WO2008033555A2 - High-pressure refolding of difficult-to-fold proteins - Google Patents

Abstract

A method of refolding denatured and/or aggregated proteins which have not previously been refolded by non-pressure-based methods is disclosed, as well as proteins refolded by such methods.

Description

HIGH-PRESSURE REFOLDING OF DIFFICULT-TO-FOLD PROTEINS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This non-provisional patent application claims priority under 35 USC §119(e) from United States Provisional Patent Application having serial number 60/845,005, filed on September 15, 2006 and titled HIGH PRESSURE REFOLDING OF DIFFICULT-TO- REFOLD PROTEINS, wherein the entirety of said provisional patent application is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] This invention relates to methods for refolding proteins under high pressure which have not been previously refolded, as well as to the proteins thus refolded and compositions containing the proteins.

BACKGROUND OF THE INVENTION

[0003] Many proteins are valuable as therapeutic agents. Such proteins include human growth hormone, which is used to treat abnormal height when insufficient growth hormone is produced in the body, and interferon-gamma, which is used to treat neoplastic and viral diseases. Protein pharmaceuticals are often produced using recombinant DNA technology, which can enable production of higher amounts of protein than can be isolated from naturally-occurring sources, and which avoids contamination that often occurs with proteins isolated from naturally-occurring sources.

[0004] Proper folding of a protein is essential to the normal functioning of the protein.

Improperly folded proteins are believed to contribute to the pathology of several diseases, including Alzheimer's disease, bovine spongiform encephalopathy (BSE, or "mad cow" disease) and human Creutzfeldt- Jakob disease (CJD), and Parkinson's disease; these diseases serve to illustrate the importance of proper protein folding.

[0005] Several proteins of therapeutic value in humans, such as recombinant human growth hormone and recombinant human interferon gamma, can be expressed in bacteria, yeast, and other microorganisms. While large amounts of proteins can be produced in such systems, the proteins are often misfolded, and often aggregate together in large clumps called inclusion bodies. The proteins cannot be used in the misfolded, aggregated state. Accordingly, methods of disaggregating and properly refolding such proteins have been the subject of much investigation. Early work in this area focused on chemical methods of disaggregating inclusion bodies (or "refractile bodies"); see, for example, U.S. Patent No. 4,511,503.

[0006] One method of refolding proteins uses high pressure on solutions of proteins in order to disaggregate, unfold, and properly refold proteins. Such methods are described in U.S. Patent No. 6,489,450, U.S. 7,064,192, U.S. Patent Application Publication No. 2004/0038333, and International Patent Application WO 02/062827. Those disclosures indicated that certain high-pressure treatments of aggregated proteins or misfolded proteins resulting in recovery of disaggregated protein retaining biological activity (i.e., the protein was properly folded, as is required for biological activity) in good yields. Particular devices and teachings regarding the use of high pressure devices are also provided in International Patent Application Publication No. WO 2007/062174. U.S. 6,489,450, U.S. 7,064,192, U.S. 2004/0038333, WO 02/062827 and WO 2007/062174 are incorporated by reference herein in their entireties.

[0007] Refolding of proteins that are unable to be properly refolded would contribute significantly to the development of protein therapeutics. Several proteins of therapeutic interest have proven refractory to refolding by chemical or other non-pressure based methods, hindering the use of those proteins in medical applications and basic and applied research. The present invention addresses these problems and provides advances and improvements in the art of refolding proteins. The current invention is based upon application of high pressure refolding methods to proteins which cannot be folded by any other methods, referred to as "difficult to fold" proteins or "never before refolded" proteins. Isolated biologically active proteins can thus be obtained from proteins which have never before been refolded in the isolated state, particularly when such isolated proteins are obtained in large quantities from recombinant processes.

SUMMARY OF THE INVENTION

[0008] This invention provides an effective and efficient method for refolding proteins which are refractory to refolding by chemical or biochemical methods. The method provides routes for overcoming the difficulties presented in refolding of proteins of this particular nature, thereby allowing production thereof. [0009] In one embodiment, the invention embraces an isolated and purified biologically active protein, wherein said protein has been isolated and/or purified in a biologically inactive form, but which has not previously been isolated and/or purified in biologically active form. The biologically inactive form can be a protein aggregate or a denatured form of the protein, or both.

[0010] In another embodiment, the invention embraces an isolated and purified biologically active protein, wherein said protein has been isolated and/or purified in a biologically inactive form, but which has not previously been isolated and/or purified in biologically active form, where the protein was converted from biologically inactive form to biologically active form by the application of high pressure.

[0011] In another embodiment, the invention embraces a method for producing biologically active protein from a mixture comprising aggregated or denatured protein which has not previously been refolded by non-pressure-based methods, comprising: subjecting the aggregated or denatured protein to from about 0.25 kbar to about 12 kbar of pressure for a time sufficient for disaggregation or renaturation of the protein, and reducing the pressure to atmospheric pressure, wherein the protein retains biological activity.

[0012] In certain embodiments, the non-high-pressure based methods with which the protein cannot be refolded include one or more of additive-introduced stepwise dialysis methods; centrifugal filtration methods; chaotrope-based methods; chaperone-assisted refolding methods (including reverse-micelle chaperone assisted refolding); column-based refolding methods, including affinity immobilization, cobalt-chelating chromatography, hydrophobic interaction chromatography, ion-exchange chromatography, nickel-chelating chromatography, oxidative refolding chromatography, protein immobilization, size exclusion chromatography with partner subunits, and size-exclusion chromatography; detergent-based methods, detergent precipitation/sequestration methods; diafiltration methods; dialysis methods; dilution methods; dilution-into detergent methods; dilution with complex partner subunits methods; dilution/column refolding combination methods; dilution/dialysis combination methods; gel filtration methods, including gel filtration with pH and denaturant gradients; matrix-assisted dialysis methods; matrix assisted refolding/ renaturing gel filtration methods; and non-detergent sulfobetaines (NDSB) based methods [0013] In certain embodiments, the non-pressure-based methods with which the protein canot be refolded include one or more of chaotrope-based refolding methods; urea- based refolding methods; guanidinium-based refolding methods; or thermal refolding methods.

[0014] In certain embodiments, the high hydrostatic pressure is from about 500 to about 10,000 bar. In some variations, the increased hydrostatic pressure is from about 1500 to about 4000 bar. In some variations, the increased hydrostatic pressure is from about 1500 to about 3500 bar. In some variations, the increased hydrostatic pressure is from about 1500 to about 3000 bar. In particular variations, the increased hydrostatic pressure is about 2000 bar.

[0015] In some embodiments, reducing the pressure to atmospheric pressure comprises stepwise pressure reductions. In certain variations, during at least one pressure reduction step the rate of pressure reduction is from about 5000 bar/4 days to about 5000 bar/sec. In some embodiments, during at least one pressure reduction step the rate of pressure reduction is about 250 bar/5 minutes. In some embodiments there are at least 2 stepwise pressure reductions. In additional embodiments, there are more than 2 stepwise pressure reductions.

[0016] In some embodiments, the step of reducing the pressure to atmospheric pressure further comprises a hold period at constant pressure after at least one of the stepwise pressure reductions. In some embodiments, the hold period is from about 2 hours to about 50 hours. In certain embodiments, the hold period is about 6 hours. In some embodiments, during the hold period the constant pressure is from about 500 bar to about 2000 bar. In certain embodiments, during the hold period the constant pressure is about 1000 bar. In certain embodiments, during the hold period the constant pressure is about one-half of the initial high hydrostatic pressure. In some embodiments, during the hold period the constant pressure is about one-third of the initial high hydrostatic pressure.

[0017] In some embodiments, the step of reducing the pressure includes a continuous rate of pressure reduction. In some embodiments, the rate of pressure reduction is from about 5000 bar/1 sec to about 5000 bar/4 days. In certain embodiments, the rate of pressure reduction is 250 bar/5 minutes.

[0018] In certain embodiments the methods further include, at any point in the method, adding one or more disulfide shuffling agent pairs to the mixture in an amount sufficient to facilitate formation of native disulfide bonds in the protein. In one embodiment, the method comprises subjecting the mixture to a further period of increased hydrostatic pressure compared to atmospheric pressure for a time sufficient for formation of native disulfide bonds in the protein.

[0019] In some embodiments, the pH of the mixture is from about pH 3 to about pH

12. In certain embodiments, the pH of the mixture is from about pH 6 to about pH 8.5. In particular embodiments, pH of the mixture is from about pH 7 to about pH 8.5.

[0020] In some embodiments, the step of subjecting the mixture to high pressure is performed at a temperature from about -2O⁰C to about 100⁰C. In some variations, the step of subjecting the mixture to high pressure is performed at a temperature from about O⁰C to about

75⁰C.

[0021] In some embodiments, the mixture further comprises one or more additional agents selected from one or more stabilizing agents, one or more buffering agents, one or more surfactants, one or more disulfide shuffling agent pairs, one or more chaotropic agents, one or more salts, and combinations of two or more of the foregoing.

[0022] In certain variations, the one or more additional agents is one or more stabilizing agents. In some embodiments, the one or more stabilizing agents is selected from one or more free amino acids, one or more preferentially excluding compounds, trimethylamine oxide, one or more cyclodextrans, one or more molecular chaperones, and combinations of two or more of the foregoing.

[0023] In some embodiments, the one or more stabilizing agents is one or more preferentially excluding compounds, hi certain embodiments, the one or more preferentially excluding compounds is one or more sugars, glycerol, hexylene glycol, or combinations of two or more of the foregoing. In some embodiments, the one or more preferentially excluding compounds is one or more sugars. In certain embodiments, the one or more sugars is sucrose, trehalose, dextrose, mannose, or combinations of two or more of the foregoing. In some variations, the one or more sugars is present at a concentration of from about 0.1 mM to about the solubility limit of the sugar.

[0024] In some embodiments, the one or more stabilizing agents is one or more free amino acids. In certain variations, the one or more free amino acids is arginine, lysine, proline, glutamine, glycine, histidine, or combinations of two or more of the foregoing. In some embodiments, the one or more free amino acids is present at a concentration of from about 0.1 mM to about the solubility limit of the free amino acid. [0025] In some variations, the one or more stabilizing agents is a cyclodextran. In some embodiments, the cyclodextran is present at a concentration of from about 0.1 mM to about the solubility limit of the cyclodextran.

[0026] In some variations, the one or more stabilizing agents is a molecular chaperone. In certain variations the molecular chaperone is GroEs or GroEL. In some variations, the molecular chaperone is present at a concentration of from about 0.01 mg/ml to about 10 mg/ml.

[0027] In some embodiments, the one or more additional agents is one or more surfactants. In particular embodiments, the one or more surfactants is selected from polysorbates, polyoxyethylene ethers, non-detergent sulfo-betaines, alkyltrimethylammonium bromides, alkyltrimethyl ammonium chlorides, pyranosides and combinations of two or more of the foregoing. In some variations, the one or more surfactants is selected from polysorbate 80, polysorbate 20, Triton X-100, Brij 35, sarcosyl, octyl phenol ethoxylate, β-octyl-gluco- pyranoside, polyoxyethyleneglycol dodecyl ether, sodium dodecyl sulfate, polyethoxysorbitan, deoxycholate, sodium octyl sulfate, sodium tetradecyl sulfate, sodium cholate, octylthioglucopyranoside, n-octylglucopyranoside, octylphenoxypolyethoxy-ethanol, polyoxyethylene sorbitan, cetylpyridinium chloride, and sodium bis (2-ethylhexyl) sulfosuccinate.

[0028] In some variations, the one or more additional agents is one or more buffering agents. In certain variations, the one or more buffering agents is an organic buffer. In particular variations, the one or more buffering agents is an inorganic buffer. In some embodiments, the one or more buffering agents is selected from phosphate buffers, carbonate buffers, citrate, Tris, MOPS, MES, acetate, and HEPES.

[0029] In some embodiments, the one or more additional agent is one or more chaotropic agents. In some variations, the one or more chaotropic agents is guanidine, guanidine sulfate, guanidine hydrochloride, urea, or thiocyanate. In some embodiments, the one or more chaotropic agents is urea in a concentration from about 0.1 mM to about 8 M. In certain embodiments, the one or more chaotropic agents is guanidine hydrochloride in a concentration of from about 0.ImM to about 8 M.

[0030] In some variations, the one or more additional agents are one or more disulfide shuffling agent pair. In some embodiments, the disulfide shuffling agent pair include an oxidizing agent and a reducing agent. In some variations, the oxidizing agent is at least one of oxidized glutathione, cystine, cystamine, molecular oxygen, iodosobenzoic acid, sulfitolysis or a peroxide and the reducing agent is at least one of glutathione, cysteine, cysteamine, diothiothreitol, dithioerythritol, tris(2-carboxyethyl)phosphine hydrochloride, or β-mercaptoethanol. In certain variations, the disulfide shuffling agent pair is present at an oxidized concentration of from about 0.1 mM to about 100 mM oxidized thiol. In some variations, the concentration is from about O.lmM to about 10 mM. In certain variations, the concentration is from about 2 mM to about 6 mM.

[0031] In some embodiments, the time during which the mixture is subjected to high hydrostatic pressure is from about 15 minutes to about one week. In some embodiments, the time is from about 15 minutes to about 50 hours. In some embodiments, the time is from about 6 hours to about 18 hours.

[0032] In some embodiments, the protein is a monomer. In certain embodiments, the protein is a dimer. In particular embodiments, the dimer is a homodimer. In some variations, the dimer is a heterodimer. In some embodiments, the protein is a trimer. In particular embodiments, the dimer is a homotrimer. In certain embodiments, the dimer is a heterotrimer. In particular embodiments, the protein is a tetramer. In particular embodiments, the tetramer is a homotretramer. In some variations, the tetramer is a heterotetramer.

[0033] In some variations, the total concentration of protein in the mixture is from about 0.01 mg/mL to about 300 mg/mL. In some embodiments, the total concentration of protein in the mixture is from about 0.01 mg/mL to about 150 mg/mL.

[0034] In some embodiments, the method further includes agitation of the mixture during the period of high hydrostatic pressure.

[0035] In certain embodiments where chaotropic agents are included, the methods further include the step of reducing the amount of the chaotropic agent present in the mixture after the high hydrostatic pressure.

[0036] In some variations, the method does not include any chaotropic agent.

[0037] The invention also embraces proteins prepared by any of the methods described herein.

DETAILED DESCRIPTION OF THE INVENTION

[0038] All publications and patents mentioned herein are hereby incorporated by reference in their respective entireties. The publications and patents disclosed herein are provided solely for their disclosure. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate any publication and/or patent, including any publication and/or patent cited herein.

[0039] The embodiments of the present invention described below are not intended to be exhaustive or to limit the invention to the precise forms disclosed in the following detailed description. Rather, the embodiments are chosen and described so that others skilled in the art can appreciate and understand the principles and practices of the present invention. [0040] By "high pressure" is meant a pressure of at least about 250 bar. The pressure at which the methods of the invention are used can be at least about 250 bar of pressure, at least about 400 bar of pressure, at least about 500 bar of pressure, at least about 1 kbar of pressure, at least about 2 kbar of pressure, at least about 3 kbar of pressure, at least about 5 kbar of pressure, or at least about 10 kbar of pressure.

[0041] As used herein, a "protein aggregate" is defined as being composed of a multiplicity of protein molecules wherein non-native noncovalent interactions and/or non- native covalent bonds (such as non-native intermolecular disulfide bonds) hold the protein molecules together. Typically, but not always, an aggregate contains sufficient molecules so that it is insoluble; such aggregates are insoluble aggregates. There are also abnormal oligomeric proteins which occur in aggregates in solution; such aggregates are soluble aggregates. In addition, there is typically (but not always) a display of at least one epitope or region on the aggregate surface which is not displayed on the surface of native, non- aggregated protein. "Inclusion bodies" are a type of aggregate of particular interest to which the present invention is applicable. Other protein aggregates include, but are not limited to, soluble and insoluble precipitates, soluble non-native oligomers, gels, fibrils, films, filaments, protofibrils, amyloid deposits, plaques, and dispersed non-native intracellular oligomers. [0042] "Atmospheric," "ambient," or "standard" pressure is defined as approximately

15 pounds per square inch (psi) or approximately 1 bar or approximately 100,000 Pascals. [0043] "Biological activity" of a protein as used herein, means that the protein retains at least about 10% of maximal known specific activity as measured in an assay that is generally accepted in the art to be correlated with the known or intended utility of the protein. For proteins intended for therapeutic use, the assay of choice is one accepted by a regulatory agency to which data on safety and efficacy of the protein must be submitted. A protein having at least about 10% of maximal known specific activity is "biologically active" for the purposes of the invention. [0044] "Denatured," as applied to a protein imthe present context, means that native secondary, tertiary, and/or quaternary structure is disrupted to an extent that the protein does not have biological activity.

[0045] In contrast to "denatured," the "native conformation" of a protein refers to the secondary, tertiary and/or quaternary structures of a protein as it occurs in nature in its biologically active state.

[0046] "Refolding" in the present context means the process by which a fully or partially denatured polypeptide adopts secondary, tertiary and quaternary structure like that of the cognate native molecule. A properly refolded polypeptide has biological activity that is at least about 10% of the non-denatured molecule, preferably biological activity that is substantially that of the non-denatured molecule. Where the native polypeptide has disulfide bonds, oxidation to form native disulfide bonds is a desired component of the refolding process.

Proteins for refolding

[0047] Proteins which can be refolded according to methods of the invention include proteins which have never been refolded before in a purified and isolated state. Such proteins can be produced recombinantly. Such proteins can also be produced by isolation from a natural source.

[0048] To be classified as a "never before refolded" protein (or, alternatively, a

"difficult to refold" protein), unsuccessful refolding of the protein should have been attempted with at least one non-pressure method. For example, if a protein cannot be refolded after solubilization in high concentrations of urea or guanidinium salts (e.g., 6M, 7M, 8M, or higher), followed by replacement of the urea or guanidinium solution with a solution amenable to protein refolding, it can be classified as a difficult-to-refold protein. [0049] A protein may be classified as a difficult-to-refold protein if the protein cannot be refolded by one or more of the following methods: additive-introduced stepwise dialysis methods; centrifugal filtration methods; chaperone-assisted refolding methods (including reverse-micelle chaperone assisted refolding); column-based refolding methods, including affinity immobilization, cobalt-chelating chromatography, hydrophobic interaction chromatography, ion-exchange chromatography, nickel-chelating chromatography, oxidative refolding chromatography, protein immobilization, size exclusion chromatography with partner subunits, and size-exclusion chromatography; detergent-based methods, detergent precipitation/sequestration methods; diafiltration methods; dialysis methods; dilution methods; dilution-into detergent methods; dilution with complex partner subunits methods; dilution/column refolding combination methods; dilution/dialysis combination methods; gel filtration methods, including gel filtration with pH and denaturant gradients; matrix-assisted dialysis methods; matrix assisted refolding/ renaturing gel filtration methods; non-detergent sulfobetaines (NDSB) methods; methods based on the Novexin kit (Novexin, Cambridge, United Kingdom); or other non-high-pressure based methods. In another embodiment, if refolding of a protein proceeds with less than about a 30%, 25%, 20%, 15%, 10%, 5%, 2%, or 1% yield based on any of these non-high-pressure based methods, the protein can be classified as a difficult to fold protein.

[0050] Proteins which can be refolded with the methods of the invention include a wide variety of polypeptide or polypeptide-containing molecules. Proteins which can be refolded include both globular and fibrous proteins; the preferred protein is a globular protein. Proteins which can be refolded include, but are not limited to, monomeric, dimeric, multimeric, heterodimeric, heterotrimeric, and heterotetrameric proteins; disulfide bonded proteins; glycosylated proteins; helical proteins; and alpha helix- and beta sheet-containing proteins. Examples of particular proteins include, but are not limited to, hormones, antibodies, enzymes, and metal binding proteins. Examples of protein structures which can be refolded include up-and-down helix bundle, Greek key helix bundle, miscellaneous antiparallel alpha helix, singly wound parallel beta barrel, doubly wound parallel beta sheet, miscellaneous parallel alpha/beta, up-and-down beta barrel (antiparallel beta), Greek key beta barrel (antiparallel beta), multiple, partial, and other beta barrel, open-face beta sandwich, miscellaneous antiparallel beta, SS-rich (disulfide-rich), and metal-rich proteins. [0051] "Heterologous" proteins are proteins which are normally not produced by a particular host cell. Recombinant DNA technology has permitted the expression of relatively large amounts of heterologous proteins (for example, growth hormone) from transformed host cells such as E. coli. These proteins are often sequestered in insoluble inclusion bodies in the cytoplasm and/or periplasm of the host cell. The inclusion bodies or cytoplasmic aggregates contain, at least in part, the heterologous protein to be recovered. These aggregates often appear as bright spots under a phase contrast microscope. [0052] Among other resources, the REFOLD database (Chow et al., Protein Expr.

Purif. 46(1):166-71 (2006), and Buckle et al., Nature Methods 2:3 (2005)), accessible at World Wide Web URL refold.med.monash.edu.au, can be consulted to determine whether a protein has been successfully refolded using non-high-pressure based methods.

Protein Purification

[0053] A wide variety of techniques are known in the art for protein separation and purification, such as affinity chromatography, high-pressure liquid chromatography (HPLC), dialysis, ion exchange chromatography, size exclusion chromatography, reverse-phase chromatography, ammonium sulfate precipitation, or electrophoresis. Several conditions for HPLC can be varied for enhancing separation, such as the stationary and mobile phases. HPLC can be used with ion-exchange columns, reverse-phase columns, affinity columns, size-exclusion columns, and other types of columns. FPLC, or "Fast Performance Liquid Chromatography," can also be used. Gel-filtration chromatography can be used at low solvent pressures. Removal of small molecules (such as chaotropes, kosmotropes, surfactants, detergents, reducing agents, oxidizing agents, or small molecule binding partners) from protein solutions can be achieved via diafiltration, ultrafiltration, or dialysis. [0054] For refolding, the protein can be present in varying degrees of purity, for example, as part of a whole cell slurry, a partially purified cell preparation, a partially purified preparation of inclusion bodies, or a precipitated partially purified protein. Further purification of the protein can be performed after refolding if necessary.

Other considerations

[0055] Several conditions can be adjusted for optimal protein refolding.

[0056] Protein Concentration: the concentration of protein can be adjusted for optimal protein refolding. One advantage of high-pressure protein refolding is that much higher concentrations of protein can be used as compared to chemical refolding techniques. Protein concentrations of at least about 0.01 mg/ml, at least aboutθ.1 mg/ml, at least about 1.0 mg/ml, at least about 5.0 mg/ml, at least about 10 mg/ml, or at least about 20 mg/ml can be used. Protein in the mixture may be present in a concentration of from about 0.001 mg/ml to about 300 mg/ml. Thus, in some embodiments the protein is present in a concentration of from about 0.01 mg/ml to about 250 mg/ml, from about 0.01 mg/ml to about 200 mg/ml, from about 0.01 mg/ml to about 150 mg/ml, from about 0.01 mg/ml to about 100 mg/ml, from about 0.01 mg/ml to about 50 mg/ml, from about 0.01 mg/ml to about 30 mg/ml, from about 0.05 mg/ml to about 300 mg/ml, from about 0.05 mg/ml to about 250 mg/ml, from about 0.05 mg/ml to about 200 mg/ml, from about 0.05 mg/ml to about 150 mg/ml, from about 0.05 mg/ml to about 100 mg/ml, from about 0.05 mg/ml to about 50 mg/ml, from about 0.05 mg/ml to about 30 mg/ml, from about 10 mg/ml to about 300 mg/ml, from about 10 mg/ml to about 250 mg/ml, from about 10 mg/ml to about 200 mg/ml, from about 10 mg/ml to about 150 mg/ml, from about 10 mg/ml to about 100 mg/ml, from about 10 mg/ml to about 50 mg/ml, from about 10 mg/ml to about 30 mg/ml, from about 0.1 mg/ml to about 100 mg/ml, from about 0.1 mg/ml to about 10 mg/ml, from about 1 mg/ml to about 100 mg/ml, from about 1 mg/ml to about 10 mg/ml, from about 10 mg/ml to about 100 mg/ml, or from about 50 mg/ml to about 100 mg/ml can be used.

[0057] As used in the present context the phrase "a period of time sufficient to form biologically active protein" and cognates thereof refer to the time needed for the protein aggregates or denatured protein to be disaggregated and to adopt a conformation where the protein is biologically active. Typically, the time sufficient for solubilization is about 15 minutes to about 50 hours, or possibly longer depending on the particular protein, (e.g., as long as necessary for the protein; for example, up to about 1 week, about 5 days, about 4 days, about 3 days, etc.). Thus, in some embodiments of the methods, the time sufficient for formation of biologically active protein may be from about 2 to about 30 hours, from about 2 to about 24 hours, from about 2 to about 18 hours, from about 1 to about 10 hours, from about 1 to about 8 hours, from about 1 to about 6 hours, from about 2 to about 10 hours, from about 2 to about 8 hours, from about 2 to about 6 hours, or about 2 hours, about 6 hours, about 10 hours, about 16 hours, about 20 hours, or about 30 hours, from about 2 to about 10 hours, from about 2 to about 8 hours, from about 2 to about 6 hours, from about 12 to about 18 hours, or from about 10 to about 20 hours.

[0058] The mixture comprising protein aggregates or denatured protein is typically an aqueous solution or aqueous suspension. The mixture may also include other components. These additional components may be one or more additional agents including: one or more stabilizing agents, one or more buffering agents, one or more surfactants, one or more disulfide shuffling agent pairs, one or more salts, one or more chaotropes, or combinations of two or more of the foregoing.

[0059] The amounts of the additional agents will vary depending on the selection of the protein, however, the effect of the presence (and amount) or absence of each additional agent or combinations of agents can be determined and optimized using the teachings provided herein. Exemplary additional agents include, but are not limited to, buffers (examples include, but are not limited to, phosphate buffer, borate buffer, carbonate buffer, citrate buffer, HEPES, MEPS), salts (examples include, but are not limited to, the chloride, sulfate, and carbonate salts of sodium, zinc, calcium, ammonium and potassium), chaotropes (examples include, but are not limited to, urea, guanidine hydrochloride, guanidine sulfate and sarcosine), and stabilizing agents (e.g., preferential excluding compounds, etc.). [0060] Non-specific protein stabilizing agents act to favor the most compact conformation of a protein. Such agents include, but are not limited to, one or more free amino acids, one or more preferentially excluding compounds, trimethylamine oxide, cyclodextrans, molecular chaperones, and combinations of two or more of the foregoing. [0061] Amino acids can be used to prevent reaggregation and facilitate the dissociation of hydrogen bonds. Typical amino acids that can be used, but not limited to, are arginine, lysine, proline, glycine, histidine, and glutamine or combinations of two or more of the foregoing. In some embodiments, the free amino acid(s) is present in a concentration of about 0.ImM to about the solubility limited of the amino acid, and in some variations from about 0.1 mM to about 2 M. The optimal concentration is a function of the desired protein and should favor the native conformation.

[0062] Preferentially excluding compounds can be used to stabilize the native confirmation of the protein of interest. Possible preferentially excluding compounds include, but are not limited to, sucrose, hexylene glycol, sugars (e.g., sucrose, trehalose, dextrose, mannose), and glycerol. The range of concentrations that can be use are from O.lmM to the maximum concentration at the solubility limit of the specific compound. The optimum preferential excluding concentration is a function of the protein of interest. [0063] In particular embodiments, the preferentially excluding compound is one or more sugars (e.g., sucrose, trehalose, dextrose, mannose or combinations of two or more of the foregoing). In some embodiments, the sugar(s) is present in a concentration of about 0.1 mM to about the solubility limit of the particular compound. In some embodiments, the concentration is from about O.lmM to about 2M, from about O.lmM to about 1.5M, from about O.lmM to about IM, from about O.lmM to about 0.5M, from about 0.1 mM to about 0.3M, from about 0.1 mM to about 0.2 M, from about 0.1 mM to about 0.1 mM, from about 0.1 mM to about 50 mM, from about 0.1 mM to about 25 mM, or from about 0.1 mM to about 1O mM.

[0064] In some embodiments, the stabilizing agent is one or more of sucrose, trehalose, glycerol, betaine, amino acid(s), or trimethylamine oxide. [0065] In certain embodiments, the stabilizing agent is a cyclodextran. In some embodiments, the cyclodextran is present in a concentration of about 0.1 mM to about the solubility limit of the cyclodextran. In some variations from about 0.1 mM to about 2 M.

[0066] In certain embodiments, the stabilizing agent is a molecular chaperone. In some embodiments, the molecular chaperone is present in a concentration of about 0.01 mg/ml to 10 mg/ml.

[0067] A single stabilizing agent maybe be used or a combination of two or more stabilizing agents (e.g., at least two, at least three, or 2 or 3 or 4 stabilizing agents). Where more than one stabilizing agent is used, the stabilizing agents may be of different types, for example, at least one preferentially excluding compound and at least one free amino acid, at least one preferentially excluding compound and betaine, etc.

[0068] Buffering agents may be present to maintain a desired pH value or pH range.

Numerous suitable buffering agents are known to the skilled artisan and should be selected based on the pH that favors (or at least does not disfavor) the native conformation of the protein of interest. Either inorganic or organic buffering agents may be used. Suitable concentrations are known to the skilled artisan and should be optimized for the methods as described herein according to the teaching provided based on the characteristics of the desired protein.

[0069] Thus, in some embodiments, at least one inorganic buffering agent is used

(e.g., phosphate, carbonate, etc.). In certain embodiments, at least one organic buffering agent is used (e.g., citrate, acetate, Tris, MOPS, MES, HEPES, etc.) Additional organic and inorganic buffering agents are well known to the art.

[0070] In some embodiments, the one or more buffering agents is phosphate buffer, borate buffer, carbonate buffer, citrate buffer, HEPES, MEPS, MOPS, CAPS, TAPS, CHES,

MES, or acetate buffer.

[0071] In some embodiments, the one or more buffering agents is phosphate buffers, carbonate buffers, citrate, Tris, MOPS, MES, acetate or HEPES.

[0072] A single buffering agent maybe be used or a combination of two or more buffering agents (e.g., at least two, at least 3, or 2 or 3 or 4 buffering agents).

[0073] A "surfactant" as used in the present context is a surface active compound which reduces the surface tension of water. Surfactants are used to improve the solubility of certain proteins. Surfactants should generally be used at concentrations above or below their critical micelle concentration (CMC), for example, from about 5% to about 20% above or below the CMC. However, these values will vary dependent upon the surfactant chosen, for example, surfactants such as, beta-octylgluco-pyranoside may be effective at lower concentrations than, for example, surfactants such as TWEEN-20 (polysorbate 20). The optimal concentration is a function of each surfactant, which has its own CMC. [0074] Useful surfactants include nonionic (including, but not limited to, t- octylphenoxypolyethoxy-ethanol and polyoxyethylene sorbitan), anionic (e.g., sodium dodecyl sulfate) and cationic (e.g., cetylpyridinium chloride) and amphoteric agents. Suitable surfactants include, but are not limited to deoxycholate, sodium octyl sulfate, sodium tetradecyl sulfate, polyoxyethylene ethers, sodium cholate, octylthioglucopyranoside, n- octylglucopyranoside, alkyltrimethylanmonium bromides, alkyltrimethyl ammonium chlorides, non-detergent sulfobetaines, and sodium bis (2 ethylhexyl) sulfosuccinate. In some embodiments the surfactant may be polysorbate 80, polysorbate 20, sarcosyl, Triton X-100, β-octyl-gluco-pyranoside, or Brij 35.

[0075] In some embodiments the one or more surfactant may be a polysorbate, polyoxyethylene ether, alkyltrimethylammonium bromide, pyranosides or combination of two or more of the foregoing. In certain embodiments, the one or more surfactant may be β- octyl-gluco-pyranoside, Brij 35, or a polysorbate.

[0076] In certain embodiments the one or more surfactant may be octyl phenol ethoxylate, β-octyl-gluco-pyranoside, polyoxyethyleneglycol dodecyl ether, sarcosyl, sodium dodecyl sulfate, polyethoxysorbitan, deoxycholate, sodium octyl sulfate, sodium tetradecyl sulfate, sodium cholate, octylthioglucopyranoside, n-octylglucopyranoside, sodium bis (2- ethylhexyl) sulfosuccinate or combinations of two or more of the foregoing. A single surfactant maybe be used or a combination of two or more surfactants (e.g., at least two, at least 3, or 2 or 3 or 4 surfactants).

[0077] Where the desired protein contains disulfide bonds in the native conformation it is generally advantageous to include at least one disulfide shuffling agent pair in the mixture. The disulfide shuffling agent pair facilitates the breakage of strained non-native disulfide bonds and the reformation of native-disulfide bonds.

[0078] In general, the disulfide shuffling agent pair includes a reducing agent and an oxidizing agent. Exemplary oxidizing agents oxidized glutathione, cystine, cystamine, molecular oxygen, iodosobenzoic acid, sulfitolysis and peroxides. Exemplary reducing agents include glutathione, cysteine, cysteamine, diothiothreitol, dithioerythritol, tris(2- carboxyethyl)phosphine hydrochloride, or β-mercaptoethanol. [0079] Exemplary disulfide shuffling agent pairs include oxidized/reduced glutathione, cystamine/cysteamine, and cysteine/cysteine. Additional disulfide shuffling agent pairs are described by Gilbert HF. (1990). "Molecular and Cellular Aspects of Thiol Disulfide Exchange." Advances in Enzymology and Related Areas of Molecular Biology 63:69-172, and Gilbert HF. (1995). "Thiol/Disulfide Exchange Equilibria and Disulfide Bond Stability." Biothiols, Pt A. p 8-28, which are hereby incorporated by reference in their entirety.

[0080] The selection and concentration of the disulfide shuffling agent pair will depend upon the characteristics of the desired protein. Typically concentration of the disulfide shuffling agent pair taken together (including both oxidizing and reducing agent) is from about 0.1 mM to about 100 mM of the equivalent oxidized thiol, however, the concentration of the disulfide shuffling agent pair should be adjusted such that the presence of the pair is not the rate limiting step in disulfide bond rearrangement. [0081] In some embodiments, the concentration will be about 1 mM, about 2 mM, about 3 mM about 5 mM, about 8 mM, about 9 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, or from about 80 mM to about 100 mM, from about 0.1 mM to about 20 mM, from about 10 mM to about 50 mM, from about 1 mM to about 100 mM, from about 50 mM to about 100 mM, from about 20 mM to about 100 mM, from about 0.1 mM to about 10 mM, from about 0.1 mM to about 8 mM; from about 0.1 mM to about 6 mM, from about 0.1 mM to about 7 mM, from about 0.1 mM to about 5 mM, from about 0.1 mM to about 3 mM, from about 0.1 mM to about 1 mM.

[0082] A single disulfide shuffling agent pair maybe be used or a combination of two or more disulfide shuffling agent pairs (e.g., at least two, at least 3, or 2 or 3 or 4 disulfide shuffling agent pairs).

[0083] Chaotropic agents (also referred to as a "chaotrope") are compounds, including, without limitation, guanidine, guanidine hydrochloride (guanidinium hydrochloride, GdmHCl), guanidine sulfate, urea, sodium thiocyanate, and/or other compounds which disrupt the noncovalent intermolecular bonding within the protein, permitting the polypeptide chain to assume a substantially random conformation [0084] Chaotropic agents may be used in concentration of from about 10 mM to about 8 M. The optimal concentration of the chaotropic agent will depend on the desired protein as well as on the particular chaotropes selected. The choice of particular chaotropic agent and determination of optimal concentration can be optimized by the skilled artisan in view of the teachings provided herein.

[0085] In some embodiments, the concentration of the chaotropic agent will be, for example, from about 10 mM to about 8 M, from about 10 mM to about 7 M, from about 10 raM to about 6 M, from about 0.1 M to about 8 M, from about 0.1 M to about 7 M, from about 0.1 M to about 6 M, from about 0.1 M to about 5 M, from about 0.1 M to about 4 M, from about 0.1 M to about 3 M, from about 0.1 M to about 2 M, from about 0.1 M to about 1

M, from about 10 mM to about 4 M, from about 10 mM to about 3 M, from about 10 mM to about 2 M, from about 10 mM to about 1 M, or about, 10 mM, about 50 mM, about 75 mM, about 0.1 M, about 0.5 M, about 0.8 M, about 1 M, about 2 M, about 3 M, about 4 M, about 5

M, about 6 M, about 7 M, about 8 M.

[0086] When used in the present methods, it is often advantageous to use chaotropic agents in non-denaturing concentrations to facilitate the dissociation of hydrogen bonds.

While a non-denaturing concentration will vary depending on the desired protein, the range of non-denaturing concentrations is typically from about 0.1 to about 4 M. In some embodiments the concentration is from about 0.1 M to about 2 M.

[0087] In certain embodiments, guanidine hydrochloride or urea are the chaotropic agents.

[0088] A single chaotropic agent maybe be used or a combination of two or more chaotropic agents (e.g., at least two, at least 3, or 2 or 3 or 4 chaotropic agents).

[0089] Agitation: Protein solutions can be agitated before and/or during refolding.

Agitation can be performed by methods including, but not limited to, ultrasound energy

(sonication), mechanical stirring, mechanical shaking, pumping through mixers, or via cascading solutions.

[0090] Temperature: The methods described herein can be performed at a range of temperature values, depending on the particular protein of interest. The optimal temperature, in concert with other factors, can be optimized as described herein. Proteins can be refolded at various temperatures, including at about room temperature, about 25 ⁰C, about 30 ⁰C, about

37 ⁰C, about 50 ⁰C, about 75 ⁰C, about 100 ⁰C, about 125 ⁰C, or ranges of from about 20 to about 125 ⁰C, about 25to aboutl25 ⁰C, about 25to aboutlOO ⁰C, about 25to about75 ⁰C, about

25to about50 ⁰C, about 50to aboutl25 ⁰C, about 50to aboutlOO ⁰C, about 50to about75 ⁰C, about 75to aboutl25 ⁰C, about 5to aboutlOO ⁰C, or about lOOto aboutl25 ⁰C. [0091] In some embodiments of the methods, the temperature can range from about -

20°C to about 100°C without adversely affecting the protein of interest, provided that prior to return to room temperature, the mixture is brought to a temperature at which it will not freeze. Thus in certain embodiments, the temperature may be from about 0°C to about 75⁰C, from about O⁰C to about 55⁰C, from about 0°C to about 35⁰C, from about O⁰C to about 25⁰C, from about 20°C to about 75⁰C, from about 2O⁰C to about 65°C, from about 2O⁰C to about 35°C, from about 2O⁰C to about 25°C.

[0092] Although increased temperatures are often used to cause aggregation of proteins, when coupled with increased hydrostatic pressure it has been found that increased temperatures can enhance refolding recoveries effected by high pressure treatment, provided that the temperatures are not so high as to cause irreversible denaturation. Generally, the increased temperature for refolding should be about 20⁰C lower than the temperatures at which irreversible loss of activity occurs. Relatively high temperatures (for example, about 60⁰C to about 125°C, about 80° C to about 110⁰C, including about 100⁰C, about 105⁰C, about 11O⁰C, about 115⁰C, about 120⁰C and about 125°C) may be used while the solution is under pressure, as long as the temperature is reduced to a suitably low temperature before depressurizing. Such a suitably low temperature is defined as one below which thermally- induced denaturation or aggregation occurs at atmospheric conditions. [0093] "High pressure" or "high hydrostatic pressure," for the purposes of the invention is defined as pressures of from about 500 bar to about 10,000 bar. [0094] In some embodiments, the increased hydrostatic pressure may be from about

500 bar to about 5000 bar, from about 500 bar to about 4000 bar, from about 500 bar to about 2000 bar, from about 500 bar to about 2500 bar, from about 500 bar to about 3000 bar, from about 500 bar to about 6000 bar, from about 1000 bar to about 5000 bar, from about 1000 bar to about 4000 bar, from about 1000 bar to about 2000 bar, from about 1000 bar to about 2500 bar, from about 1000 bar to about 3000 bar, from about 1000 bar to about 6000 bar, from about 1500 bar to about 5000 bar, from about 1500 bar to about 3000 bar, from about 1500 bar to about 4000 bar, from about 1500 bar to about 2000 bar, from about 2000 bar to about 5000 bar, from about 2000 bar to about 4000 bar, from about 2000 bar to about 3000 bar, or about 1000 bar, about 1500 bar, about 2000 bar, about 2500 bar, about 3000 bar, about 3500 bar, about 4000 bar, about 5000 bar, about 6000 bar, about 7000 bar, about 8000 bar, about 9000 bar. [0095] Reduction of pressure: Where the reduction in pressure is performed in a continuous manner, the rate of pressure reduction can be constant or can be increased or decreased during the period in which the pressure is reduced. In some variations, the rate of pressure reduction is from about 5000 bar/1 sec to about 5000 bar/4 days (or about 3 days, about 2 days, about 1 day). Thus in some variations the rate of pressure reduction can be performed at a rate of from about 5000 bar/1 sec to about 5000 bar/80 hours, from about 5000 bar/1 sec to about 5000 bar/72 hours, from about 5000 bar/1 sec to about 5000 bar/60 hours, from about 5000 bar/1 sec to about 5000 bar/50 hours, from about 5000 bar/1 sec to about 5000 bar/48 hours, from about 5000 bar/1 sec to about 5000 bar/32 hours, from about 5000 bar/1 sec to about 5000 bar/24 hours, from about 5000 bar/1 sec to about 5000 bar/20 hours, from about 5000 bar/1 sec to about 5000 bar/18 hours, from about 5000 bar/1 sec to about 5000 bar/16 hours, from about 5000 bar/1 sec to about 5000 bar/12 hours, from about 5000 bar/1 sec to about 5000 bar/8 hours, from about 5000 bar/1 sec to about 5000 bar/4 hours, from about 5000 bar/1 sec to about 5000 bar/2 hours, from about 5000 bar/1 sec to about 5000 bar/1 hour, from about 5000 bar/1 sec to about 1000 bar/min, about 5000 bar/1 sec to about 500 bar/min, about 5000 bar/1 sec to about 300 bar/min, about 5000 bar/1 sec to about 250 bar/min, about 5000 bar/1 sec to about 200 bar/min, about 5000 bar/1 sec to about 150 bar/min, about 5000 bar/1 sec to about 100, about 5000 bar/1 sec to about 80 bar/min, about 5000 bar/1 sec to about 50 bar/min, or about 5000 bar/1 sec to about 10 bar/min. For example, about 10 bar/min, about 250 bar/5 minute, about 500 bar/5 minutes, about 1000 bar/5 minutes, about 250 bar/5 minutes, 2000 bar/50 hours, 3000 bar/50 hours, 40000 bar/50 hours, etc. In some embodiments, the pressure reduction may be approximately instantaneous, as in where pressure is released by simply opening the device in which the sample is contained and immediately releasing the pressure.

[0096] Where the reduction in pressure is performed in a stepwise manner, the process comprises dropping the pressure from the highest pressure used to at least a secondary level that is intermediate between the highest level and atmospheric pressure. The goal is to provide an incubation or hold period at or about this intermediate pressure zone that permits a protein to adopt a desired conformation.

[0097] In some embodiments, where there are at least two stepwise pressure reductions there may be a hold period at a constant pressure between intervening steps. The hold period may be from about 10 minutes to about 50 hours (or longer, depending on the nature of the protein of interest). In some embodiments, the hold period may be from about 2 to about 30 hours, from about 2 to about 24 hours, from about 2 to about 18 hours, from about 1 to about 10 hours, from about 1 to about 8 hours, from about 1 to about 6 hours, from about 2 to about 10 hours, from about 2 to about 8 hours, from about 2 to about 6 hours, or about 2 hours, about 6 hours, about 10 hours, about 20 hours, or about 30 hours, from about 2 to about 10 hours, from about 2 to about 8 hours, from about 2 to about 6 hours. [0098] In some variations, the pressure reduction includes at least 2 stepwise reductions of pressure (e.g., highest pressure reduced to a second pressure reduced atmospheric pressure would be two stepwise reductions). In other embodiments the pressure reduction includes more than 2 stepwise pressure reductions (e.g., 3, 4, 5, 6, etc.). In some embodiments, there is at least 1 hold period. In certain embodiments there is more than one hold period (e.g., at least 2, at least 3, at least 4, at least 5 hold periods). [0099] In some variations of the methods the constant pressure after an initial stepwise reduction may be at a hydrostatic pressure of from about 500 bar to about 5000 bar, from about 500 bar to about 4000 bar, from about 500 bar to about 2000 bar, from about 1000 bar to about 4000 bar, from about 1000 bar to about 3000 bar, from about 1000 bar to about 2000 bar, from about 1500 bar to about 4000 bar, from about 1500 bar to about 3000 bar, from about 2000 bar to about 4000 bar, or from about 2000 bar to about 3000 bar. [00100] In particular variations, constant pressure after the stepwise reduction is from about four-fifths of the pressure immediately prior to the stepwise pressure reduction to about one-tenth of prior to the stepwise pressure reduction. For example, constant pressure is at a pressure of from about four-fifths to about one-fifth, from about two-thirds to about one- tenth, from about two-thirds to about one-fifth, from about two-thirds to about one-third, about one-half, or about one-quarter of the pressure immediately prior to the stepwise pressure reduction. Where there is more than one stepwise pressure reduction step, the pressure referred to is the pressure immediately before the last pressure reduction (e.g., where 2000 bar is reduced to 1000 bar is reduced to 500 bar, the pressure of 500 bar is one-half of the pressure immediately preceding the previous reduction (1000 bar)). [00101] Where the pressure is reduced in a stepwise manner, the rate of pressure reduction (e.g., the period of pressure reduction prior to and after the hold period) may be in < the same range as that rate of pressure reduction described for continuous reduction (e.g., in a non-stepwise manner). In essence, stepwise pressure reduction is the reduction of pressure in a continuous manner to an intermediate constant pressure, followed by a hold period and then a further reduction of pressure in a continuous manner. The periods of continuous pressure reduction prior to and after each hold period may be the same continuous rate for each period of continuous pressure reduction or each period may have a different reduction rate. In some variations, there are two periods of continuous pressure reduction and a hold period. In certain embodiments, each continuous pressure reduction period has the same rate of pressure reduction. In other embodiments, each period has a different rate of pressure reduction. In particular embodiments, the hold period is from about 8 to about 24 hours. In some embodiments, the hold period is from about 12 to about 18 hours. In particular embodiments, the hold period is about 16 hours.

[00102] Combinations of the above conditions: Various combinations and permutations of the condition above, such as agitation of the protein under high pressure at an elevated temperature in the presence of chaotropes and redox reagents, can be employed as desired for optimization of refolding yields.

High Pressure Devices and Considerations

[00103] Commercially available high pressure devices and reaction vessels, such as those described in the examples, may be used to achieve the hydrostatic pressures in accordance with the methods described herein (see BaroFold Inc., Boulder Co.). Additionally devices, vessels and other materials for carrying out the methods described herein, as well as guidance regarding the performing increased pressure methods, are described in detail in U.S. Pat No. 6,489,450, which is incorporated herein in its entirety. The skilled artisan is particularly directed to column 9, lines 39-62 and Examples 2-4. International Pat. App. Pub. No. WO 02/062827, incorporated herein in its entirety, also provides the skilled artisan with detailed teachings regarding devices and use thereof for high hydrostatic pressure solubilization of aggregates throughout the specification. Particular devices and teachings regarding the use of high pressure devices are also provided in International Patent Application Publication No. WO 2007/06217, which is hereby incorporated by reference in its entirety.

[00104] Multiple-well sample holders may be used and can be conveniently sealed using self-adhesive plastic covers. The containers, or the entire multiple-well sample holder, may then be placed in a pressure vessel, such as those commercially available from the Flow International Corp. or High Pressure Equipment Co. The remainder of the interior volume of the high-pressure vessel may than be filled with water or other pressure transmitting fluid. [00105] Mechanically, there are two primary methods of high-pressure processing: batch and continuous. Batch processes simply involve filling a specified chamber, pressurizing the chamber for a period of time, and repressurizing the batch. In contrast, continuous processes constantly feed aggregates into a pressure chamber and soluble, refolded proteins move out of the pressure chamber. In both set ups, good temperature and pressure control is essential, as fluctuations in these parameters can cause inconsistencies in yields. Both temperature and pressure should be measured inside the pressure chamber and properly controlled.

[00106] There are many methods for handling batch samples depending upon the specific stability issues of each target protein. Samples can be loaded directly into a pressure chamber, in which case the aqueous solution and/or suspension would be used as the pressure medium. Alternately, samples can be loaded into any variety of sealed, flexible containers, including those described herein. This allows for greater flexibility in the pressure medium, as well as the surfaces to which the mixture is exposed. Sample vessels could conceivably even act to protect the desired protein from chemical degradation (e.g., oxygen scavenging plastics are available).

[0100] With continuous processing, scale-up is simple. Small volumes under pressure can be used to refold large volumes the sample mixture. In addition, using an appropriate filter on the outlet of a continuous process will selectively release soluble desired protein from the chamber while retaining both soluble and insoluble aggregates. [0101] Pressurization is a process of increasing the pressure (usually from atmospheric or ambient pressure) to a higher pressure. Pressurization takes place over a predetermined period of time, ranging from 0. 1 second to 1 0 hours. Such times include 1 second, 2 seconds, 5 seconds, 10 seconds, 20 seconds, 1 minute, 2 minutes, 5 minutes, to minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, and 5 hours. [0102] Repressurization is a process of decreasing the pressure, from a high pressure, to a lower pressure (usually atmospheric or ambient pressure). Depressurization takes place over a predetermined period of time, ranging from 10 seconds to 10 hours, and may be interrupted at one or more points to permit optimal refolding at intermediate (but still increased 30 compared to ambient) pressure levels. The repressurization or interruptions may be 1 second, 2 seconds, 5 seconds, 10 seconds, 20 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, and 5 hours. [0103] Degassing is the removal of gases dissolved in solutions and is often advantageous in the practice of the methods described herein. Gas is much more soluble in liquids at high pressure as compared to atmospheric pressure and, consequently, any gas headspace in a sample will be driven into solution upon pressurization. The consequences are two-fold: the additional oxygen in solution may chemically degrade the protein product, and gas exiting solution upon repressurization may cause additional aggregation. Thus, samples should be prepared with degassed solutions and all headspace should be filled with liquid prior to pressurization.

[0104] The disclosures of all publications, patents, patent applications and published patent applications referred to herein by an identifying citation are hereby incorporated herein by reference in their entirety. United States Patent Nos. 6,489,450 and 7,064,192, and United States Patent Application Publication Nos. 2004/0038333 and 2006/0188970 are specifically incorporated herein by reference in their entirety. In particular, the exemplary proteins for refolding, the experimental techniques for refolding found in those documents, and the examples are incorporated by reference herein.

[0105] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is apparent to those skilled in the art that certain minor changes and modifications will be practiced. Therefore, the description and examples should not be construed as limiting the scope of the invention.

Claims

WHAT IS CLAIMED IS:

1. An isolated and purified biologically active pressure refolded protein, wherein said protein has been isolated and/or purified in a biologically inactive form, but which has not been isolated and/or purified in biologically active form.

2. The protein of claim 1, wherein the biologically inactive form is a protein aggregate.

3. The protein of claim 1, wherein the biologically inactive form is a denatured form.

4. A protein of claims 1, 2, 3, or 4, wherein said protein cannot be refolded by using non-high-pressure based refolding methods.

5. A protein of claim 4, wherein the non-high-pressure based refolding methods include one or more of additive-introduced stepwise dialysis methods; centrifugal filtration methods; chaotrope-based methods; chaperone-assisted refolding methods (including reverse- micelle chaperone assisted refolding); column-based refolding methods, including affinity immobilization, cobalt-chelating chromatography, hydrophobic interaction chromatography, ion-exchange chromatography, nickel-chelating chromatography, oxidative refolding chromatography, protein immobilization, size exclusion chromatography with partner subunits, and size-exclusion chromatography; detergent-based methods, detergent precipitation/sequestration methods; diafiltration methods; dialysis methods; dilution methods; dilution-into detergent methods; dilution with complex partner subunits methods; dilution/column refolding combination methods; dilution/dialysis combination methods; gel filtration methods, including gel filtration with pH and denaturant gradients; matrix-assisted dialysis methods; matrix assisted refolding/ renaturing gel filtration methods; and non- detergent sulfobetaines (NDSB) based methods.

6. A protein of claim 5, wherein method is a urea-based or guanidine-based refolding method.

7. An isolated and purified biologically active protein, wherein said protein has been isolated and/or purified in a biologically inactive form, but which cannot be isolated and/or purified in biologically active form in greater than 20% yield from the inactive form using non-high-pressure based methods.

8. The protein of claim 7, wherein the non-high-pressure based methods include one or more of additive-introduced stepwise dialysis methods; centrifugal filtration methods; chaotrope-based methods; chaperone-assisted refolding methods (including reverse-micelle chaperone assisted refolding); column-based refolding methods, including affinity immobilization, cobalt-chelating chromatography, hydrophobic interaction chromatography, ion-exchange chromatography, nickel-chelating chromatography, oxidative refolding chromatography, protein immobilization, size exclusion chromatography with partner subunits, and size-exclusion chromatography; detergent-based methods, detergent precipitation/sequestration methods; diafiltration methods; dialysis methods; dilution methods; dilution-into detergent methods; dilution with complex partner subunits methods; dilution/column refolding combination methods; dilution/dialysis combination methods; gel filtration methods, including gel filtration with pH and denaturant gradients; matrix-assisted dialysis methods; matrix assisted refolding/ renaturing gel filtration methods; and non- detergent sulfobetaines (NDSB) based methods.

9. A method for producing biologically active protein from a mixture comprising aggregated or denatured protein which has not previously been refolded by non-high- pressure-based methods, comprising: subjecting the aggregated or denatured protein to from about 0.25 kbar to about 10 kbar of pressure for a time sufficient for disaggregation or renaturation of the protein, and reducing the pressure to atmospheric pressure, wherein the protein retains biological activity.

10. The method of claim 11, wherein the non-high-pressure based methods include one or more of additive-introduced stepwise dialysis methods; centrifugal filtration methods; chaotrope-based methods; chaperone-assisted refolding methods (including reverse-micelle chaperone assisted refolding); column-based refolding methods, including affinity immobilization, cobalt-chelating chromatography, hydrophobic interaction chromatography, ion-exchange chromatography, nickel-chelating chromatography, oxidative refolding chromatography, protein immobilization, size exclusion chromatography with partner subunits, and size-exclusion chromatography; detergent-based methods, detergent precipitation/sequestration methods; diafiltration methods; dialysis methods; dilution methods; dilution-into detergent methods; dilution with complex partner subunits methods; dilution/column refolding combination methods; dilution/dialysis combination methods; gel filtration methods, including gel filtration with pH and denaturant gradients; matrix-assisted dialysis methods; matrix assisted refolding/ renaturing gel filtration methods; and non- detergent sulfobetaines (NDSB) based methods.

11. A protein produced by the method of claims 9 or 10.