c») United States cz) Reissued Patent Shadle et al. (34) ANTIBODY PURIFICATION (75) Inventors: Paula Shadle, Lafayette, CA (US): John C. Erickson, Research Triangle Park, NC (US); Robert G. Seott, King of Prassia, PA (US); Thomas M. Smith, King of Prussia, PA (US) (73) Assignce: SmithKline Beecham Corporation, Philadelphia, PA (US) (21) Appl. No. 11/140,825 (22) Filed: May 27, 2005 Related U.S. Patent Documents Reissue of (64) Patent Nox 5,429,746 Issued Tal 4, 1995 Appl. No 08/200,126 Filed: Feb. 22, 1994 (51) Inc BOLD 1508 (2006.01) BID 1532 (2006.01), COTK 1600 (2006.01) cork 1/00 2006.01) us. 210/635; 210656; 5300387.1: '530/300.5; 5301413; 530/417 (68) Field of Classification Search 21011982, 210V635, 656; 530/387.1, 390.5, 413, 417 See application file for compete search history. 66) References Cited USS. PATENT DOCUMENTS. 4629513 A ¢ 1/1987 Hou eta. s30°3905 458.722 A © 1/1991 Bloom et a S30 3871 SMISIOL A * $1992. Bloom et 53038825 5122373 A * 61992 ible a ‘241771 SuosasT A * 111992 Kothe tal 530380 1 S190952 A © 31903 Moller ta M761 S210 99 A © G1003 Stak ea 3033905 al 5268306 A © 121903 Borger etl SSri700 A © 11/1906 Grande etal (10) Patent Number: 4s) Date of Reissued Patent: USOORESOO70E US RE40,070 E Feb. 19, 2008 FOREIGN PATENT DOCUMENTS w A2084193 ——-¥/1990 » AS01152 2/1904 Wo woorm00 © DI9T wo wooNo2I0s 21993 (OTHER PUBLICATIONS Whatman web article (Mar: 2007). Hakala, etal, “Purification of monoclonal antibodies ‘ised against prostate acid phosphatase for use in vivo in rrdioimaging of prostaie cancer” Journal of Inmunologt fal Methods (1989) 117: 131-136, ‘be, ct al, "Purifcation of monoclonal antibodies with light-chain heterogencity produced by mouse hybridomas rised with NS-I-myelomas: application of bydrophobi interaction high-performance guid chromotography, Journal of Biochemical and Biophysical Methods (1993) 27:215-227, Danielsson, eal “One step purification of monoclonal laG antibodies from mouse ascites," Journal of Immunological Methods (1988) 115:79-8. * cited by examiner Primary Examiner—lohn Kin (74) Attorney, Agent, or Firm Andrea V. Lockeoour award R. Ginnm on ABSTRACT This invention relates to the application of hydrophobic interaction chromatography combination chromatography the purification of antibody molecule proteins. REEXAMINATION RESULTS The questions raised in reexamination request, 091006,966 filed Mat. 12, 2004 have boen considered and the results thereof are reflected inthis reissue patent which constitutes the reexamination certificate required by 35 U.S.C. 307 as provided in 37 CFR 1.5706), for ex parte reesaminations, or the reexamination cerificate required by 35 USC. 316 as provided in 37 CER 1.997(e) for inter partes reexanina 31 Claims, 1 Drawing SheetU.S. Patent Feb. 19, 2008 US RE40,070 E Conditioned Media Protein A Affinity Chromatography 1 1 I Viral Inactivation #1 (pH 3.5) \ 1 ! Cation Exchange Chromatography 1 ' 1 Viral Inactivation #2 (guanidine) 1 ' ' Hydrophobic Interaction Chromatography ' 1 1 Concentration and Diafiltration 0.2 micron Filtration ' ' 1 Purified RSHZ-19 for Further Manufacture FIGURE 1US RE40,070 E 1 ANTIBODY PURIFICATION Matter cnelosed in heavy brackets [ ] appears in the original patent but forms no part of this reissue specifi ‘cations matter printed in italics indicates the additions made by reissue. More than one reissue application has been filed forthe reissue of Pat. No. 5,429,746. The reissue applications are “application Ser No. 11/140,525 (the present reissue) and 11/804,729 which isa continuation of the reissue applica- tion Ser No. 11/140,325 FIELD OF THE INVENTION ‘Tis invention relates to the Hild of protein purification More specifically, this invention relates to the application of Hydrophobic Intention Chromatography (HIC) to the separation of Immunoglobulin G monomers and t0 the integration of TIC into a combination chromatographic protocol fr the putification of TgG antibody molectles BACKGROUND OF THE INVENTION Historically, protein purification schemes have been predicated on diferences inthe molecular properties of size, ‘charge and solubility between the protein tobe purified and undesired protein contaminants. Protocols based on these parameters include size exclusion chromatography, ion ‘exchange chromatography, differential precipitation and the like. Size exelusion chromatography, otherwise known as gel ‘ration oF gel permeation chromatography, relies on the penetration of macromolecules in a mobile phase into the pores of stationary phase particles. Differential penetration {s a function of the hydrodynamic volume ofthe particles. Accordingly, under ideal conditions the larger molecules ee ‘excluded from the interior of the particles while the smaller molecules are accessible to this volume aad the onder of ‘elution ean be predict by the sizeof the protsin because 3 Tincar relationship exists between elution volume and the log ‘of the molecular wight. Chromatographie supposts based on crosslinked dextrans e.g. SEPHADEX®, spherical agarose beads e.g SEPHAROSE® (both commercially available from Phar= macia AB. Uppsala, Sweden), based on crosslinked poly- sctylamides eg. BIO-GEL ® (commercially available from BioRad Laboratories, Richmond, Cali.) or based on elhyl- ‘ene glycol-methaerylate copolymer e.g. TOYOPEARL Wes (commercially availabe from Toso Laas Co, Tokyo, Japan) are useful in forming the various chromatographic ‘columas for size exclusion, or HIC chromatography in the practice of certain aspects ofthis invention. Precipitation methods are predicated on the fact that ia cenide mixtures of proteins the solubilities of individual proteins are likely to vary widely. Although the solubility of f protein in an aqueous medium depends on a variety of factors, for purposes of this discussion it can be said enerally that a protein willbe soluble if is interaction with the solvent is stronger than its interaction with prot ‘molecules ofthe same or similar kind. Without wishing to be bound by any particular mechanistic theory describing pre- cipitation phenomena, i is nonetheless believed that inter- ‘action herwcen a protein and water molecules can occur by hydrogen bonding with several types of uncharged groups andor eleerostatcally, as dipoles, with charged groups and that precipitants such as salts of monovalent cations (eg. unoaium sulfate) compete with proteins for water mol. 0 o 2 ecules. Thus at high salt concentrations, the proteins becon “dehydrated” reducing their interaction with the aqueous enviroament and increasing the aggregation with like or similar proteins, resulting in precipitation from the medium. Ton exchange chromatography involves the interaction of | charged functional groups in the sample with jonie fune- tional groups of opposite charge on an adsorbent surface Two general types of interaction are known. Anionic exchange chromatography is mediated by negatively charged amino acid side chains (eg, aspanic acid and slutamic acid) interacting with positively charged surfaces And cationic exchange chromatography’ is mediated by pos tively charged amino aid residues (ex. lysine and aginine) interacting with negatively charged surfaces. ‘More recently affinity chromatography’ and hydrophobic interaction chromatography techniques have been developed to supplement the more waditional size exclusion and ion cexchange-chromatographie protocols. linity chromatogra- phy relies on the specific interaction of the protein with an immobilized Iigand. The ligand can be specific for the particular protein of interest in which case the ligand is @ Substrate, substrate analog, inhibitor, receptor or antibody. ‘lteratively, the ligand may be able to ret with a number ‘of related proveins. Sul group specific ligands as adenosine ‘monophosphate, adenosine diphosphate, nicotine adenine inveleotide or certain dyes may be employed to recover a particular class of proteins. With respect to the purification of antibody molecules, both specific and generalized affinity teclnigues are app cable. The most specific choice of ligand for the allinty purilication of an antibody is the antigen (or an epitope thereo!) to which desited antibody reacts. Many of the well-known immunosorbent assays such as the enzyme- Jinked immunosorbent assays (FLISA) are prediceted on sch specific antigen/antibody'aiiniy interactions However, generalized afinity techniques are also useful Por example, Staphylococcal Protein A is known to bind ies of the IgG class (See: Ey, P-L. etal Tmunochemistry 15:429-36 (1978) Alternatively, ani era raised in heterologous species (e.g. rabbit anti-mouse antisera) can be used fo separate general groups of antibod- ies, (See, Current Protoools ia Molecule Biology Supr Chap 11.) Hydrophobic interuction chromatography was frst devel- ‘oped following. the observation that proteins could be igined on alfinty gels which comprised. hydrocarbon spacer arms but lacked the alinity ligand, Although in this field the term hydrophobic chromatography is sometimes used, the tem hydrophobic interaction chromatography (HIC) is prefered because it i the interaction between the solute anc the gel that is hydrophobic not the chromato graphic procedure. Hydrophobic interactions are strongest at high ionic strength, therefore, this form of separation is conveniently performed following salt prcipitations or ion fexchange procedures. Piston from HIC supports ean be effected by alterations in solvent, pH, ionie strength, or by the addition of chaotropie agents or organic mosifers, such as ethylene or propylene glycol. description of the peneral principles of hydrophobic interaction chromatography can be fond in U.S, Pat, No, 3,917,527 and in US. Pat. No. 4,000,008. The application of HIC 19 the purification of specific proteins is exemplified by reference to the following disclosures: human growth hannone (U.S. Pat. No. 4,332, 717), toxin conjugates (US. Pat. No. 4,771,128), ant hemolytic factor (US. Pat. No. 4,743,680), tumor necrosis factor (US. Pat. No. 894,439), interleukin-2(U.S. Pat. No.US RE40,070 E 3 4.908.434), human lymphotoxin (USS, Pat, No. 4,920,196) and lysozyme species (Fausnaugh, J. and FE. Regnier J ‘Chromatog. 359:131_ 146 (1986) and soluble complement receptors (US. Pat. No, $,252,216). HIC in the context of high performance liquid chromatography (HPLC) has beens ‘sed to separate antibody fragmens (eg, F(ab), fom intact aniibody molecules ina singe step protocol. (Morimoto, K. ‘tal. J. Biochem. Biophys. Meth. 24:107-117 (1992) Inaddition to ality and FIC techniques, one or more of the traditional protein purification schemes have been applied to antibody purification. For example, Hakala, L. ‘et a, J. Immunol. Meth. 117:131-136 (1989) disclose a protocol employing two successive jon exchange chromato- tziphie sleps or one employing a single ion exehange step followed by a HIC step. Danielsson A. etal. Immunol. 1S Methods 115:79.88 (1988)) compare single step provocols based on anion exchange, cation exchange, ehromatofoeus- ing and HIC respectively. ‘Although Protein \ affinity column chromatography is ‘widely used, itis also appreciated that elution of antibody fom such columns can result in leaching of residual Protein AA from the suppor. Size exclusion HPLC (Das et al, Analytical Biochem, 145:27-36 (1985) and anion exchange ‘chromatography (EPO345S49, published Dee. 13, 1989) have been suggested as means for dealing with this problem. has now been surprisingly discovered that 11IC ean be usefully employed to remove contaminating Protein A from IgG mixtures eluted from Protein A chromatographic sup- por. This invention relates tothe application of HIC to the Separation of monomerie IgG from mixtures containing same and othe integration of HIC into a protocol combining Projein A and ion exchange chrometography’ forthe pu ceation of immunoglobulin G molecules. BRIEF DESCRIPTION OF THE INVENTION ‘This invention relates to @ method for separating IgG ‘monomers fom aggregates in mixtures containing same hy ‘contacting said mixture with a hydrophobic interaction ‘chromatographic suppor and seletively eluting the mono- ‘mer from the support. In another aspect the invention provides forthe purifica tion of an IgG antibody fom conditioned cell culture ‘mim containing same comprising sequentially subjecting the medium to (a) Protein A, (b) ion exchange ‘chromatography, and (c) hydrophobic interaction chrom ‘ogra, In another aspect the invention provides @ method for removing Protein A from a mixture comprising Protein A tnd antibodies comprising contacting said mixture with @ hydrophobic interaction chromatography support and selec- tively eluting the antibody from the suppor. DETAILED DESCRIPTION OF THE FIGURES FIG. 1 illustrates a flow diagram of one process for purifying an antibody aecording to this invention DETAILED DESCRIPTION OF THE INVENTION wo ‘This invention relates (© proven purification techniques ‘which have application to the large scale purification of immunoglobulin molecules. The inveotion 4s particularly tsefl because it permits the recovery of monomeric 1aG of 395% protein purty. The invention may be applied to the Purification of a number of different immunoglobulin G molecules 4 Antibody ike proteins are proteins which may be purified by the protocol described herein, such protocol being med tied if necessary by routine, non-inventve adjustments that ddo not ental undue experimentation. Such proteins include isotypes. allotypes and alleles of immunoglobulin genes, truncated forms, altered antibodies, such as chimeric antibodies, humanized antibodies and the like, chemically ‘modified forms such as by PEG treatment, and fision proteins containing an immunoglobulin moiety. These pro- ‘eins are referred to as antibody-like because they possessor ‘tain sullcient immunoglobulin protein properties ex. F determinants) to admit t© purification by the process of this invention. Unless specifically identified otherwise, the term Antibody or immunoglobulin protein als includes antibody like proteins. ‘The immunoglobulin molecules of this invention ean be isolated from 1 number of sources, including. without imitation, serum of immunized animals, ascites ud, hybri- doma or myeloma supematans, conditioned media derived from culturing a recombinant cell line that expresses the immunoglobulin molecule and from all cell extracts of {immunoglobulin producing cells. This invention i partiew- ‘ioned cell culture media of a variety of antibody producing recombinant cell lines. Although one may expect some variation from call fine to call line and among the various ly produets, based on the disclosure herein, iis well the purview of one of ornary’ skill in this an to adapt the invention herein toa particular combination of antibody protein and producing cel line Generally, genes encoding proweins such as antibodies may be cloned by incorporating DNA sequenees coding for the desired regions of the polypeptide into @ recombinant DNA vehicle (eg, vector) and transforming or transfecting suitable prokaryotic of eukaryotie host. Suitable prokary- ‘otic hosts include but are not limited to Escherichi Steptomyees, Bacillus and the like. Suitable eukaryot hosts include but are not limited fo yeast, such as Sacchi omyees snd animal eels in culture such 3s VERO, HeLa, mouse C127, Chinese hamster ovary (CHO), WI-38, BHK, COS, MDCK. myeloma, and insect cell lines. Particularly preferrad hosts are CHO eel ines deficient in dihydrofolate rednetase such #s AFCC CRE. 1793, CRI. 9096 and other cell lines described herein below. Such recombinant techniques have now beoome well known and are described in Methods in Enzymology; (Academie Press) Volumes 65 and 69 (1979), 100 and 101 (1983), and the references cited therein. ‘An extensive technival discussion embodying most com: ‘monly used recombinant DNA methodologies ean be found in Maniatis, etal, Molecular Cloning Cold Spring Harbor Laboratory’ (1982) or Current Protocols in Molecular Biology, Greene Publishing, Wiley Interscience (1988, 1991 1993), ‘One way of obtsining a DNA fragment eneoding a desired polypeptide such as an antibody: molecule is via eDNA. loning. In this process, messenger RNA (mRNA) is isolated from cells known or suspected of producing the desired protein. Through a series of enzymatic reactions, the mRNA. population ofthe cells i copied into a complementary DNA. (@DNA). The resulting eDNA is then inserted into cloning vehicles and subsequently used 10 transform a suitable prokaryotic or eukaryotic host. The resulting ‘duced oF cultured. For example » chimeric monoclonal antibody to CDA was also purified by the process of this, The purified antibodies obtained by practicing the process of this invention have the following properties: 1) arester than 97% antibody protein by weight; 2) stable to provelytic degradation at 4°. for at feast three months; 3) low (<0.1 F.U/mg protein) endotoxin; 4) Tow ( TSH A mynd ligrams protein per milliter. This yaration used the same ProSep A eluate as Tablet summaries he columa peramcies fr hs gg IMR_prnion wel th ame Pusey Alu ‘example. The product and protein recovery data for each — Scaled-up to accommodate approximately 40 grams af step are shown in Table 5, along with the Procin A content, pyiein wing TOYOPEARL. Phenyl ss0M. a the HIC ‘xpretsed as nanograms Protein A per milligram IgG (og) 25 fedur. The preparation ofthe CM SEPHAROSE Load i tmp) Altboogh the recovery of gC i lower coupared to dasribed in Example 1A above. 78 liters of pH 3.5 teed Example 18.7% 94%) the rocin A content is rediced an fire ProSepA elite were load dtl ono a 42 sppeokimtely 20-old using Bul 680M as an AIC step. ler QS em diameters8.S om length) column of CM cae Using TABLE 4 (una Pane a eau LN Shine i ep Loa bow Bass 0 (icy (en) _talo (cut) (aio Guserinnose 022 4x18 ou ggeainpe 5 sta ee Dayan oo 32030 Woagpmetner 0 2 tha Soe SEPHAROSE FF which had heen previously equilib TABLE 5 ‘with CM Equitation bile at 1-2 Uni. Afr loading the * column was washed at 1.2 L/min with approximately 8 liters ‘aan Say fr Ee FCM Fauiibaton Buller The IgG was luted by applying ae CCM Floion Buffer at 1.2 Umin. The IgG came off the column after approximately [hed volume of Baton Buller tt atom had passed. ‘The entre peak was. collected. as CM oume RHZ Tl, Sep Prin sp SEPHAROSE chante: The column fiagtons of the CM ‘non-bound and eluate were collected and analyzed for IgG content, toa protein content, and Prowein A content as Coe a rs described previously. The eluate was approximately. §.7 sen (ites) (Grams) Grime) 60) a) a a 7 + ayn its it volume, and contained approximately 6-7 mil resp A ems moe 98302 gg grams pon pr milter, re ee "To 345 ftom of CM SEPHAROSE cluste was added ROSE! Loe (slowly with constant stirring) total of 28 lites of Guani- CASEI 016 201410148 ne Stock Solution (3.2 kilogram by weiaht). This brovaht ose ete Co the guanidine concentration to 2M for vital inatvation, at Beso xt Ort a6) 4 a volume of 83 liters. While sting the suaniine-teated Lea ot oay on ome ay ag SOMO, total OF 83 Titers (6.7 ey weigh) of 26M Eee g Ammonium Sulfate Stock Solution was ads The resing an oa on) om aap Soon was TOM in guanidine and 13M in ammonium forts bss aD ‘ie sulfite, with » final volume of 16.7 fiers. The ammonim Comuave 6 sulfite treated solution was applied toa 46 liter column (8 nevey ém diameter em length) of TOYOPEARL Pheay-680M, previously equilibrated with Phenyl Equilibration BulerUS RE40,070 E 17 The flow rate was 0.5-066 L/min throughout the run, Afier Joading, the column was washed with approximately 14 Jiters of Phenyl Equlibrtion Buffer. The IgG was eluted by spplying a linear gradient starting st 100% Equilbration ulfer and ending at 100% Gradient buffer, in 20 column volumes. This represents a starting ammonium slate con- ‘centration of approximately I-3M and an ending concentra tion of OM. The slope ofthis gradient was approximately & 5% increase in elution bufler per column volume, ‘or =0.065M ammonium sulfate per column volume. The IgG ‘began eluting from the eolunin at approximately 7 column volumes and ended at approximately 12 column volumes Py 64M oo 18 Table 6 summarizes the column parameters for this ‘example, The produet and protein recovery data for each sep are shown in Table 7, along with the Protein A content, expressed as nanograms Provein A per milligram IgG (ng! ‘mg) and the IgG aggregate content, expressed as % of total IgG. As seen in Table 7, the Protein A. reduction over Pheny!-650M is approximately 3-fold, and the recovery is approximately 902%, IgG aggregates were reduced from (0.5% in the CM SEPHAROSE eluate to 0.06% in the ormulated product ABLE 6 (Gime Pane a ga imei neh Lond How Ras (ite) te) Baio (ene) i) 2 SSKES Sepmenpe IS liebe vohine 46 Wx U0 gpmempe 406 ther fe wohe TABLE 7 —Puesion Summary for eam) gm se Teal Toul, Sep ke ome RSHZI* Prot VeK Proein AY Agsepte {liters _"iGaune) (Gone) ‘test O) "Ay Abo ot 200 om © 127 emg at “Only pin of he tt Psep A Hate was cared orard “Prt Aa Ta agen pate prin eth a raion into the gradient (ammonium sulfate concentration of approximately 0.85 to 05M), The eluate fraction was eol- lected until the UV absorbance on the tailing side ofthe peak decreased 10 20% of the peak height, then collection was switched to another vessel (tail). Fractions of the Phenyl non-bound, eluate and til and strip fractions were collected and analyzed for IgG content, total protein content, and Protein A content as described previously. The eluste was approximately 15.4 L in volume, and contained epproxi- mately 2.2 milligrams protein per milter “The Phenyl Eluate was concentrated to approximately 16 mg/ml, using a tangential low ultrafiltration apparatus (CUP, Millipore Corp.) equipped with 30,000 MWCO ‘Omega membranes (Filton Carp.) and buffer exchanged by ‘continuous diafiltration against suitable formulation butler o Example ID. RSHZ-19 Purification at 125 Gram Seale Using TOYOPEARL Phenyl-650M ‘A'5.5 liter (20 em diameter by 18 em length) ProSep A afinity column was equilibrated with PBS (see Table 1) at 48 liteeimin, 430 liters of conditioned culture medinm containing 094 grams per liter of RSHZ-19 monoclonal antibody Was clarfed by mierofiltration as described above, fd applied in four separate 90-95 liter portions and one Titer porn wo the column ata flow rate of 4.8 iter min (and so throughout), Each eyele on the columa ran as follows: After the load, approximately 17 liters of PBS/glyeine was ‘applied tothe column atthe same flow rate. The IgG was led by applying 15-20 lites of ProSep A Elution buller. Fractions ofthe non-bound peak and the elution peak were collected and assayed for IgG content using a HPLC assay. The eluate from each evele was approximately 9 Titers inUS RE40,070 E 19 volume, and contained approximately $-10 milligrams pro- tein per milliliter. Immediately after elution, the ProSep A eluates were adjusted to pH 3.5 by the addition of 2.5M. hydrochloric acid, held for approximately 30 minutes, and adjusted to pH 5.5 by the addition of approximately 250 nilites of IM Tris base. Aer nutralizing to pH 5.5, the ‘tustes were pooled together, and filtered through a 0.1 micron Polygard CR filter in tandem with 3 sterile 0.2 :ieton Millipak 200, in 5 Iter aliquots in sterile containers. “The filtrate was stored at 4° C, Samples ofthe filrate were anilyzed for IyG content using an HPLC assay, and for total protein by absorbance at 280 nanometers. The samples were ‘also analyzed for Proven A coatent by an ELISA proce, ‘and 1gG aggregates by HPLC. The downstteam steps were scaled-up to accommodate approximately 120-140 grams of protein, 1633 liters of pl 35 treated and filtered ProSep 4 eluate containing approx mately 130 grams of protein was loaded directly ontoa 44 Titer @S em diameter em length) column of CM SEPHAROSE EP at 24 Limin, which had been previously ‘equilibrated with CM Pauilibration buffer. ARer loading, the ‘colluma was washed at 2.4 L/min with approximately 45 liters of CM Faqilibration Bfer The IgG was eluted by “applying CM Elton Buster st 2.4 Lin, The IgG began to ‘elute from the column after approximately 1-2 bed volumes ‘of Elution Buffer had passed. The entire peak was collected a CM SEPHAROSE eluate. Fractions of the CM non- bound and eluate were collected and analyzed for TaG ‘content, ttal protein content, and IgG aggregate, The este ‘was approximately 21 Titers in vohime, and contained approximately 120 grams protein To 1944 liter: of CM SEPHAROSE eluate was added (slowly with constant stirring) a total of 9.7 liters of Guan ‘ine Stock Solution. This broveht the gusnidine concent tion to 2M for viral inactivation, ata volume of 29.1 liters While stirring the guanidine-teated solution, total of 29.1 liters of 2.6M Ammonium Sulfate Stock Solution was ‘added. The resulting solution was 1.0M in guanidine and 13M in ammonium sulfate, with a final volume of $8.2 Jiters, The ammonium sulfate treated solution was applied 10 1244 Titer column (30 em diameterx18 cm length) of 20 TOYOPEARL Phenyl-650M, previously equilibrated with Phenyl Equilibration Buller. The flow rate was 11-13 Lia. throughout the run, After loading, the column was washed with approximately 37 liters of Phenyl Equilbration Buller. The IgG was eluted by applying a linear gradient starting at 80% Pquiibration Buler20% Gradient Rue and ending at 20% Equilibration Buffer/80% Gradient buffer, in 12 column volumes. This represents a starting ‘ammonium sulfate concentration of approximately. 1.0M and an ending concentration of approximately 0.26M. The slope of this gradient was approximately a 5% increase in Gradient bulle per column volume, or =0.085M ammoninm sulfate per column volume, The IgG came off the columa essentially in the middle of the gradient, with the peak containing opproximately OSM ammonite sulfate, The eluate fraction was collected until the UV absorbance on the tailing side of de peak decreased to 20% ofthe peak height, then eolletion was switched to another vessel (al), Fine 1s of the Phenyl non-bound, eluate and tail and strip {actions were collected and analyzed for IgG content, foal protein content, and IgG agareyate. The eluate was appeoxi- mately 29 L in volume, and contained approximately 100 rams protein, The Phenyl Eluste was concentrated to approximately 10 ‘mg/mL. using @ tangential flow ultrafiltration apparatus (CUF, Millipore Corp.) equipped with 30,000 MWCO ‘Omega membranes (Filton Corp) and butler exchanged by continuous diafleation against a suitable formulation bute ‘The prodict was analyzed for IgG content, total protein, Protein A and IgG aggregate Table 8 summarizes the column parameters for this ‘example. The product and protein recovery data for each sep are shown in Table 9, along with the Protein A content, expressed as nanograms Protein A per milligram IgG (ng! ‘mg) and the TgG aguregate content, expressed as % offal IgG. As seen in Table 9, the Protein A reduetion over CM SEPHAROSE and Phenyl-650M is approximately 7-fld, fad the cumulative recovery is approximately 70%, IgG ‘aggregates were reduced from 0.4% in the CM SEPIIAROSE clite to 0.06% in the formulated proc, TABLE 8 lune Parr a 3S gan Votine i log Land ow Rate hey (em) _ Rao (exit (Ua) perlite bet one TABLE 9 Zunfaon Sums fr anol ID 28 mae Teal Toul, Sep ke Whine RSHZ-I* Pree Ye) Pein A” Agaeate Lom) Gras) (Grams) ()_ asi o,US RE40,070 E 2 2 TABLE 9-continned oane RSHZIN Pasi Yd Pein AY Agente sen (tes) (Grams) (Game) 05) gm) 00) CEM SEPHAROSE* assy css Let ‘Cumisive Resse » “ty Renee HPLC Yay Abotence at 280 am © 27 ag! cn! “ty Bead ay “Downstream poses apacy& apoxiatly 140 grams cly prion of thet Prep {650M resin (lot #65PHMOI H), The flow rate was mine ‘TABLE 10 sy ined at 2 mlinin fr all steps. The corn was oi ee rated with 20 mf. Equation aller (IM smmonion ee ‘sulfate, 50 mM sodium phosphate, pH 7.0), The product was Ageresse Pig’ Avis! prare fr oding on the column by taking $m partly so Ro, csottenl amy Purfied CHACDS monoclonal stbody (17 mm concen- cer Teramceie aa ee se 56 tration by absorbance at 280 nny, adding 25 mL 6M eee es ie MS guanidine HT, $0 mM sodium phosphate, pH 7.9, mixing ee ras ed "3 holding for 30 mists, al hen ang 78 mil. 2M ammo- Fede ie oF 105s slits, $0 aM socio phospbst, pt 7.0. The fl EGRET wy Ammonium sulfite eonsentation ofthe fea was IM, the eee oun o : final avanidine HC concentration was TM, the inl volume Sista le iy eee Sve HS Wns ssf mpl wae taken wot 12 mL, and the final concen FERRIED LERSTS RATT FAR io nsz-i9 oom wation of product was 5.6 mg/ml. This material was then sono HC Jean th column at 2 mln an then cht with 10 4s column volume linear gradient of Bguiibraton bute o $0 SM sodium phospinte, p70, Fractions of appoints EXAMPLE I 4m were ken asthe prot etd fom the column ‘The resus are shown in Table 1. Al of the prod seventy elt in the gradient. Protein A aso uted and ‘The sni-C4 monoclonal antibody CH-CDI4 wos made as eid a aor tons foe radon To achieve by cal culture tesniqus and partially prfedvsing Pro- _nedaton in Protein inthe final prod it was necessary tein and ion exchange cromstograpy’inafshionsinilar__ to exclde some ofthe factions at the end ofthe gradint, to thot used in Example I except that an anion exchange thus ruin the yield of prsuc. A ewo-fld reduction in resin was employed. An Amicon column (-em dameer hy Protein A was possible with an 86% ld of product an 0 1Wcm high) as packed with mLof Phenyl TOVOPEARL thre-foldrodvton was possible wih a S0% ye TABLE 1 Se oem) tet) opme ad Far erUS RE40,070 E 23 2 TABLE L-continned Cusine Frasion Volume Probate Pr A Pdit Rema Nol) mgmt) owl) om Med Fac “Remonl forecasted Wy poli ease Tacos fen sat of eho dei {nce an ding tal Pl Aaa by Py Ang poe hate fac ‘om, For vamp the fctor alcatel ty dv he am of Pein. a> aoe he mmo poi i ig -3 0 ge ogg Ths mer he ive by 39 magi he eo a EXAMPLE IIB: CH-CD4 monoclonal antibody was partially purified, and prepared for loading on the HIC columa as described ia ‘example IA. The same columa, flowrate, equilibration and yy Toading described in example TA were used. In this ‘example, after the column was loaded and washed with Equilibration butler, it was washed with 18 ml. Wash buller (IM ammonium sulfate, SO mM sodium cite, pl 3.5). “This was followed by another washing with 13.5 mL. Equil bration buffer (deserbed in example I). The column was ‘luted with « gradient and then washed with water as described in example ILA. The eolumn eluate was divided into 14 mL trations which were then analyzed for product _4y and Protein A. ‘The results are presented in ‘Table 12. Protein A could be reduced by 6 fold 0% yield, and by 8 fold at 78% yield. EXAMPLE 1IC Tie example was performed similar to example IB, ‘except thatthe scale was increased. Equilbration and Wash bullers are deseribed in example IIB, The colun ws 5 diameter and 28 em high and the flowrate was 50 amin, CH-CD4 monoclonal antibody was prepared and partially purified, as deseribed in example TAA. The partially purified product (440 mL.) was mixed with 220 ml. 6M guanidine HCI for 31 min. Then, 660 mL 2M ammonium sulfate, 30 ‘iM sodium phosphate, pl 7.0 was added. The final wamo- ‘ium sulfare concentration was IM and the final antibody concentration was 5.3 mgiml. Aer sampling, the load volume was 1,290 mi ‘The column was equilibrated with 2 cotamin volumes of [Equilibrio buller snd then loaded on the eoluma. The column was then washed sith 630 ml. of Pquilbration buffer, 1,000 mf. of Wash buffer, 800 ml. of Equilbration buffer, and then eluted with a § column volume gradient TABLE 12 ‘Specie Cmubtive Pain A Freton Volume Podut Praia Pci “Prat = Renov Comparing the results of example TB with example TA it can be seen that Protein A was reduced by 6 08 fold with about 80% yield when the pH 3.5 wash was included, but reduction was only about 2 fold with 80% yield of CH-CDH ‘without the pH 3.5 was. from 0.75M ammonium sulfate, $0 mM sodium phosphate, 1pH70, to 50mM sodium phosphate, pH 7.0. Fractions were collected during elution, ‘The results are presented in Table 13. Protein A. was seduced 100 fold with a yield of 70%, or by 30 fold with an antibody yield of 80%.US RE40,070 E 25 TABLE 13, Cusine Frution Volume Frat Proeina ean A Prt, Now ml) pray aw ow Md EXAMPLE ID Partially purified CH-CD4 monoclonal atibody was pre= pared as shown in example TIA. Equilibration and Wash bles are described in example I1B. Four milliliters of 6M guanidine HC, SO mM sodium phosphate, pH 7.0 was added to 8 ml of a 95 mpl solution of partially purified ‘CH-CD# monoclonal antibody and incubated for 30 min- utes. Then, 12 mL of 2M ammonium sulfate, 50M sodium phosphate, pl 7.0 was added slowly. The column was 0:5 ‘em in diameter and 20 em high (4 mL.) and the flowrate for fall stops was 0-5 miLmin, The colnmn was rinsed with 2 column volumes each of water and Fquiibration buffer. ‘Then, 22 mL of the load solution was passod through the columa, followed by 2 column volumes of Equilibration bull, followed by Wash buffer until the pH of the eoluma ‘ellluent was 3.5. This was followed by Eguiibraion bute ‘until the elluent pH was 7.0, The column was eluted with ‘03M ammonium sulfate, $0 mM sodium phosphate, pH 7.0. Aer the UV trace started t0 rise, 126 mL of eluate were collected and analyzed for prove and Protein A. The yield ‘of product was 80% andthe Protein A was reduced from 28 ngimg to 6 ng/mg, «reduction of 47 fold, ‘What is claimed is: [1. A method for purifying monomeric IgG antibody from ‘a mixture comprising sid monomeric antibody and atleast ‘one of immungglobulin aggregates, misfolded species, host cell protein or protein A comprising contacting said mixture ‘witha hydrophobic interaction chromatographic support and Selectively eluting the monomer from the support 2, [The method according to claim 1] 4 method for purfsing monomeric 1gG antibody from a mixture compris Ing said monomeric 1G antibody and at Teast one of immunoglobulin aggregates, misfolded species, aed protein 4 wherein said method comprises the steps of (i) contact. ing said mixture with a hydrophobic interaction chromato- ‘graphic support and (i) selectively eluting the monomeric 4BgG antibod from the support wherein the monomeric IgG Js selected fom the group consisting of anti-RSHZ-19 and CHECDs 3. The method according to claim 1 wherein the HIC suppor is selected from the group consisting of alkyles.cx agarose, arylagarose, alkyl-silica,ary-silca alky! onganic polymer resin and aryl organic polymer resin] [4. The method according to lsim 3 wherein the support js selected from the group consisting of butl-, phenyl and ‘tylagatose and butyl, phenyl- and ether- organic polymer resin] 26 enol 0 o Fare [S. The method sccondng to claim 4 wherein the support is phonyl-ogunie polymer resin] 6 The method according wo claim 4 wherein the suppot js buiylorganie polymer res 7. The method according to claim 1 wherein the antibody fs selectively eluted with aw salt bullet The method accoring to clsim [7] 2 whercin the antibody is sletvely cuted witha gradient decreasing in salto 80 mM phosphate, pH 9A method fr the purifcaion of an 1gG antibody fom conttioned ell culture medium conaining sae compe ng sequensally subjecting the medium to) Protein A aint chromatography. (b) ion exehange ehromstography, an (6) hydrophobic interaction ehromatogephy. [H. The melted secordng to claim 9 wherein the ion exchange chromatograpy erploys a support selected from the group consisting of CMA28—~, CM32-,CM.S2- cell Jose; CNeand, SPctos-fiaked dextrans, CM and Sangose, CNt-onganie polymer resin, DEAE-QAF-Qeross-litked dextians; DEAFQAEQ. linked agarose; and. DEAE nganie polymer reins and is by 2 buted sl soho] Ti. {he method according io claim 10 shersin. the support is CMagarose Fast Flow andthe salt NaCl] Ez. The method according to claim 10. wherein the bulered sat sotution i 40 aM eltate containing, 100 mM, NaCl pit 60] 13. The meihod according wo claim 9 wherein the hydko- rhobie interaction chromatographic employs @ support Felcted fom the group consisting of llyleyq-nzarse rylagarose.alkylsilics, arya, alkyl-agasse polymer ‘resin and ary-organe polymer resi, 14. The method according to claim 18 wherein the support, fs selected fom the group consisting of butyl, phenyl and cety-agaose and but, phenyl- and etherorganie polymer 15. The method according claim 14 wherein the support is phcyl-onganic polymer rein or buty-organic polymer 16. The method seconting to claim 9 wherein the support, js pheny- or butyonganic polymer resin andthe antibody Fs Selectively eluted wih alow sal bull 17. The method according to claim 16 wherein the an toy is selovtvely luted witha gradiem decreasing to 0 ‘mM sodium phosphate baer, pH 7.0. 18. The method aecorting to csi 9 wherein the Protein, A chromatography employs asa support Protein A linked to outrolled pore glass and clon is by ako pl Bulle,US RE40,070 E 27 19. The method aevording fo claim 18 wherein said bulfer js 25 mM citrate, pH 3.5 20. A method for purlying antibody from a conditioned cell mestiam comprising: (@) adgorbing the antibody onto & Protein A chromato- _graphie support () washing the adsorbed antibody with at least one butler; (6) eluting the antibody from step (6): (@) adsorbing the antibody from step (¢) onto an ion ‘exchange chromatographie suppor: (@) washing the absorbed antibody with a Teast one baler; () selectively eluting the antibody fom step (e; (@) adsorbing the eluate of step (1) onto a hydrophobic intervtion chromatographic support (8) washing the adsorbed antibody with at lewst one bater; (@ luting the adsorbed antibody; and ) recovering the antibody 21. The method according to claim 20 which includes one ‘or more optional steps of inactivating virsses if present. 22. The method aecording to claim 21 wherein a viral inactivation step is performed after stop (f) and before step @. 23. The method according o claim 22 wherein sai viral, ingctivation step comprises treatment of the eluate with ‘auanidine hydrochloride for a period of time sufficient t0 Ingctivate virus followed by the addition of an ammonium sulfate solution, 24. The method acconding to claim 28 wherein the guani- dine hydrochloride is present at 2.0M and following treat ment eluate from step (fis adjusted to 1.3M ammonium sulle 26, The method according to claim 22 wherein an a tional viral inetivation step is performed after step (e) and before step (, 26, The method soconding to claim 28 wherein said additional vial inactivation step comprises treatment ofthe chaste of step (e) with acid 27. The method according to claim 26 wherein the pl of the eluate is adjusted to pl] 3.5 and maintined at at pl for a period of time suficent to inactivate virus, and termina ing the treatment by adjusting the pH 10 5.5 28. The method aeeording to claim 20 wherein the ion ‘exchange support of step (Q) is selected from the group, ‘consisting of carboxymethyl (CM), sulfoethyl (SE), sufo- propyl (SP), phosphate(P) dithylaminoethy! (DEAE), qna- temnary aminoethyl (QAE), and quatemary (Q), substituted 28 cellulosie resins, cross linked dextrans, sgarose snd organ polymer resins. 29, The method acconting to claim 28 wherein the: ionic support is CM-agarose '30. The method according o claim 20 whereia the bydro- phobic interaction chromatographic seppot is selected from the group consisting of alkyl > cyragarose, aryhagarose, alkylsitica, arysilica, alkylonginie polymer resin and arylorganie polymer resins ‘31, The method according wel js selected ffom the group consisting of butyl, phenyl- and cctyl-agarose and phenyl. ether- and butyL-organie polymer 132. The method aocording wo claim M where the support, fs phenyl- or butyl-organie polymer resins 33. The method according to claim 20 wherein said protein is recovered by pooling and concentrating the pr ‘ein containing fractions trom chromatography step (i) by ‘uleafiration, 34, The method according to claim 20 wherein the ehro- smatographic support of step (a) is Protein A linked to controlled pore glass. 38. The method according to claim 20 wherein the absorbed antibody of step (b) is washed with two buffers, @ list equibraton bulfer and a second low pHT wash bulfer, 36. The method second to claim 38 wherein the pH of the second butler is less than 4.0. 37. The method according to claim 36 whersin the socond buifer is IM ammonium sulfite, $0 mM sodium citrate, pH 35, [38 A method of removing Protein A from a mixture comprising Protein A and IgG antibodies comprising con- ‘acting the mixture with a hydrophobic interaction chrom ‘ography support and selecting eluting the antibody from the support] 39, [Ihe method according to claim 38] A method for removing Protein from a misture comprising Protein A and IgG antibodies comprising contacting said misture with a Inydrophobie interaction chromatography support and selec ‘vel eluing the antibody from the support which includes ‘washing the support prior #0 elution with a buffer having & PH less thas 7.0 40. The method aeconding to claim 39 wherein the pli of the wash buller i less than 40 41, The method according to claim 40 wherein the buffer is (QM ammonium sulfate, 50 mM sodium citrate, pl 35D)