AU2005310305A1 - Methods for refolding polypeptides - Google Patents

Description

WO 2006/060083 PCT/US2005/038404 A-1056 -1 METHODS FOR REFOLDING POLYPEPTIDES This application claims the benefit of U.S. Patent Application (number not yet assigned, attorney docket number 01017/40170A) filed October 21, 2005 and 5 PCT Patent Application (number not yet assigned, attorney docket number 01017/40170A PCT) filed October 21, 2005 which claim the benefit of the U.S. Provisional Application No. 60/621,295, filed October 22, 2004, and U.S. Provisional Application No. 60/701,762 filed July 22, 2005, and each of which are hereby incorporated by reference. 10 Background of the Related Art The advent of genetic engineering brought with it the promise of production of large quantities of polypeptides from genetically-engineered organisms. Early research focused on expression in prokaryotes due to ease of use 15 of these organisms. However, in prokaryotes, oftentimes recombinant eukaryotic polypeptides are not subject to post-translational modification and/or are not correctly folded upon expression. This will frequently result in accumulation of the polypeptide in insoluble inclusion bodies which require denaturation and renaturation of the protein in order to recover it, frequently with only partial or 20 little success. Many important target proteins are at best inefficiently expressed in soluble form in prokaryotic cells, due at least in part to the complexity of the protein folding process in vivo (Houry et al., Nature, 402: 147-154, 1999). Eukaryotic, and in particular mammalian, expression systems and vectors have been improved over recent years to maximize levels of polypeptide 25 expression from eukaryotic host cells. However, while correct folding is seen more often than in prokaryotic expression systems, not all of the polypeptide expressed and secreted from the cells is modified correctly and in the desired conformation and thus, there is a need to maximize yields of biologically active polypeptides.

WO 2006/060083 PCT/US2005/038404 A-1056 -2 Polypeptides, and in particular antibodies, can be post translationally modified in undesired ways leading to heterogeneity in structure, which can correspondingly lead to a reduction in function. Some of these modifications include incomplete galactosylation and fucosylation of the two N 5 linked biantennary oligosaccharides attached to the second constant domain (CH2) of the heavy chain, N-terminal glutamine residue of the heavy chain conversion posttranslationally to pyroglutamate, C-terminal lysine modification, in addition to the following modifications: oxidation, deamidation, isomerization of aspartic acid, disulfide bond scrambling, cleavages, dimer formation, and others 10 (Dillon et al., Journal of Chromatography A, 1053 (2004) 299-305 and references cited therein). Recent improvements in RP-HPLC separation and detection techniques reveal that there is detectable conformational heterogeneity in polypeptides that were previously thought to be homogeneous (U.S. Patent 15 Application No: 11/040,659, publication no. 2005/0161399 to Dillon et al. filed January 21, 2005, incorporated herein by reference in its entirety). As discussed in the noted application, the nature of the heterogeneity is due at least in part to disulfide scrambling. One way of improving the yield of a correctly and homogeneously 20 folded, biologically active polypeptide is to improve the purification processes used to isolate the polypeptide, whether it has been expressed in a prokaryotic or a eukaryotic system. Prior methods have been provided for obtaining a correctly folded population of fusion polypeptides, e.g., Sassenfeld et al., WO 02/068455. However, the present inventors have developed a method that results in improved 25 efficiency of obtaining a desired conformation of a polypeptide that is captured on a column, during a commercial scale up process. SUMMARY OF THE INVENTION The present invention is directed to providing efficient and 30 economic production and purification of active polypeptides. More particularly, WO 2006/060083 PCT/US2005/038404 A-1056 -3 the invention describes methods to improve purification of a correctly folded and more active polypeptide by virtue of a refolding process. As described in further detail below, the addition of reduction/oxidation (redox) agents alone or in combination with mildly denaturing conditions, i.e., with a chaotropic agent, to a 5 polypeptide that is captured on a column during a purification process can facilitate the folding of a population of polypeptides where disulphide bonds are more uniform and thus result in a greater percentage of structurally homogeneous and therefore active forms of the molecule. Accordingly, one aspect of the invention is directed to methods of 10 modifying an Fc domain containing polypeptide on a column comprising mixing a redox reagent alone or in combination and a mildly denaturing reagent with an Fe domain containing polypeptide that has been immobilized on a column. The redox reagent, with or without the denaturing agent, then promotes conformational changes of the Fc domain containing polypeptides to a more homogeneous 15 structural conformation than would be present without the treatment. The methods of the invention broadly comprise producing an Fc domain containing polypeptide, isolating the polypeptide on a column and contacting said polypeptide with a reduction/oxidation reagent at a pH of about 5 to about 11; and optionally further contacting said preparation with a chaotropic 20 agent before, after or concurrently with said contacting with said reduction/oxidation reagent. The methods further comprise eluting and isolating the treated polypeptide. In certain aspects the redox reagent can be any suitable redox reagent, and in one embodiment comprises cysteine/cystine. The concentration of 25 the redox reagent can be up to 30 mM, and can be in a range of 30 mM to 1 mM, or 25 mM to 5 mM or 15 mM to 5 mM. In one embodiment where cysteine and cystine are both added as the redox reagents, the ratio of cysteine to cystine can be 50 to 1, 40 to 1, 33 to 1, 25 to 1, 20 to 1, 17 to 1, 12 to 1 or 10 to 1 or even less depending on the reaction condition that best suits the Fc domain containing 30 polypeptide. The best ratio of cysteine to cystine can be determined with routine WO 2006/060083 PCT/US2005/038404 A-1056 -4 experimentation by one of skill the art. Furthermore, the pH of the redox reagent can be from about 7 to about 10, or more specifically from about 7 to about 9. In one specific, non-limiting exemplary embodiment the pH of the redox reagent is about 8. The method can be performed at a temperature of from about 5C to 5 about 40'C, and is more preferably at about 18*C to about 25'C (e.g., at or near room temperature). The methods of the present invention may further comprise an additional step of contacting the isolated polypeptide with a chaotropic agent. In some embodiments, the chaotropic agent can be selected from the group 10 consisting of urea, SDS and guanidine hydrochloride. In particular embodiments, the chaotropic agent is guanidine hydrochloride present in the reaction mixture in a final concentration of about 0.1 M to about 1.5 M. The step of contacting with the redox reagent and/or a chaotropic agent with the Fc domain containing polypeptide on the column may be 15 performed over a sufficient time period to allow the desired conformational changes to occur. In one embodiment, the contacting step with the redox agent, and/or the chaotropic agent is performed for about 1 to about 48 hours. In other aspects, the incubation period is more than 2 hours and less than 24 hours. In a certain embodiment, the incubation is about three to four hours. 20 Capturing the Fc domain containing polypeptide on a column may be done using conventionally methods. This step may comprise prior steps to purify the polypeptide, such as one or more techniques selected from the group consisting of HPLC, size-exclusion chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, affinity 25 chromatography, and capillary electrophoresis, followed by capture on a protein A, protein G or protein L column. While captured on a column, the concentration of the polypeptide for use in the methods of the invention may be any concentration that is amenable to refolding. In specific embodiments, the concentration of polypeptide in the reaction mixture is from about 1 g/L to about 30 50 g/L, from about 10 g/L to about 40 g/L, or from 20 g/L to about 30 g/L.

WO 2006/060083 PCT/US2005/038404 A-1056 -5 Preferably, the methods of the invention are characterized in that the contacting with the redox reagent produces an increase in the biological activity of the polypeptide as compared to the same polypeptide that has not been prepared using the methods of the invention. By treating the polypeptide 5 according to the invention the relative concentration of the desired conformation of the polypeptide is increased compared to untreated polypeptide (enriched or increased abundance), thereby increasing the activity of the recovered polypeptide. The methods of the invention produce a polypeptide population 10 which may further be processed by formulating into a sterile bulk form. In other embodiments include formulating the polypeptide used in the methods of the invention into a sterile unit dose form. Also encompassed by the present invention is a population of Fc domain containing polypeptides prepared according the method of any of claims 15 described herein. Other features and advantages of the invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating embodiments of the invention, are given by way of illustration only, because 20 various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE FIGURES The following drawings form part of the present specification and 25 are included to further illustrate aspects of the present invention. The invention may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

WO 2006/060083 PCT/US2005/038404 A-1056 -6 Figure 1. Comparison of refolding results using different experimental refolding conditions listed as 'ref. These results correspond with the conditions described in Table 1. Figure 2. Comparison between refolding on the column and in 5 solution. DESCRIPTION OF THE EMBODIMENTS The present invention is directed to addressing a need for commercial scale methods of producing a homogeneous conformation population 10 of polypeptides, and more particularly, homogenous mammalian-cell produced Fc domain containing polypeptides. The present inventors have discovered that contacting an Fc domain containing polypeptide while it is captured on a purification column with redox agents, with or without a chaotropic agent, can facilitate the formation of correct disulphide bonds and structure of the 15 polypeptide. These polypeptides have then been shown to have improved biological activity relative to untreated polypeptides. The reduction/oxidation reagent is a source of reducing agents. Preferred reducing agents are free thiols. The reduction/oxidation reagent can be selected from the group consisting of cysteine and cystine, reduced and oxidized 20 glutathione, dithiothreitol (DTT), 2-mercaptoethanol, hydrogen peroxide (oxidizer) and dithionitrobenzoate. For ease of use and economy, reduced glutathione and/or reduced cysteine can be used. The redox reagent may be added to the fermentation media in which the cells producing the polypeptide are grown. In additional embodiments, the reagents also may be added to the liquid 25 chromatography (LC) mobile phase during the LC separation step for separating the polypeptide. In a particular embodiment, the antibody isotype of IgG2 has shown using LC methods to elute as three major peaks from HPLC. The use of the reduction/oxidation reagent and/or chaotropic agent while the polypeptide is on a column can promote conformational changes such that there is a more 30 uniform, single peak. It is contemplated that this more uniform peak, i.e., as WO 2006/060083 PCT/US2005/038404 A-1056 -.7 exemplified by peak 3 shown in the examples, may be isolated as a more homogeneous, and therefore more active, preparation of IgG2. The reduction/oxidation reagent is present at a concentration sufficient to increase the relative proportion of the desired conformation. The 5 optimal concentration of the reduction/oxidation reagent depends upon the concentration of protein and number of disulfide bonds in the protein. Generally, the concentration of free thiols from the reduction/oxidation reagent can be from about 0.05 mM to about 50 mM, more preferably about 0.1 mM to about 30 mM, and still more preferably about 0.2 mM to about 20 mM. Likewise, the total 10 concentration of the redox reagent can be up to 35 mM, and can be in a range of 30 mM to 1 mM, or 25 mM to 5 mM or 15 mM to 5 mM. In addition, the reduction/oxidation reagent can contain oxidized thiols at approximately higher, equal or lower concentrations as the reduced thiol component. For example, the reduction/oxidation reagent can be a combination of 15 reduced glutathione and oxidized glutathione. A ratio of reduced glutathione to oxidized glutathione of from about 1:1 to about 100:1 (reduced thiols:oxidized thiols) can function equally well. Alternatively in another embodiment, the reduction/oxidation reagent can be cysteine or a combination of cysteine and cystine. Thus, when oxidized thiols are included in the initial reduction/oxidation 20 coupling reagent, the ratio of reduced thiols to oxidized thiols can in a preferred embodiment be from about 1:10 to about 1000:1, more preferably about 1:1 to about 500:1, still more preferably about 5:1 to about 100:1, even more preferably about 10:1. In a certain embodiment the redox reagent comprises a mix of 25 cysteine/cystine. In this embodiment, the reduction/oxidation reagent can be from about 0.1 mM to about 30 mM cysteine and from about 0.05 mM to about 15 mM cystine. In other embodiments, the cysteine and cystine can be in a cysteine:cystine ratio from about 1:50 to alternatively about 50:1. In specific, non-limiting exemplary embodiments, the reduction/oxidation reagent comprises 30 about 20 mM cysteine and about 1 mM cystine, i.e., a ratio or 20:1. In more WO 2006/060083 PCT/US2005/038404 A-1056 -8specific embodiments, the ratio of cysteine to cystine is from about 15:1, to 40:1. Even more specifically, the ratio of cysteine to cystine is 17:1. Even more specifically, the ratio of cysteine to cystine is 33 to 1. In one example, the latter ratio can be achieved where it contains 20 mM cysteine and 0.6 mM cystine. It is 5 contemplated that 10 mM cysteine and 0.3 mM cystine may also be suitable to achieve the desired ratio. One of skill in the art will readily appreciate that the desired ratio of cysteine to cystine can be 50:1, 40:1, 33 :1, 2 5 :1, 20:1, 17:1, 12:1 or 10:1 or even less depending on the reaction condition that best suits the Fc domain containing polypeptide. In other embodiments, the redox reagent 10 comprises glutathione and the ratio of reduced glutathione to oxidized glutathione in the reduction/oxidation reagent can be from about 1:1 to about 100:1. In other embodiments, the refolding can be performed using other redox reagents including copper. Furthermore, chaperones and different buffer, temperature and time compositions can be used. The refolding step will thus 15 triple the production of polypeptide, and reduce by three times the protein concentration need in formulation solutions to achieve the same activity. The methods of the present invention may further comprise contacting the isolated polypeptide with a chaotropic agent. In exemplary embodiments, the chaotropic agent is selected from the group consisting of urea, 20 SDS and guanidine hydrochloride. In specific embodiments, the chaotropic agent is guanidine hydrochloride. The concentration of the guanidine hydrochloride may be varied according to particular conditions. IN certain specific embodiments, the concentration of guanidine hydrochloride can-be from about 0.1 M to about 2 M. In particularly embodiments, the concentration of guanidine 25 hydrochloride in the reaction mixture is from about 0.5 M to about 1.5 M. In still other exemplary embodiments, the concentration of guanidine hydrochloride in the reaction mixture is from about 0.8 M to about 1.2 M. The methods of the invention contemplate the refolding on a column of a Fc domain containing polypeptide. As such, the polypeptide will be 30 purified from expression systems on columns such as affinity resins such as WO 2006/060083 PCT/US2005/038404 A-1056 -9 concanavalin A-agarose, heparin-toyopearl@ or Cibacrom blue 3GA Sepharose@. It is also possible to utilize an affinity column comprising a polypeptide-binding polypeptide, such as a monoclonal antibody to the polypeptide, to affinity-purify expressed polypeptides. Other types of affmity purification steps can be a Protein 5 A, Protein G or Protein L column (e.g., MabSelect Sure@, ProSepvA@ or ProSep vA Ultra@, Red-Oxi), which affinity agents bind to proteins that contain Fe domains. In a particular embodiment, the preparation of polypeptide is purified and treated according to the invention on a Protein A affinity column. 10 Upon treatment according to the invention, the polypeptides can be removed from an affinity column using conventional techniques, e.g., in a high salt elution buffer and then dialyzed into a lower salt buffer for use or by changing pH or other components depending on the affinity matrix utilized, or can be competitively removed using the naturally occurring substrate of the affinity moiety. 15 It is also contemplated that the methods of the invention result in a higher degree of purity of the treated polypeptide. In the case of an antibody, it has been found that the redox and refold step on the column reduces the amount of free light chain in the mixture as it is washed out of the column after the treatment step. The resulting antibody preparation is then more pure in that there is less 20 contaminating species of proteins that are not a full length antibody, i.e., light chains, and also the antibody is more correctly folded. The invention provides methods of increasing the recovery of active polypeptides. In particular, the invention involves promoting a desired conformation of a protein in preparations of a polypeptide. Using the methods of 25 the invention on preparations of polypeptide results in a higher percentage, or higher relative fraction, of the polypeptide in the preparation with a desired conformation. A desired conformation for a polypeptide is the three-dimensional structure of a protein that most closely resembles, and/or duplicates the function of, the naturally occurring domain of that protein.

WO 2006/060083 PCT/US2005/038404 A-1056 -10 The desired conformation of a polypeptide may have other tertiary structure characteristics. For example, a desired conformation may be a monomer, dimer, trimer, tetramer, or some other higher order form of the protein. For the purposes of the invention, the "conformation" of a protein is its three 5 dimensional structure. Two different structures of a polypeptide with the same primary amino acid sequence are "conformers" of each other when they have different conformations corresponding to energy minima, and they differ from each other only in the way their atoms are oriented in space. Conformers can be interconverting (referring to the rotational freedom around bonds to the exclusion 10 of breaking bonds). Two different structures of a polypeptide with the same primary amino acid sequence are "configurational isomers" when they have different conformations corresponding to energy minima, they differ from each other in the way their atoms are oriented in space, and they are non interconvertible without the breaking of a covalent bond. In the practice of the 15 invention, configurational isomers can be interconverted by, for example, breaking and optionally reforming disulfide bonds. Thus, in one aspect, the invention contemplates contacting a preparation of polypeptide that is made up of a heterogeneous mixture of least two configurational isomers of the polypeptide to a reduction/oxidation reagent for a 20 time sufficient to increase the relative proportion of the desired configurational isomer and determining the relative proportion of the desired configurational isomer in the mixture. In another aspect, the invention contemplates contacting a preparation of a polypeptide that has been produced by mammalian cells with a reduction/oxidation coupling reagent, at a pH of about 7 to about 11, and isolating 25 a fraction of the preparation of the polypeptide with a desired conformation. Polypeptides can be glycosylated polypeptides such as, e.g., those produced by eukaryotic cells. In certain aspects, the methods of the present invention are used to reduce the conformational heterogeneity that is induced by disulphide scrambling. 30 In more specific aspects, conformational heterogeneity is present in antibodies and WO 2006/060083 PCT/US2005/038404 A-1056 -11 more particularly, IgG2 antibodies. It should be noted that the term "configuration" is used interchangeably with the term "conformation" herein throughout and is intended to mean a protein that has a different secondary, tertiary or quaternary structure from another protein that has the same primary 5 structure (the same amino acid sequence). Using the redox reagents either alone or in combination with the further processing using chaotropic agents, it is possible to produce a more structurally homogeneous, and more therapeutically active polypeptide compared to a sample of the same protein produced in the same manner but for the presence of the redox reagents and/or chaotropes. 10 The methods of the invention find particular use in treating proteins that have at least about 3 cysteine residues. As discussed elsewhere in the present application, it is noted that proteins such as antibodies are high molecular weight moieties and the methods described herein are particularly useful for the production of uniform conformations of such high molecular weight proteins. An 15 antibody is understood to include polyclonal and monoclonal antibodies as well as humanized or chimeric forms. An Fc domain containing polypeptide is understood to include those that include portions of an antibody Fc domain such that the fusion polypeptide can be purified on a column in a manner similar to an antibody. 20 A "domain" is a contiguous region of the polypeptide chain that adopts a particular tertiary structure and/or has a particular activity that can be localized in that region of the polypeptide chain. For example, one domain of a protein can have binding affinity for one ligand, and one domain of a protein can have binding affinity for another ligand. In a thermostable sense, a domain can 25 refer to a cooperative unfolding unit of a protein. Such proteins that contain more than one domain can be found naturally occurring as one protein or genetically engineered as a fusion protein. In addition, domains of a polypeptide can have subdomains. The inventive compositions and methods are useful for preparation 30 of polypeptides, including immunoglobulin molecules or portions thereof, e.g., Fc WO 2006/060083 PCT/US2005/038404 A-1056 -12 fusion polypeptides, and chimeric antibodies (e.g., an antibody having a human constant region coupled to a murine antigen binding region) or fragments thereof produced from any desired expression system. Numerous techniques are known by which DNA encoding immunoglobulin molecules can be manipulated to yield 5 DNAs capable of encoding polypeptides such as single chain antibodies, antibodies with enhanced affinity, or other antibody-based polypeptides in eukaryotic and prokaryotic expression systems (see, for example, Larrick et al., 1989, Biotechnology 7:934-938; Reichmann et al., 1988, Nature 332:323-327; Roberts et al., 1987, Nature 328:731-734; Verhoeyen et al., 1988, Science 10 239:1534-1536; Chaudhary et al., 1989, Nature 339:394-397). Certain embodiments contemplate expression systems that are mammalian cell based such that the Fc domain containing polypeptides are modified in a manner most like natural human proteins. Preparations of fully human antibodies (such as are prepared using transgenic animals, and optionally further modified in vitro), as 15 well as humanized antibodies, can also be used in the invention. The term humanized antibody also encompasses single chain antibodies. See, e.g., Cabilly el al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent No. 0,125,023 BI; Boss et al., U.S. Pat. No. 4,816,397; Boss et al., European Patent No. 0,120,694 B1; Neuberger, M. S. et al., WO 86/01533; 20 Neuberger, M. S. etal., European Patent No. 0,194,276 BI; Winter, U.S. Pat. No. 5,225,539; Winter, European Patent No. 0,239,400 B1; Queen et al., European Patent No. 0 451 216 B1; and Padlan, E. A. et al., EP 0 519 596 Al. The method of the invention may also be used during the preparation of conjugates comprising an antibody and a cytotoxic or luminescent substance. Such substances include: 25 maytansine derivatives (such as DM1); enterotoxins (such as a Staphlyococcal enterotoxin); iodine isotopes (such as iodine-125); technium isotopes (such as Tc 99m); cyanine fluorochromes (such as Cy5.5.18); and ribosome-inactivating proteins (such as bouganin, gelonin, or saporin-S6). In a particular embodiment an Fc domain containing polypeptide is a 30 soluble form of the TNF receptor fused to an Fc domain (TNFR:Fc), however, it is WO 2006/060083 PCT/US2005/038404 A-1056 -13 to be understood that any polypeptide containing an Fe domain is suitable for use in the instant formulation. A commercially available TNFR:Fc is known as etanercept (Enbrel@, Immunex Corporation), which is a dimeric fusion polypeptide consisting of the extracellular ligand-binding portion of the human 75 5 kilodalton (p75) tumor necrosis factor receptor (TNFR) linked to the Fc portion of human IgGI. The Fc component of etanercept contains the constant heavy 2 (CH2) domain, the constant heavy 3 (CH3) domain and hinge region, but not the constant heavy 1 (CHI) domain of human IgG1. It is to be understood that an Fe domain can contain one or all of the domains described above. 10 An Fe domain fusion polypeptide substantially similar to one of the following polypeptides is suitable for use in the present invention: a flt3 ligand, a CD40 ligand, erythropoeitin, thrombopoeitin, calcitonin, Fas ligand, ligand for receptor activator of NF-kappa B (RANKL), tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), thymic stroma-derived lymphopoietin, 15 granulocyte colony stimulating factor, granulocyte-macrophage colony stimulating factor, mast cell growth factor, stem cell growth factor, epidermal growth factor, RANTES, growth hormone, insulin, insulinotropin, insulin-like growth factors, parathyroid hormone, interferons, nerve growth factors, glucagon, interleukins I through 18, colony stimulating factors, lymphotoxin-P, tumor 20 necrosis factor (TNF), leukemia inhibitory factor, oncostatin-M, and various ligands for cell surface molecules ELK and Hek (such as the ligands for eph related kinases or LERKS). Polypeptides suitable for use according to the invention also include recombinant fusion polypeptides comprising an Fe domain of an antibody plus a 25 receptor for any of the above-mentioned polypeptides or polypeptides substantially similar to such receptors. These receptors include: both forms of TNFR (referred to as p55 and p75), Interleukin- 1 receptors (type 1 and 2), Interleukin-4 receptor, Interleukin- 15 receptor, Interleukin- 17 receptor, Interleukin- 18 receptor, granulocyte-macrophage colony stimulating factor 30 receptor, granulocyte colony stimulating factor receptor, receptors for oncostatin M and leukemia inhibitory factor, receptor activator of NF-kappa B (RANK), WO 2006/060083 PCT/US2005/038404 A-1056 -14 receptors for TRAIL (TRAIL receptors 1, 2, 3, and 4), and receptors that comprise death domains, such as Fas or Apoptosis-Inducing Receptor (AIR). Other polypeptides suitable for use in the present invention include differentiation antigens (referred to as CD polypeptides) or their ligands or 5 polypeptides substantially similar to either of these, which are fused to an Fc domain of an antibody. Such antigens are disclosed in Leukocyte Typing VI (Proceedings of the VIth International Workshop and Conference, Kishimoto, Kikutani et al., eds., Kobe, Japan, 1996). Similar CD polypeptides are disclosed in subsequent workshops. Examples of such antigens include CD27, CD30, 10 CD39, CD40, and ligands thereto (CD27 ligand, CD30 ligand, etc.). Several of the CD antigens are members of the TNF receptor family, which also includes 41BB ligand and OX40. The ligands are often members of the TNF family, as are 41BB ligand and OX40 ligand. Accordingly, members of the TNF and TNFR families can be formulated according to the present invention. 15 Enzymatically active polypeptides or their ligands can also be used according to the invention. Examples include recombinant fusion polypeptides comprising an Fc domain of an antibody fused to all or part of one of the following polypeptides or their ligands or a polypeptide substantially similar to one of these: metalloproteinase-disintegrin family members, various kinases, 20 glucocerebrosidase, superoxide dismutase, tissue plasminogen activator, Factor VIII, Factor IX, apolipoprotein E, apolipoprotein A-I, globins, an IL-2 antagonist, alpha-l antitrypsin, TNF-alpha Converting Enzyme, ligands for any of the above mentioned enzymes, and numerous other enzymes and their ligands. The methods of the invention can also be used on compositions 25 comprising antibodies, human antibodies, humanized antibodies, chimeric antibodies, i.e. antibodies having human constant antibody immunoglobulin domains coupled to one or more murine variable antibody immunoglobulin domain, and/or non-human antibodies, or fragments thereof. While the present examples relate to antibodies described in detail in published US application 30 US2004/0097712 Al (U.S. Application No. 10/656,769, incorporated herein by WO 2006/060083 PCT/US2005/038404 A-1056 -15 reference in its entirety) one of skill in the art will readily be able to determine by routine experimentation what other Fe domain containing polypeptides will be suitable for use in the methods of the invention. The methods of the invention can also be used for compounds comprising 5 an Fc domain containing polypeptide conjugated to a cytotoxic or luminescent substance. Such substances include: maytansine derivatives (such as DM1); enterotoxins (such as a Staphylococcal enterotoxins); iodine isotopes (such as iodine-125); technetium isotopes (such as Tc-99m); cyanine fluorochromes (such as Cy5.5.18); and ribosome-inactivating polypeptides (such as bouganin, gelonin, 10 or saporin-S6). Examples of other polypeptide-substance conjugates contemplated for use in the invention include those that recognize one or more of the following antigens: CD2, CD3, CD4, CD8, CDl la, CD14, CD18, CD20, CD22, CD23, CD25, CD33, CD40, CD44, CD52, CD80 (B7.1), CD86 (B7.2), CD147, IL-4, IL-5, IL-8, IL- 10, IL-2 receptor, IL-4 receptor, IL-6 receptor, IL- 13 15 receptor, PDGF-3, VEGF, TGF, TGF-2, TGF-31, EGF receptor, VEGF receptor, C5 complement, IgE, tumor antigen CA125, tumor antigen MUC1, PEM antigen, LCG (which is a gene product that is expressed in association with lung cancer), HER-2, a tumor-associated glycoprotein TAG-72, the SK-1 antigen, tumor associated epitopes that are present in elevated levels in the sera of patients with 20 colon and/or pancreatic cancer, cancer-associated epitopes or polypeptides expressed on breast, colon, squamous cell, prostate, pancreatic, lung, and/or kidney cancer cells and/or on melanoma, glioma, or neuroblastoma cells, TRAIL receptors 1, 2, 3 and 4, the necrotic core of a tumor, integrin alpha 4 beta 7, the integrin VLA-4, B2 integrins, TNF-a, the adhesion molecule VAP- 1, epithelial 25 cell adhesion molecule (EpCAM), intercellular adhesion molecule-3 (ICAM-3), leukointegrin adhesin, the platelet glycoprotein gp Ilb/IIla, cardiac myosin heavy chain, parathyroid hormone, rNAPc2 (which is an inhibitor of factor VIIa-tissue factor), MHC I, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), tumor necrosis factor (TNF), CTLA-4 (which is a cytotoxic T lymphocyte-associated 30 antigen), Fc-y-1 receptor, HLA-DR 10 beta, HLA-DR antigen, L-selectin, IFN-y, WO 2006/060083 PCT/US2005/038404 A-1056 -16 Respiratory Syncitial Virus, human immunodeficiency virus (HIV), hepatitis B virus (HBV), Streptococcus mutans, and Staphylococcus aureus. The methods of the invention can also be used for modifying the conformation of anti-idiotypic antibodies, or substantially similar polypeptides, 5 including but not limited to anti-idiotypic antibodies against: an antibody targeted to the tumor antigen gp72; an antibody against the ganglioside GD3; or an antibody against the ganglioside GD2. Additional examples of Fc domain fusion polypeptides include proteins expressed as a fusion with a portion of an immunoglobulin molecule, 10 proteins expressed as fusion proteins with a zipper moiety, and novel polyfunctional proteins such as a fusion proteins of a cytokine and a growth factor (i.e., GM-CSF and IL-3, MGF and IL-3). WO 93/08207 and WO 96/40918 describe the preparation of various soluble oligomeric forms of a molecule referred to as CD40L, including an immunoglobulin fusion protein and a zipper 15 fusion protein, respectively; the techniques discussed therein are applicable to other proteins. Any of the above molecules can be expressed as a fusion protein including but not limited to the extracellular domain of a cellular receptor molecule, an enzyme, a hormone, a cytokine, a portion of an immunoglobulin molecule, a zipper domain, and an epitope. 20 By "partially purified" means that some fractionation procedure, or procedures, have been carried out, but that more polypeptide species (at least 10%) than the desired protein or protein conformation is present. One of the advantages of the methods of the invention is that the preparation of polypeptide can be at a fairly high concentration. Examples of such concentration ranges are 25 0.1 to 100 mg/ml, from 0.5 to 40 mg/ml, and from 1 to 10 mg/ml. The preparation of polypeptide can be prepared initially by culturing recombinant host cells under culture conditions suitable to express the polypeptide, in the presence of the redox reagents as described herein. The polypeptide can also be expressed as a product of transgenic animals, e.g., as a 30 component of the milk of transgenic cows, goats, pigs, or sheep which are WO 2006/060083 PCT/US2005/038404 A-1056 -17 characterized by somatic or germ cells containing a nucleotide sequence encoding the polypeptide. The resulting expressed polypeptide can then be purified, or partially purified, from such culture or component (e.g., from culture medium or cell extracts or bodily fluid) using known processes. While fractionation 5 including but not limited to one or more steps of filtration, centrifugation, precipitation, phase separation, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction chromatography (HIC; using such resins as phenyl ether, butyl ether, or propyl ether), HPLC, or some combination of above may be used herein, the advantageous methods of the present invention 10 may employ LC fractionation and purification of the high molecular weight therapeutic proteins as described below. To the extent that these additional separation methods also may be useful herein, such methods are discussed in further detail elsewhere herein. Some or all of the foregoing purification steps, in various 15 combinations, can also be employed to prepare an appropriate preparation of a polypeptide for use in the methods of the invention, and/or to further purify the recombinant polypeptide after contacting the preparation of the polypeptide with a reduction/oxidation coupling reagent. The polypeptide that is substantially free of other mammalian polypeptides is defined as an "isolated polypeptide". The 20 specific LC methods that may be combined with the redox reagent-based methods described herein are described in further detail below. The polypeptide can also be produced by known conventional chemical synthesis. Methods for constructing polypeptides by synthetic means are known to those skilled in the art. The synthetically-constructed polypeptide 25 sequences can be glycosylated in vitro. The desired degree of final purity depends on the intended use of the polypeptide. A relatively high degree of purity is desired when the polypeptide is to be administered in vivo, for example. In such a case, the polypeptides are purified such that no polypeptide bands corresponding to other polypeptides are 30 detectable upon analysis by SDS-polyacrylamide gel electrophoresis (SDS- WO 2006/060083 PCT/US2005/038404 A-1056 -18 PAGE). It will be recognized by one skilled in the pertinent field that multiple bands corresponding to the polypeptide can be visualized by SDS-PAGE, due to differential glycosylation, differential post-translational processing, and the like. Most preferably, the polypeptide of the invention is purified to substantial 5 homogeneity, as indicated by a single polypeptide band upon analysis by SDS PAGE. The polypeptide band can be visualized by silver staining, Coomassie blue staining, and/or (if the polypeptide is radiolabeled) by autoradiography. By "contacting" is meant subjecting to, and/or exposing to, in solution. The protein or polypeptide can be contacted with the redox reagents 10 while also bound to a solid support (e.g., an affinity column or a chromatography matrix). Preferably, the solution is buffered. In order to maximize the yield of protein with a desired conformation, the pH of the solution is chosen to protect the stability of the protein and to be optimal for disulfide exchange. In the practice of the invention, the pH of the solution is preferably not strongly acidic. Thus, 15 preferred pH ranges are greater than pH 5, preferably about pH 6 to about pH 11, more preferably from about pH 7 to about pH 10, and still more preferably from about pH 7.6 to about pH 9.6. In one non-limiting embodiment of the invention, the optimal pH was found to be about pH 8.6. However, the optimal pH for a particular embodiment of the invention can be easily determined experimentally 20 by those skilled in the art. Contacting the preparation of polypeptide with a reduction/oxidation reagent is performed for a time sufficient to increase the relative proportion of the desired conformation. Any relative increase in proportion is desirable, but preferably at least 10% of the protein with an 25 undesired conformation is converted to protein with the desired conformation. More preferably at least 20%, 30%, 40%, 50%, 60%, 70% and even 80% of the protein is converted from an undesired to a desired conformation. Typical yields that have been achieved with the methods of the invention range from 40 to 80%. The contacting may be performed by providing the redox reagent to the 30 fermentation medium in which the protein is being generated. Alternatively, the WO 2006/060083 PCT/US2005/038404 A-1056 -19 contacting takes place upon partial purification of the protein from the cell culture in which it is generated. In still other embodiments, the contacting is performed after the protein has been eluted from the HPLC column but before any further processing. Essentially, the contacting may be performed at any stage during 5 preparation, purification, storage or formulation of the antibody. If the contacting step is performed on a partially or highly purified preparation of polypeptide, the contacting step can be performed for as short as about 1 hour to about 4 hours, and as long as about 6 hours to about 4 days. It has been found that a contacting step of about 4 to about 16 hours or about 18 hours 10 works well. The contacting step can also take place during another step, such as on a solid phase or during filtering or any other step in purification. The methods of the invention can be performed over a wide temperature range. For example, the methods of the invention have been successfully carried out at temperatures from about 4 0 C to about 37'C, however 15 the best results were achieved at lower temperatures. A typical temperature for contacting a partially or fully purified preparation of the polypeptide is about 4*C to about 25*C (ambient), but can also be performed at lower temperatures and at higher temperature. The preparation of polypeptide can be contacted with the 20 reduction/oxidation reagent in various volumes as appropriate. For example, the methods of the invention have been carried out successfully at the analytical laboratory-scale (1-50 mL), preparative-scale (50 mL-10 L) and manufacturing scale (10 L or more). Thus, the methods of the invention can be carried out on both small and large scale with reproducibility. 25 The presently described reaction can be quenched in any way known to those of skill in the art. For example, the reduction/oxidation reagent can be removed and/or it can be chemically inactivated by, e.g., acidifying the solution. Typically, when the reaction is quenched by acidification, the pH of the solution containing the reduction/oxidation reagent will be brought below pH 7, 30 and generally the pH is reduced to between about pH 2 and about pH 7.

WO 2006/060083 PCT/US2005/038404 A-1056 -20 Determining the conformation of a protein, and the relative proportions of a conformation of a protein in a mixture, can be done using any of a variety of analytical and/or qualitative techniques. For example, highly sensitive LC and LC/MS methods described in U.S. Patent Application No: 60/548,302, 5 Dillon et al. filed February 27, 2004 and Dillon et al., Journal of Chromatography A, 1053 (2004) 299-305 and references cited therein can be used. Determining the conformation can also be done by way of an activity assay (e.g., binding to a ligand, enzymatic activity, biological activity, etc.). In certain examples, bioassays for polypeptide activity may be assessed 10 using a chondrocyte activity assay by monitoring IL-6 and MMP13 levels in the presence and absence of the refolded polypeptide. The EC50 values in such assays may be determined using techniques known to those of skill in the art. From such assays, it was determined that refolding in the presence of a reduction/oxidation reagent produced an antibody that had over three-times 15 greater activity than a control that had not been refolded in the presence of a redox reagent and a polypeptide refolded in the presence of a redox reagent and a chaotropic agent as described herein produced material that had an activity over six times greater than the activity of the control. By the term "isolating" is meant physical separation of at least one 20 component in a mixture away from other components in a mixture. Isolating components or particular conformations of a protein can be achieved using any purification method that tends to separate such components. Accordingly, one can perform multiple chromatography steps in addition to the RP-HPLC described above, including but not limited to HIC, hydroxyapatite chromatography, ion 25 exchange chromatography, affmity, and SEC. Other purification methods are filtration (e.g., tangential flow filtration), electrophoretic techniques (e.g., electrophoresis, electroelution, isoelectric focusing), and phase separation (e.g., PEG-dextran phase separation), to name just a few. In addition, the fraction of the preparation of polypeptide that contains the protein in the undesired conformation WO 2006/060083 PCT/US2005/038404 A-1056 -21 can be treated again in the methods of the invention, to further optimize the yields of protein with the desired conformation. Analytical methods particularly suited for serving the purpose of the initial separation step include size-exclusion chromatography, ion-exchange 5 chromatography, hydrophobic interaction chromatography, isoelectric focusing and capillary electrophoresis. Ion-exchange chromatography relies on the affinity of a substance for the exchanger, which affinity depends on both the electrical properties of the material and the relative affinity of other charged substances in the solvent. Hence, bound material can be eluted by changing the pH, thus 10 altering the charge of the material, or by adding competing materials, of which salts are but one example. An additional useful example of a preparatory column is a size exclusion column, otherwise known as gel filtration or gel permeation chromatography, relies on the penetration of macromolecules in a mobile phase 15 into the pores of stationary phase particles. Differential penetration of the macromolecules is a function of the hydrodynamic volume of the particles. Size exclusion media exclude larger molecules from the interior of the particles while the smaller molecules are accessible to this volume. The order of elution can be predicted by the size of the protein as a linear relationship exists between elution 20 volume and the log of the molecular weight of the protein being eluted. Yet another example is hydrophobic interaction chromatography. Certain proteins are retained on affinity columns containing hydrophobic spacer arms. This observation is exploited in the technique of hydrophobic interaction chromatography (HIC). Hydrophobic adsorbents now available include octyl or. 25 phenyl groups. Hydrophobic interactions are strong at high solution ionic strength, as such samples being analyzed need not be desalted before application to the adsorbent. Elution is achieved by changing the pH or ionic strength or by modifying the dielectric constant of the eluant using, for instance, ethanediol. A further detailed description of the general principles of hydrophobic interaction 30 chromatography media may be found in U.S. Patent No. 3,917,527 and in U.S.

WO 2006/060083 PCT/US2005/038404 A-1056 -22 Patent No. 4,000,098. The application of HIC to the purification of specific proteins is exemplified by reference to the following disclosures: human growth hormone (U.S. Patent No. 4,332,717), toxin conjugates (U.S. Patent No. 4,771,128), antihemolytic factor (U.S. Patent No. 4,743,680), tumor necrosis 5 factor (U.S. Patent No. 4,894,439), interleukin-2 (U.S. Patent No. 4,908,434), human lymphotoxin (U.S. Patent No. 4,920,196) and lysozyme species (Fausnaugh, J. L. and F. E. Regnier, J Chromatog. 359:131-146 (1986)) and soluble complement receptors (U.S. Patent No. 5,252,216). Suitable hydrophobic interaction chromatography media include, Pharmacia's phenyl-Sepharose, and 10 Tosohaas' butyl, phenyl and ether Toyopearl 650 series resins. Affinity chromatography is another chromatographic technique that may be used herein. Examples of commonly used affinity chromatography include immobilized metal affinity chromatography (IMAC), sulfated affinity chromatography, dye affinity chromatography, and heparin affinity. In another 15 example, the chromatographic medium may be prepared using one member of a binding pair, e.g., a receptor/ligand binding pair, or antibody/antigen binding pair (immunoaffinity chromatography). In certain embodiments, affinity chromatography is used to selectively separate IgG antibodies from a mixture of proteins by using protein A or G as a functionality of the stationary phase. 20 It should also be understood that the methods of the invention can be used to prepare a protein formulation for use in a patient where the preparation involves mammalian cell production of the protein, purification of the protein from that mammalian cell culture, refolding of the purified protein using the refolding methods described herein, exchanging the buffer of the composition thus 25 produced to formulation buffer and producing a single dose formulation that may be used in the patient. Alternatively, the preparation of formulation involves the steps of mammalian cell production of the protein, purification of the protein followed by refolding of the protein as described herein, followed by isolation of desired form of the protein, after which the buffer of the composition thus WO 2006/060083 PCT/US2005/038404 A-1056 -23produced is exchanged to formulation buffer and producing a single dose formulation that may be used in the patient. The invention also optionally encompasses further formulating the proteins. By the term "formulating" is meant that the proteins can be buffer 5 exchanged, sterilized, bulk-packaged and/or packaged for a final user. For purposes of the invention, the term "sterile bulk form" means that a formulation is free, or essentially free, of microbial contamination (to such an extent as is acceptable for food and/or drug purposes), and is of defined composition and concentration. 10 The term "sterile unit dose form" means a form that is appropriate for the customer and/or patient administration or consumption. Such compositions can comprise an effective amount of the protein, in combination with other components such as a physiologically acceptable diluent, carrier, and/or excipient. The term "pharmaceutically acceptable" means a non-toxic material that does not 15 interfere with the effectiveness of the biological activity of the active ingredient(s) see for example, U.S. Pat. No. 6,171,586, which is incorporated herein in its entirety. Formulations suitable for administration include aqueous and non aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of 20 the recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. In addition, sterile bulk forms and sterile unit forms may contain a small concentration (approximately 1 microM to approximately 10 mM) of a reduction/oxidation reagent (e.g., glutathione, cysteine, etc.). 25 The polypeptides can be formulated according to known methods used to prepare pharmaceutically useful compositions. They can be combined in admixture, either as the sole active material or with other known active materials suitable for a given indication, with pharmaceutically acceptable diluents (e.g., saline, Tris-HCl, acetate, and phosphate buffered solutions), preservatives (e.g., 30 thimerosal, benzyl alcohol, parabens), emulsifiers, solubilizers, adjuvants and/or WO 2006/060083 PCT/US2005/038404 A-1056 -24 carriers. Suitable formulations for pharmaceutical compositions include those described in Remington's Pharmaceutical Sciences, 16th ed. 1980, Mack Publishing Company, Easton, Pa. In addition, such compositions can be complexed with 5 polyethylene glycol (PEG), metal ions, and/or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, dextran, etc., or incorporated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, 10 sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat. No. 4,235,871; U.S. Pat. No. 4,501,728; U.S. Pat. No. 4,837,028; U.S. Pat. No. 4,737,323. Sustained-release forms suitable for use include, but are not limited 15 to, polypeptides that are encapsulated in a slowly-dissolving biocompatible polymer (such as the alginate microparticles described in U.S. Pat. No. 6,036,978), admixed with such a polymer (including topically applied hydrogels), and or encased in a biocompatible semi-permeable implant. The following are included to demonstrate embodiments of the 20 invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes 25 can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. EXAMPLE 30 Refolding on Affinity Column versus in Solution WO 2006/060083 PCT/US2005/038404 A-1056 -25 A human antibody to interleukin 1 receptor has been developed. This antibody is described in detail in US application US2004/0097712 Al (U.S. Application No. 10/656,769, incorporated herein) and is an IgG2 isotype. When this antibody was produced and purified using convention column 5 chromatography methods, it had an elution profile using the HPLC/MS analysis of Dillon et al., Application No. 11/040,659, of three peaks. The following procedures and reagents were used to run this antibody preparation onto a protein A column. 10 Resin: MabSelect Protein A (GE) Operating temperature: Room temp (approximately 15 - 25'C) Step Buffer Volume (CV) Equilibration 20 mM Tris, 100 mM NaCl, pH 7.4 3 Load Clarified Cell Culture Supernatant 10 - 30 g per CV Wash 1 20 mM Tris, 100 mM NaCl, pH 7.4 5 Wash 2 Redox Solution 0.5 - 12 hours Wash 3 20 mM Tris, 100 mM NaCl, pH 7.4 5 Elution 50 mM Acetate, pH 3.4 3 Equilibration: 20 - 50 mM Tris in 50 - 500 mM NaCl, pH 6.5 - 8.0 15 20 - 50 mM Citrate-Phosphate in 50 - 500 mM NaCl, pH 6.5 - 8.0 Phosphate buffered saline, pH 7.0 - 7.5 Wash 1 and 3: Same as Equilibration buffer Elution: 20 - 200 mM Acetate, pH 3.0 - 4.0 20 - 200 mM Citrate, pH 3.0 - 4.0 20 20 - 200 mM Glycine-HCl, pH 3.0 - 4.0 When the three peaks shown on the HPLC/MS analysis were separated and tested for bioactivity, peak 3 demonstrated the most activity. The antibody was then subjected to refolding on the column. Table 1 25 below sets out the experimental conditions and some results. Results of the refolding are also shown in Figure 1 which demonstrates a comparison of the conditions described in Table 1. The data in Figure 1 correlates with the Table 1 information such that, for example, 'ref 7' indicating 'refolding-7' conditions. The conditions described result in refolding the antibody to bias the peak 3, WO 2006/060083 PCT/US2005/038404 A-1056 -26 namely, the most active fraction, and refolding method 7 and 8 have the best profiles showing that the ratio of redox can be manipulated to improve antibody refolding on an affinity column. Figure 2 shows the comparison of experimental results from affinity 5 column refolding versus solution refolding. M588 is the result of refolding in solution, whereas M589 is the same antibody refolded on an affinity column. These data show that the affinity column provides a more homogenous peak 3 than the materials generated in solution refolding. 10 Table 1. Refolding conditions for affinity column process Data Summary: RP-HPLC Refolding 20 mM Cysteine, 0.6 mM Cystine, pH 8.0 Buffer: 10 mM Cysteine, 0.6 mM Cystine, pH 8.0 20 mM Cysteine, 0.6 mM Cystine, 1.2 M Gu-HCI, pH 8.0 10 mM Cysteine, 0.6 mM Cystine, 1.2 M Gu-HCI, pH 8.0 Experimental Conditions Batch Sample Load Pk3 to Pk1 SEC Record Name Factor :Cysteine/Cystine GuHIRefolding Ratio (% Main) (mg/mL Ratio Time (hr) resin) 30021905-1 Load N/A -N/A N/A 3.1 99.8: ---~------------- L------------ --------------------------- Refolding - Pool 20 33 N 12 0. Reolding-4 Fol 10 17 N 2 0.5 99.6 Refolding -5 Pool 10 17 N 12 0. 99.6 Refolding -5 Pool 20 Refolding -6 Pool 20 33 Y 2 Refolding -7 Pool 20 ~3 6 31.2 Refolding -9 Pool 1d 10 ~ ~ Refolding -10 Pool 10 17 Y 12 1 13.399 15 While the examples provided herein are directed to Fc domain containing polypeptides on a column, it is contemplated that the methods may readily be adapted and used for any polypeptide that undergoes post-translational refolding and exhibits heterogeneity due to presence of disulfide bond that are WO 2006/060083 PCT/US2005/038404 A-1056 -27 amenable to scrambling, varying glycosylation patterns and the like while on a column. However, the methods described herein may be especially useful for the production of other Fc domain containing polypeptides, and particularly IgGs such as for example, IgGI, IgG3 and IgG4 antibodies which may exhibit 5 heterogeneity. All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of certain or specific embodiments, it will be apparent to 10 those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described 15 herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. The references cited herein throughout, to the extent that they 20 provide exemplary procedural or other details supplementary to those set forth herein, are all specifically incorporated herein by reference.

Claims (17)

1. A method comprising: contacting a Fe domain containing polypeptide already bound to a column with a reduction/oxidation coupling reagent at a pH of about 7 to about 11, and isolating a fraction of the preparation of the polypeptide with a desired conformation.

2. The method of claim 1, where the column is a Protein A or Protein G column.

3. The method of claim 1, where the Fc domain containing polypeptide is a fusion polypeptide.

4. The method of claim 1, where the Fe domain containing polypeptide is an antibody.

5. The antibody of claim 4, wherein the antibody is of the IgG2 isotype.

6. The method of claim 1 wherein the pH is from about 7 to about 10.

7. The method of claim 6 wherein the pH is about 7.5 to about 9.

8. The method of claim 7, wherein the pH is about 8. WO 2006/060083 PCT/US2005/038404 A-1056 -29

9. The method of claim 1 wherein the reduction/oxidation coupling reagent comprises glutathione.

10. The method of claim 1 wherein the reduction/oxidation coupling reagent comprises cysteine.

11. The method of claim 1 wherein the contacting step is performed for about 4 to about 16 hours.

12. The method of claim 1 wherein the contacting step is performed at about 4 to about 25'C.

13. The method of claim 1 wherein the contacting step is quenched by acidification.

14. The method of claim 1 wherein the isolating step comprises one or more chromatography steps.

15. The method of claim I wherein the polypeptide concentration is from about 1 to about 40 mg/ml.

16. A method comprising formulating into sterile unit dose form a polypeptide that has been produced by mammalian cells, contacted with a reduction/oxidation coupling reagent while on a column, and isolated from the fraction of the polypeptide having an undesired conformation. WO 2006/060083 PCT/US2005/038404 A-1056 -30

17. A pharmaceutical composition of an antibody produced by the method of claim 16.