AU2013213691A1 - Capture purification processes for proteins expressed in a non-mammalian system - Google Patents

Abstract

) Methods of purifying proteins expressed in non-mammalian expression systems in a non-native soluble form directly from cell lysate are disclosed. Methods of purifying proteins expressed in non-mammalian expression systems in a non-native limited solubility form directly from a refold solution are also disclosed. Resin regeneration methods are also provided.

Description

CAPTURE PURIFICATION PROCESSES FOR PROTEINS EXPRESSED IN A NON MAMMALIAN SYSTEM The present application is a divisional application of Australian Application No. 2010266093 which is incorporated in its entirety herein by reference. This application claims the benefit of U.S. Provisional Application No. 61/220,477 filed June 25, 2009, which is incorporated by reference herein. FIELD OF THE INVENTION The present invention relates generally to processes for purifying proteins expressed in non-mammalian systems in both non-native soluble and non-native insoluble forms, and more particularly to the direct capture of such proteins from a refold mixture or a cell lysate pool by a separation matrix. BACKGROUND OF THE INVENTION Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field. Fc-containing proteins are typically expressed in mammalian cells, such as CHO cells. The use of affinity chromatography to purify Fc-containing proteins is documented (see, e.g., Shukla et al., (2007) Journal of Chromatography B 848(1):28-39) and is successful, in part, due to the degree of Fc structure observed in proteins expressed in such systems. Fc-containing proteins expressed in non-mammalian cells, however, are often deposited in the expressing cells in limited solubility forms, such as inclusion bodies, that require refolding, and this has been a limiting factor in selecting non-mammalian systems for expressing Fc-containing proteins. A drawback to the use of Protein A, Protein G and other chemistries is that in order for a protein comprising an Fc region to associate with the Protein A or Protein G molecule, the protein needs to have a minimum amount of structure. Often, the requisite amount of structure is absent from proteins expressed recombinantly in a soluble, but non-native, form and consequently Protein A chromatography is not performed in a purification process.

In the case of a protein expressed in an insoluble non-native form, Protein A chromatography is typically not performed in a purification process until after the protein has been refolded to a degree that it can associate with the Protein A molecule and has been subsequently diluted out of its refold solution. This is because it was believed that after apotein has been eIbided it as necessrI y to di kte or remove the components of the refold mixtnr in a wash step, due to the tendency of the componnts that typically make up a retold solution to disrupt intemetions between thetarget prteinand he Protin .A molecules (Wang et . (1997,Biachem. . 325(Pat 3007-710; This d&uMdon step can consume time and resources which, when working at a manufactuing scale of thousands of litersof culure, can he costly. The present disclosure addresses these issues by providing simplified methods of purifing proteins comprising Fe regions that are expressed in non-mammalian expression systems in a non-native soluble forn or in a non-native insoluble form, SUMMvtARY OF' HE IN ENTION A method of purifying a protein expressed in a non-naive sotuhle form in a non mmiaan expresion stems pvide. in one enbodint the nmethodomesa lysing a non-mammaianh el in wichte prtein is expressed in a non-native soluble form to generate a ell ysate; (h contating the elllyate with an separation matrix under condition suitable for the protein to associate wit th searation mati; (c washing the separationatx; and d eluing the protein rom the separain matrix The protein an he a complex protisuh as aprotein is selected fromnhe oup consisting ofa muhieric protein antibody and an Fe fusion protein. The non mammalian expression system can compose bacteria or yeastes. Theseparan matrix can be an afinity resin such as an affindy resin selected from he group insisting of Protei A Prote and a synthetic mietic affinity resin oitan be a non-afiniresin uch as a non-affmityrsin selected from the group cnsisting of ion exchange mixed mode, and a hydrophobic interacton resin. The cell lysate can be filwred befre i is contacted wih thesparaion matrix.Athoughnot reqiredthe method can further compose refcdding the protein to its ative n after tis elutnd from thceparation mai A method of purifying a protein expressed in a non-native united solubility form in a non-mammalian expression system is provided, In one embodiment that method comprises (a) expressing a protein in a non-native limited solubility formn in a non mammalian cel (b) lysing a non-mammahan cell; (el solubiiring the expressed protem a solubiiztion solution comprising oner more nte Ioi l a denaturanii) a redinat; and ii a Nurfactant (id" fAming a refold sohuton corising the soubilizatnion solution and a refol1d butlbr, h folci h"tor com-prising .. o"ne or mnore of he fbloing( a denature () an aggregation suppressor (ii) a protein stabilizer and (iv)a redox component; e app ing the reAl solon to a searaon matrix under conditions suiable fo e prote to associate ikh the matrix( ashig thesepartion matrix; and (g) eluting the protein from the separation matrt The nonnnatie lmite soabiliy fbrm can be a component of an indusionbody, The pomtin can be a complex prtein. such as a compile proten selected fAm the gmup consisting of a multiteric protein an antibody, a peptibody, and an I fusion protein. The nonvnWmi a expression system can be bacteria or yeast e The denaturant can compose one or moreo"urea, guanme hlurea and hyla the redu t can comprise one or mr of etysine, DT beta mereaptoethanol and glutathione he surfactacan comprise one or more of sarcosy I and sodium dodeyIsulfate, the aggregation suppressor can be selected fom the group considng of arinine proine polyethylene glycols. nondonic surfatants, ionic surfactants, polyhydc alcohols, gycerol, srose, sorbito, glucose trisodium stufat potasim sulfate and osnuolytes, the protein stabilize can comprise one or more f arginine, prone, polyethylene glycol non-ionic surf actant ionic surfactants, poyhydic alohols, gcero, escrose sorhitolglucosers sodi slfhte potassim suffate and osmolyes and the redox component can comprise one or more of glutathioneredued gath ione-oxitized, cysteine. cystne, Cysteamnhe vstaine and etamercaptoethanal. The separation matix can be an affinity resin such as an affity resin selected fron the group cnsistng of Protein A. Protein ( and syntheticnumed affrity rtifn or the separation matrx can beon afndtyresin sdncted fromithegroup consisdng of jot exchangemixed mode, and a hydrophobic interaction resin. In other mbodinents, the disclosed methods cn further comprise the steps of (i) washing the separaio matrix with a regeneration reagent and (b) regenerating the separation matriZx Thirgenerati agent can be one of a strong base, such as$ sodium hydroxide oar strong acid, such as phosphoic acid. [h regeneradng can comprise washing the separation matrix with a solution comprising one or both of a chaOtrnpe present at a concentration of 4- M and a reluctant. The chaotrope can be one of urea, dim-ethyl rea, methylurea, ethylurea, and guanidinium, and th reductant can be one of cysteine, DTT, beta;mrcaptoethanol and gutathione, In a particular embodiment the regenerating comprises washing the separation matrix with a solution comprising 50mM Tris, 10mM citrate, 0M urea, 50mM DTT at pH 7,4 BRIEF DESCRIPTION OF THE DRAWiNGS Figure t is a plot demonstatngthe binding offded, non-mCamnialian non ative limited solubilit fraiAn complex protein to Protin A media; in the figurethe X denotes resin loading at a 9m2ummresidentimesar denotes resin loading at an 6 niinreidence and solid circles denote resin loading at a 6 mi ence time Figure 2 is a table demnstating purfication of a complexproteinacomprising an Ec domain using Protein A resin. Figure 3 is a table demonstrating the reusability of Protein A resin when used to capture a non-mammalian non-native limited solubility complex protein over 150 cycles using the disclosed nethods. Figr'e 4 is a plot demonstrating the binding profiles of a refolded non mammalian non-native lmtd solubility comply prtoti to six diffeention excange resins (EX Resins 1, 2, 3. 4. 6 corresponding tooyopear SP$500CN ypea 3P650Mrm. GigaCAP S't POROS H50M Toyopeard SP60CTM and GE Healtheare $Pxm respectively) and a mxedomode resin (vtMRCesUn 1. (E Hcaltheare MM lowing captureusing thediscosed methods figure s table demonstratng purifation levels achieved for a protein comprising an Fe domain using one anion exchange resin (Fractogel TMAE") and one ation change rein (Fractogel M% DETAILED DESCRIPTION OF THE INVENTION The present disclosure provides methods of capturing on a separation matrix non ntie proteins produced b nirluhia ells n the se ofthe directcaptreof aprotein expresed in a non-native soluble form the advantages of the present invention over typical process include enhanced protein concentration, owume reduction, and 4 inr recovery overstraditional mlthods. improd protein stabili and ultimatdy process costsavings. In the ase of the direct capture of a protein expressed in a non-native limited lubility Arm, the advantagesf h1, in o pical proesses inclide the eliminaton of the need to dilute! the protei out of a refold xolution prior to eaptunng it on a separation matrix Another advantnge ofhe disdoed methods i khat they may be performed a rangeof sales, from laboratory scale Oypicay miilt e erorer scae a pio plant sc ae (tpicaly hundreds of lierK) or on an industrial scal (icalldhosands iters'The application of te disclosed mehods on large scales may be particularly desirable due to the potenalSavings in time and resources Nonw-nannahan, e;, microbial, els can natural produce, or can be engneered to produce, protins that are expressedin the a slle o nnd solUbil trn. "Most oen engineered non-mammalian cells wil deposit the recombinant poteins into large limited solubility aggregates ea led inelsion bodies. However, cerain Cellgoth conditions r temperatror pH) can be modified to drive the recombinantprotins to be expressed as intracndellulr, slble monomers. A man alternative to producing a protein of interest in cels in which the protein is expessedin the m i oflted sAolubilitndclusion bodies, e growth condition cam be modified such that proteins are expressed in a non-native yet soluble form. The els can thn be ysed ad the protein can be isolated by capAingidireedyfron ell lysate using on exchange chromatography, affinity chromatography orn xed node chroNatography, as described herein. The method can e particlar useM for purifying proteins comprisingan Fe region. In one aspect, therefore e prset disclosure relates to inethod of isolating a protein of iaerest comprising an Fe region that is expressed in a non-mammalian eHin a non-nativee at sobile fon Am a pol of sate genated from the el in which the protein was expressed. The method employs a separation matrixsuch as ein A One beneficial aspect of the discoAed me odis that it elininates the need for a refoding step before he protin is applied to the sepation matix. That is, non-mamalian clls expressing the protein of interesin a nonnative solule form can be lysed e lysate applid drealy to the separation matrix and the protein subsquently dWted from the eparationmatix. hs process alows the separation of proteins from cell culres in highly concentrated pools that can be subsequentiyrefolded at highconcentrations and can be of benef hen producing large quantities of potein particuladSrlyince the method is scalable om Mbench scale, which involves cultmes on the order of several lters topoducion scalehich in ov+ es cultures of thousands of liters. Following violation by the separation matrix, the protein of interest can optionally be subsequently refolded using any technique known or suspected to wdor well fbr the protein of mterest, in another aspect, the present invention rates to a method isolating a protein of interest comparing an Fe region that expressed in a nonmativeiied s hiy form. for example in inclusion bodies that needs to be refolded and isolated front the reifod Ixturem Comxnon a refold solution conains denatran g., ra o, other chaotrope orgaic solvent or strong deergent an aggrgation suppresor leg, a mild detergent. arginie orow concentrations of PEG a protein stabilizer e glyerol sucsse or other osmo salts) andor a redox cmponent geg, i cys e, ystin sntmine ysteanmine .utathone While oen benefica forrefolding proteins these comnponentsa inhibit purification (Oee e A; wang ci a (1997) BioLmc ona/ 32 art 3007-710) and A is necessary to isolate or dilute the prein from these conponentx o thrther processig, particuarly befr appying the protein to a separasonanrx. In one emnbodimfet ofihe disclosed method, purification is achieved y diey capping aprotein of interest, which is resent ia refoid mixture, to a eparation mat I this approch, following a refold step the entire rild ixte including the protein Of tere is applied directly to paaonatrix, suh as a protein A G rsn The protein of interest amsociates with the matrix in the presence Of thecomponents of retold buffer, impuritis are washed away and the protein is eluted, Since the method onits the need for removing any components of the refod mixture beforete refold mixture is applied to a separation matrn the method an have thoet of saving steps tne and resoes at regypicany nded on i n he protein froneiding and dilution 6 buffers in purification processes in some cases, the method can also reduce or eliinate the need for subsequent purification steps, The disclosed methods can also be employed to purify proteins expressed in a non-native soluble and non-native limited solubily forms in a non-mammalian expression system that have subsequently been derivatized, For example, following expression a protein comprising an Fc region can be associated with a small molecule, such as a toxin. Such conjugates can be purified using the methods described herein. L Meinition As used herein the terms -' and an" mean one or more unless specifically indicated otherwise As used heren the term non-mamrmalian expression systems means a system for xpr ngprotein ineneivd froim aganism othe an a mammal, incuding but not limitedt rokaryotes including bacteria sueh l as and yeat Often a non mammalian expression systems deployed to press a eonbinant protein of interest while in ther instances a protein of interest i an endogenous otein thatis expressed by a non-ammaincell For purposes of the present disclosureegardless of whether a protein of' interest is endogenous oecominant, if the protein is expressed in a non mammahan ce then that cell is a oniammali expression system Siilady a nonWaana n el i a c delved firom an or anism other than a mammal examples of which include bacteria or yeast. As used herein the term denaturan" means any compound having the ably to remo e some or all a protein s econdar and tertiary sttue when paced in contact with the protein. The term denaturant refe to particular chemia compounds that affect detuatdion s yeu as sluhions comprising a particular compound that affect denat uraion. Examples of denatuants dhat can be employed in the disposed method include, buts are not imited to urea, guanidinihum salts, dimethy urea, miethera. ethyl urea and combinaions thereof As used herein the term aggregation suppressor means any compound having the ability to disrupt and decrease or eliminate interactions between two or more proteins. Examples of aggregation suppressors can include, but are not limited to, amino acids such as argninst, prolinc. and glycine; polyo and sugars such as glycerio sorbitol surose, and trehakose; surtants such as, polysorbate0 CRAPS riton X-100, and dodeev maltoside; and combinations thereof As uoed herein the term proteinn stabilizer" means any compound having the ably to change a protein's reaction equilibrium stae such thatthe naave state of the protein is unproved or favd Exampes ofpoen stabilizers can include but are not imied to, sugars and pohedric alcohos suelas glycerol po.mei such as polyethylene glyo (PEG) and z-yelodetmi; aino acids salts suh as arginine, proline and g4yin; osmolye and certain [Honeister satsuch as ssdim stbtc and potassiun sulfate; and combinations thereof. Asused herein, the terms '10 and WFc region" are used interchangeably and mean a Ragment of an anybody that compares human or nonhuman (ag, marine) and Ca inmnoglobu in domains, or which comprises two contiguous regions which are aleast 90%identica to hPuman or nonhuAman Q, and , inamunoglobui domins An c can but need not have the ability to interact with an Foreceptor Segn Hasemann & Capra 'lmnoglbhns Stce and Funcin? inWiami.Paled. Pundarncntt.1 hmun Scoond Edition, 209, 210-218 0 989). which iscorpoated byference herein in its entirety As used herein, the tems "protein d Tpolpepde arc usednterchangeaby and mean any chainot. least e natural or non-naturaly oeurrin amino acids linked by pepd tonda As used herein~ the term complex nioleculemens any protein that is (a)large tan 2,000 MW or composes greater than 250 amino add residues, and (b comprises tw n ormre disulfide bonds in its natie form compl molncue can but need not fomnn limersn Examples of complex molecules include but are not lmited to. antibodies, peptibodies and polypeptides comprising an Pc donain and other large proteins. Pepubodies are described In US Patent No 660. ,US Patent No 738370 and US latent No 7.5 .012 As used herein, the. therm "pepibody" refers to a polypeptide comprising one or more bioactive peptides joined together, optionally via linkers, with an Fc domain. See US Patent No US60 US Patent No 710370 and US Patent No 7j11,012 fr examiple fpeptibodies As used herein, the terms "Fe fusion" amd "Fc fusion proten are useri interchangeably and rSfer to a peptide or potypep tide covalently attche to an Fe domam,. As used herein the term "Protein Nean my protein identical or substantial similar to staphylococcal Prtein A. icuinug commnerciay aaable and/or reombia ibrms oProtin A Forte purpose of this ientin Protein A spcifically includes engineered Protein A derived media suci as Mab SelectSuRe media (GE HeIdthcare), in whih a l unit (eOghe R subunit) is replied two or more imes and joined in a continuous sequence to form a in Pinnn A molecule, and other non.inaturally occurrng Protein A molecules. As used herein, rie erm Prtein G, mans any pIotin identical or substantially similar to Snreptococcal Protein uding commercialy available awd/or recombinant forms of Po.n G As used herein, t erm ubstantiay similar? when used in te context of a proteiniudingProein A, means proteins theatre atleast eera aleast0% identical to each other i amino acid sequence and maintinnr alemita desirable manner the biological aiiy of the undered protein. ded in anino acids considered identical for tie purpose of dterminig whether potent ae substantially similarae amino acids that are conservave substtutionsunlikel o affect biological activity. inckding the foli.g S N r, Val fOr lle Asp for Gin. Thr for s Ala fmrCOY Ala frTrcr fror Asn, Aa for Val, Secr for GIy. Tyr for Ple Ala fIPro, Lys or Arg. Asp for Asn, Leu for or k eM ,a for Glu, Asp for ly and these changes in the eese See , e at .laPan sdli P'orke (1979).The percent identity of two amino sequences can be determined by visualinspection and athematiacalculatio, or more preferably, the comparison is done by comparing sequence information usng a computer pograni such as the Genetics Computer Group (CC, Madison. Wis.) Wisconsi package versON 10 program, AP (Devereu i aL 1984. /wthc Aidw Rcs. 12: 387) or other comparable computer programs. The preferred defaut parameters for the 'tAP" program indes: (1 the weighted amino acid comparison matrix of Gribskov and Burgess ((1986b NcA AcM Rem 14: 6745h as described by Schwartz and Dayhof, eds, Atlas of Poiypeptide Scquece and Structure. National Biomedical Research Foundation, pp. 353-35S (1979% or other comparable comparison iatices; (2) a penalty of 30 for each gap and an additional pemlty of I for each symbol in each gap for amino acid sequences; (3) no penahy for end gaps and (4) no aimum penalty for long gaps. Other programs used by those skilled in the art of sequenecomparisncan alo be used, As used herin the terms "isolate and "purify" are used terhangeay and mean to reduce Q 1%, 2% 3% , 4%, 5,% 5% 20%, 25 %, 0 90-35% 40145%, 50% 35%, 60%165%, 700 751 80% 85%, 09% 95% or orethe aount of heteronoueleents, for example biological macromeules sach piois or DNA, that may be presenin a sample comprising a protein of interest. The presence of heterogenous proteins can be assay d any appropriate method including igh performance Liquid thro matography (HPL'I gel electrophoresis and stain ing andior ELSA assay. The prsenceof DNA a other nuclic acids can be assayed by any a -propdate method including gel eetrophoresiand staining andr assays employing polymerase chain reaction. Asused herein, the term "separtio maix" mns any adsobienmnateil that utilizes specific, reversible interactions between synthetic and/or biomolecles, g the property of Protein A to bind to an Fe regin of an IgO antibody or other Fc-eontakng poein, in order to effectteseparation of the protein from its environment In other embodiments the specific, reversible interactions can be base ton a pIoperty such as isckettic point. iydrophobicity. or siz. In one particular moment a separation manix comprises an adsobent suc as Protein A, affixed to a solid support. See, eag. Ostove (1990) in "Guide to Prote 1Pfi onAehadgin/Snzmov8259, which is incorporated herein in is entrety. As used here, the terms onnative" and onnaive forM" are used interchangeably and when used in the context of a protein of interest, such a protein comnpning a edomnt, mean that the protein lacks at least one formed strduicre tribute found in a form of the protein that is biologically active in an appropriate in vivo or in vwm assay designed to assess the protein's biological activity, Examples of 10 strucural features that can be lacking in a non-native form of a protein can include, but are not inited to, a disulfide bond, quaternary structure, disrupted secondary or tertiary sncture or a state that makes the protein biologically inactive in an appropriate assay. A protein in a non-native form can but need not form aggregates, As used herein the term "non-native soluble form" when used in the comext of a protein of interest, suht as a protein comprising a Fe domain, means that the protein lacks at least one formed structure attribute found in a form of the protein that is biologically active in an appropiatein rivc or in vitro assay designed to assess the protein's biological activity. but in which the protein is expressed in a form or state that is soluble intracellulrly (for example in the cell's cytoplasm) or extracellularly (for example, in a vysate POW), As used herein the term nonnative imited soinihtv tonn hen used in the contest of a protein of interest, such as a protein comprsng a Fe domain man any forn or state in which the protein lacks at least one formed structure eatre found in a form of the protein that (a) is biologically active in an appropriate in N or in Ono assa designed t assess the protect's biological activity and/or (b) forms aggregates that require atment, ch as hemicatreatmen, to becomesoluble. Ihe tem specifically nds proteins existing in inclusion bodies, such as those sometimes found whei a recombinant protein expressed in a non-mammaian expression system. iAsed herein, the erm "soluble form" when usdin 6the cotext of a protein of interest such as a protein comprising a Fe doman, broadly refers toa form or state in which the proteinexpressed ina frm ithatissoluble in a intracelularly(forexample in the Ae cytoplas) or extracelhdary(for exanein acl ysate pool) in a Nonnmaa lian Expression System One advantage of the didlsed method over typical purification methods is the elimnAation of the need for a refolding step before the soluble protein is applied to the separation atrix, hat is, a protein solublied ielysate cnbe diectlgapied to the sepration matrix hisi advantageous because the method does nt require any initial prification efforts, although an initiltato step may be desirable in someeases. 11 In the case of a protein comprising a R dominhe 1k region must have a certain eeof st re to be bound by p'otain A (Wang c a,,,, 1991) ie 32J. Pa # 0i1 ThisA et has limited the application of separation matrices for purinyg proteins that are expessd in aon-alive ble form particularly proteinscniprsing an Fe region, because it is commonly beheved that a soluble nonnatie Fe-contaunmg oteinwou odt have the requisite structural elements required to associte with a separationmatix Furhenmore the Pc region of an antibody spontaneously forms a homodimer under nonreducng condition and prior to the instant disclosure it was exeted t obser that e in heh ducie environment of the ell, the c conjugated protins an peptides not Only form enough structure for protein to bind to the at'ytesn but th he idividual peptide chains ready fo ed non-covalet dimenrs even though the proteins had not yet been completely refoded to nativefom Smiew oF evailing beif the success ot' the discloAd eod as spring and unantipated because it was not expected tat a non-mamalianu mcrobial cell fermentatn old be induced to produce a pteinthatn as solle, yet stdl had enough statue toasociatevhhe afdfnity separaion matri The cloned method can be employed to pudfy a protein oneret that is expressed i a non native soluble form in a non nmaniiazan cell expression system. Ihe protein of interest can be produced by liing host cells that ether natundy produce the proin or that have bee getically enginered toduce th poti hods of genetically engineering cells to produce proteins are known in the art See eg Ausabel i aL eds (190Current Protocolsin Molecular Bio (WileyNew Yotk} Such methods inLde introducing uncle acids that encode and alow expression of the protein into living host cel In the context of the present disclosurea host cell will be a onninliancell, such as bacterial cells, funga l yeast ces, and insect cel. Bactetil hostcells includebut re not limited to,E scer pcob Ex ps o able F. strains include: 811101 Dt,5M2929. IM109. KW25 1, NM53K NIM39, and any F strain that faailsto ceae foreign DNA. Fungal ost eils a can be used incud but arecno limited to Sa arvnvsce wie/topastas and Apergus ells New cell lines can be established using methods known those skilled in the arn (eg by irnsfomaion vind infection, and/or selection. t is noted tha 12 the method caneperformed on protein that are endogenously expressed the non mammalian c as Mwl. Durng the production of a non-manmahan cultu, growth conditonscan be identified and employed so as to favor the prodution of a protein of interest in ;an intracellular soluble fhm, Such conditons an be identified by sstenmtic empirical opturivation of the cultue condition parameters, such as mperature or tNH This optimization can be achieved using anaysis of mulidfactoriamates For example. a natrx axor es of multiactoria matrices can be evauaed to optimize te- perature and pi- conditions for production of a desired speie , a non-nativeoble form) An optimization screencan be set up to systematically evauateemnperature and pH in a or partial factoial matith each ompnent Varied over a range of at least thre temperature or pH levels with all other parameters kept constaT The protein can be expired and the yild and quality of prot expressed in the desired fom can be evaluated using standard muiivariate statisicatools iniay non-mammalian elS that express a partieuar prtein of interest are grown to a desired target density under conditions designed to induceepression of the protein in a souble form. Inone em bodiment, the ellskexpress a wild type protein of interest.In another embodiment, the cells ca be engineered ing standaGrd molecular biology techniques to recombinantly express protein of interest ananduced to produ the protein e of tret. Th rotein nest can be any protein, fo eanmple a protein thatcomprises an Fc moiety. Such a protein can be, for example an antibod a peptibody or an Fe fusion protein, any of which can be joined to an Pc moiety via a linker. Once the desired target density is reached, the nonnmmmalian cellarseparated frm the growt media. One convenent way of achieving separation is by centrifugationhowever filtration and other clarification methods can also be use The cels are then collected and aresupended to an appropriate vohue a resuspension solution. Examples of resuspension solutions that can be used in the disclosed methodsinclude phosphate buiOe saline, Tris bured saline or water. The sectin of an appaplate buffer ill be detemned, in part, by te propeies of the moleculle of interest as well as any vokme or concentration constraints. 13 Following resuspensionthe non-mamnalian cels arc lsed to releasete protein which will be present ihe cell lysate in a non-native solubleform to generate a cel lysate The lysis can be performed usingany convenient meansuch as ceding he cen suspension through a high pressur hmnogenizer or by employing a chemicalIs process. Whichever lvtie process is scleced, the %nction of the lysis step is to break open the cells and tobra down DNA Theysis can be perfmied in multipe yces to achieveamore complete lysisar to accommodate rgevolimes ocel suspension. For example, the celI suspension cen edeld through a mnehaical honogenizerseveties This process releases the intracelular contents, including the protein of interest, and forms a pool of cel lysate. Fllointhe is proced ,the cell can optionally be fitew Filtation can remove particlate matter andor purities, uch as nucic acids analpid and may be desire n soe cases, such as w one suspectsa direc applatin oF the cel lysae to the chromatography equipment or media may lead to fouling or clogging or vwhen the separation matrix is sensitive to fouling or difficult to clean inplsee The beneit of fihering the cl late prior to conting it wth the separation mtrix can be detennined on a easease asis After the lysis procedure the cell lysate can optionally be incubated for an approprie amount of time ithe presence ot air or oxygen, or exposed to a redox component or reox th ipr The b can a and/r e formateo of the mininal secondary stmeture required to faciliate an associaton witha separation arx The particllngth of the incubaton can vary with the protein but istypicaly less Uhan 72 hour (g 0, OS 1, 2. 3, 5. 7, 10. 12, 24, 36, 4 72hours When an Inubation is performed, te length of incubaton time can be determined by enpirieal analysis forah tei which in some cass ill be shorternottedad other cases longer. Following the incubation period the cel lysate which comprisesthe rdeased protein of interes is contacd with a separation matrix under conditions suitable for the rteinto associte with a binding elementofhe separation mtrin Regresentave conditions conducive to the association of a protein with an afinitv matrix are provided n the Examples. The separation natrix can be any media by which the protein of interest 14 can beseparaed frcmn the components of the resuspension and/or dysisbderihlding impurities such as host cell pains. DN lipids and chemical imaurities introduced by the comoients of the resuspension and/or ysis bufe Proteins A and C areQien pnployed to purify antibodies peptibodies ad other fusion proins comprising a Fe region by affinity chromatography Seg Vola cia (0994),a Biph. 244252 Aybay and nir (20002 b w4m erhody 233(i 2 7741 Ford e at (20011 Rhtog 74: 427-435. Proteins and are useful in this regard because they bind to the Fe region of these types of proteins Recon! bnan fusion poeins comprising an Fe region of an antibody can be purified sing similar methods. Protein A and G can be employed in Ahe disclosed methods an adsorbent component of asepaiation man Thus examples of separation matrics that can be employed in the present invention include Protein A resin, which is known to be, and is commonly employed as, an eecive agent for puriying moinlecules comprising an Fe moiety, as welas Protein G and syntheicimetdc affnity resins, such as 1MEP HperCe@ chromatographyresn After the protein of interest has ben associated withte sepanaion natix byl contacting the esllysate containing the protein with te sepanaton mat thereby alowing the protein to associate with the adsorbent component f he epaation msatrix the separation matrix is washed to remove unbound e im npurities. The ash bu fe can be of any composiion. as g lo ng the composition and pH4 of the wash buffer is compatible with both the protein and he matrix, and mantainshe iteraction between the protein and the matrix; Examplefutable washbffers that can be employd include sohmtions containing gleine. ris, curater phosphate; typically at levels of 5100 mM(cg. 5, 10. 15. 20, 25. 30, 35, 40. 45 50. 75 or 100 mlW These nuxnis can also contain an appropuat salt iOn, such as chloride, aniator acet at evels of 5-500 mvI ( , 1 32 40. 50, 70, 00, 90. 100. 150, 200. 250, 300, 350, 400. 450 or 500 mM The resin can be washed oice or'an number of times. The exact composition ofa wash buffet will vary wth the protein being purified. Afer the sepaation matix with whih the protein has associated has been washed the protein of finest iseated from the matix using an appropriate solution The proin of interest can be hued using a solution thatnterferes wih he binding of 15 the adsorbent componnt of th sqparation natix to de protein for example by d listing the interations between the arationatix and the protein of interest T is souion can include an agent that can either increase or decrease pH, and/or a salt For eIxnle, the pH can be lowered tdbout 4.5 or lessAfor eanple to between about 3.3 and about 44 . 3.3, 34, 35, 34 1 3 , 39, 444 i, 4 42, 4. 4 or 40. A hsolution compsing citrate or acetate. for example an be employed to lowethe p Other nethods oflon are also known, such as via the use ofhotropes (see Eg ni aet A (2005) Anabeal Bhentt 345(5 257 or auino acid salts (see. ntg akawa i at M 004Poen xpesion & Purwau 62r244248) Protocols for such ainity chromatography are well known thi ae atSee g Miller and Stone 0978) .. iunu Meho 24(1-2: 1.1125 Conditions fr binding and eliting can be ready optimized by hose skiNed in the art The exact composition of ank eaion bu"rwiliy with n protein being purified. The protein can hen emotionally be fhrdei puried rom the eluton pool and Aflded as necessary In otherstuatns t1e protein need not be furdier puried and instead can be refoldd direct fronthe ation pool. Refolding directly from the nation pool may or may not require denaturadon or reduction of the protein pror to incubaton in a refolding olNdon and wi depend in part on theipropeties of the prin In some eases it will be desirable to provide the separation matrix in a column format In such cases a chromatography column can be prepared and then equihbrated before the cel suspension is loaded, Techniques for generating a chronatography column arewell known and can be emp loyd. Ar option ptparaton and equilration stp can comprise thie icoumnaith a Wofeh an appropria 11 Rand salt condion that is conducive topotein matrxinteraction. This step Can provide the benefit of renoving impuities present in the searation matrix and Can enhance the binding of the protein to be isolated to the adsorbent component of a separatonmatrix As noted, tc separadion matrix can be disposed in a column The column Can be run with or without pressure and fom top to bottom or bottom to top, The diredcon of the flow offld in the colan can be versed during the purification process. Purifications can also be carried out using a batchpocess inuchich the solid supporais separated front the luid used to load wash, anddate te sample by any stable means 16 ng vit, ctigation, or filtraion. Moreover, purifications can also be carried out by contacting the sample with a filter that adsorbs or retains some molecues in the sample more strongly than otheM buch as anion exchange membrane Ifdesired, the protn COncentraton ofa sample ainy given step of the diclosed method can be deternined, and any suitable method can be enpkye Sudi methods are wel known in the rt and include co wimetric methodssud as the Lory assay the Badfd asay, the Snith assay, and the colloidal godassay; I') ethos uinthe ! absorption propertes ofproteins; and 3)isu estimation based on stained protein bads on gels rying on comparison with protin standards of' known quantity on the osme gel. See,, Stostehsk (1990),"'Quantitarion of Pim 'in "'uide to Protein Purificaion Mhods in Em 2 Periodic eterminations of protein concetationcan beustid for moitorin progress oftheneiod a t is before It is noted that any or all steps of the disOosedehds can he cared out manualy or by any convnient automated eans, suc as by employing automated or computer-controuled systems S DirectCapture of NoiNativenimited Soubditv Protein FormsFrom a Refid Solutina ollowina Exression nNotnsAammaia mCels In another se of the present disclosure a methd of idtying a proein expressed in a non-natielinited sohibiiity form in a nonammalian expression system is disclosed. An advanOaf die disclosed method is that the method liinatesh need for removing Or dihini the refid solution befbr applying the protein to a se"paationmai tereby saving the time and resources associated With what is a typical step in a pa ion process for isoladng pritenexpressed in a n aion- native li niied solubility for, No aammaliacellsg. n iab cels can produce recombinant proteins theatre expressed intracelhdary in either a soluble or a limited solabiity fOrm. When the growth conditons are not dieted to foceepressionf th protein in a soiubno the cells may deposit the onbinant proteins into large rdatively insohble aggregates. such as inclusion bodies. These aggegates comprise proten ta is typically not biologically active or less active than the completely folded native form of the protein, In order to produce a funcnamd protein, these imclusion bodies oten need to be carefully denatured so Ota! the protein of imerest can be extracted and refolded into a biologically activeform. hn typical appmaches, the inclusion bodies need to be captured, washed,exposed to a denatUnri g andtroreducing sokbiIauon solution and the donating Soltion is then dilUted ith a solutio n o generate a condition thatallows the protein to refOld into an activ iOrm and form a structure that is found int native protein Subsequently it is necessary to remove the components of the diluted denaturing solution from the inmediate location ofthe protein, In order to do this, the refold solution comprisng the olih ation solution and te refded protein is ypically dilted with a baffeied soluton before it is applied to a separation mati, such as a Protein A ion exchange or other mixedmode adsombeas This process cane i& ostnin and our ntensietalso significantly incass the volumes that need to be handed as wel as the associated tankage r e which can become limnitinwhenm gonlage smlea The disclosed ieod elimines nhened tbr such a dilution step Theisclosed methodispaicularlyusefu for purifying protein ofinterest that is expressed in anonnaie limited solubility form in a noneamma ll expression system. The protein of interest can be produced by living host els thateither naturally produce the protein or that have b eneticaly g red to podete protein Methods of genetically engineering cells *to produce proteins are well known in he at Se eggAusbe et al, eds (1990). Crrentdrotocolsin Mlecular Biology (Wey. New oA Such methods include intoducing nucleic acids haencode and allow expression of the protein into living host cells. in the conetof the present disclosure. these h ot es ll be no an liacells, Such as bacterial elsfngal cells Bacterial host els iclde but are not limited to .Echelchia co/i cels Examples of able strains include: I1, DHtGb,9 9 M 109. KW25 I, NM535 NM A39, and any . d strain that fails to cleave foreign DNA. Mnga host els M at can be used include, but are not limited to. Saccharces cerewsae, Pchta pastoris and Aspergilus cells. New cell lines can be established using methods well know by those skilled in the art (e., by transformation, viral infection, and/or selection). t is 8 S noted that the method can be performed on endogenous proteins that are nuraly expressed by the non-mammaliance as wel Initially, nonmamaian cels that express a particular protein of interest are grown to a desirearg density In one embodimnrt the cels can be xpnessing a parieular wild type microbiaprotein of interest. in another embodiment, thle cells can be engineered using standard molecular biology techniques to ieombinautyepress a protein of interest, and in this conte they can be induced to overpoducethe protein of interest, The protein of niteretan bean y proten, for example a protein that comprises an Fe moietySue a protein can befrexaiple an antibody. a pepibody or an Fe fuion proteinany of which can he joined to 'n Fe noiety via a tinker Once the deird tagt density is reachedhe nnannaaiia cel can be separated from the growth media. One convenient way of achieving separation is b cenrifugaioni however filtration and other c fiction ietho c aso be uAsed The cells ar then co leeted and are r pendedo an appoprite volume n a resuspension solution. Examples of esuspensinsutionstat can be used inte preent iveno include phosphate febufred sine, ds offered salineorater The seletion of an appropriate buffer will be determine in pat, by the propertie of te molecde of ntest as wel as anyoume or concentration constraints. in order to release the limited solubility non-native protein fom the cells, the non mammalian cells are lysed to form a cell lysate comprising the released the limited solubility nonnadve protein The ys en be performed in any convenient way such as feeding the cell suspension. through a high pressure honmenizer or by employing a chemical ysis process. Whichever Wis prces i SAeetedi, te function of the lysiwsep -S to break open the cells and to break down DNA. The lysis can be performed in gmitki cycles io chieve a moe cole lysis or to accommodate largevolums of el pension. For example the cell suspension cn be fed through a mechanied homnogcnerseverltimes. This process eleases the intracellular contents, including the natundl-occan.ing or recombinant Proien of nearest, and forms a pool of celI lysate. Next, the limed sohubility non-natve protein is separated lon the rest of the lysis pol. This can be done, for example, by centifugation. Representative conditions for a centrifuge-mediated separaton o washing typically ilude removal ofvexcess i19 water rom the lysite suspension otthe resulting slury in a resuspens solution 1his washing process nay be performed oncoro uiple times Exanples of typical eentrnge types include, but are not limited to, diskastack cotinuous discharge, and tube bowl Example of resuspension solutions aha t can be used in the present invention mcl ude phosphate feedsfeared sainted sale, or water and can include other agents, such as EIDA or other sahts The section of an appropriate bu ffer wil be determned, in par, by the properetis of the molecule of interest as well as any volume or concentration constraints The eact coIpOsiion of an resuensi bf wil vary wih the protein being puri fied, The expressed protein is then h dsolubilza in a sbilizaion solution comprising one o. ore of( a denat (ii) ;a redutiat and (i) a surfattan The denatuntt can be included as a means of unfolding the limited solubihty protein, thereby removing any xising tucture posing buried residues and making the protein nmor soluble. Any denatuant can be employed in the so ubilizaton solution Examples of somte common denaturants that can be employed in the refold buffe include urea, guanidinium dimethyl urea, metylurea or ethylua. The spec ie, concentradon of the denaatrant can be determnedbyroutine optnmaon The redutant can be included as a means to reduce exposed residues that have a propent to form covalentntra or intermolecuar-protein bonds and minimize non spcific bond frmation Examples of suitale ridu ns include, but are not united to, eysteine, DY betanimrcaptoethanoa md gdmathione. The specific concentration of the reductant can be determined by routine optimization. t surfacant can be included as a means ofunfolding the limited .soiubilitv non natie protein, thereby exposing buried residues and making the protein more soluble Eampoes of suitablsurfacants include but are not limited o, sarcosyl and sodium dodecyilsuifatedh.Tie specific concentration of the surfactant canbedeterined by routine optimization. Although te composition of a solubilizaion soltionwilvary wih the protan being Pried in one particular enb thesoh 0 diation sohion ComprIses 4 M guanidine. 0 mM JUT Gontining a efok solution comprising he solubilizadion solution (which cornlpises the proingand a reflbd buffr isfned The refbldbffer comprises eoneor more of i) a denaturant; 0i an ggregtion suppressor;ilia protein stabjzer and (iv" aedc component The denaturant can be included as a ieans of modilving the thermodynamics of the soluion, thereby shiing the equilibrium towards an optimal ban o nae fori.The aggregatin supgressr ca be included a a means of preventing nonOspeic associan fone protein wh another, or wit one region of0a protein with another region of the same prein. The proten stabiercan be included as a means of promoting stable native protein struetreand mayalso suppress aggregation hn various embolden th dentuman in the reold buffer can be selected from the group conisting of urea, guanidinum sals urea, methyhwrea and ethyhorea, Invarious embodimentshe protein stabilzerin the refold buffer can be selected uim the grup Consiting of ha pr i nen polythylene ges non-ionc suramants, ionic surtiaants, poyhydrie a Kcohos glycero, suerose, sorbito lucose, Tris sodium sulfate, potassium sulfate and osmolytes In various embodiments, the aggregation suppressor can be selected trom the group consisting of arginine, prolino, polyethylene glycols, nonionic surfactants, ionic surfactants, polyhydric alcohols, glycerol, sucrose, sorbitol glucose, Tris sodium sulfate, potassium sulfate ad osmolytes. hi vario enbodimenits the thiol-pairs can comprise at least one componnt selected from he group consisting of giutathioneweduced. glutathioneoxidized. cysteine, The specficoncentrations of the0components of a retold buffer ean be detemined by routine optimzation For example, a matix or sees of mutiaorial maisri ca be evaluate to optimize the refoldingw ee fibfor conditions that optinze yivhd and distributions of desired species. An optim on screen can be setup to systnaictellenate dearantgreation spressoprtein stabier ad redo componentconcentrations and proportions in a full or parial fatoria matrix, with each component vared over a range of concentratons with all other parameters keptcnstant The completed relations can be evaluated by IRPIL and SETIPLC analysis for yield and product qualiy standard mutivaaiestatisticaltoos 21 The function of the hfecomponentof the refldolution s to mantain the pH of the refoki solution and can conmpoise any buffer that buffers |i the appopiae r range. Exampes of the buffering component ofa reld buffer thatcan be empoycd in themeithod include but are notd ted to phosphate b Oft itte buTers. tris butffir glycineufferCAPS CES and arginincshased uliers, typically ateves oi10 a Wg, 5, 10, 1 20. 2$ 30, 35, 40, 45, 50 55, 60, 6 I 75 80. 81 9t 95 or 100 mMY Although the composition of an refoid buffer witl vary with the protein being purified, in one embodiment a rebld buffer comprises arginine, uroa, glycerol, cysteine and cystamine. The nrld solution can then be incubated for a desired period of time The incubation period can be o any length but is typically ctwcn 0 and 2 ours ( 0 05 1, 2, 32 5, 7, ,2 8 24, 36. or 7hour after an appropriate incubation timehe refold solution is then applied to a separation matix under conditions suitable fr the protein to associate with the matrix. The separation matrix can be any media by which the protein ofiterest can he separated Om the components of the suspensionandor lysis buffr, including impues such as host cA proteins DNA and cheniced impurites introduced by the components of the sohiiton andor lysis buffer. Proteins A and (1 e often employed to puri eseptibodies and other fion proteins comprising a e region by affinity chromatography See eg" Vola t a (1994p 24-21 27Wd6 Aybay and m-ir (2000) .J lmmunof Mthods233 21 77-1. Ford er At (2001), onug 754: 427-435, Proteins A and G are useful in this regard because they bind to the Ec region of these types of proteins. Recombinant fusion proteins comspriing an Fogignof an IgO antibody can be purified sing similar methods. Proteins A and 6 can be employedin the disclosed methodsan adsoentcomponenf a separation matix Thus, examples of affinity separation maticestatcan be employed inthe present inventonincld Pnotein A resin, which inow to be, and is commonly cop toyed a, an effective agent or purifying oleculeNs comprising an Fe moiety. aswell as protein G and synthid nmetic finy resins. Otermateals ta ean be employed include MC and ion exchange resns (see Example depending on the properties of the protein to be puriied, It is noted that when pedomung the method the tetold solution comprdaine refolded protein of interest is ap plied dircty to the separation matrix, without the need fording or removing the components of the soluton requed for retoldingthe proven. ThIS is an advantage of the disclosed method. Initially, it was eted that the highly onic and/or chaotropic compounds ad various other components of the ebfoid solution would inhibit the association of the proteinwith theeparatioa un attix However, in contrast to reports in the literature (g Wang et at (199) lackna/ ournat 325Pan K07 0) it was smprising to observe that the protein was in fact able to associate with th separationatr then esence of the comtponents ofthe retb soaion The unexpectedfining that the protein could assoiatewiththe separation matrixn the presence ff the componens o'the retold usoion fauciliates theichlmnation of a dilution step or buffer exchange operatonproviding a savigs otme and resources After the protein of interest has associated with the separation matrix the spaation mtrx is washed to remove unbound protin.lysate, impurities and unwanted components of the refold solution, The wash bufer can be of any composition, as long as the oznposition and pH of the wash buffe is comptible wAi both the protein and. the matrix. Examples of suiable washbufeix hatca;.n include, brut are limied to, Wout=n containing glvcine, iris, citrate, or phosphe. These solutions may also contain an appropriate salt Suitable salts incIde, but are not imi ted t sodium, potassiun ammonium, agnesium calcum, chloride fhiordek acetate phosphc, ant or aatc. The pHi rangenichosen to optimze the chromatography conditon preserve protein binding, and to retain the desired characteristicsof the protein of interest. Thesin can be washed oncee or any nuaber of times. The exact composition of a wash buffe illW vary with the proteinung purified. After the separation matr wi whih the protein has associated has een washed the protein of interest is eluted using an appropdate solution g.a low p buffered solution or a sat sodion) to Snm an elution pool comprisig the protein of intere 2. 31 The protein of interest can be euted using a soltion that interfes with the b6dinng of the adsorbent component of the separation matrix to the protein fr eample by disrpting theteactions between rotein A and theFe region of a potein of interest This sohition may included gena t that can either increase or decrase pH, andora ai la various embodiments, the elnuon solAion can comprise acecacdd gyne or citne ac d. Euion can be achieved by loweng the PH. For anple, the pi can be lowered to about 4.5 or less, or example to between about 33 to abou 4 3egg 4, 5 30 37, 31 3., 4,0. 4,1 or 42, usig a solution comprising citrate or aetateamongother gossilities. Income siuaion the protein can then e further purified frondhe eiution pool and can be further refoed. if necessary. In other situats the pota need not be funher purified and stead can be further folded directly in the eitan pooli necessary. Protocols forsuch afaniy chRi aeraph arc known in fi art. Seee uer and Stone (1978} tLjimummotA ha ho 24(12): 11 123 In the eases that udize ton exchange, nixednode, or hydrophobic neteretianchrmatographheconcentationeo sa4 can be increased or decreased to disruptionic intention between bound protein and aeparatinmatrixSoltions appropiate to effect such elutions can include, but are not Sied to, sodium, potassium ammoun.n magneseim, calcinm chloridefuoridle acetate, phosphate, and/or citrate. Other methods of euon are also known. Conditions for binding and elutin, can be readily optinized by those skilled in the art. The eat composition of an elution buf r W vary with the protein being pudied and the separaion tixbeing employed in some cases wil be desirable to situate the separation matrix in a column format In such cases a cwnn can & greard and then cqiilibrated before the ell suspension is loaded Techniques for generaig a chromatography column are well known and can be employed. The optional preparation and epqilibraton step can comprise washing the column with a buffer having an appropriate pH and composition that will prepare the media to bind a protein of interest. This step has the benet of removing impudties present in the separion ntax and can enhance the binding of the protein to be isolated to the adsorbetacomponentof separating matri 24 It is noted that any or all steps of the invention can be carried out by any mechanical rneans. As noted, the separation matrix can be disposed in a column. The column can be run with or wihout pressure and from top to bottom or bottom to top. The direction of the flow of ihd in the column an beeversed during the puriteatiotn process. Puriications can also be carried out using a bath process in which the solid support is separated fro the hqtud used load, wash, and Qeut the sample by any suitable means including gravity centifugation, or titrntion. Moreover purifications can aso be cared out by contacting the sample with a filterthat adsorbs oi-etans some molecules in the sanple more strongly than others. If desired, the protein cncentration of a sample at any gIven step of the disclosed method can be determined by any itabmethod Such metho are wl knw n the arand include: 1) colo etic methods such as the Lowry assay, the Bradfod assay the SmA assay, and the coloidal gold assay 2) methods ruling the Uv absortion properies of proteins and 3) visual estimaton based on stained potein bands on gels relying o comparison with protein standards of known quantity on the sanet. Se eg Stosehek (1990 Quantain of Protein. in "Gidedt Ptein urication Me~hds nmakIo1 2: 50 , Perdic deteminaions of protein concenrain can be useful fortmonitoring the progress of the nbethod as itis prtormcd | is noted that any or al steps of the disclosed methods canbe ca rried out manually or by yonveient autoated eanssh as by employing utoad or computerscontrolled systems. 1. Column Cleaning i another aspect the present disclosure relates to the observation that in many casesthe seaationatrix employed in the inthods provided herein can be cleaned after multiple separations and reused) This unnexpected property of the method provides a signinieanr cost and resourcsavings particulafly on ithe mnanfaturing saale. sncae the separation matri need not be discarded after a separation is compete Common wisdom in the industry suggests that after a separation mtari such as Protein A. is repeatedly exposed to highly heterogenous feedstocks comprising high lipid and host protein content it becomes irreversibly con aminated and unusable when treated with the mild regeneration solutions conntly utilized for potnybased afni resins The disclosed methods however avoid this station and exnd the usable 1fetime of a separation matrix In the context ofaarge scale manufaauring process this can translate into a masurable savings of tmn and mnoney Moror, dhe aing step can he peorfred as disclosed in te Examples. in-place and with no need to xtrac Mthe separaion matrix from a cohmn or other mtix rtainin device r ckeang, thus saving time and resources, In one embodiment of a cleaning operation of a separation matrix, following a separation employing the disclosed method the separation matrix is washed with a regeneration reaent, such as sodium hydroxide, or an acidic reagent, such as phosphoric acid. In one particular embodiment of a caning operation, Protein A is the separation matrix and a column containing Protein A resin is washed with 5 column volumes of 1 0 mM phosphoric acid and held for 5 minutes over the column. Following the wash whithe acid the olumn can be flushed with water regeneratedi w olumn volumes of 50mM Trs1mM etrae i urea, 50mM 61DT: pH subequently washed ih water and hen pushed wih 3 colan volumes of 150 mM phosphoric acid Ti, s cleaning protocol has beenutilzed to achiee over 200 cycles of protein Aesin% Figure 3 highlightshe results achievable using the disclosed cleaning methods. EXA MPLES The following examples demonstrate embodiments and aspects of the present invemionTd and are not intended to be limiting, Directtature of Pteins Epresed in a Soluble Forn Using Protein AA i t Chromatozraphv Thne Jfbiowin xermn dio'srk that a prti opiiga pluraliiyof polypeptdes joined to an Fe moiety can be separated from an E cni cell lysateh suy usiga Protein A affinity media. 26 A. protein compriing a plurality of polypptis joined to an #c moity was expressed in an E. fermentation induced at 3MC and driven to express solbleifrm protein produt.. The femmetaton broth was centrifu.g, te liquid fraction removed. and the Cell paste was collected. The celA were resuspended ih a 10 nIN potassn phosphate. 5 mM DIA pI 6 buter solution to approximately 10% of the original okin The cells werethen lysed by means of three passes through a high pressure homogenizer. After the cellswere lysed, the cell lysate was filered through a 0.1m ler to reduce particulate evels Thematerialwas thenstored in a closed bottle for - 24 hours at approximately 5C, hn a separate operation, a packed column comprising GE Healthcae Mab Select" Protein A aftiniy resin was prepared and equilibrated with 5 column volumes (CV) of 10mM Tris; pH ST, An aliquot of a protein comprising an Fe moiety was sampled directly from a lysate. The protein mixture was loaded to approximately .02 millimoles total proteiw resin at a 6-10 minute residence time. See Figure *l which correlates protein bound and protein loaded as a function of residence time, After ioadin, the column was washed wh 10 mMIris; pH J.0, or CA, at up to 220 emnhr The protein ofintest was recoved front the resin by elution ith 50mM sodium acetate pH 1at up to2 em r TheS eluion pool yielded greater than 90% rcovery of the soluble material in de initial cell broth The colleted protein in the elation pool was stored at 2Ik 0 C until the next pmification step was erred out. Following the separation, the resin media was cleaned in-place by flowing 5 CV of'6 M Guanidine, pH $ AO at 220 em/hr. The results of this separation demonstrated that a souble proteinexpressed in a nnmammaliansystemn can be captured and purified ith high yield, directly fion cell lysae broth without having to efold the protein prior to application to a separation matrx Exanmol 2 Capture of a F~e~ontainine Protein Eixpressed in a Limited So lubility Form From a Refold Mixture Uine Protein A Affinity Chromatoaraphy The following experiments demonstrate that an FC-containing protein can be separated frm a refold mixture comprising glycerol, uanidine,. and aine uing Protein A affinity media. In one expeientn a recombinant protein compsng a biologically active peptide linkedA to the Qterminus of the Fe moiety of an Ig I molecule i lnkeand haing a molecular weight of about 57 kDa and comprising8 dislfide bonds in a non mammalian expression system namely E coW harvested, refded under appropriate condition and captured using ProteinA affinity media, The growth media in which the cels were growing was centriuged and tecid factionre ed leavinghe d as a paste ellwereresuspendedin water to approxmatey 60% of the original volnne. The ells were lysed by means of tree passes though a highpressure homogenized After the cells ere lysed.the lysate was cntrifuged in discstack cenmrifuge to collect the protein in he sold fractionhch was expressed in a limiedaslbitnons native fAm, amely as inclusion bodies. The protein sIurry was washed multiple times by resuspending the slurry in water to between 50 and 80% of the original fermentation broth volume, mixing, and centrifxuation to collect the protein in the solid fraction. The concentrated protein was then combined in a solubilization solution e protein, guanidine, urea and DTT, After incubation fo one hour heprotein solution was dilutedA in to a refeld buffer conining agrorate levels of arginine rea, glycerol. cysteine, nd cystamign In a separate operation a packed cohn comprising PoSepVA Ultra Protein A finity resin dimensions of . c ntenmal diameter and 25cm heightwas prepared and equAibrated with column volumes(CVof 25,M 'his10mM sodium chloride; pH 7,4, or similar buffered solution, An aliquot of a protein comprising an FC moiety fmm the refold. solution was filtered through a series of depth and/or membrane filter to remove particulate. The 28 condoned and iered protein mixture was loaded to approimaly 3 millimols total prolin/L resin at a 6-0 minute residence tie. See Igue wich coreates protein bound and protein loaded as a function of residence time. Aer Oading, the cAlun was washed widh 25mM TriM 00m siodium chloride: pHN 7A or sindiar buffered solution for 4.5 CV a up to 400 cir 'The ccontiaing protein was recorded fromthe rei, bn eaion withl00mM sdim acetate . a up o 300 Mhr The average eve of purity achied shown in Fiore 3. wng the separation, the resin ddi was cleaned e by floOwing 5 ('V of 150 M phosphoric acid. The conunwas regeerated with V of 50mN Iris In 0 itrate, M urea nd 50mM DTT pH 7, washed wh water, and then hushed with 3C of50 M phosplhon acid. The resuaofthisseparaton demonstrate tha aninsoluble protein expressedia nogmmrna nsystema can be prfied directly .lon a refbId havhithout thing to dilte the rentbd buffer prior to application to a separation matrix for morethan 1 ce s. agsindicated by thetie presented inigure 3. hn nother separation, he Protein Acolunwas cycled with the above procedure 1 thaimes and then the fnal eycle was run a foows: he nediawasequilibrated with 5 column voimes (C%) of 25mM Iris, 100mM sodium chloride; pH 7.4. or sinlar buffered solution. An aliquot of proteinIsampled dirctly from a refoId buffer was e red through aeie of depth andeorm ban fir to remov paries. The conditioned and fltered protein mixture was then loaded on the column to 0.35 nilnmoles total protein rsin at a 6-10 minute residencetime.ee Fure which correlates protein bound and protein loaded as a function of residencetme. After oadinghe coumn was washed wih 25imM ids. 100Md sodiun chloride: pH 7.4. orimilar ffred i for 4.5 CV at up to 400 cm/hr o Theprotein of interests recovered from the resin by eluing with 100mM sodm acetatepH 37 a up to 300 Ehr The en media was leaned inpae by fowing 5 CV of' 150 M phosphoric acid over nally, the column was flushed wit water regenerated wit 5C of50mM rs Al 10cM ata, M urea, and 50mM DTT pH 7,4, washed with water, and then flushed with 3C? of 150 u phosphoric acid. Subsequent analysis of 29 the resin showed no protein carry-over between ces demotanthe ability to re use hrsin after both leaning methods Example 3 Senaration oflan Ee-containine Protein fom a Re fold Mixtre Using Caion Exehanize Chnnatoeranhv lie tonowing experiments demonstratehat an Fc-eonaining protCi can be separate om a refold ixta comprising glycerol guanidine ur and arginine usng cation exchange media. in one x ermentarecombinantprotein corprisng a biologically active pepdde linked to te C terminus f the F dety of an IG nolecule via a 1inkerand having moledcuar weight of about 57 kDa and comprising disufide bonds was exposed in a non- niammrd[ian expression system namelyfe £ conharvested reolded under private condions, and captured sing cation exchange media, The grottmledia in which the cells are grown was centifuged and the liquid fraction remnvedeaving the cells as a past The cells er suspended in watr The tenis were lysed by meansof multipl passes through a high pressure homogenizerAfter the cels weir lysed tHe lysate was enrifuged to colct the protin which was expressed in a limited solbiity no-native form, namely as inclusion bodies. The protein ay sln !as washed mulple times by euspcnding the y n water miing, and centriNgation to colctthped roti. Tcoprotein was then transferred to sOiIZaion buffer containing guaidine and P71. After incubation forne hour, tOhe protein solution was diluted inato a refold buffer onaining appropriateevelsof arginine. urea. glycerol cysteinead cystaanene In a separate operaon a packed column comprisingIg D Fractoge SO ca exchange resin with dimensions of 1.1cm intemal dameter and 20cm height, was prepared and equilibrated with5 column volumes of 30mM ME2S; p4A 4.5 buffered solution. An aliquot of a protein cornprisng an Fe moiety was sampled directly from a refold solution, was diluted 3-fold with water, titrated with 50% hydrochloric acid to -pH 4.5 and was fihered through a series of depth and/or membrane fiter to remove particulates. The conditioned anflteredproteinmixturewas loaded to appximatedy 096 mniuimolus total protein/i at 60cn/hr Aer loading the column was washed with 30mM MIES; pH 4,5, for 3 CV it 60 cm/hr. thenwashed ith an additional 3 CV of 30nM MESpH 6"0 The protein of rest was recoveredfrom the resinb grade euhnion over25C between (A" NIES: pH 60 and 30mM, IS.500 M NaCI; PH 6.0 at 60 cm/hr Thecollcted protein in the chiton pool Was stored at 2.ItC untihe next purificaton step was carried out. Purtyees acved as determined by SEC and RP-PLC are shown inFigure Following the separation, the resinmedia was leaned place by floing 3 CV of I N sodurn hydroide, at 120 cmnhr and heid r 60 minutes prior an additional 3-V wash with I n sodium hydroxide. The rsuls of this separation demonstrate that an insoluble protein expressed in a non-mam mal ian system can be captured and purified from a refold bufer with a variety of separation matrices, including an ion-exchange separation mais Example 4 Re-usabditv of Protein A Affinity Resin Used to Isolatea Fe-containine Protei Directdy from Refold BRuffer lv init Chromatographv In another aspect of the method, a range of column caning Mehods can b employed in conjuction with the methods described herein, allowing t chromatography resins to be reused to an extent that make the methodeconomically feasible As described in Exmples 2 and 3 fore case of Prtein A affinity ren leaning prtocds have been developed and demonstrated to remove product and non product contruninantsfo hesino allkw reuse. 'The ci caning ientsinclude caustic (cg. sodium or potassium hydroide detergents(g 55 or on-100), deaturants Mg. ura or ganidine-derivatiesN and redutants og T, orho hiweolates These agents can be used in combination or alone. in order to demonstrate the remusabilty of column resins following application of the direct capture methods described, an aliquot of pH adjusted and filtered e-containing protein was loaded on new, unused resin and resin that had been previously cycled 94 31 times to evalae the cleaning of the Protein A resinand the effiet on purfication binding and separation of anFoniuing protein with regard to resin history The mediawas equilbrated with 5 column volume(CV) of25mM rs. 100ma sodiumchkoride; pH 7A, orilir buffered solution An aliquot of protein sarnfled directly from a retbid butr wasitered though a series ofdepth and/or membrane iler to remove particuates. The conditoned and fitcred proteinnAmture was thn loaded on the colum. to approximately 35 milimoles total prteinimL resin ata 640 mine residence tinm See iure ,which correlates protein bound and protein oaded as a function of residence time. Ater loadig, the colun waswashed w ith 25mM Tris, 1OtN0m sodium chlolide; pH7. or imar buff.ed solution, for 4.5 CV at up to 400 cmhr Theproein of interest was recovered tilm the resin by eating with iOmMsodium acetatep 37 a up to 300 cWhr Each column waregenerad using SCV phosphoric acid and 5 CV of an ackiicd n containing 50mM N ON ION citrate. 6M ura.and DT pH 7.4. This procedure was repeated for greater than 100 cycles. Selected samples rm this rouse study were submitted for SEC-HPLC analysis. The goal was to track the %MP purity, % HMW and % dimer species from the pools as well as to understand the change of purity level from the load. No major differences were observed between the used columns and rn colhmus This Example demonstraes that not only can a complex protein be captured front a complex chemical sotion, but that the rsin can be cycled repeatedly and cleaned and reused reproducibly over a number of industriallvrelevant cycdes.

Claims (16)

1. A method of purfying a protein expressed in a nonnative soluble form in a nonwmamnmlian expr ession system comprising: (a) losing a non-mannmalan cell in which the protein is expressed in a non native sohble form to generate a celi lysate: t) contacting the cell lysate wit an separation mnatrix under conditions suitable for the protein to associate with the separation matrix; (c) washing the separation matrix; and i ciuting the protein front he eparation matrix, 2, The method of claim wherein the protein is a complex protein. 3 The method of claim 2, wherein the complex protein is selected from 0 c group consisting of a nultirneric protein, an antibody and an Fe fusion protein.

4. The mehod of claim wherein the nonmmamnmalian expression system conpriss baceia or yeas cells.

5. The method of claim L. wherein the separation matrix is an affnity resin selected frum the group consisting of Protein A, Protein G and a synthetic mimetic affinity resin, 6 The method of claim 1 wherein the separation matrix is a non-afniy resin selected from the group consisting of ion exchange mixed mone, and a hydrophobic interactiorresin

7. The mehod of Clain . wherein he cell lyie is 4iiered before is ontacd withthe separatdon matrix.

8. Theinethod of daim I furdhi comprising refoding the protein to is nativefm afte it is eluted. 9, A method of purifying a protein exprssed in a non-native limited solubility form in a non-mammalian expression system comprising: (a) expressing a protein i a non-native limited sohubiibty form im a non mammal ian cell; (b) ysi6ng a non-mammalian cell (e solubilizing the expressed protem in a solubilization solution comprising one or more of the following: I) a denatuant, i a reduetant; and na tuaant (d) forming, a reold solution eompnsig the solubiizatiom solution and a refold buffer, the refld buffer compring one or more of the follow ing: (il a denaturnt; (i) an aggregation suppressor; ii a proein stabilizer; and ina redox component; (e) applying the refokd solutiontoa separation iatrix tinder condition suitale for the protein to associate with the matrix: ) shingthe separation matri; and ( u) eh1tinga protein fromn the separaion matdx

10. The method of cAS 9 wherein the no-natinve hined sohility fobr is a component of an inclusion body, 1 L. The method of claim 9. wherein the protein is a complex protein, 34 I I The method of claim 9, Wherein the complex protein is selected from the group consisting of a ultimeric protein, an antibody, a peptibody, and an Fe fusion protein. 12 NThe method of claint 9) wherein the nonmammalmi expression system v bacteria or yeast cells 114 The method of claim 9. wherein the denaturant comprises one or more of urea, guanidinium saits. dimetwhy urea, methylurea and ethylurea. 5 The method of claim 9. wherein the reductant comprises one or mon of cysteinc. DTT, beta-mercaptoethanoi and gltathione.

16. The method of claim 9, wherein the surfttant comprises one or more of sareos. and sodium dodeeisul fate,

17. The method of claim 9 wherein the aggregation suppressor is seleted front thego insisting of argnne, prone polyethylene glycols non ionic surfactantsionic surfaeants polyhydric alcohol, glycerol sucros, sorbitoL glucose Tri. sodim slfate potasinulfte anolytes. Is, The method of claim 9 where the protein stabilizr compose one or more of arginine, pline. polyehylene lycol non ionic surfacants ionic surfactants. poyghydic alohols gycer, sucose, sorbtl, glucose. rissodun sufdepotassan sulfate and osmolytes.

19. The method of claim 9, wherein the redox component comprises one or more of glutathione-reduced, giutatione-oxidized, cystemne, cystine, cytstea&imiicytstaridne and beta-nmercaptothanol.

20. The method of clAim 9, wherein the separation matrix is an aMity resin, seected front the group consisting of Protein A, Protein G and synthetic mimetic affinity resin. 2 1. The method of claim 9, wherein the separation matrix is a non-affnity resin Selected from the group consisting of ion exchange, mixed mode, and a hydrophobic interaction resin.

22. The method of claims I or 9, further comprising the steps of (a) washing the separation matrix with a regeneration reagent; and bIS regnerating the searation matri.

23. The method of claim 22, wherein th.e regeneration reagent is one of a strong base or a strong aid. 24, The method of claim 23, wherein the strong acid is phosphoric acid.

25. The mthod of ain 23 wheein the strngage is sodium hydroxide.

26. The method of claim 22, wherein the regenerating comprises washing the separation matrix with a solution comprising one or both of a chaotrope present at a concentration of 46 M4 and a reluctant

27. The metod ofaim26 wherein the chaotope is one of urea ditnethyl ureaethhj~ira; ethylurcaand guandinam. 2$. 'The method of claim 26, wherein the reductant is one of cysteine, DIT, betaamercaptoethanol and ghuathione

29.Ih mhod o cai 22 wcrinthereenraingco~piss wshngth