CN101120017A - Affinity-plus ion exchange-chromatography for purifying antibodies - Google Patents

Abstract

A novel method for purifying antibody and other product protein concomittant with removing aggregates made up from single product protein species is devised.

Description

The affinity-plus ion exchange-chromatography that is used for antibody purification

The present invention relates to the protein field, relate in particular to the antibody purification in the biotechnology production.The objective of the invention is to describe the novel method of this proteinoid of a kind of purifying or antibody.

Because can be used for an antibody (normally IgG) that goes on foot in the purifying cells culture supernatant almost completely, the A protein chromatographic has been widely used in the industrialization production of antibody.After using repeatedly, the part that A albumen affinity column takes place to a certain degree inevitably comes off.This part is because the A albumen on the post is hydrolyzed to shear to come off, and in the antibody industrialization of field of medicaments is produced, goes up reason for management and must not add protease inhibitor cocktail.Unfortunately, this A albumen or A protein fragments pollutent still keep their avidity to IgG, and they and antibody purified form mixture and be difficult to remove.This two class in the antibody purification must be removed by the heterodimer mixture of different macromole (composition), harmful immune response can be caused because A albumen is bacterioprotein; Have and report and externally A albumen is added the formed model mixture of monomer (monomelic) IgG can activate the white corpuscle that carries Fc and complement system and produce oxidation and anaphylatoxin activity (Balint etc., Cancer Res., 44,734,1984).Balint etc. (the same) and other people (Das etc., 1985, Analyt.Biochem., 145,27-36) proof can be separated this IgG-albumen composition and the IgG that does not form mixture by gel-filtration.The shortcoming of this method is the low and antibody loss of treatment capacity.

As US6,399,750 is described, and the up-to-date commercially produced product of the reorganization A protein formulation that links to each other with base for post matter through a thioether bond has higher A albumen column capacity.But simultaneously, be connected the natural A albumen substrate with the traditional multiple spot that obtains by the CNBr coupling and compare, the expulsion rate of this reorganization A albumen substrate often sharply rises.

US 4,983, and 722 have described by the antibody preparation mixture with contaminative A albumen and A protein purification and are adsorbed onto anion-exchange material, and wash-out antibody separates the selective separation method of these two kinds of compositions with A albumen successively under ionic strength rising condition.This resolution method height depends on the pI of antibody, but for given antibody, its resolution pole is different.In addition, obtain the required salt gradient slope of good separation and limited treatment capacity.

Except removing the A albumen composition, another issues of purification that relates to antibody (but can expand to any other type biological medicine albumen) is the homogeneous dimer and the formation of higher coacervation aggressiveness.Opposite with A albumen formation mixture (this mainly also can form based on avidity even natural protein), spontaneity or salt or pH inductive at least a portion protein denaturation make solvent to cause antibody or analogous protein to be subjected to the driving of homogeneous chemistry mass law (chemical mass law) to begin to form aggregate near the water repellent region exposure on surface.Therefore, form on the contrary with mixture based on avidity, non-specific cohesion is driven by solvent repulsive interaction (solvent exclusioneffect) mainly, is similar to the crystal growth behavior in this.Still soluble at first condensation product increases in time, and then protein precipitates from solution.After medicine gave, the contaminative condensation product of low per-cent also can cause deleterious immune response.Up to now, can remove condensation product reliably and almost have only size exclusion chromatography (SEC); Yet SEC is the bottleneck of purifying, compares with other chromatographic technique, and its a large amount of treatment time of needs, material expensive and load capacity are low.

One of purpose of the present invention is the homogeneous condensation product that the another kind of method of design is separated A albumen or A protein fragments and antibody (preferred IgG) and/or separation antibody condensation product or other product albumen, and this method has been avoided the shortcoming of prior art.Adopt the inventive method to realize this purpose.

The antibody purification method of the present invention's design may further comprise the steps:

At first, by A albumen affinitive layer purification antibody, wherein A albumen is natural A albumen or its functional deriv; Second step, under contaminative A albumen or its functional deriv and ion-exchange material bonded condition (the antibody condensation product that this condition can circulate by classification in the liquid is not differentiated circulation liquid with the antibody monomer that forms mixture with A albumen or A protein derivatives), the antibody purification that will contain antibody condensation product and A albumen or A protein derivatives is loaded on the ion-exchange material; Three, the 4th step be classification should circulation liquid collection of ions exchanger at least a antibody monomer fraction in circulation liquid, this fraction with load on ion-exchange material before the antibody composition compare its A albumen or A protein derivatives and the reduction of antibody condensation product content.

The inventive method preferably can be reduced to condensation product content in the antibody purified monomer like this below 1.0%, and more preferably the condensation product of final all antibody of collecting is lower than 0.5% in described or first ion-exchange step circulation liquid.Therefore, by the analytical volume-exclusion measurement in chromatography that the technician knows, the antibody monomer that obtains after the inventive method ion-exchange step is at least 99%, more preferably is at least 99.5%.

Also preferably collect to be recovered in the fraction and load at least 70%, more preferably at least 80% of antibody total amount on the ion-exchange material at described ion-exchanger circulation liquid, most preferably at least 90%, and contaminative A albumen or A protein derivatives keep combining with ion-exchange material.

Condensation product of the present invention is interpreted as the non-covalent combination of same protein, preferably with at least 10 ^-7The binding equilibrium constant combination of M or following (the low expression of this numerical value is in conjunction with tightr), described protein can be made of the homogeneous or heterogeneous many polypeptide of single chain protein matter or covalent attachment (for example passing through disulfide-bonded).The same with its monomer of deriving, condensation product water soluble solution of the present invention.For example, " monomer " of IgG antibody of the present invention relates to the standard tetramer antibody that contains two identical glycosylation heavy chains and light chain respectively.So, for example the dimer condensation product is the non-specific binding of two IgG molecules.The formation of condensation product is closely related with the sex change influence (factor) of and quaternary structure of protein folding to natural protein; For example heating can cause cohesion with the sex change of pH inductive protein folding.Therefore, given proteic cohesion rate has high degree of specificity, energy stability (the Chiti etc. that depend on each protein folding particular attack, 2004, " sudden change is to the reasonable dismissal of protein condenses rate action " (Rationalization of the effects of mutations on protein aggregation rates), Nature, 424:805-808).

A albumen is the cell surface protein of finding in the streptococcus aureus (Staphylococcus aureus).It has in conjunction with Mammals antibody, particularly the characteristic in IgG antibody-like Fc district.In a given antibody-like, avidity can be according to source of species and antibody subclass or allotype and slightly different (summary is seen Surolia, A. etc., 1982, and " A albumen: natural universal antibody " (Protein A:Nature ' s universal, antibody), TIBS, 7,74-76; Langone etc., 1982, " staphylococcal protein A and relevant immunoglobulin receptor " (Protein A of staphylococcus aureus and related immunoglobulinreceptors, Advances in Immunology), 32:157-252).A albumen can directly separate from secretion A proteic streptococcus aureus culture, perhaps be not difficult recombinant expressed in intestinal bacteria (E.coli) (Lofdahl etc., 1983, Proc.Natl.Acad.Sci., USA, 80:697-701).Prior art is described A albumen in detail at antibody purification, and the particularly application among the mono-clonal IgG (for example, Langone etc., the same; Hjelm etc., 1972, FEBS Lett., 28:73-76).For being used for A albumen affinity chromatography, can be with the A albumen coupling in solid substrate, for example crosslinked no electric charge agarose (Sepharose does not have the contained electrically charged component of natural not refined agarose), crosslinked dextran or the earth silicon material of trisacryl.This link coupled method is well known in the art, for example is coupled to the CNBr activated substrate by proteinic primary amine functional group.A albumen combines with the Fc part of IgG with high-affinity and high specific, i.e. the Cy2-Cy3 interface region of (1982, the same) described IgG such as Langone.Particularly A albumen can be with people's allotype or subclass IgG1, IgG2, IgG3 and mouse allotype or subclass IgG2a, IgG2b, IgG3 is strong combines.A albumen also shows the gene family to VH, and there are avidity (Sasso etc., 1991, J.Immunol, 61:3026-3031 in the IgF ab district of VH III coding; Hilson etc., J Exp.Med., 178:331-336, (1993)).The proteic gene order of its coding A shows two zone (Uhlen etc., J.Biol.Chem., 259:1695-1702, (1984) that function is different; Lofdahl etc., Proc.Nutl.Acad.Sci., (USA), 80:697-701, (1983)).Its amino terminal region contains 5 height homologous IgG binding domainss (being called E, D, A, B and C), and the C-terminal zone is anchored on this protein on cell walls and the cytolemma.With the proteic B structure of A is example, as Deisenhofer etc. (1981, Biochemistry, 20:2361-2370) and Sauer-Eriksson etc. (1995, Structure, shown in crystalline structure 3:265-278), proteic all 5 the IgG binding domainss of A combine with the Fc district of IgG, comprise for example human IgG-Fc residue 252-254,433-435 and 311.(1998, Biochemistry, NMR-solution 37:129-136) studies confirm that Gouda etc., find two main binding sites that adjoin basically of Fc part.One of the proteic IgG-binding domains of A A-E is enough to the Fc part in conjunction with IgG on principle.In addition, some human allelotrope of finding VH3 structural domain family can be chosen protein mediated (Ibrahim etc., 1993, J.Immunol., the 151:3597-3603 of combining with human IgG by A wantonly; " the V-district combines with human IgG by A is protein mediated " (V-regionmediated binding of human Ig by protein A)).In this application, another purpose of the present invention, every mentioning is applicable to antibody Fc zone and A protein binding, is applicable to the combination of antibody through the A-of VH3 family protein binding equipotential volume too, if the Fc of this antibody zone itself is can not be with high-affinity and A protein binding the time.Can think that this is the equivalent embodiments of the main Fc method of the present invention; Further described the Fc method with the lower section.

IgG antibody of the present invention is interpreted as can be with high-affinity mode and the protein bound alloantibody of A.In addition, except that the antibody Fc relevant with A protein binding part, this antibody is not necessarily corresponding to the antibody of natural generation.Particularly in its variable region part, it can be as the engineered chimeric antibody of this area conventional design or CDR grafted antibody.In brief, IgG antibody of the present invention is interpreted as IgG type antibody.

The feature of A albumen of the present invention or A protein fragments functional deriv is that the binding constant to mouse IgG2a or human IgG1's Fc part is at least K=10 ^-8M, preferred K=10 ^-9M.The interaction that this paper will meet this binding constant value is called " high-affinity combination ".The proteic this functional derivatives of A preferably contains at least a portion of the proteic functional IgG binding domains of wild-type A, and this structural domain is selected from natural structure territory E, D, A, B, C or it has kept the engineered mutain of IgG combined function.As described in US6013763, the example of this derivative is functional 59 amino acid whose " Z "-fragments of the proteic B structural domain of A, and this B structural domain can be used for antibody purification.Yet antibodies fragment of the present invention preferably contains the complete Fc binding domains of two this section definition at least.Its example is a reorganization A protein sequence, for example described in the EP-282 308 and EP-284 368 of Repligen Corporation.

Also preferably (make up separately or with above-mentioned A albumen or A protein functional derivative) thereby the A protein derivatives that engineered energy single-point is connected.Single-point connects the expression protein portion and links to each other through the chromatography support material of a covalent linkage with A albumen affinity chromatography.It is by suitable reactive residue that this single-point connects, and described residue preferably is arranged near the exposure amino acid position of N-or C-end (i.e. ring) or is positioned at the periphery of this protein folding.Suitable reactive group is for example sulfydryl or amino.This reorganization A albumen or its functional fragment more preferably comprise halfcystine in its aminoacid sequence.Described halfcystine most preferably is included in the section that last 30 amino acid by reorganization A albumen or its functional fragment C-end constitute.In such another preferred embodiment, at least 50% of reorganization A albumen or its functional fragment links to each other through the chromatography upholder or the substrate material of thioether sulfide linkage with A albumen affinity chromatography medium.An example of this embodiment is described in, the US 6399750 of Pharmacia for example, and can be according to the character of used supported matrix with trade(brand)name Streamline ^TMOr MabSelect ^TMAvailable from Amersham-Biosciences.In this article, thioether bond is interpreted as-S-bonding mode with answering narrow sense, and its chemical ingredients no matter has deviation with the conventional chemical term in this; For example, of the present invention described-S-" thioether " key bridge can be than macoradical, for example a thioesters or a part of mixing acetal have deviation with the conventional term that the chemist is named according to reactivity in this.The thioether bond bridge is preferred thioether bond bridge on its conventional narrow sense chemistry connotation.This bridging sulfide group can be, for example the epoxide group reaction that contained of sulfydryl by making the proteic cysteine residues of A and activatory chromatography support material produces.Utilize the terminal cysteine residue under the condition that is suitable for unique sulfydryl that coupling protein matter only exposes, to carry out this reaction, thereby only cause this proteinic single-point to connect.

In a particularly preferred embodiment, A albumen of the present invention or functional A protein derivatives are the described reorganization of US6399750 A albumen, its proximal end (juxtaterminal) contains an engineered cysteine residues, and preferred at least 50%, more preferably at least 70% sulphur atom by described cysteine residues is as single tie point and the coupling of chromatography support material.Also the activation that preferably mediates by epoxide realizes this coupling, more preferably adopt 1,4-two-(2, the 3-glycidoxy) butane activation, for example agarose matrix (is used the crosslinked sepharose 4B of epoxy chloropropane as SepharoseFast Flow, Amersham Biosciences, Britain) or adopt the epoxy chloropropane activation, agarose matrix for example is as Sepharose FF.In conjunction with the above-mentioned preferred embodiment of this section, also preferred first ion-exchanger is an anionite, particularly quaternary amine anionite, for example Sepharose Q ^TMFF (Amersham-Biosciences/Pharmacia), more preferably coupling has the anionite of function exchangeability group Q on the matrix upholder, described group Q is N, N, N-trimethylammonium amino-methyl, most preferred anionite are the Sepharose Q of Pharmacia/Amersham Biosciences ^TMFF.Quaternary amines is that the pH to loading/lavation buffer solution changes insensitive strong exchanger.The basis of stream (fast flow) exchanger is the 45-0165 μ m sepharose 4B of the higher physical stability of high-crosslinking-degree fast; Also have this sepharose (sepharose) thus even removed the non-specific matrix absorption that antibody can not take place the electrically charged sulfating numerator component of natural agar sugar yet under the high carrying capacity condition of antibody.The example of this embodiment is seen the experiment chapters and sections.

The functional IgG of any kind that contaminative A albumen of the present invention is above-mentioned A albumen or its functional derivatives is in conjunction with product (offspring), available from the A albumen affinity column during wash-out institute binding antibody.For example, peptide bond hydrolysis can produce this contaminative A proteic substance, and this hydrolysis may take place utilizing in the effect of enzyme, particularly industrial production very much.The A protein chromatographic is applicable to the early stage step of downstream processing, and this moment, the fresh product solution of refining purifying did not still contain suitable protease activity.The cell of checkmating in the cell culture fluid or in preliminary centrifugal or filtration step the disruptive cell emit free proteolytic enzyme probably; Opposite with biochemical research practice, for the Quality Control purpose common before downstream processing or during do not add the cell culture fluid that contains proteinase inhibitor.(proteinase inhibitor) example is phenyl-methyl-SULPHURYL CHLORIDE (PMSF) or e-caproic acid.This chemical reagent is deleterious additive in bio-pharmaceutical production.The quaternary structure that depends on the A protein folding, its recombination function derivative or fragment also may be not so good as wild-type A albumen to the tolerance of proteolytic enzyme.In case the sum of binding domains reduces, the amino acid section that connects each IgG binding domains may expose.Contact between structural domain has and helps the folding stability of structural domain.Because institute's inductive conformational change during antibodies, A albumen or its described functional derivatives and antibodies also may influence or promote the susceptibility to the proteolytic enzyme effect.Wild-type or total length A albumen or its functional engineered fragment also may show difference.It is conjugated protein that contaminative A albumen of the present invention preferably is still functional IgG, therefore when on the ion-exchange separating medium of the present invention that it is loaded on subsequently can with the antibodies of A protein purification.Contaminative A albumen is the reason that is difficult to separate effectively the two why with the high-affinity combination of antibody purified.

According to the present invention, preferred collection antibody to be purified from cell culture fluid earlier is by the described antibody of A albumen affinitive layer purification.More preferably described cell culture fluid is the mammalian cell nutrient solution.Mammalian cell has and is called lysosomal big compartment, wherein contains in necrocytosis or cytoclasis discharges when collecting degrading enzyme.Specifically, described cell culture fluid can be myeloma cell's nutrient solution, for example NSO cell (Galfre, G. and Milstein, C., Methods Enzymology, 1981,73,3).The myeloma cell is the plasmoma cell, i.e. lymphocyte pedigree cell.Exemplary NSO clone is, for example can be from Britain, and WiltshireSP4 OJG, Salisbury, using microbe and research centre (Centre for Applied Microbiology ﹠amp; Research), No. the 85110503rd, the clone ECACC that (ECACC) freely obtains of European cell culture preservation institute (European Collection of Cell Cultures).Found that NSO can produce high product output, if when particularly being used to produce recombinant antibodies.And then find to compare with other host type cell that is used for some A albumen affinity chromatography system at least, the NSO cell can produce higher levels of contaminative A albumen with reappearing, and it may be that fragment is shortened in the proteic reorganization of wild-type A that single-point connects that described system adopts.The example of this system has Streamline ^TMReorganization A albumen affinity chromatography resin (AmershamBiosciences; US 6,399, the reorganization A albumen that the 750 described single-points of thioesters basically connect).Utilize Streamline ^TMReorganization A albumen affinity column can obtain approximately or surpass the level of 1000ng contaminative A albumen/mg antibody.The inventive method difference with the prior art is and can effectively contaminative A albumen be reduced to＜1ng/mg antibody from described high level in a step fast purifying step, promptly decontamination factor about 1000 *.

The antibody of stand-by A albumen affinitive layer purification also preferred (separately or with leading portion combination) thus do not handle when collecting or collect the back inactivated proteases, more preferably antibody does not mix with at least a exogenous proteinase inhibitor of adding after collection.In this article, proteinase inhibitor be can selectivity the arrestin enzyme, simultaneously can the chemically modified antibody protein three grades and/or quaternary structure or to its harmless any kind of chemical reagent (not being proteolytic enzyme); The example of proteinase inhibitor is a sequestrant; for example may think chelating to the EDTA and the hydrochloric acid N-[(2S of the important metal ion of metal proteinase activity, 3R)-3-amino-2-hydroxy-4-phenyl butyryl radicals]-L-leucine [bestatin] (equivalent anti-metal proteinase activity) is this class inhibitor.Most preferably described proteinase inhibitor is selected from PMSF and Laskowski etc. (1980, " protein inhibitor of proteolytic enzyme " (Protein inhibitors ofproteinases), Ann.Rev.Biochem., 49,593-626) described specific proteins enzyme inhibition peptide.Example is bright Trypsin inhibitor,Trasylol and aprotinin.

Extensively described the operation of A albumen affinity chromatography in the technical literature, need not to give unnecessary details.Other example except that above-mentioned is, for example Duhamel etc. (J.Immunological Methods, 31, (1979), 211-217) described from the last pH gradient elution of A albumen-Sepharose human IgG1, IgG2 and IgG4.

Certainly, to contact with the condition of acidic pH of about pH3-4.5 during wash-out,, also be certain to form condensation product even be about pH7.5 with buffer exchange immediately and then.Early stage in the eighties in 20th century, the A protein chromatographic begins widespread use and awares this problem when making comparisons with traditional chromatography training.In other preferred embodiment, pickling is carried out viral inactivation treatment after taking off the A albumen substrate, again antibody purified like this is loaded on first ion-exchanger, wherein viral inactivation treatment more preferably is included in pH3.5-pH4.5 and hangs down pH and cultivated about 50-90 minute, preferably at least 30 ℃ of temperature, more preferably at least 45 ℃, or via hole diameter is less than 1 μ m, preferably reduce strainer and filter less than the animal virus of 0.25 μ m.Therefore, reducing before condensation product content removes contaminative A albumen or A protein derivatives simultaneously, should insert another important treatment step and grow to guarantee condensation product and form again and to guarantee condensation product; Described processing can be, for example (heat) during acid pH is attacked the protein capsid sex change that makes virus or made it assembling and dissociates (de-assemble), perhaps available ultrafiltration step, and this step also is subjected to the influence of film Denaturation.Preferred low pH incubation step is handled in the virus minimizing, is easy to obtain the log virus minimizing that coefficient is about 6-8.

In an embodiment preferred, utilization be lower than 5mS/cm, preferably be lower than 3mS/cm, more preferably less than 2mS/cm, 1.2mS/cm or lower low conductivity elution buffer wash-out antibody from the A protein chromatographic post most preferably from about, preparation 1 * buffered soln is used for wash-out antibody product albumen from the A albumen post.Sort buffer liquid equally preferably should have at least 0.1mS/cm, preferred 0.5mS/cm at least, the minimum specific conductivity of 0.8mS/cm at least most preferably.Be unexpectedly, of the present invention aspect this, prove that this low conductivity damping fluid (not relying on the chemical property of used buffering salt) the consistent demonstration: i. condensation product content during wash-out from A albumen post is low, ii. in acid or low pH inactivation of virus step subsequently (then immediately with the pH re-adjustment to about neutral pH, be pH6.5-7.5) the most of appropriateness risings of condensation product content, still significantly reduce with the log of iii. virus during acid pH is handled, the general log that obtains after 60 minutes in contact reduces coefficient and is about 7.This common benefit of low conductivity damping fluid is not still understood.Should notice that the character of buffering salt influences the increase of condensation product content strongly when high conductance (about 5-20mS/cm).Should notice that specific conductivity is that 5-10mS/cm and above Citrate trianion cause that the condensation product ratio greatly raises in the acid pH inactivation of virus step.Therefore, in other embodiment preferred (separately or with other embodiment combination of above-mentioned low conductivity elution buffer), A protein chromatographic elution buffer utilizes monovalent carboxylic and/or its corresponding monocarboxylate of the about 3-4 of pKa value, for example its alkali or alkaline earth carboxylate salt more preferably utilize formate/formic acid as buffering salt.Randomly, preferred described monocarboxylate or carboxylic acid are unit price a-amino acid, and charged any group when not having pH4 in its side chain is except H ₄N ⁺-CHR-COO ^-Outside the group (head group), wherein R is a side-chain radical, and described amino acid does not have mercapto functional group, is water-soluble when pH4 preferably, and concentration is at least 5mM, more preferably 10mM at least, the pKa value (pKa of also preferred its carboxylic acid functional ₁) be pH2-3.Described amino acid can be natural or alpha-non-natural amino acid, preferred natural amino acid.The pKa value of natural amino acid carboxyl head group is seen Dawson etc., " biochemical research data " (Data for BiochemicalResearch), second edition, 1-63 page or leaf, Oxford University Press (1969).More preferably described amino acid is selected from: glycine, L-Ala, C1-C5 alkyl hydroxy amino acid, for example Serine or Threonine or C1-C5 alkoxyalkyl or possible poly-alkoxyl group (polyoxyalkyl) amino acid.The strong preferred buffering amino acid that uses glycine as the elution buffer of preparation A protein chromatographic step of the present invention.

In the circulation liquid of first ion-exchanger, contaminative A protein concentration should be reduced to＜10ng/mg antibody, more preferably＜4ng/mg antibody, most preferably＜1ng/mg antibody, wherein antibody should be interpreted as and refer to IgG.Experimental section has described the Elisa measuring method of verifying these threshold values in detail, and it is most important to the proteic content of A that accurate mensuration comes off to note that sample is acidified to pH=4 (preferably having gentle stain remover exists).This threshold value will of course be appreciated that to can not surpassing the protein bound load capacity of the first ion-exchanger A, (otherwise) will cause contaminative A albumen to spill (break-through) inevitably.US 4,983, and 722 have described the suitable Elisa method of measuring A albumen or A protein fragments.Suitable anti-A protein antibodies can be buied, for example available from Sigma-Aldrich.Specifically, when containing the A protein derivatives of extra sulfydryl when utilizing engineered, it is most important suitably to keep these protein standard substance.Checking is most important as the monomer feature of this pure A protein derivatives of specimen quantitative criterion product, the result because covalent dimer that forms by-S-S-bridge or polymer can lead to errors.The SDS-PAGE that is not difficult to carry out this area routine under reduction and non-reduced condition analyzes and verifies.Therefore, utilize DTT or beta-mercaptoethanol to reduce this A protein derivatives-standard solution and help to prevent measuring error in the elisa technique.

In the methods of the invention, also preferably from the circulation liquid of ion-exchanger, be recovered to and load at least 70%, more preferably at least 80%, most preferably at least 90% of antibody on first ion-exchanger.Preferably (the no matter sugar form of same antibody and the final variant of handling) only exists a kind of (animal) type antibody by A albumen affinity chromatography, then through ion exchange chromatography purifying of the present invention in starting mixt.For example, when according to purifying people of the present invention or people-mouse mosaic type or primates or primatesization IgG antibody, in the cell culture fluid of supplemented serum, should there be the ox IgG that may bring into from serum.In other words, the inventive method should be used for collecting the rough unpurified antibody of serum-free cell culture medium.

First ion-exchanger of the present invention is an anionite-exchange resin; As described in EP-289129 B1, A albumen can with two types of resin-bonded.Ion exchange resin can be immersed the soft sample solution that stirs, adopt then to filter and replace liquid medium, thereby with chromatography column mode and definite flow velocity, or operate this first ion-exchanger or anionite with batch operation mode.Can determine to load the suitable pH and the ionic strength conditions of first ion-exchanger according to the present invention and the PI that consider given antibody, described condition antibody can be remained in the circulation liquid and simultaneously the A protein pollutant in conjunction with (in exchanger), thereby remove antibody.As mentioned above, the inventive method can sharp separation antibody and contaminative A albumen.The example of the functional group of this first anionite that links to each other with the matrix upholder is, for example primary amino, secondary amino group, particularly uncle's amino or season amino, as amino-ethyl, diethylamino ethyl, trimethylammonium amino-ethyl, trimethylammonium amino methyl and diethyl-(2-hydroxypropyl)-amino-ethyl.The suitable chromatography supported matrix of anionite known in the art.Example is agarose resin and pearl, dextran bead, polystyrene bead and polystyrene/divinyl-benzene resin.Can also use ion-exchange membrane adsorbent (for example, the Sartobind Q of Sartorius) equally.Obviously in order to improve flow velocity and to shorten disengaging time, substrate material can be pouring material (perfusion material), and this is another preferred embodiment.Described material can (cp. is Afeyan etc. for example by the refining perfusion pearl matrix that contains ceramic substrate, 1991, J.Chromatography, 544,267-279) constitute, perhaps can be monoblock type (monolithic) pouring material, for example the commodity of Sepragen Inc. (Hayward, California/U.S.) sale be called the fast flow velocity material of SepraSorb .SepraSorb  is the substitute of special exploitation as pearl matrix.It is crosslinked sponge sample material of regenerated cellulose, has successive intercommunication perforate (50-300 μ m) structure.This monoblock type matrix has the approaching surface that is easy to of non diffusible ion exchange functional group (DEAE, QM, CM and SE) that is not difficult on it.Flow through the different of pearl on every side with feeding liquid in conventional media, in fact feeding liquid stream flow into the intercommunicating porosity of this continuous matrix as perfusion.Than the pearl medium, SepraSorba has many advantages in industrial scale.It is not difficult to bear 100ml/ minute flow velocity, and back-pressure seldom surpasses 1 crust (14.5psi).Monoblock type matrix is highly susceptible to operation and configuration, has avoided dress post heavy and consuming time.This matrix can prevent to stop up, channel and anti-fragmentation, therefore can extend working time and recycles number of times.

Most preferred ion-exchanger is the anionite that is connected with season amino on the agarose matrix, for example the SepharoseCL-6B of Amersham-Biosciences/Pharmacia or Sepharose Fast Flow (FF).The example of this exchanger is the Sepharose Q of Amersham-Biosciences/Pharmacia ^TMAlso preferred (coupling first anionite) antibody of the present invention is the monoclonal antibody of iso-electric point (pI) than at least two pH units of the proteic pI height of used A in the above-mentioned A albumen affinity chromatography step, i.e. this antibody meta-alkalescence more; For example the proteic pI of natural A is about 5.0, and liquid stream (in) the proteic pI of reorganization A about 4.5.The preferred iso-electric point of antibody of the present invention (pI) is at least 6.5 or higher, and more preferably 7.0 or higher, most preferably pI at least 7.5 or higher monoclonal antibody.Should notice that this pI refers to the pI of actual cut and antibody purification, be not the pI that only estimates from aminoacid sequence.Also can modify the polypeptide backbone of actual antibody purified molecule, for example glycosylation, thus described modification can be to add the pI that charged part changes this molecule.Measure the pI of product antibody by isoelectric focusing (IEF) after, antibody protein, for example the product antibody of each glycosylation form of small unhomogeneity of monoclonal antibody protein translation post-treatment formation causes wideer pI scope, all these antibody form in the IEF gel and are similar to band in blocks but not single band, and most of at least products have specific (pI) numerical value.According to the present invention, in above-mentioned this preferred embodiment, " pI of antibody " refers to that pI is in the common pI of antibody product molecule in the above-mentioned pI preferable range.Other definition of all of this specification sheets, the antibody ratio % that for example reclaims behind given purification step only refers to the pI that described antibody is total.(approx) the average pI digital value of " smear " scope of measuring also should be interpreted as pI of the present invention or average pI, supposes the distributed number that this has rationally represented glycosylation form (antibody) liberally.

With regard to the common purpose of removing condensation product and contaminative A albumen or A protein derivatives simultaneously, preferably will load and clean the used pH of buffer of first ion-exchanger be set to avoid contacting damping fluid the time charged groups of ion-exchange material and A albumen or A protein pollutant and antibody to be purified between on the whole direct repulsion.If the A proteic substance is combined with ion-exchanger static, antibody debond under identical buffer condition simultaneously, then should consider the pI of antibody and A albumen or A protein derivatives, so first ion-exchanger should be the anionite of operating usually under the pH that is close to or higher than antibody pI to be purified.So the surface charge of antibody can be for 0 or for negative, but directly (bluntly) thus for just repelling.The A protein binding is most important to realizing the antibody debond suitably to regulate ionic strength conditions then.Yet this belongs to above-mentioned average pI value really; Therefore with regard to the antibody of glycosylation form, this does not mean that the rarer common pI value of glycosylation form antibody or the pI (scope) that is not close is arranged.In addition, in view of removing condensation product, should consider to differentiate monomer and condensation product, but partial action is at least owing to the of short duration and weak ion adelphotaxy and the ion-exchange of non-binding mode with circulation style; The surface charge and the pI of monomer and condensation product show fine difference; Therefore the inventive method at least to some antibody success effectively, even (for example) when utilizing anionite the scope of pH of buffer (vice versa than low 0.5 the pH unit of the pI of antibody at most, in view of only removing condensation product, when utilizing cationite the pH of damping fluid should be than the pI of antibody high 0.5 pH unit, see following), afterwards the explanation of this phenomenon be because only contain difference can near the surface and finally be neutral or even electronegative monomer rather than the condensation product ionic forces that is subjected to ion-exchange material initiatively repel.Yet, implement pI and uncertain cohesion characteristic thereof that this scheme height of the present invention depends on condensation product, thereby can not be used to estimate any given antibody.According to another embodiment preferred, for optimal separation contaminative A albumen and condensation product, utilize antibody to be purified average pI that pH of buffer is set is undesirable; Under non-binding chromatography condition of the present invention, produce the loading that contains antibody product peak circulation liquid of collecting and preferably be set to be higher than the monomeric pI of antibody, more preferably be set to be higher than the monomeric pI+0.5 of this an antibody pH unit with the pH of buffer of cleaning first anionite.

The operating method of the present invention's first anionite need with the acid of the equalizing and buffering fluid exchange A albumen affinity chromatography step of first anionite or in and elution buffer.Level pad is identical with the loading damping fluid in the methods of the invention.The conventional ultra-filtration equipment that uses (for example Amicon or Millipore sell) can be advantageously used in this purpose; Those devices can be avoided diluting effect energy usefulness, for example lower molecular weight porous filtering matrix, for example Sephadex G-25 simultaneously.The salt concn scope of the preferred displacer of level pad of the present invention (for example, sodium-chlor) is 1-150mM, more preferably 5-110mM, most preferably 20-100mM.The preferred pH6.5-pH9.0 of the pH of level pad, more preferably pH7.5-pH8.5, most preferably pH7.9-pH8.4.The pKs value that should note proteinic N-terminal amino functional groups is about 9.25, so contaminative A albumen and any other electronegative protein and anionite can combine under the pH of meta-alkalescence more strongly; With regard to given application, the pH that loads damping fluid be may need to finely tune and a pair of given antibody and the proteic combination of contaminative A and non-binding (situation) distinguished with the best, described antibody has different pI values and different halfcystine and Histidine side chain content with contaminative A albumen, and causes electric charge change in selected pH zone.In addition, because ionic strength increases, can disturb A albumen-antibody to interact than alkaline pH; Equally, need the fine setting ionic strength to prevent antibodies with balance, this is to remove the protein bound needs of contaminative A.The ionic strength that the technician knows this damping fluid certainly usually and the pH value be retrocorrelation; A albumen (depending on pH) combines strong more with anionite, prevent that antibodies from needing the salt of tolerance many more with disturbing potential A albumen-antibody interaction.Therefore, it is relevant that pH is interpreted as with the above-mentioned scope of displacer salt: pH is low more, and it is few more to implement the salt that above-mentioned preferable range of the present invention allows.The pH buffer substance is added other salt load, further increased the ionic strength of solution.Can be by detecting the conductance measurement ionic strength of level pad.Term " specific conductivity " refers to the ability of aqueous solution conduction current between two electrodes, can weigh total ion concentration, has also considered electric charge and mobility of ions.Therefore, along with aqueous solution ions content increases, solution will have higher specific conductivity.The unit of weighing specific conductivity is mS/cm (a milli Siemens/cm), with the conductivity meter that can buy, for example Topac Inc. (Hingham, MA/U.S.A.) or the conductivity meter of Honeywell detect.In this application, all numerical value belong to 25 ℃ concrete specific conductivity.The loading of first anion exchange step or the specific conductivity of level pad are 0.5-5mS/cm, more preferably 1-3mS/cm, most preferably 1.25-2.5mS/cm.Specific conductivity preferably is about 2mS/cm.

The example of suitable buffering salt is seen Good, and N.E. (1986, Biochemistry, 5:467-476).For example, conventional Tris.HCl damping fluid or the sodium hydrogen phosphate damping fluid that uses is suitable buffer substance.The concentration of buffer substance is generally, for example the 10-40mM buffering salt.At the negatively charged ion that may be used for designing damping fluid, the certain strength of preferred anionic elutriant is lower than cl anion, and low eluting power performance roughly is retrocorrelation and proportional with ionic size with ionic charge density.The experience of negatively charged ion eluant strength is relatively seen the form in the biological chemistry teaching material.Buffer substance of the present invention is more preferably phosphate buffered saline buffer.Hydrophosphate has low eluting power, if especially the pH of Cai Yonging was equal to or less than 8 o'clock, low especially can make it more outstanding from fluidity.

In another preferred embodiment, first anionite is the ceramic substrate anionite, for example Biosepra-branded HyperD  anionite more preferably contains the ion of quaternary ammonium (=quaternary amine) and the ceramic substrate anionite of matrix bonded functional group.These exchangers are exceedingly useful to therapeutic scale purifying.Season (amine) ceramic anionite Q-ceramic substrate anionite most preferably, (preferred especially) Britain for example, Guildford/Surrey, Ciphergen Biosystems Ltd. is with the Q-HyperD  anionite-exchange resin of trade(brand)name " Biosepra " sale.Above-mentioned and hereinafter mention antibody pI, the also preferred and the present embodiment coupling of the preferred embodiment of protein loading and pH of buffer, except when utilize Q-Hyper D  material, when particularly utilizing Q-Hyper D -F ion-exchanger, the preferred specific conductivity of damping fluid is 0.5-2mS/cm preferably, more preferably 0.6-1.7mS/cm is most preferably 1-1.5mS/cm.With regard to regard to removing contaminative A albumen or its fragment the product albumen, this specific conductivity has been guaranteed best purification result.The ceramic HYPERD sorbent material that utilizes the rigidity porous bead to make, it uses functionalization hydrogel dressing and infiltration.This gives outstanding hardness of these pearls and flowing property, and rare mass transfer and dynamics.Pottery HYPERD sorbent material is highly susceptible to using.Their higher density makes it be easy to adorn post and can be used in the very large post.They do not shrink or swelling thereby need not to adorn repeatedly post/tear open post fully.At present, utilize post more than 500 liters as treatment with the preparation type chromatography of molecule.The ceramic HYPERD ion-exchanger that is used for preparation process also can obtain 50 μ m levels (F level), and their heavy body and low back-pressure make 50 grades can perfect be used for acquisition procedure and common downstream processing.The ceramic character of pearl makes it chemically highly stable, can adopt the most frequently used sanitising agent, comprises the 0.5MNaOH cleaning.

Above-mentioned condition has been set up framework for removing contaminative A albumen in order to circulation style.For further from given antibody monomer, removing condensation product simultaneously, need in the general ranges such as specific conductivity, pH and identity of above-mentioned buffering salt, further carefully test the condition that allows to determine, with regard to given antibody, these conditions can be removed A albumen simultaneously and be differentiated condensation product.As mentioned above, this can not do further definition with generic term to given antibody high special.Further for example understand this research at experimental section, demonstration is operated anionresin matrix in non-binding mode can cause condensation product and monomer classification, the part thereby the non-binding protein peak that makes the condensation product of resolution mainly be present in antibody in the circulation liquid partly descends.This surprising discovery not only can be applicable to the monomeric purifying of antibody product albumen, and does not consider A albumen.By careful selection with merge each fraction, can reduce the condensation product level in the main elution peak of first ion-exchanger circulation liquid.This discovery did not appear in the newspapers in scientific literature in the past, because unexpected to circulation liquid-moving phase and solid phase, promptly ion-exchange material can obviously interact and not predict these discoveries.The most surprising is, circulation liquid condensation product tailing effect takes place at the pH of circulation damping fluid during away from the monomeric average pI of product albumen or antibody, and this state of charge (chargewise) makes that ion taking place at static under in conjunction with the unallowed ionic strength of institute attracts.Discovery negatively charged ion and cationite can be differentiated the condensation product in the circulation liquid fraction in this way or make it hangover.Infer and be not subjected to the constraint of this explanation, as mentioned above, can propose afterwards with ion-exchanger a little less than some but the ion of dynamic is attracted to small part promotes this effect.The matrix support material may produce other effect.Separate in condensation product and product albumen monomer or the monomeric appropriateness of antibody (temptatively) preferred embodiment with circulation style on ion exchange resin of the present invention, the substrate material of first anionite is polymeric polyvalent alcohol or polysaccharide.The avidity of described resolution effect is subjected to the expansion with binding site more than needed on a part, and condensation product is big more may be big more to this contribution.Dimer condensation product (for example two IgG antibody constitute) still can be successfully be separated with the monomer I gG antibody of the inventive method so.

Although available batch mode operation, the preferred first anionite step is the column operation mode.In this case, the preferred flow velocity that adopts about 10-60ml/ hour.The loading concentration of institute's antibody applied is preferably 10-30mg antibody/ml exchange resin.The technician knows that certainly the extremely rare sample of utilization can reduce the productive rate of antibody.Antibody to be purified is collected in the circulation liquid (low-through) of application of sample operation, comprises with identical level pad and washs about 1 column volume.With the pH regulator of circulation liquid to neutral to improve stability and to prevent the new cohesion and/or the precipitation of antibody protein.

It should be noted that the antibody that the inventive method can not be applied to produce generally at the entrained epi-position of A albumen.Abandon this antibody, though this is the significant limitation to the technician.The implication that should also be noted that " first " of the present invention ion exchange chromatography step is open definition, and it has only considered the time sequence of each step of the present invention; Should not get rid of any insertion ion exchange chromatography step of carrying out albumen to be purified or antibody protein with traditional combination and type of elution.

The most attracting feature of the inventive method be with non-binding or circulation style through the anionite antibody purification, the treatment capacity that the capacity of post can limiting material; This capacity only determines the proteic minimum content of the contaminative A that is kept.This has saved many treatment times and raw material, can remove the A protein pollutant very effectively simultaneously.

Above-mentioned another purpose of the present invention of partly mentioning is the universal method of removing condensation product from the monomer of product albumen to be purified already, it may further comprise the steps, the solution that will contain product albumen earlier under the condition of differentiating wherein said product albumen condensation product and described product albumen monomer (preferably not forming mixture with second protein ligands) by classification circulation liquid is loaded on the ion-exchange material, and described product albumen comprises described proteic monomer and cohesion form; Secondly further in classification circulation liquid collection of ions exchanger circulation liquid (with loading be used for product albumen on the ion-exchange material of purifying form compare) at least a product albumen monomer fraction that reduces of product albumen condensation product content.

More than definition also is applicable to this paper, the actually operating of the ion exchange chromatography that particularly circulates; Therefore, condensation product is interpreted as containing the given proteinic non-specific dimerization or the high poly-solubility condensation product of one or more covalently bound protein chain.Condensation product preferably includes the object lesson of above definition antibody and the proteic dimer of same products and the high-order condensation product of IgG institute example, and the ion exchange chromatography step of the present invention that defined all this type condensation products can be carried out with circulation style is removed.Discovery negatively charged ion and cationic exchange can be implemented the present invention; More surprising is, find that described method works when the monomeric pI of product albumen to be purified and when causing ion between product albumen monomer and the ion-exchange material to attract the pH of buffer of (for example the positive charge of exchanger and protein surface attracts), though since the specific conductivity of damping fluid do not allow product albumen to combine and do not cause the productivity combination with ion-exchanger.In brief, for removing condensation product, with regard to product albumen to be purified, this cationite should work when adopting the cationite of non-binding mode, but will utilize pH to be about or be lower than loading and cleaning (the loading the back) damping fluid of the average pI of product albumen; Vice versa, and when utilizing anionite, this anionite only works when one or more loadings that are about or are higher than the average pI of product albumen with pH and cleaning (loading the back) damping fluid.As explain in the preamble antibody purification but have the general meaning, pH of buffer preferably is not arranged on the pI of product albumen.Above-mentioned to glycoprotein pI explanation and the glycosylation form distributes and the experiment of measuring pI is equally applicable to this purpose.Described cleaning must be deferred to the non-binding operating method that given product albumen monomer is set up certainly with the loading damping fluid, in set limited field, with regard to composition, described cleaning buffer solution can be identical or different with the loading damping fluid, perhaps even can use several different cleaning buffer solutions successively, though be unaware of do so obviously beneficial.For simplicity's sake, the loading of sample product and cleaning, for example utilizing forever, identical damping fluid carries out.Yet, and just will operate liquid sample goods that the damping fluid that allowed suspends and be poured on the uniform ion exchange column and compare with non-binding mode, sensation can adopt meticulousr loading regime to carry out chromatography purification method of the present invention.

Preferably will circulate liquid the classification of antibody peak or be divided at least two fractions and discard hangover level and assign to realize classification.Finally can obtain the monomer of the monomer per-cent purity of collected antibody in this way, replace loaded down with trivial details gel-filtration (permation) or size exclusion chromatography method or equal complicated costliness shunting or the precipitation technology of small throughput, machinery with the high throughput of widespread use and ion exchange chromatography extremely fast simultaneously in gross product protein content at least 99%.Directly collecting from ion exchange column circulation liquid is the fastest treatment process, and it need not extra loaded down with trivial details washing, wash-out and regeneration step.

Experiment

1.A albumen Elisa

Test A albumen or the proteic many Elisa methods of reorganization A (referring to US 4983722 and reference as herein described) have been described.All working hereinafter described adopts simple sandwich Elisa, wherein is coated on flat 96 hole titer plate (Nunc ^TM) on the anti-A protein antibodies of catching property can keep A albumen here.Utilize then biotinylation anti--A Protein Detection antibody detects bonded A albumen, described detection antibody can also be in conjunction with the horseradish peroxidase (Amersham#RPN 1231) of coupling Streptavidin.Be used to catch buy anti-A-albumen rabbit antibody (producing) with natural staphylococcal protein A immunizing rabbit can be available from Sigma-Aldrich (#P-3775).Adopt this antibody in this research.Detect with rabbit antibody also available from Sigma-Aldrich (#3775).Behind this (antibody) albumen of non-specific adsorption method bag quilt, utilize this coating protein, promptly catching property of specificity A albumen antibody is kept A albumen here, utilizes the anti-A albumen of biotinylation rabbit (antibody) and Streptavidin-horseradish peroxidase to detect then.Tetramethyl benzidine is as producing the look substrate.Read the concentration of unknown sample with proteic parental generation A albumen of contaminative A to be detected or A protein derivatives (making) typical curve.Prove most important with acid pH bag quilt and suitable standard substance.Particularly through the engineered reorganization A albumen that carries extra cysteine residues, for example Streamline protein A ^TM(Amersham Biosciences, Pharmacia originally), discovery need be with reductibility sulfhydryl reagent pre-treatment standard solution to guarantee the free state of protein standard substance solution.

On the contrary, wild-type A protein standard substance can be available from many companies, and for example Sigma-Aldrich/ Switzerland (#P6031) or Pharmacia (#17-0770-01) need not this pre-treatment.Just observe contaminative A albumen from Streamline ^TMThe following stated experiment that comes off on the matrix can adopt the not link coupled reorganization A protein sample that derives from the manufacturer as standard substance.

1.1 the A protein standard substance of Cys is rich in pre-treatment

At Streamline ^TMContaining freeze dried pure reorganization A albumen-Cys in A albumen affinity chromatography (Amersham Biosciences) column material can be available from Pharmacia/Amersham Biosciences.Protein dissolution is reached 20mg/ml in containing the 0.1M Tris pH8 of 0.5M NaCl, 1mM EDTA and 20mM dithiothreitol (DTT), cultivated 15-30 minute in room temperature, with disposable PD-10 gel-filtration column desalination (AmershamBiosciences).Bag before is used to handle all damping fluids of this standardized solution and is used N ₂Handle in case sulfhydryl oxidase.Preferably just preparing protein standard substance before by titer plate with this standard substance bag.The storing solution of optional preparation 1mg/ml is kept in-65 ℃ of refrigerators, after the thawing sample aliquot is added on irreducibility SDS-PAGE and goes up the proteic monomer feature of test A.With Bradford test (Bradford etc., 1976, Anal.Biochem., 72:248-254; Splittgerber etc., 1989, Anal.Biochem., 179:198-201) and the automatization amino acid analysis measure the concentration of protein standard substance.Fig. 1 shows by irreducibility 10%SDS-PAGE detection staphylococcal protein A,SPA standard substance (swimming lane 1: natural A albumen; Swimming lane 2: after the pre-treatment) and pure not coupling Streamline ^TMReorganization A albumen (Pharmacia, present Amersham-Biosciences is so kind as to give; Swimming lane 4: recombinant natural A albumen; Swimming lane 5: after the pre-treatment) this pre-treatment result.Swimming lane 1 is the molecular weight marker thing, and corresponding molecular weight is labeled on the longitudinal axis.The Pharmacia reorganization A albumen that contains extra Cys residue is moved to about 34kD place after being reduced to lower molecular weight, be single stronger band, is derived from obviously that disulfide-bonded is dimeric dissociates.

1.2 Elisa

1.2.1 preparation sample

1: 200.000 diluent of the A protein standard substance stock solution by two step dilution preparation 1mg/ml obtains the 50ng/ml standard substance.Thereby the diluent of preparation 0.2ng/ml is used for the typical curve test.In addition, utilize the diluent (" admixture solution ") of standard substance to come admixture duplicate product sample to be tested to get rid of existing of interfering substance in the sample.

For final product sample test, every duplicate samples is divided into isopyknic two parts of 500 μ l.The admixture solution of a admixture 1000ng/ml, or 10 μ g/ml solution (if suitable) are so that final A protein content is every milligram of antibody 10ngA albumen.The sample buffer of second half admixture equal volume; Therefore eliminated the coefficient that causes the product diluted sample because of admixture.Below this two based article is called " admixture sample ".Sample buffer is made of 7.51g glycine (alkali), 5.84g NaCl, 0.5ml Triton X-100, with deionized water or distilled water volume is complemented to 1L.

For the best is accurately measured, adopt the antibody concentration in the conventional Elisa working sample well known in the art in advance.The known standard antibody that A albumen is had suitable constant region avidity of another part standard solution admixture equivalent, measure the effect of acidification step and remove any possible systematic error in the mensuration (because of antibody and A protein binding make A albumen from catch (antibody) come off cause).

Acidifying: in the sample of 450 μ l admixtures or standard substance, add 200 μ l 0.2M Citrate trianion/0.05%Triton X-100 damping fluids, pH3.0.All samples carries out triplicate acidifying.In addition, prepare all dilution of sample liquid and, because the optimum antibody concentration of test is 1mg/ml-0.2mg/ml with triplicate test.In this test, acidification step is most important for discharging contaminative A albumen or A protein fragments, otherwise described albumen or protein fragments can combine with the excessive antibodies that exists in the sample solution.

1.2.2 use the antibody sandwich titer plate

Bag is cushioned liquid by 1.59g/L Na ₂CO ₃, 2.93g/L NaHCO ₃Constitute with the 0.20g/L sodium azide.With the pH regulator of damping fluid is pH 9.6.Add the 100 μ l antibody-solutions that anti-body contg is not enough to saturated A protein standard substance to every hole.Use the plastic film wrapper plate, place moist cell.37 ℃ of about 18 hours of cultivations of spending the night.With at least 300 μ l lavation buffer solution [NaCl 5.8g/L, Na ₂HPO ₄1.15g/L, NaH ₂PO-H ₂O 0.26g/L, EDTA 3.7g/L, tween 20 0.2g/L, butanols 10ml/L, pH7.2] clean institute porose 3 times, pat dry.[bag that contains 0.5% casein hammarsten is cushioned liquid, cultivates 2 hours with desktop track shaking table (rotating speed is 120rpm) in room temperature to add 250 μ l sealing damping fluid to each hole.Clean institute porose 3 times with at least 300 μ l lavation buffer solutions, pat dry.

1.2.3 sample is cultivated and is detected

With 100 μ l/ holes standard substance and sample (comprising the admixture sample) are added on the plate.Use the plastic film wrapper plate, cultivated 90 minutes with desktop track shaking table in room temperature.Clean institute porose 3 times with at least 300 μ l lavation buffer solutions, pat dry.The anti-A albumen of biotinylation rabbit (antibody) is diluted to pre-determined optimum dilution degree.Add with 100 μ l/ holes, use the plastic film wrapper plate, cultivated 90 minutes with the track shaking table in room temperature.Clean once more.

With coupling buffer [Na ₂HPO ₄1.15g/L, NaCl 5.84g/L, NaH ₂PO ₄.H ₂O 0.26g/L, EDTA 3.73g/L, Triton X-100 0.05% (v/v), pH7.2] Streptavidin-horseradish peroxidase is diluted to the optimum dilution degree of prior mensuration.Add with 100 μ l/ holes, use the plastic film wrapper plate, cultivated 45 minutes with the track shaking table in room temperature.Clean once more.Tetramethyl benzidine (TMB, the ICN production number 980502) substrate solution that adds 100 μ l prepared fresh.Preparation substrate solution as follows: 10mg TMB is dissolved in 1ml DMSO prepares storing solution.In 2.05% (w/w) aqueous sodium acetate solution that is adjusted to pH0.6 with the 0.5M citric acid, add 10 these storing solutions of μ l and 10 μ l H ₂O ₂Be used to prepare this water of testing any reagent yes extra best best, promptly deionized ultrapure water or be distilled water at least.

Cultivated substrate solution 8-11 minute in room temperature with shaking table.In every hole, add 50 μ l stop buffer [13%H then ₂SO ₄] with termination reaction.After adding stop buffer, in 10 minutes, utilize the 450nm absorbancy in each hole of dull and stereotyped reading spectrophotometric determination.The limit of detection of this Elisa is a 0.2ng/ml A albumen, and working range is 0.2-50ng/ml.Test bay difference is less than 10%.

Fig. 2 has shown Streamline ^TMThe proteic level of reorganization A that comes off in the antibody liquid of reorganization A protein chromatographic wash-out, the single-point that described chromatography utilizes thioether bond to connect connects A albumen.Cycle index refers to reusable number of times behind 1M sodium-chlor wash-out and the reequilibrate.Although coming off of the cell cultures meat soup of Hybridoma Cell Culture is generally the 500ppm level, the level of other cell type is up to 1000ppm.Table 1 has been summed up the expulsion rate of different sources matrix; Operation instruction according to the manufacturer is carried out chromatography.

Table 1

Matrix	Upholder	The coupling chemical agent	P.p.m typically comes off	Working capacity (mg/ml)	Flow velocity (cm/ hour)
						Natural A Protein S epharose 4FF	Amersham-Bioscience	The CNBr that multiple spot connects	10-20	5-20	30-300
Rmp A Protein S epharose F	Amersham-Bioscience	Multiple spot connects	10-20	5-20	30-300
						Porous A heavy body	Amersham-Bioscience	Multiple spot connects	10-50	10	500-1000
A albumen pottery HyperD	Biosepra	Multiple spot connects	The highest by 300	10-20	200-500
						Reorganization A Protein S epharose	Amersham-Bioscience	The thioether bond that single-point connects	50-1000	20-40	30-300
MabSelect	Amersham-Bioscience	The thioether bond that single-point connects	50-1000	20-40	500
						STREAMLINE reorganization A albumen	Amersham-Bioscience	The thioether bond that single-point connects	50-1000	20-40	200-400

The contaminative A albumen that Fig. 3 has come off during providing the A albumen affinity chromatography with identical avidity substrate material to operate does not repeatedly have the substantive data that reduce; As described in 2.1 parts hereinafter, Sepharose 4 FF (Amersham-Biosciences) that use wild-type A albumen multiple spot to connect repeatedly, by above-mentioned Elisa measure elutant further handled before wherein contaminative A protein level.

2.A albumen is removed condensation product with Sepharose Q chromatography/different time stages

2.1 use Streamline ^TMA albumen affinity chromatography

By centrifugal, filter and the cell culture supernatant of ultrafiltration and concentration preliminary purification NSO myeloma cell culture deeply, also adopt ultrafiltration nutrient solution to be replaced with PBS, pH7.0.The titre of No. 5 antibody that cell produced is 0.2mg/ml, and the total heap(ed) capacity of supernatant liquor of having changed damping fluid is 1L.The pI of No. 5 monoclonal antibodies is 8.5.A Protein S treamline ^TMPost (5.0ml volume) is used the 50mM glycine/glycinate of 10 column volumes, pH8.8,4.3M NaCl balance in advance; Flow velocity is 200cm/ hour.Be to load, to operate this post in 50cm/ hour; Load capacity is about the 20mg/ml substrate material.The glycine level pad of having added extra 200mM NaCl and 0.1% tween 20 with at least 10 column volumes washs this post earlier, uses 0.1M glycine/HCl again, the elution buffer wash-out that the pH4.0 damping fluid constitutes.Use capacity 0.5M TrisHCl behind the wash-out immediately, the pH7.5 neutralization contains each elutant fraction at antibody peak, adopt the saturating filter device of Amicon to replace damping fluid to prevent to contact for a long time acid pH with the loading/level pad (10mM Tris/HCl pH8.0,50mM NaCl) of the follow-up anion exchange step of the present invention.

Measure antibody concentration and contaminative A protein concentration as mentioned above.Be 1434ng/mg antibody before the saturating filter of the proteic level of contaminative A in the elutant, saturating filter back is a 1650ng/mg antibody.According to the titre antibody rate of recovery that is replaced the buffering supernatant liquor before loading is 81%; The concentration of antibody is 3.6mg/ml in the solution of saturating filter.

2.2 the Q-Sepharose FF anion exchange step of non-binding mode

The non-antibody purification of further processing 2.1 parts as described below: the Q-Sepharose FF post (Amersham-Biosciences) for preparing 5.0ml with 10ml 0.1M NaOH, use 0.1 Tris pH8 (washing) of two column volumes then, with the 10mM Tris pH8/50mM NaCl balance of 10 column volumes, flow velocity is 75cm/ hour.After the balance, flow velocity was reduced to 50cm/ hour.The antibody-solutions of the saturating filter of 6ml is loaded on the post, collects circulation liquid and be for further processing; To continue with pure loading or level pad (10mM Tris pH8/50mM NaCl) through post behind the preceding 6ml loading upper prop, continue to collect circulation liquid and return back to baseline until the 280nm place absorbancy of the detect liquid that circulates.

The antibody that reclaims in the circulation liquid is 23mg (87%) altogether.The proteic level determination of contaminative A is＜3ng/mg antibody.With Sephacryl S-300 gel-filtration (size exclusion chromatography, SEC) further handle this Q-Sepharose antibody purified, with 10mM phosphoric acid salt pH7.0,140mM NaCl damping fluid, flow velocity is 10cm/ hour, LOADING RATES is every ml gel 15mg antibody, finds not change basically this trace contamination A protein level.Rule of thumb, can adopt SEC that the proteic level of contaminative A is reduced to about 1-5ng/mg from about 30-100ng/mg.Therefore, for trace A albumen, the decontamination factor of SEC is extremely low, and possible explanation is to exist avidity to interact between antibody and contaminative A albumen.Yet because inevitably diluted sample slowly makes antibody protein that certain degraded is arranged with handling, SEC can only reclaim 70% of institute's antibody applied amount.This explanation SEC causes material damage inevitably, and the while is time-consuming more.

As mentioned above, for reusing available 2M NaCl wash-out and balance regeneration Q-Sepharose post.

2.3 the multiple spot with customization carries out Streamline in conjunction with A albumen ^TMA albumen affinity chromatography

This multiple spot bonded Streamline ^TMA albumen affinity chromatography matrices customizes, and is provided by PharmaciaBiotech (being Amersham-Pharmacia now).The manufacturer will have the identical 34kD Streamline of terminal Cys residue ^TMType reorganization A albumen coupling is made this affinity chromatography matrices in identical Sepharose substrate material, but adopt activation of traditional CNBr chemical process and coupling, and without the activation of epoxide mediation and selective reaction condition only by-SH group coupling (seeing manufacturer's product information).Experiment 2.1 has repeated this method, and the proteic level determination of contaminative A is a 353ng/mg antibody.Therefore, but but the reorganization A albumen affinity matrix albumen that inference A albumen is connected with the coupling mode partial interpretation heavy body single-point of substrate material come off and why increase; Compare with total length wild-type A albumen, in this reorganization A albumen, introduce amino acid sequence modifications and also significantly cause the protein increase that comes off.

3. parallel testing: with the comparison (US4,983,722) of Miles method

The patent the (the 4th of Miles, 983, No. 722) declare DEAD Sepharose is used as second chromatographic step with combination, the A protein content that comes off in the elutant can be reduced to (scope of A albumen (content) is a 0.9-14ng/mg antibody) below the 15ng/mg antibody with salt gradient (0.025M-0.25M NaCl) wash-out.

Table 2:

Relatively A is proteic residual in the A protein chromatographic matrix purifying 6A1 antibody elutant sample that is connected with multiple spot with single-point

Matrix	Sample	A protein level (ng/mg)
			Reorganization A Protein S epharose (single-point connection)	A albumen elutant	20.2
Rmp A Protein S epharose (multiple spot connection)	A albumen elutant	2.16
			Natural A Protein S epharose (multiple spot connection)	A albumen elutant	＜2.0

The purpose of these experiments is these results that confirmation utilizes MabSelect (the reorganization A albumen substrate that new single-point links to each other) and the lower antibody (pI6.5-7.5) of pI to obtain, and the patented method of directly more non-binding Q-Sepharose method (with different balances/loading damping fluid) and Miles.Collect the 6A1 antibody of NSO cell, the various cell culture processes of expression and collection antibody are described in detail in hereinafter experimental section 7.

Used method:

The purifying of 6A1 antibody (pI6.5-7.5) comprises two step chromatographies: MabSelect A albumen step, carry out Q-Sepharose anion-exchange chromatography (non-binding) or DEAE Sepharose chromatography (combination) step then.

MabSelect A protein chromatographic:

Base for post matter: Mab Select reorganization A albumen (rPA that single-point connects)

Column dimension: the 1.6cm internal diameter * 15cm height of bed

Column volume: 30mL

Operate flow velocity: 500cm/ hour (16.80mL/ minute)

Cleaning: 6M Guanidinium hydrochloride (2 column volumes)

Load capacity: 35mg/ml matrix

Balance: 50mM glycine/glycinate pH8.0/250mM NaCl (8 column volumes)

Load after scouring: 50mM glycine/glycinate pH8.0/250mM NaCl (8 column volumes)

Elution buffer: 100mM glycine pH3.50 (6 column volumes)

Washing; 100mM citric acid pH2.1 (2 column volumes)

MabSelect post (30ml) purifying that utilization links to each other with AKTA FPLC system contains the culture supernatant of 6A1 antibody.Used condition is as above shown described.Utilize 0.1M glycine pH3.5 wash-out antibody.Behind the wash-out, the elutant pH regulator to pH7.0, is divided into 5 equal portions then; Each equal portions is made anion-exchange chromatography with the saturating filter of different damping fluids.

First equal portions are done Q-Sepharose chromatography running 1 with 50mM TrisHCl pH8/75mM NaCl diafiltration.Second equal portions are done Q-Sepharose chromatography running 2 with 50mM TrisHCl pH8/100mM NaCl diafiltration.C grade part is done Q-Sepharose chromatography running 3 with 20mM sodium phosphate pH6.5/80mM NaCl diafiltration.Quarter and the 5th equal portions damping fluid are replaced with 25mM TrisHCl pH8.0/25mMNaCl assess the described DEAE Sepharose of Miles patent combining method.The difference of running between 4 and 5 is that the main peak of running 4 is collected with standard phosphate-buffered saline diafiltration post analysis as a fraction, and operates in 5, and phosphoric acid buffer diafiltration according to the described preparation of Miles patent is also used in the peak of this elutant of classification.

5 kinds of post runnings condition has separately hereinafter been described:

Q-Sepharose chromatography: running 1

Base for post matter: Q-Sepharose Fast Flow (Amersham Biosciences)

Column dimension: the 1.6cm internal diameter * 8cm height of bed

Column volume: 16mL

Post preparation: use 0.1M sodium hydroxide with 150cm/ hour dress post

Operate flow velocity: 100cm/ hour (3.35mL/ minute)

Cleaning: 0.1M sodium hydroxide (2 column volumes)

Load capacity: 15mg/ml matrix

Balance: 50mM TrisHCl pH8.0/75mM NaCl (8 column volumes)

Load after scouring: 50mM TrisHCl pH8.0/75mM NaCl (5 column volumes)

Strip buffer: 2M sodium-chlor (2 column volumes)

Washing: 0.1M sodium hydroxide (2 column volumes)

Q-Sepharose chromatography: running 2

Base for post matter: Q-Sepharose Fast Flow (Amersham Biosciences)

Column dimension: the 1.6cm internal diameter * 8cm height of bed

Column volume: 16mL

Post preparation: use 0.1M sodium hydroxide with 150cm/ hour dress post

Operate flow velocity: 100cm/ hour (3.35mL/ minute)

Cleaning: 0.1M sodium hydroxide (2 column volumes)

Load capacity: 7.5mg/ml matrix

Balance: 50mM TrisHCl pH8.0/100mM NaCl (8 column volumes)

Load after scouring: 50mM TrisHCl pH8.0/100mM NaCl (5 column volumes)

Strip buffer: 2M sodium-chlor (2 column volumes)

Washing: 0.1M sodium hydroxide (2 column volumes)

Q-Sepharose chromatography: running 3

Base for post matter: Q-Sepharose Fast Flow

Column dimension: the 1.6cm internal diameter * 8cm height of bed

Column volume: 16mL

Post preparation: use 0.1M sodium hydroxide with 150cm/ hour dress post

Operate flow velocity: 100cm/ hour (3.35mL/ minute)

Cleaning: 0.1M sodium hydroxide (2 column volumes)

Load capacity: 7.5mg/ml matrix

Balance: 20mM TrisHCl pH6.5/80mM NaCl

Load after scouring: 20mM TrisHCl pH8.0/80mM NaCl (5 column volumes)

Strip buffer: 2M sodium-chlor (2 column volumes)

Washing: 0.1M sodium hydroxide (2 column volumes)

Q-Sepharose chromatography: running 4

Base for post matter: Q-Sepharose Fast Flow (Amersham Biosciences)

Column dimension: the 1.6cm internal diameter * 8cm height of bed

Column volume: 16mL

Post preparation: use level pad with 150cm/ hour dress post

Operate flow velocity: 100cm/ hour (3.35mL/ minute)

Cleaning: 0.1M sodium hydroxide (2 column volumes)

Load capacity: 7.5mg/ml matrix

Balance: 25mM TrisHCl pH8.6/25mM NaCl (8 column volumes)

Load after scouring: 25mM TrisHCl pH8.0/25mM NaCl (5 column volumes)

Elution buffer: 25mM TrisHCl pH8.6/25mM NaCl to 25mM TrisHCl pH8.6/250mM NaCl (2 column volumes)

Washing: 2M sodium-chlor (2 column volumes)

DEAE Sepharose combining method: running 5 (Miles methods)

Base for post matter: DEAE Sepharose

Column dimension: the 1.6cm internal diameter * 8cm height of bed

Column volume: 16mL

Post preparation: use level pad with 150cm/ hour dress post

Operate flow velocity: 100cm/ hour (3.35mL/ minute)

Cleaning: 0.1M sodium hydroxide (2 column volumes)

Load capacity: 7.5mg/ml matrix

Balance: 25mM TrisHCl pH8.6/25mM NaCl (8 column volumes)

Load after scouring: 25mM TrisHCl pH8.0/25mM NaCl (5 column volumes)

Elution buffer: 25mM TrisHCl pH8.6/25mM NaCl to 25mM TrisHCl pH8.6/250mM NaCl (2 column volumes)

Washing: 2M sodium-chlor (2 column volumes)

The different damping fluid performances that are used for this research see Table 3.

Detected the A protein level of the elutant sample of 5 secondary ion exchange operations generations with rPA ELISA.The results are shown in Table 4.

Table 3:

The damping fluid that this institute uses

Level pad	The running numbering	Specific conductivity (ms/cm)	Resin	pH
					50mM TrisHCl pH8.0/75mM NaCl	1	10.74	Q-Sepharose (non-binding)	8.00
50mM TrisHCl pH8.0/75mM NaCl	2	12.85	Q-Sepharose (non-binding)	8.01
					20mM sodium phosphate pH6.5/80mM NaCl	3	10.20	Q-Sepharose (non-binding)	6.50
25mM TrisHCl pH8.6/25mM NaCl	4/5	3.35	DEAE-Sepharose (combination)	8.60
					25mM TrisHCl pH8.6/250mM NaCl *	4/5	24.54	DEAE-Sepharose (combination)	8.61

^*The gradient elution damping fluid

Collect each fraction of the wash-out situation of DEAE-Sepharose running 5 (Miles methods), analyze, the results are shown in Table 5 with reorganization A protein ELISA.

Table 4: reorganization A protein ELISA result: ^*CV represents column volume

Sample ID	Reorganization A protein level (ng/mg)	Antibody concentration (mg/ml)	Reclaim %	Elution volume (CV)
					Q-Sepharose elutant running 1	＜0.4	1.42	82	4.5
Q-Sepharose elutant running 2	2.94	1.49	70	3.5
					Q-Sepharose elutant running 3	0.73	1.86	85	3.4
The DEAE-Sepharose elutant merges 4 (merging all parts) of running	1.72	2.16	75	2.5
					The DEAE-Sepharose elutant merges running	1.55	1.83	73	3

5 (merging the 2-6 part)

Table 5:

Combination DEAE-Sepharose separates (Miles method); The proteic level of reorganization A in each fraction of elutant of the elution peak that obtains during the running 5

Numbering of part	Reorganization A protein level (ng/mg)	Absorbancy (A 280)
			1	3.33	0.018
2	0.4	0.108 ＊
			3	0.4	0.22 ＊
4	0.4	0.169 ＊
			5	2.01	0.092
6	16.7#	0.042
			7	6.38	0.016

The method of Miles, table 5: though main albumen and antibody peak position (start from random numbering in the fraction 2-4 of elutant; Described fraction is marked with *), but A albumen lags behind in rapid rising peak and washes out (fraction that # marks); In view of the acceptable threshold value of A protein contamination is the highest is 2ng/mg antibody, finishes antibody at the place, end of fraction 4 and reclaims and can remove most of condensation products, though fail to reclaim about 35% antibody of finding in the above-mentioned elutant.

On the contrary, for A protein content standard, the running 1-3 of non-binding mode can reclaim antibody well.Non-binding method can obtain the antibody protein spike same with traditional combining method gained, and peak shape is out of shape without any characteristic.Certainly should note the loaded volume deficiency so that the antibody sample moves and flows out from the exchanger post, because the void volume of post is much larger than loaded volume.Therefore, the moving phase that adds after the loading of this non-binding method is also referred to as " loading after scouring " in aforesaid method.Consider column void volume, migrate into the head of post, produce the circulation liquid of collecting from chromatography column that contains antibody product peak along with loading the damping fluid front end.Therefore, before collecting the product albumen mass peak, effusive one times of column volume liquid does not contain product albumen matter.The inventive method need not elution buffer, for non-binding method of the present invention, as running 1-3 institute example, though used term is similar, but this loading after scouring still can not make antibody or the combination of product albumen static certainly, and this loading after scouring buffer conditions with Miles is opposite; In theory,, load after scouring damping fluid even can be with to load damping fluid different for the inventive method, as long as kept the requirement of above-mentioned non-binding condition, certainly can be more not useful but do so yet.Then, all sort buffer liquid flow through the circulation liquid that can produce collection behind the post.Therefore, for simplicity's sake, the loading of running 1-3 is identical with loading after scouring damping fluid.In the circulation method, the antibody peak of running 1-3 is generally at about 1-2, and normally the void volume place of 1.5 posts flows down.Even but in the circulation liquid of about 2-3 column void volume, produce under the non-binding condition at product peak " wash-out " (data not shown), do not observe the peak yet and broaden or trail, show that non-binding condition has consistence in operation.Non-binding side of the present invention and with the experiment instruction of this section in, used term " wash-out " volume is opposed mutually with the real combination of Miles and the term of wash-out operating method.Under non-binding condition, this antibody (6A1; PI6.5-7.5) high-recovery (85%) is to go up at Q-Sepharose (post) with 20mM sodium phosphate pH6.5/80mM NaCl damping fluid (corresponding to running 3) to obtain.Running 1 has also shown the good rate of recovery (82%), yet " elutriant " volume of this running is slightly high, does not have substance to broaden though observe the antibody protein peak; Do not analyze the distribution of glycosylation form.Improve NaCl concentration (running 2 is compared with running 1) and reduced the proteic clearance rate of reorganization A, therefore operate 3 and 1 used Laemmli buffer system Laemmli and be more suitable in this antibody.Our observation in the past is that running 1 used Laemmli buffer system Laemmli is more suitable for antibody in high pI, operates 3 used Laemmli buffer system Laemmlis to neutral or slightly tart antibody is more useful.In view of running 1 data, can estimate with this non-binding method in addition have higher capacity (＞30mg/ml).Compare with the Miles method, non-binding method is easier to scale operation, because except the major defect (needing promptly to note carefully that the classification elutant is to avoid just A protein peak classification) that can overcome the Miles method, also can be applicable to higher capacity etc.; In case need to observe simultaneously the combination of a plurality of parameters (condensation product adds A protein pollutant threshold value), the Miles method will be more difficult (if possible).

To operate 5 (Miles methods) is example, the reorganization A protein fractions of having observed in main elution peak as shown in table 5.Therefore, need careful each fraction that merges to guarantee the good reorganization A albumen of removing.This is influential to the rate of recovery (70%), even in this example, does not also obtain with the identical good clearance rate of non-binding method gained.Therefore, in the situation of observing high coming off (for example the matrix that links to each other with single-point obtains usually), the more difficult realization of Miles method is for the good clearance rate and the high-recovery of clone/antibody.

The data represented result's condition described that adopts the Miles patented method to be obtained of running 5 with it.

The summary of method comparison and resulting data see the following form 6.

Table 6.1

The reorganization A protein level of the different steps of antibody purification is summed up ^*

Attention:Shown the proteic level of reorganization A [ng/mg] in the bracket; The supernatant liquor of noting not all NSO cloned cell line all obtains similar A protein contamination level.

^*All embodiment load anionite with 7.5mg/ml to carry out

Similar to the running 2 in left side in the table 6.1, operate 1 in non-binding mode, but also further reduced ionic strength (table 6.2), thereby remove contaminative A albumen capitally with the load capacity of 15mg/ml:

Table 6.2 (running 1)

Discovery utilizes anionresin Q-Sepharose, and this splendid result who also utilizes different for example high pI antibody to obtain can reappear fully.

6. utilize ceramic Q-HyperD  F as the anionite purifying

Basically as carrying out the non-binding anion-exchange chromatography of the elutant of Mab-Select A protein chromatographic as described in the above comparative experiments 3.Q-HyperD  F (Biosepra board chromatography upholder) is available from Britain, Guildford, Ciphergen Biosystems Ltd..Basically as described in embodiment 5, adopt Mab-Select A albumen affinity chromatography to handle the pI 8-9 antibody of NSO cell expressing.Then basically as described in the embodiment 5 (running 1-3), A albumen affinity column elutant is implemented the Q anion-exchange chromatography with circulation style, except under different damping fluid salt, pH of buffer and specific conductivity condition, replacing the running of comparing property of Q-Sepharose with Q-Hyper DF (Biosepra ).Scheme shown in the table 7 has been summarized each condition; For at utmost removing contaminative A albumen and obtaining and use the identical result of Sepharose Q material, confirm to use utmost point low conductivity less than 2mS/cm, promptly about 1.26mS/cm is better.Also observing the circulation fraction during analysis has condensation product hangover (data not shown), and applied damping fluid is also depended in the extension of hangover.The type that depends on elementary object and ion-exchange material is necessary for given separation task and determines defined top condition of damping fluid or compensation condition.Yet for large-scale commercial production, the advantage of ceramic HyperD material is life-span long, firm and incompressible (treatment time, a flow velocity).Therefore, for different column materials, specific conductivity is a most important parameters to be tested and that optimize.Should notice that also specific conductivity very seriously influences the pollution level of polyanion DNA.By finely tuning buffer condition suitably, can reduce contaminative DNA and A protein level simultaneously as far as possible.

Table 7

Q-Hyper D -assessment

A albumen 0.78ng/mg A albumen 1.06ng/mg A albumen 3.3ng/mg

For comparing: utilize identical antibody, Q-Sepharose obtains 0.4ng A albumen/mg antibody, and the contaminative dna level of mensuration is a 10.9pg/mg antibody; Yet the result who utilizes the Q gained utilizes very different damping fluid (20mM TrisHCl/50mM NaCl pH8.00, the specific conductivity of mensuration is 6.1mS/cm) to obtain.

7.A reduce condensation product and A albumen simultaneously with non-binding mode ion exchange chromatography behind the protein chromatographic

The purpose of these experiments be assessment when operating ion exchange chromatography in non-binding mode condensation product-monomeric separating effect (utilize from cloned cell line NSO-6A1 and collect, pI is the cB72.3 IgG antibody of 6.5-7.5, and described clone contains glutamine synthetase (GS) and Xin Meisu selective marker and energy constitutive expression antibody).The matrix that selection is used to assess is the Q-Sepharose anionite (Amersham Biosciences) that operates under two kinds of different buffer conditions.

Described NSO-GS cell and the method (seeing same applicant's WO 03/027300 and WO 03/064630) of collecting B72.3 antibody of cultivating in detail.According to budapest treaty, production clone NSO-6A1 on August 30th, 2002 by Britain, Salisbury/Wiltshire SP4 OJG, Porton Down, using microbe and research centre, Europe cell culture preservation institute (ECACC) represents Andy Racher (LonzaBiologicals, 224 Bath Road, Slough, Berkshire, SLl 4DY, Britain) with accession number 02083031 preservation, code " 6A1-Neo "; The Britain that gives one's address as, the CompanyAddress of Lonza Biologies pic, this trust is entrusted by Lonza Biologies pic and is had all vest rights of Lonza Biologies pic.In addition, Andy Mr. Racher is visual sometimes to do legal preservation people, its present private address is a Britain, Reading/Berkshire RG7 4UY, Aldermaston, 5Kingfisher Close, this legal explanation according to the preservation file, Mr. Racher authorizes the applicant unconditional and irrevocablely, and LonzaBiologies pic submits the preserved material applied for to, make it for the public can with and specified all titles of preservation thing to the applicant.

Whittle etc. (Protein Eng., 1987,6:499-505) gene structure of mouse-people's mosaic type antibody cB72.3 has been described with (Colcher etc., Cancer Research, 49,1738-1745, (1989)).This antibody also can be from the NSO-6A1-Neo expression of cell lines.The purification process of NSO 6A1 antibody (cB72.3) comprises two chromatographic step: rmp A Protein S epharose (chromatography), carry out non-binding Q-Sepharose anion-exchange chromatography then.

Rmp A Protein S epharose chromatography

Base for post matter: rmp A Protein S epharose (Amersham Biosciences)

Column dimension: the 1.8cm internal diameter * 15cm height of bed

Column volume: 30.1ml

Operated flow velocity: 150cm/ hour

Cleaning: 6M Guanidinium hydrochloride (2 column volumes)

Load capacity: 35mg/ml matrix

Balance: 50mM sodium phosphate pH7.0/250mM NaCl (8 column volumes)

Load after scouring: 50mM sodium phosphate pH7.0/250mM NaCl (8 column volumes)

Elution buffer: 0.1M glycine/0.1M NaCl pH3.0 (6 column volumes)

Peel off: 0.1M citric acid pH2.1 (2 column volumes)

Rmp A albumen post (30ml) purifying that utilization links to each other with ATKA FPLC system contains the cell culture supernatant of 6A1 antibody.Used condition is as above shown described.Utilize 0.1M glycine/0.1M NaCl pH3.0 wash-out antibody.Behind the wash-out, the elutant pH regulator to pH3.7, was kept 60 minutes, be neutralized to pH6.5 then.Need carry out two-wheeled.The elutant of the first round is concentrated into 25mg/ml, and damping fluid replaces with 20mM sodium phosphate/80mM NaCl pH6.5, is loaded on the Q-Sepharose post under " running 1 "-elution requirement as follows.Second elutant of taking turns is concentrated into 25mg/ml, and damping fluid replaces with 20mMTris HCl/75mM NaCl pH8.0, is being loaded on the Q-Sepharose post as described in hereinafter operating 2.

Collect each component of debond fraction (merchantable thing), adopt gel infiltration (permation)-HPLC to analyze.Reclaim with elution volume as shown in table 1.GP-HPLC the results are shown in Table 2.The condensation product distribution situation is shown in Figure 4 and 5, and the elutriant distribution situation is shown in Fig. 6 and 7.

Q-Sepharose chromatography: running 1

Base for post matter: Q-Sepharose FF (Amersham Biosciences)

Column dimension: the 1.0cm internal diameter * 15cm height of bed

Column volume: 12mL

Operated flow velocity: 100cm/ hour

Cleaning: 0.1M sodium hydroxide (2 column volumes)

Load capacity: 50mg/ml matrix

Balance: 20mM sodium phosphate/80mM NaCl pH6.5

Load after scouring: 20mM sodium phosphate/80mM NaCl pH6.5

Peel off: 20mM sodium phosphate/2M NaCl pH6.5 (2 column volumes)

UV-monitoring color atlas is seen Fig. 6.

Q-Sepharose chromatography: running 2

Base for post matter: Q-Sepharose FF (Amersham Biosciences)

Column dimension: the 1.0cm internal diameter * 15cm height of bed

Column volume: 12mL

Operated flow velocity: 100cm/ hour

Cleaning: 0.1M sodium hydroxide (2 column volumes)

Load capacity: 50mg/ml matrix

Balance: 20mM Tris HCl/75mM NaCl pH8.0

Load after scouring: 20mM Tris HCl/75mM NaCl pH8.0

Peel off: 20mM Tris HCl/2M NaCl pH8.0 (2 column volumes)

The running numbering	Reclaim (%)	Effluent volume (cc)
			Q-Sepharose operates 1 elutant	73	5.8
Q-Sepharose operates 2 elutants	77	11

UV-monitoring color atlas (OD at 260nm place) is seen Fig. 7.

For gel infiltration/size exclusion chromatography, use Feng Yusan joint inspection survey (redundant triple detection) (RALS, Viscometer and Refractive Index) and detect the effusive protein fractions of gel column.Light scattering detector is detection molecules amount and need not to carry out the post verification directly.Viscosmeter can (mensuration) direct visible textural difference.It also can measure the molecular size in the whole distribution.Another advantage that three joint inspections are surveyed is to adopt a kind of narrow sense and a kind of generalized standard to come the determining instrument parameter.Three joint inspections are surveyed can measure " definitely " molecular weight, inherent viscosity and molecular size in one-time detection.Branch, conformation, structure and cohesion that it can provide polymer samples are for information about.

Chromatography condition:

Solvent: 0.2M sodium phosphate buffer, pH=7.0

Flow velocity: 0.7ml/ minute

Volume injected: 100 μ l

Post/detector temperature: 29 ℃

Post: Superdex 200HR

Detector:

Three joint inspection colour examining spectrograms of sample show that this detector has splendid signal to noise ratio.The circulation ratio at monomer peak is very good.For all samples, molecular weight is about 140k-147k dalton, and limiting inherent viscosity IV is 0.065-0.079dl/g approximately, and water power radius (hydrodynamic radius) Rh is 5.3-5.5, weight fraction 80-99%.The second peak molecular weight is about 300k, and IV is 0.08dl/g, and Rh is 7nm, and this is consistent with dimeric result.

Fig. 8-17 has shown that being selected from each fraction of table 2 surveys the duplicate GP color atlas (seeing the concentration data of cross reference) that operates with three joint inspections

Fig. 8/fraction 1.2:Q-FT-01-F2 concentration=3.58mg/ml

Concentration=the 15.5mg/ml*7.75mg/ml of Fig. 9/F.1.5:Q-FT-01-F5

Concentration=the 7.41mg/ml*3.71mg/ml of Figure 10/F.1.11:Q-FT-01-F11

Concentration=the 1.38mg/ml of Figure 11/F.1.15:Q-FT-01-F15

Concentration=the 0.334mg/ml of Figure 12/F.1.19:Q-FT-01-F19

Concentration=the 5.9mg/ml*2.95mg/ml of Figure 13/F.2.2:Q-FT-02-F2

Concentration=the 15.5mg/ml*7.75mg/ml of Figure 14/F.2.5:Q-FT-02-F5

Concentration=the 8.95mg/ml*4.48mg/ml of Figure 15/F.2.10:Q-FT-02-F10

Concentration=the 1.57mg/ml of Figure 16/F.2.20:Q-FT-02-F20

Concentration=the 0.37mg/ml of Figure 17/F.2.33:Q-FT-02-F33

^*Do 1/2 dilution with phosphoric acid buffer.

Table 2:GP-HPLC analytical results: condensation product distribution situation

Numbering of part	Protein concn mg/ml	Condensation product %
			Running
1 loads	18.37	3.8
			2	3.58	2.6
5	15.5	2.4
			11	7.41	1.8
13	3.92	3.6
			15	1.38	9.7

17	0.62	15.4
			19	0.33	19.3
21	0.23	22.7
			23	0.17	25.0
The elutant that merges	6.36	2.9
			Running 2 loads	20.7	3.9
2	5.9	0.6
			5	15.5	0.99
10	8.95	0.6
			14	3.59	0.3
17	2.44	0.4
			20	1.57	0.5
27	0.61	1.6
			33	0.37	2.9
45	0.19	6.8
			The elutant that merges	3.4	0.9

In two Q-Sepharose with 20mM sodium phosphate/80mM NaCl pH6.5 and the running of 20mM Tris HCl/75mM NaCl pH8.0 damping fluid, the monomer of high level is present in the fraction in early stage, and most condensation product wash-out in the hangover fraction.With compare with the running 2 of 20mM Tris HCl/75mM NaCl pH8.0 damping fluid, contain the condensation product of higher level in the hangover fraction of running 1 (with 20mM sodium phosphate/80mM NaCl pH6.5 damping fluid).Merge the suitable protein peak fraction that does not contain condensation product, avoid (condensation product) peak fraction.Size exclusion HPLC show to merge does not contain the contained monomeric igg of condensation product fraction＞99.1%.Measured the proteic level of contaminative A in the monomer fraction that merges simultaneously, after measured in the merging fraction of Xuan Zeing the proteic level of contaminative A be＜＜1.5ng/mg antibody.

The PCT/RO/134 table

Explanation about microbial preservation

(detailed rules and regulations 13 two)

A. to specification sheets 42Page or leaf, the 10-22The microorganism of the described preservation of row or the explanation of other biological material
		B. the preservation item more is deposited in additional page explanation
Zooblast preservation center (ECACC), depositary institution title Europe
		Depositary institution address (comprising postcode and name of the country) applied microbiology and research centre, southern Bowden, the Sai Er Regensburg/SP4 OJG of Wiltshire, Britain
Protect a surname's August 30 2002 date	Preserving number 02083031
		C. (in case of necessity) more information in additional page remarks additionally
Enclose the separate statement about all relations of law of being born in 2003 earlier December 22 signed by Racher A..The acquisition of institute's preservation biomaterial should only limit to by the European patent expert that the 28th (4) bar and Australian Patent method the 3.25th (3) approved that reaches an agreement on.Under the situation that the application is abandoned or rejects, should continue to use or be suitable for each relevant regulation of above-mentioned law or criterion, and in day termination of the information announcement of mandate of the present invention.
		D. this explanation is done (if explanation is not done for all designated states) for following designated state
Be only applicable to be used for the expert of EP and AU
		E. remark additionally (in case of necessity)
Following explanation will provide (writing out the classification of explanation, for example: " numbering of preservation ") to international office subsequently

PCT/RO/134 table (in July, 1998, reprint in January, 2004)

Claims (21)

1. antibody purification, the method for preferred IgG antibody said method comprising the steps of:

1). by A albumen affinitive layer purification antibody, wherein A albumen is natural A albumen or its functional deriv,

2). contaminative A albumen or its functional deriv are combined with ion-exchanger material and differentiate by the classification of circulation liquid circulation in the liquid the antibody condensation product and not with A albumen or the monomeric condition of A protein derivatives compound antibody under, the antibody purification that will contain antibody condensation product and A albumen or A protein derivatives is loaded on the ion-exchange material, with

3). classification circulation liquid and from ion-exchanger circulation liquid, collect with load on this ion-exchange material before antibody form and compare at least a antibody monomer fraction that A albumen or A protein derivatives and antibody condensation product content all reduce.

2. method as claimed in claim 2 is characterized in that, thereby described A albumen is the reorganization A albumen that is connected with the column material single-point through engineered.

3. method as claimed in claim 2 is characterized in that, contains halfcystine in the proteic aminoacid sequence of described reorganization A.

4. method as claimed in claim 3 is characterized in that, described halfcystine is included in the section that terminal last 30 amino acid of C-of described reorganization A Argine Monohydrochloride sequence constitute.

5. method as claimed in claim 3 is characterized in that, described reorganization A albumen at least 50% links to each other with the chromatography support material of A albumen affinity chromatography by the thioether sulfide linkage.

6. method according to any one of the preceding claims is characterized in that, the concentration of A albumen or its functional deriv is reduced to＜1ng/mg IgG described in the ion-exchanger circulation liquid.

7. method according to any one of the preceding claims is characterized in that, the monomer ratio of collected antibody is at least 99%, and this ratio is that antibody peak by the liquid that will circulate is classified as at least two fractions and discards the tail level and assigns to realize.

8. method according to any one of the preceding claims is characterized in that, described antibody is monoclonal antibody, preferred IgG antibody, and wherein said IgG antibody can be the IgG antibody of mosaic type or CDR grafting.

9. method according to any one of the preceding claims is characterized in that, collects described antibody from cell culture, then by the described antibody of A albumen affinitive layer purification.

10. method according to any one of the preceding claims is characterized in that, collects described antibody from mammalian cell cultures.

11. method according to any one of the preceding claims is characterized in that, the antibody of stand-by A albumen affinitive layer purification does not preferably mix with at least a proteinase inhibitor without the processing of inactivated proteases.

12. method according to any one of the preceding claims is characterized in that, described proteinase inhibitor is selected from PMSF, proteolytic enzyme inhibiting peptide, e-caproic acid or reductibility sulfhydryl compound.

13. the proteic method of purified product said method comprising the steps of:

1). differentiating the described product albumen condensation product in the circulation liquid by the classification of circulation liquid and preferably do not forming under the monomeric condition of described product albumen of mixture with second protein ligands, the solution that will contain product albumen is loaded on the ion-exchange material, and described product albumen comprises described proteic monomer and cohesion form; Further

2). classification circulation liquid and from ion-exchanger circulation liquid, collect with load on the ion-exchange material that is used for purifying on product albumen form and compare at least a product albumen monomer fraction that product albumen condensation product content reduces.

14. method as claimed in claim 13 is characterized in that, the product albumen peak classification by the liquid that will circulate or be divided at least two fractions and discard the tail level and assign to realize classification, and the monomer ratio of collected antibody preferably is at least 99%.

15. as claim 12 or 13 described methods, it is characterized in that, be lower than at least one second fraction of described first fraction according to the assessment of the monomer ratio monomer degree of product albumen wherein of discarding at least.

16. method as claimed in claim 15 is characterized in that, loads with a kind of damping fluid at least and the washing ion-exchanger, wherein constitutes the circulation liquid that contains the product albumen peak from the effusive at least a damping fluid of ion-exchanger.

17. method as claimed in claim 16 is characterized in that, the pH of described damping fluid is made as monomeric pI of product albumen to be purified or average pI, and its scope is described pI ± 0.5 a pH unit.

18. method as claimed in claim 16, it is characterized in that, the pH of described damping fluid is made as and is different from monomeric pI of product albumen to be purified or average pI, described pH makes the product albumen monomer have surface charge, when contact or when being immersed in the described damping fluid described electric charge cause ion attraction between the charged groups of product albumen monomer and ion-exchange material.

19. method as claimed in claim 18 is characterized in that, when adopting cationite, the pH value of described damping fluid is made as and is lower than the monomeric average pI of product albumen to be purified, preferably value is made as the low 0.5-3 pH unit than described average pI.

20. method as claimed in claim 18 is characterized in that, when adopting anionite, the pH value of described damping fluid is made as and is higher than the monomeric average pI of product albumen to be purified, preferably value is made as the pH unit than the high 0.5-3 of described average pI.

21. method as claimed in claim 13, it is characterized in that, for combining of product albumen monomer and ion-exchange material, described condition is non-binding condition, thus can from by reclaim the circulation liquid of ion-exchange material (w/w) more than 70%, more preferably more than 80% (w/w) load on product albumen on the ion-exchange material.

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