 1.0mg/ml people's polyclone IgG of the neutral PH of in two posts of product as described in being equipped with, packing into as the isolation medium of preparation as described in the embodiment 1.Determine described ability at 10% breakthrough point, the result as shown in Figure 1.

Device

The filling of post

Post (2) XK16/20 Amersham Biosciences

Fill pond XK16/20 Amersham Biosciences

Pump 25ml/ minute, P-900 Amersham Biosciences for example

Safety valve 0.3MPa Amersham Biosciences

Pressure gauge

Chromatogram

AKTA ^TMExplorer10 or AKT FPLC ^TM(Amersham Biosciences) spectrophotometer, dual-beam

Chemical reagent

Ethanol 99.5% spectrophotometric

Sodium chloride pa

Sodium dihydrogen phosphate pa Baker M=137.99g/mol

NaOH pa Prolabo M=40.00g/mol

Natrium citricum pa Merck M=75.07g/mol

Hydrochloric acid pa

Gammanorm 165mg/ml Octapharma

Solution

Buffer solution

Filling liquid 1: 20% (v/v) ethanol that contains 0.25M NaCl

Filling liquid 2:20% ethanol

Adsorption-buffering liquid: the 0.020M NaH that contains 0.15M NaCl ₂PO ₄, PH is adjusted to 7.4 ± 0.05 with strong caustic

Stripping liquid: with hydrochloric acid PH is adjusted to 3.0 ± 0.05 0.1M natrium citricum.

The IgG sample solution

Sample solution with adsorption-buffering liquid dilution Gammanorm preparation 1.00 ± 0.01mg/ml.Should pass through the metric measurement absorbance at the 280nm place, the concentration of sample for reference solution (1+1 dilutes the back).Adopt 1.38ml/mg*cm as the correct concentration of absorbance coefficient calculations.

The filling of post

The gel preliminary treatment

The 50ml gel that is positioned on the glass filter tunnel was cleaned 5 minutes with filling liquid 1.Adopt vacuum to blot 5 minutes.The careful mixing, take by weighing two parts of 14.3g and be put in two beakers.Adding～25ml filling liquid 1.

Fill

To fill the pond is mounted on the post by connector.Measure and the required bed height of mark.Quantitatively shift gel, and the filling liquid 1 of packing into.Filled 5 minutes with 25ml/ minute with filling liquid 2, the maximum pressure that flows downward is 0.3MPa.The height of mark bed in flow process.Remove and fill the pond, top sub is installed on gel, with 10ml/ minute flow rate more than 5 minutes.If desired, with the bed height adjustment or heavily be filled to 10.0 ± 0.3cm.

Program

The control of dress post

By injecting the filling of acetone soln, calculate the symmetry at resulting peak by post inspection post.The solution of preparation 100mg acetone/ml adsorption-buffering liquid.Xiang Zhuzhong was with 5ml/ minute injection 50ul acetone soln.Adopt the AKTA system, or according to following description evaluation or calculate the peak dissymmetry factor:

The peak dissymmetry factor is calculated as the absolute value of B/A, and wherein A and B are the retention volume that the retention volume of maximum peak height deducts 10% peak heights.If symmetry is between 0.80-1.60, then described post is approved.

Determine the ability of leaking

Described analysis should be based on two posts under the room temperature of control, 23 ± 1 ℃.With the phase speed that flows is to determine the ability of leaking in 250cm/ hour.Check flow velocity according to 6.3 parts.Adsorption-buffering liquid is passed through bypass position, until reaching stable baseline.Automatic zero set adds 35ml IgG solution by bypass, to obtain stable 100% signal.Importantly, flow velocity is consistent in The whole analytical process.After adsorption-buffering liquid arrives the steady baseline of bypass position once more, with the described post of adsorption-buffering liquid balance of 5 column volumes.Automatic zero set is with the IgG solution of 100 column volumes of phase speed 250cm/h (8.38ml/ minute) Xiang Zhuzhong adding that flow.

Calibration

Must carefully calibrate described chromatographic system with respect to the volume sendout.Regularly calibrate, and before each the analysis, the pump that is used for application of sample is checked flow velocity according to the instrument handbook.

Estimate

Estimate dynamic binding ability at 10% breakthrough point q10%.Detect the UV absorbance at the 280nm place.Determine and record 100%UV signal (A _100%), and corresponding to the UV signal (A of the IgG subclass that does not have combination (determining from application of sample 60ml) _Sub).Column volume (V _c) and the concentration (C of sample raw material _o) (two-decimal) also be used for calculating.

Dynamically binding ability is calculated as: the IgG concentration in effluent is the IgG that packed in post in the 10% o'clock amount of IgG concentration in the raw material.IgG amount by post before the amount of being packed into spills 10% is calibrated.

${\mathrm{<figure-callout\; id="10"\; label="q"\; filenames="A20058004305000211.png"\; state="\{\{state\}\}">q</figure-callout>}}_{10\%}=\frac{C_0}{V_C}[V_{\mathrm{app}}-V_{\mathrm{sys}}-\underset{V_{\mathrm{sys}}}{\overset{V_{\mathrm{app}}}{\&Integral;}}\frac{A\left(V\right)-A_{\mathrm{sub}}}{A_{100\%}-A_{\mathrm{sub}}}*\mathrm{dv}]$

C wherein ₀Be material concentration, V _cBe column volume, V _AppFor leaking the volume of out-of-date adding, V until 10% _SysBe the dead volume (dead volume that does not comprise post) of system, described integration spills until taking place 10% for the total amount of IgG in the eluent that flows out from post, and A (V) is the absorbance of given adding volume, A _SubAbsorbance contribution for uncombined IgG subclass.It is gel filled that the difference of the dynamic binding ability between the single post should not surpass 1.2mg/ml.

Accuracy

The relative standard deviation of described ability is 2%.

List of references

Handbook of Process Chromatography, A Guide toOptimization, Scale-up and validation (1997), Academic Press, San Diego.Gail Sofer﹠amp; Lars Hagel edits, ISBN 0-12-654266-, 308-310 page or leaf.

Embodiment 3: calculated for gel phase breadth coefficient

Principle

Determine gel phase breadth coefficient by gel filtration according to the particle of embodiment 1.The HR16/30 post of two glucans by filling that will have different size.Detect the retention volume of each glucan, and be used for calculating K _Av, this value is used to describe the particle volume part that can use for specified molecular weight.From K _AvValue has been reported the K of Mp110000 _AvValue.

Device

Fill:

Post HR16/30

Fill pipe HR16/30

Pump AKTA ^TMP-900 pump *

Selectivity test

AKTA explorer 10 * of system

Contrast UNICORN ^TM

Sample is annotated Autosampler A-900

Sample loop 200ul

Pump P-900

Detector Shimadzu RI-detector

* or equivalent

Chemical reagent

Flowing of filling is distilled water mutually, and flowing of selectivity test is the NaCl that is arranged in distilled water of 0.20M mutually.

Flowing be to inject 2% acetone in the post of distilled water mutually, and the coupled columns filling detects.

The glucan that adopts in selectivity test is:

Natural glucan 5mg/ml Amersham Biosciences

Mp＝196300 10mg/ml Pharmacosmos

Mp＝66700 8mg/ml Pharmacosmos

All glucans dilute except Mp=196300 uses 0.25M NaCl with 0.20M NaCl dilution, and it is as the label of the cumulative volume of post.

The safety indication

Do not need to take special safety precaution.

Sample pretreatment

With 0.20M NaCl the gel that is positioned on the glass filter is cleaned, and dry, ftracture until gel.Then the 60ml xerogel is dissolved among the 60ml 0.20M NaCl, forms gel slurry.

Calibration

According to separately handbook used instrument is contrasted and calibrates.

Program

An analysis comprises fills tested twice pillar, and promptly each glucan injects twice.

The dress post

According to following method HR16/30 post (Amersham Biosciernces) is adorned post: adopt and fill connector, this post is connected to by bottom fitting fills pipe.Post is placed on the column holder, fills pipe and be positioned at the bottom.To fill pipe joint and be connected, and bit by bit fill 0.5cm water with pump.Be transferred to gel slurry, fill 0.2M NaCl, filter and bottom fitting are put on the post.

Apply 22ml/ minute flow velocity, with pillar dextrorotation (turn the column right), uninterrupted pumping 10 minutes.Remove unnecessary gel with the Pasteur pipette.Filter is put in the top, opens top sub, adjust the bed surface.Apply 10ml/ minute flow velocity, stable until bed, adjust joint once more, and then apply the stability of flow velocity with examination couch.If do not observe the further compression of bed, think that then it has been stablized.

Detect dress post quality by injecting 2% acetone soln (at distilled water).Do not need to reach balance before injecting, its reason is that flowing does not change mutually.During 1.2CV with 150cm/h (5ml/min) wash-out acetone.Calculate plate number and dissymmetry factor from resulting peak.Accept standard: plate number＞2400N/m, asymmetry 0.7-1.3.

The calculating of plate number: N/L

N ＝5.54*(t _R/W _h) ²

t _R=retention time

W _h=half peak width when high

L=post height (m)

Measure dissymmetry factor at 10% peak height place.

Selectivity test

After accepting dress post standard, can move selective test method, it may further comprise the steps:

1. column equilibration, 1.5CV 0.20M NaCl at least

2. inject the 200ul glucan with the automatic application of sample instrument of A-900

3. carry out wash-out mutually with flowing of 1.3CV.

For each glucan or glucan mixture * repeating step 2 and 3.

* can mix glucan according to following method, but also can a shot one.

Mix 1: natural glucan+Mp66700

Mix the Mp196300 among the 2:0.25M NaCl

Error source

The air that exists in the pump can be given the flow velocity that makes mistake, and therefore controls in running so that pressure stability is very important.

Estimate

Obtain the retention volume of each glucan from the RI curve of resulting chromatogram, wherein the peak is defined as the RI maximum of described glucan.Calculate the K of glucan then from following formula _Av:

K _av＝(V _R-V ₀)/(V _C-V ₀)

Wherein:

V _R=the retention volume adjusted for the additional post volume through the glucan of wash-out, ml

V ₀=the interstitial volume (retention volume of natural glucan) adjusted for the additional post volume (ml)

V _CThe geometric volume of=post (height of bed, the surface area of cm post, cm ²)

Then glucan is carried out logMp to K _AvThe value mapping.Each glucan has two values and cause that four values are arranged on each schematic diagram.Two glucans are carried out linear interpolation obtain K corresponding to 110000 molecular masses of being reported (Mp value) _AvValue.

Claims (27)

1. an isolation medium of being made up of the porous carbon carbohydrate particles is fixed with the antibody binding proteins part on the described particle, and wherein said ligand density is 5.0-10mg/ml; And median particle diameter is 65-84um.

2. isolation medium as claimed in claim 1 is the glucan of 11kDa for size wherein, and it is expressed as K _AvThe gel phase breadth coefficient greater than 0.65.

3. isolation medium as claimed in claim 1 or 2 is the glucan of 11kDa for size wherein, and it is expressed as K _AvThe gel phase breadth coefficient be 0.65-0.85.

4. as before any described isolation medium of claim, wherein said ligand density is 5.5-9.0mg/ml.

5. as before any described isolation medium of claim, wherein said median particle diameter is about 75um.

6. as before any described isolation medium of claim, wherein said particle comprises the cross-linked polysaccharides material.

7. isolation medium as claimed in claim 6, wherein said carbohydrate materials are Sepharose.

8. isolation medium as claimed in claim 7, wherein said gelose polymer is allylation before gelation.

As before any described isolation medium of claim, when it provides retention time to be 2.4 minutes greater than the dynamic binding ability of 40mg antibody/ml isolation medium.

10. as before any described isolation medium of claim, wherein said part comprises that Fc is in conjunction with albumen.

11. isolation medium as claimed in claim 10, wherein said Fc is an albumin A in conjunction with albumen.

12. isolation medium as claimed in claim 11, wherein said Fc is the recombinant protein A that the nonmammalian source is produced in conjunction with albumen.

13. as before any described isolation medium of claim, wherein said part comprises monomer, dimer or the polymer of albumin A functional domain.

14. isolation medium as claimed in claim 13, wherein one or more described albumin A functional domains are undergone mutation.

15. one kind comprises the chromatographic column as one of any described isolation medium of claim 1-14.

16. the kit of an extensive capture antibody from raw material, described kit comprise, is positioned at the chromatographic column as any described isolation medium of claim 1-14 of being filled with of different chamber, the printed instructions of one or more buffer solutions and its use.

17. method by the affinity chromatography antibody purification, this technology comprise with raw materials technology with contact as any described isolation medium of claim 1-14, the optional step that the antibody that is adsorbed on the isolation medium is washed, add the eluent that antibody is discharged from isolation medium and from eluent, reclaim antibody.

18. method as claimed in claim 17, wherein said raw materials technology comprises zymotic fluid.

19. as claim 17 or 18 described methods, wherein said raw material with before isolation medium contacts through mechanical filter.

20. as any described method of claim 17-19, wherein said antibody is monoclonal antibody.

21. as any described method of claim 17-120, wherein said isolation medium is present in the chromatographic column, described raw materials technology and eluent are by described chromatographic column.

22. one kind by the method for affinity chromatography from one or more antibody purification liquid, this technology comprise with described liquid with contact as any described isolation medium of claim 1-14, with absorbing antigen, and reclaim purified liquid.

23. method as claimed in claim 21 wherein discharges described antibody by adding eluent from isolation medium, thereby the isolation medium that preparation is used to reuse.

24. the rapid technology of multistep that antagonist carries out purifying, this technology comprise any described step of catching as claim 17-21, one or more steps of carrying out intermediate purification and/or refining afterwards for antagonist.

25. technology as claimed in claim 24, the wherein said step of catching is hydrophobic reciprocation and/or ion-exchange chromatography afterwards.

26. as the described technology of claim 120, the wherein said step of catching is the anion of various ways or cation-exchange chromatography afterwards.

27. as any described technology of claim 24-26, the wherein said step of catching is at MabSelect ^TMCarry out on the Xtra.

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