MX2008008037A - Diaminophenothiazine compositions and uses thereof - Google Patents

Abstract

Cell senescence is delayed by contacting a cell specifically determined to be in need of delayed cell senescence with an effective amount of a diaminophenothiazine.

Description

RECOMBINANT PRODUCTION OF HEPARIN-PROTEIN PRODUCTS RELATED APPLICATION This application claims the priority and benefit of US Provisional Application of Minutes No. 60 / 753,615, filed on December 22, 2005, and US Provisional Application of Minutes No. 60 / 807,432. , presented on July 14, 2006, whose descriptive memories are incorporated in their entirety to this report. FIELD OF THE INVENTION This invention relates to methods for obtaining heparin binding proteins produced in cell cultures. The invention includes methods for recovering and purifying renatured heparin-binding proteins that have been produced in prokaryotic host cells and are present in these cells, typically in the periplasmic or intracellular space. Heparin binding proteins produced in prokaryotic host cells can also be found as soluble proteins or a mixture of soluble and insoluble proteins. BACKGROUND It is known that a wide variety of naturally occurring biologically active polypeptides bind to heparin. Such heparin binding polypeptides include cytokines, such as platelet factor 4 and IL-8 (Barber et al., (1972) Biochim. Biophys. Acta, 286: 312-329, Handin et al., (1976) J. Biol. Chem., 251: 4273-422, Loscalzo et al., (1985) Arch. Biochem. Biophys. 240: 446-455, Zucker et al., (1989) Proc. Nati Acad. Sci. USA, 86: 7571-7574, Talpas et al., (1991) Biochim. Biophys. Acta, 1078: 208-218, Webb et al., (1993) Proc. Nati Acad. Sci. USA, 90: 7158-7162) heparin-binding growth factors (Burgess and Maciag, (1989) Annu. Rev. Biochem., 58: 576-606, Klagsbrun, (1989) Prog. Growth Factor Res., 1: 207-235), such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF). ), the basic fibroblast growth factor (bFGF), the acidic growth factor of fibroblasts (aFGF), the vascular endothelial growth factor (VEGF) and the hepatocyte growth factor (HGF) (Liu et al., ( 1992) Gastrointest. Liver Physiol. 26: G642-G649), and selectins, such as L-selectin, E-selectin and P-selectin (Norgard-Sumnicht et al., (1993) Science, 261: 480-483). See also Muñoz and Linhardt., (2004) Arteposcler Thromb Vasc B ol., 24: 1549-1557. International publication No. WO 95/07097 describes heparin-binding protein formulations that include heparin-binding growth factors such as VEGF, with purified native heparin or other polyanionic compounds for therapeutic use. The oligosaccharides derived from heparin and various other polyanionic compounds have been shown to stabilize the active conformation for heparin-binding growth factors (Barzu et al., (1989) J. Cell Physiol. 140: 538-548, Dabora et al. al., (1991) J. Biol. Chem. 266: 23627-23640) and affinity chromatography has been employed in various purification schemes (see generally, International Publication No. WO 96/02562). Many of the heparin binding proteins of mammalian origin have been produced by a recombinant technology and are clinically relevant (Muñoz and Linhardt, (2004) Arterioscler Thromb Vasc Biol., 24: 1549-1557, Favard et al. (1996) Diabetes and Metabolism 22 (4): 268-73, Matsuda et al., (1995) J. Biochem. 118 (3): 643-9, Roberts et al., (1995) Brain Research 699 (1): 51-61). For example, VEGF is a potent mitogen for vascular endothelial cells. It is also known as the vascular permeability factor (VPF). See, Dvorak et al., (1995) Am. J. Pathol. 146: 1029-39. VEGF fulfills important functions in vasculogenesis, the development of the embryonic vasculature and angiogenesis, the process of formation of new blood vessels from pre-existing ones. See, for example , Ferrara, (2004) Endocrine Reviews 25 (): 581-611, Risau et al., (1988) Dev. Iiol., 125: 441-450, Zachary, (1998) Intl. J. Biochem Cell Bio : 1169-1174, Neufeld et al., (1999) FASEB J. 13: 9-22, Ferrara (1999) J. Mol. Med. 77: 527-543, and Ferrara and Davis-Smyth, (1997) Endocri. Rev. 18: 4-25. Clinical applications of VEGF include those where the growth of new capillary beds is indicated as, for example, the promotion of wound healing (see, for example, International Publication No. WO 91/02058, and File No. P2358R1). , entitled "Healing of wounds" presented on June 16, 2006), for the promotion of tissue growth and repair, eg. , liver (see, eg, W02 003/0103581), bone (see, eg, WO2003 / 094617), etc. See also, Ferrara, (2004) Endocrine Reviews 25 (4): 581-611. Typically, therapeutically relevant recombinant proteins are produced in a variety of host organisms. Most proteins can be expressed in their native form in eukaryotic hosts such as CHO cells. Animal cell culture generally requires prolonged growth times to obtain maximum cell density and ultimately a lower cell density is obtained than in prokaryotic cell cultures (Cleland, J. (1993) ACS Symposium Series 526, Protein Folding: In Vivo and In Vitro, American Chemical Society). Additionally, animal cell cultures often require expensive media containing growth components that can interfere with the recovery of the desired protein. The expression systems of the host bacterium provide a cost-effective alternative for the scale production of recombinant proteins. There are numerous US patents on general bacterial expression of recombinant proteins, which include U.S. Patent Nos. 4,565,785, 4,673,641, 4,795,706, and 4,710,473. The greatest advantage of the production method is the ability to easily isolate the product from cellular components by centrifugation or microfiltration. See, for example , Kipriyanov and Little, (1999) Molecular Biotechnology, 12: 173-201, and, Skerra and Pluckthun, (1988) Science, 240: 1038-1040. Recombinant heparin-binding growth factors such as acidic fibroblast growth factor, basic fibroblast growth factor and vascular endothelial growth factor have been recovered and purified from a variety of sources including bacteria (Salter DH et al., (1996) Labor, Invest. 74 (2): 546-556 (VEGF), Siemeister et al., (1996) Biochem. Biophys. Res. Commun. 222 (2): 249-55 (VEGF ), Cao et al., (1996) J. Biol. Chem. 261 (6): 3154-62 (VEGF), Yang et al., (1994) Gaojishu Tonqxun, 4: 28-31 (VEGF), Anspach et al. al., (1995) J. Chromatogr., A 711 (1): 129-139 (aFGF and bFGF), Gaulandris (1994) J Cell, Physiol. 161 (1): 149-59 (bFGF), Stape and Rhesus. 1996) Biotech, Tech. 10 (7): 481-484 (bFGF), McDonald et al., (1995) FASEB J. 9 (3): A410 (bFGF)). However, bacterial expression systems such as E. coli lack the cellular machinery to facilitate the appropriate renaturation of proteins and generally do not produce the secretion of large proteins in the culture medium. Recombinant proteins expressed in bacterial host cells are often found as inclusion bodies consisting of dense masses of partially folded and misfolded reduced protein. In this way, the recombinant protein is generally inactive. For example, the predominant active form of VEGF is a homodimer of two polypeptides of 165 amino acids (VEGF-165). In this structure, each subunit contains 7 pairs of intrachain disulfide bonds and two additional pairs that effect the covalent attachment of the two subunits (Ferrara et al., (1991) J. Cell, Biochem 47: 211-218). It has been shown that the native conformation includes a strongly basic domain that readily binds to heparin (Ferrara et al (1991) supra). Covalent dimerization of VEGF is needed for receptor binding and effective biological activity (Potgens et al., (1994) J. Biol. Chem. 269: 32879-32885, Claffey et al., (1995) Biochim. et Biophys. Acta 1246: 1-9). The bacterial product potentially contains several misfolded intermediates and mixed disulfides. In addition, renaturation with frequency, trimerosy n multimeros badly folded and joined with disulfides. (Morris et al., (1990) Biochem. J., 268: 803-806, Toren et al., (1988) Anal. Biochem., 169: 287-299). This phenomenon of association is very common during the renaturation of the protein, particularly at higher protein concentrations it often seems to involve association by the hydrophobic interaction of the partially folded intermediates (Cleland and Wang, (1990) Biochemistry, 29: 11072-11078 ). The misfolding occurs both in the cell during fermentation or during the isolation procedure. Proteins recovered from the periplasmic or intracellular space must be solubilized and the soluble protein renatured to the native state. In vitro methods to renature the proteins in the correct biologically active conformation are essential for obtaining functional proteins. Typical downstream processing of proteins recovered from the inclusion bodies includes dissolution of the inclusion body with a high denaturant concentration such as urea followed by dilution of the denaturant to allow renaturation to occur (see, US Patent Nos. 4.512, 922, 4,511,502, and 4,511,503). See also, eg. , Rudolph and Lilie, (1996) FASEB J. 10: 49-56, and Fischer et al., (1993), Biotechnology and Bioengineermg, 41: 3-13. Such recovery methods are considered universally applicable, with minor modifications for the recovery of biologically active recombinant proteins from inclusion bodies. These methods have been applied to the heparin binding protein such as VEGF (Siemeister et al (1996) supra). These methods attempt to eliminate random binding by disulfide bonds before the recombinant protein adjusts to its biologically active conformation through its other stabilizing forces and can not remove the misfolded intermediates inappropriately or provide homogeneous populations of properly folded products. Inverse micelles chromatography or ion exchange have been used to assist in the renaturation of denatured proteins by capturing a single protein within the micelles or isolating them on a resin and then removing the denaturant (Hagen et al., ( 1990) Biotechnol, Bioeng 35: 966-975, Creighton (1985) in Protein Structure Folding and Design (Oxender, DL Ed.) Pp.249-251, New York: Alan R. Liss, Inc.). These methods have been useful to prevent protein aggregation and facilitate proper renaturation. To alter the speed or degree of renaturation, specific renaturation of conformation with ligands and antibodies has been performed for the native structure of the protein (Cleland and Wang, (1993), in Biotechnology, (Rehm H.-J., and Reed G. Eds.) pp 528-555, New York, VCH). For example, creatine kinase was renatured in the presence of antibodies to the native structure (Morris et al., (1987) Biochem. J. 248: 53-57). In addition to antibodies, ligands and cofactors have been used to improve renaturation. These molecules probably should not interact with the folding of the protein after the formation of the native protein. Consequently, the folding equilibrium could be "directed" to the native state. For example, the renaturation rate of ferricytochrome c increased by the extrinsic ligand for the axial position of heminic iron (Brems and Stellwagon, (1983) J. Biol. Chem. 258: 3655-3661). Chaperone proteins have also been used to assist in the folding of proteins. See, for example , Baneyx, (1999) Current Opinion in Biotechnology, 10: 411-421. New and more effective methods of folding and / or recovering heparin-binding proteins from a host cell culture are needed, eg, for the efficient and economical production of heparin binding proteins in bacterial cell cultures, which provide the elimination or reduction of biologically inactive intermediates and improve the recovery of an appropriately renatured, biologically active and highly purified protein that is applicable for the manufacturing scale production of the proteins. The invention relates to these and other needs, as will be apparent after review of the following description. BRIEF DESCRIPTION OF THE INVENTION The invention provides a method for recovering and purifying renatured heparin binding proteins from a cell culture. In particular, the invention provides a method for recovering a heparin binding protein from prokaryotic host cells, for example. bacterial cells. For example, a method comprises the steps of (a) isolating the unpaired hepapin binding protein from the periplasmic or intracellular space of said bacterial cells, (b) solubilizing said insoluble heparin-binding isolated protein in a first buffered solution comprising a chaotropic agent and a reducing agent, and c) incubating said solubilized heparin-binding protein in a second buffered solution comprising a chaotropic agent and sulfated polyanionic agent for such time and under such conditions as to produce renaturation of the hepapin-binding protein, and (d) recovering said renatured hepapin-binding protein, where there is a 2- to 10-fold increase in the concentration of protein recovered by incubation with a sulfated polyanionic agent as compared to a control. In one embodiment, the second buffered solution further comprises arginine. In one embodiment, the second buffered solution further comprises cistern or a moderate reducing agent.

In one embodiment of the invention, there are, e. , a 2-8 fold increase in protein concentration recovered from biologically active renatured protein, or 2-5 fold increase in protein concentration recovered from biologically active renatured protein or 3-5 fold increase in concentration of protein recovered from the biologically active renatured protein or a 2-3 fold increase in the concentration of protein recovered from the biologically active renatured protein. In another embodiment of the invention, there are, e. , an increase of more than 2.0 times, 2.5 times, 2.8 times, 3.0 times, 5 times, 6 times, 7.0 times, 8 times, 9 times, etc., in the concentration of Protein recovered from the biologically active renatured protein. In one embodiment of the invention, there is a 3 to 5 fold increase in the concentration of protein recovered from the biologically active renatured VEGF. The processes of the invention are broadly applicable to heparin-binding proteins and especially to hepapine-binding growth factors and in particular, vascular endothelial growth factor (VEGF). In certain embodiments of the invention, the sulfated polyanionic agent is between about 3,000 and 10,000 daltons.

In one embodiment, the sulphated polyanionic agent used in the production processes is a dextran sulfate, sodium sulfate or heparin sulfate. In one aspect, dextran sulfate is between 3,000 daltons and 10,000 daltons. The invention further provides processes and methods for the purification of heparin-binding proteins both alone and in connection with the recovery of the hepapin-binding protein as described herein. In a particular embodiment, purification methods include contacting said renatured heparin-binding protein with a hydroxyapatite chromatographic support, a first hydrophobic interaction chromatographic support, a cation chromatographic support and a second hydrophobic interaction chromatographic support and eluting in selective form the heparin binding protein of each support. In another embodiment, a purification method comprises contacting said renatured heparin binding protein with a cation exchange support, a first hydrophobic interaction chromatographic support and an ion exchange chromatographic support or a mixed media chromatographic support eluting at selective form the heparin binding protein of each support. It is contemplated that the recovery steps can be performed in any order, eg. , successively or altering the order of the chromatographic supports. In certain embodiments of the invention, methods are provided for recovering and purifying renatured heparin binding proteins from cell culture on a manufacturing or industrial scale. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates a chromatography of VEGF produced by bacterial strain W3110 loaded on a POROS HE2 / M column (4.6 x 100 mm, PerSeptive BioResearch Products, Cambridge, MA). For example, the POROS HE / 2M column is equilibrated in 10 mM sodium phosphate, pH 7 containing 0.15 M sodium chloride. The column is eluted by a linear gradient of 0.15-2 M sodium chloride in phosphate of sodium 10 mM, pH 7 for 10 minutes. The eluent is monitored at 280 nm. The protein recovered in each peak corresponds to VEGF however only peak 3 corresponds to an appropriately renatured biologically active VEGF.

Figure 2 illustrates a graph representing the stabilization of native VEGF appropriately renatured by heparin. VEGF is suspended in 50 mM HEPES, pH 8, containing 5 mM EDTA, 0.2 M NaCl and 10 mM cysteine. Figures 3A-3D illustrate chromatographies of VEGF produced by bacterial strain W3110 and incubated with 12 μg / ml dextran sulfate 5, 000 dalton (Figure 3A), 12 μg / ml dextran sulfate 8,000 dalton (Figure 3B), 12 μg / ml dextran sulfate 10,000 dalton (Figure 3C) or 25 μg / ml heparin (Figure 3D), 3,000 daltons and loaded onto a POROS HE2 / M column (4.6 x 100 mm, PerSeptive BioResearch Products, Cambridge, MA). For example, the column is equilibrated in 10 mM sodium phosphate, pH 7 containing 0.15 M sodium chloride. The column is eluted by a linear gradient of 0.15-2 M sodium chloride, 10 mM sodium phosphate, pH 7 for 10 minutes. The eluent is monitored at 280 nm. The protein recovered in each peak corresponds to VEGF, however, peak 3 corresponds to an appropriately renatured biologically active VEGF. Figure 4 illustrates the effect of the scale on the renaturation of VEGF. Figure 5 illustrates the effect of heparin, of low molecular weight (PM) and high MW, and dextran sulfate of 10,000 daltons, on the renaturation of VEGF. Peak 3 corresponds to an appropriately renatured biologically active VEGF. Figure 6 illustrates the effect of sodium sulfate on the renaturation of VEGF. Peak 3 corresponds to an appropriately renatured biologically active VEGF.

Figure 7 illustrates the effect of heparin, heparin, low molecular weight (MW) and high MW, and dextran sulfate of 5,000 daltons, 8,000 daltons and 10,000 daltons, on the renaturation of VEGF. Peak 3 corresponds to an appropriately renatured biologically active VEGF.

Figure 8 illustrates the effect of heparin and dextran sulfate on the renaturation of VEGF. Peak 3 corresponds to an appropriately renatured biologically active VEGF. Figure 9 illustrates an effect of urea and DTT on VEGF extraction from bacterial inclusion and v bodies. Figure 10 illustrates an effect of urea and DTT on the renaturation concentration of VEGF. Figure 11 illustrates the amino acid sequence of VEGF165 with the indicated disulfide bonds.

Figure 12 illustrates the effect of the presence of charged amino acids. At a concentration of 0.75 M in the second buffered solution both argimine and lysine are beneficial whereas histidine has little additive effect compared to the buffered solution without it. In addition, arginine was shown to have a similar effect in concertations from 0.1 to 1M. Figure 13 illustrates the effect of dilution on the% efficiency of renaturation where, although the concentration of total VEGF is lower as the dilution increases, the% efficiency of renaturation is higher with more dilution. DETAILED DESCRIPTION Definitions "Heparin" (also called heparinic acid) is a heterogeneous group of highly sulfated straight chain ammonium mucopolysaccharides, called glycosaminoglycans. While others may be present, the main sugars in heparin are: aL-? Duron acid? Co-2-sulfate, 2-deoxy? -2-sulfammo-a-glucose-6-sulfate,? -D-glucuronic acid , 2-Acetamido-2-deoxyria-A-glucose and L-iduronic acid. These and optionally other sugars are joined by glycosidic bonds, forming polymers of various sizes. Due to the presence of covalently attached sulphate and carboxylic acid groups, heparin is strongly acidic. The molecular weight of the hepapna varies from approximately 3,000 to approximately 20,000 daltons depending on the source and method of determination. Native heparin is a constituent of various tissues, especially liver and lung and mast cells in several mammalian species. Hepapna and hepapna salts (hepapna sodium) are available in the market and are used mainly with anticoagulant in various clinical situations. "Dextran sulfate" is a dextran sulfate whose main structure is a D-glucose polymer. Glucose and optionally other sugars are bound by α-D (1-6) glycosidic linkages, and form polymers of various sizes. Due to the presence of covalently bound sulfate, dextran sulfate is strongly acidic. The sulfur content is generally not less than 10%, and typically from about 15% -20% with up to 3 sulfate groups per glucose molecule. The average molecular weight of dextran sulfate is from about 1,000 to about 40,000,000 daltons. Examples of dextran sulfate which can be employed in the invention include the dextran sulfate produced by microorganisms such as Leuconostoc mesen teroides and L. dextrani cum. "Polyanionic agent" as used in the scope of the invention is intended to describe preparations and purified native heparin compounds available on the market that are capable of binding to heparin binding proteins that include other "polyanionic agents" such such as sodium sulfate, heparin sulfate, heparan sulfate, pentosan (poly) sulfate, dextran, dextran sulfate, hyaluronic acid, chondroitin, chondroitin sulfate, dermatan sulfate and keratan sulfate. Particularly useful within the context of the invention is a "sulphated polyanionic agent" such as, for example, a sulfated derivative of a polysaccharide, such as heparin sulfate, dextran sulfate, the sulfates of the cyclodextrin produced by microorganisms such as Bacill. us ma cerans described in U.S. Patent No. 5,314,872 in addition to sulfates or other glucans such as sulfates of β-1,3 glucan, with β-1,3 glucan being produced by microorganisms belonging to the genus Alcaligenes or Agroba cteri um, and chondroitin sulfate in addition to sulfated heparin fragments.

The aforementioned agents are generally available and are known to skilled professionals. For example, sulfated heparin fragments can be obtained from a library of heparin-derived oligosaccharides that have been fractionated by gel permeation chromatography. The preparation of affinity fractionated heparin-derived oligosaccharides was reported by Ishihara et al., (1993) J. Biol. Chem., 268: 4675-4683. These oligosaccharides were prepared from a commercial porcine heparin after partial depolymerization with nitrous acid, reduction with sodium borohydride and fractionation by gel permeation chromatography. The resulting mixtures of di-, tetra-, hexa-, octa-, and decasaccharides were successively applied to an affinity column of human recombinant bFGF covalently linked to SEPHAROSE ™ 4B, and further fractionated into submixes on the basis of their Elution of this column in response to sodium chloride gradients. This produced five mixtures, designated Hexa-1 to Hexa-5, whose structures and biological activities were further evaluated. The structure of Hexa-5C and its 500-MHz NMR spectra are shown in Figure 4 of Tyrell et al., (1993) J. Biol. Chem., 268: 4684-4689. This hexasaccharide has the structure [Ido (2-OS03) l-4GlcNS03 (6-OS? 3) l-4] 2IdoA (2-OS03) l-4AManR (6-OS? 3) - All heparin-derived oligosaccharides described above, in addition to other heparin-like oligosaccharides, they are suitable and can be used according to the invention. In one embodiment of the invention, hexasaccharides and polysaccharides of hepapine of larger unit size (eg hepta-, octa-, nona- and decasaccharides) are used. In addition, oligosaccharides derived from hepapna or heparin-like with a large net negative charge, for e. Due to the high degree of sulfation, they are used with advantage. The term "heparin binding protein" or "HPB" as used herein refers to a polypeptide capable of binding to heparin (as defined herein above). The definition includes the pre, pro-pro, and pro-mature forms of the native heparin binding proteins produced recombinantly. Typical examples of heparin-binding proteins are "heparin-binding growth factors" including but not limited to epidermal growth factor (EGF), platelet-derived growth factor (PDGF), the basic factor of fibroblast growth ( bFGF), acidic fibroblast growth factor (aFGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) (also known as dispersed factor, SF), and nerve growth factor (NGF), IL -8, etc. As used herein, "vascular endothelial growth factor" or "VEGF" refers to a mammalian growth factor originally derived from bovine pituitary follicular cells having the amino acid sequence described in Castor, CW, et al. al., (1991) Methods in Enzymol. 198: 391-405, together with their functional derivatives having the qualitative biological activity of a corresponding native VEGF, including but not limited to the amino acid sequence of human VEGF as reported in Houck et al., (1991) Mol. . Endocpn. : 1806-1814. See also, Leung et al. (1989) Science, 246: 1306, and Robinson & Stringer, (2001) Journal of Cell Science, 144 (5): 853-865, U.S. Patent No. 5,332,671. The predominant form of VEGF is a homodimer of 165 amino acids having sixteen cistern residues that form 7 intramolecular disulfide bonds and two intermolecular disulfide bonds. Alternative splicing has been implicated in the formation of multiple human VEGF polypeptides consisting of 121, 145, 165, 189 and 206 amino acids, however the variant VEGF121 lacks the heparin binding domain of the other variants and consequently is not included in the definition of heparin binding protein disclosed herein. All isoforms of VEGF share a common amino-terminal domain, but differs in the length of the carboxyl-terminal portion of the molecule. The preferred preferred active form of VEGF, VEGF165, has disulfide bonds between the amino acid residues Cys26-Cys68, Cys57-Cysl04, Cys61-Cysl02, Cysll7-Cysl35, Cysl20-Cysl37, Cysl39-Cysl58, Cysl46-Cysl60 of each monomer. See Figure 11. See also, eg. , Keck et al., (1997) Archives of Biochemistry and B ophys cs 344 (1): 103-113. The VEGFi? S molecule is composed of two domains: an amino-terminal receptor binding domain (amino acids 1-110 disulfide attached to the homodimer) and a carboxyl-terminal heparin binding domain (residues 111-165). See, for example , Keyt et al., (1996) J. Biol. Chem., 271 (13): 7788-7795. In certain embodiments of the invention, isolated and purified VEGF165 is not glycosylated at residue 75 (Asn). See, for example , Yang et al., (1998) Journal of Pharm. & Experimental Therapeutics, 284: 103-110. In certain embodiments of the invention, isolated and purified VEGF165 is substantially uninnate in the AsnlO residue. In certain embodiments of the invention, isolated and purified VEGF165 is a mixture of a deamidated (at the AsnlO residue) and non-deamidated protein, typically with the majority of the non-deamidated protein. Since VEGF165 is a homodimer, deamination can occur in one or both of the polypeptide chains. As used herein, VEGF or other HPB and the like "properly folded" or "biologically active" refer to a molecule with biologically active conformation. The skilled professional will recognize that misfolded intermediates and disordered disulfides have biological activity. In such a case, the VEGF or other HPB and similarly folded appropriately or biologically active correspond to the native folding pattern of VEGF (described above) or another HBP. For example, appropriately folded VEGF has the disulfide pairs indicated above, in addition to the two intermolecular disulfide bonds of the dimeric molecule, however other intermediates can be produced by the culture of bacterial cells (Figure 1 and 3A-3D). For properly folded VEGF, two intermolecular disulfide bonds are produced between the same residues Cys51 and Cys60, of each monomer. See, for example , patent W098 / 16551. The biological activities of the VEGF include, dog without limitation to, by e. , promotion of vascular permeability, promotion of vascular endothelial cell growth, binding to the VEGF receptor, binding and signaling by the VEGF receptor (see, e.g., Keyt et al., (1996) Journal of Biological Chemistry, 271 (10): 5638-5646), induction of angiogenesis, etc. The terms "purified" or "pure HBP" and the like refer to the free material of the substances that normally accompany it as it is found in its recombinant production and especially in cell cultures of prokaryotes or bacteria. Thus the terms refer to a recombinant HBP that is free of contaminating DNA, host cell proteins or other molecules associated with its m if your environment. The terms refer to a degree of purity that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 98% or more. The terms "inclusion bodies" or "shrink bodies" refer to intracellular masses of polypeptide aggregates of antibodies, which constitute a significant portion of the total cellular protein, which includes all the components of the cell. In some cases, but not in all cases, these aggregates of the polypeptide can be recognized as bright spots visible within the cell envelope ba or a phase contrast microscope with magnification less than 1,000 times. As used herein, the term "misfolded" protein refers to precipitated or aggregated polypeptides that are contained in the shrinking bodies. As used herein, VEGF or other "miscible" or "misfolded" HPB refers to precipitated or aggregated VEGF that is contained in the pepplásmic or intracellular space of prokaryotic host cells, or otherwise associated with the prokaryotic host cell and assumes a biologically inactive conformation with mismatched or unformed disulfide bonds. The HBP insoluole is generally, but not necessarily, contained in retractable bodies, that is, it may or may not be visible with a phase contrast microscope. As used herein, a "chaotropic agent" refers to a compound which, in a suitable concentration in aqueous solution, is capable of changing the configuration or spatial conformation of the polypeptides by altering the surface of the same to take soluble to the polypeptide in aqueous medium. Alterations can occur due to changes, for ex. , of the hydration state, the solvent environment or the solvent-surface interaction. The concentration of the chaotropic agent will directly affect its strength and effectiveness. A strongly denaturing chaotropic solution contains a chaotropic agent in high concentrations which, in solution, effectively deploys a polypeptide present in the solution, and effectively removes the proteins from the secondary structure. The unfolding will be relatively broad, but reversible. A moderately denaturing chaotropic solution contains a chaotropic agent, which in sufficient concentrations in the solution, allows the partial folding of a polypeptide from any twisted conformation that the polypeptide has assumed through soluble intermediates in solution, in the spatial conformation in which it is formed. it finds when it operates in its active form under endogenous physiological conditions and its homologs. Examples of chaotropic agents include guanidine hydrochloride, urea and hydroxides such as sodium or potassium hydroxide. Chaotropic agents include a combination of these reagents, such as a mixture of hydroxide with urea or guanidine hydrochloride.

As used herein, "reducing agent" refers to a compound which, in a suitable concentration in aqueous solution, maintains free sulfhydryl groups so that the intra- or intermolecular disulfide bonds are chemically altered. Representative examples of suitable reducing agents include dithiothreitol (DTT), dithioerythritol (DTE), beta-mercaptoethanol (BME), cistern, cysteamine, thioglycolate, glutathione, Tris [2-carboxyethyl] phosphine (TCEP), and sodium borohydride. As used herein, "buffered solution" refers to a solution that resists pH changes by the action of its acid-base conjugated components. The "bacteria" for the purposes of the present specification include eubactepas and archaebacteria. In certain embodiments of the invention, eubacteria are used, which include gram-positive and gram-negative bacteria in the methods and processes described herein. In one embodiment of the invention, gram-negative bacteria are used, e. Enterobacteriaceae. Examples of bacteria belonging to Enterobactepaceae include Escheri chia, En teroba cter, Erwinia, Klebsiella, Proteus, Salmonella, Serra tia, and Shigel la. Other types of suitable bacteria include Azotoba cter, Pseudomonas s, Rhi zobia, Vi treosci la, and Para coccus. In an embodiment of the invention, E is used. coli The E. Suitable host hosts include E. col i W3110 (ATCC 27.325), E. col i 294 (ATCC 31,446), E. col i B, and E. coli X1776 (ATCC 31,537). These examples are illustrative rather than limiting, and W3110 is an example. Mutant cells of any of the above-mentioned bacteria can also be used. Obviously it is necessary to select the appropriate bacterium taking into consideration the replicability of the replicon in the cells of a bacterium. For example, the species of E. coli, Serra tia, or Salmonel the species can be used in a suitable manner well known plasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to provide the replicon. See below the respective examples of suitable bacterial host cells. As used herein, the terms "cell" "cell line" "strain" and "cell culture" are used interchangeably and all these designations include the progeny. Thus, the words "transformants" and "transformed cells" include the main subject cell and the cultures derived therefrom without taking into account the number of transfers. It is also understood that all progeny can not be exactly identical in DNA content, due to deliberate or unintentional mutations. Mutant progeny that have the same function or biological activity selected from the originally transformed cell are included. Where different designations are required, it will be clarified from the context. As used herein, "polypeptide" generally refers to peptides and proteins from any cellular source having more than about ten amino acids. The "heterologous" polypeptides are the foreign polypeptides for the host cell used, such as a human protein produced by E. col i. Although the heterologous polypeptide can be prokaryotic or eucapotic, it is preferably eukaryotic, more preferably mammalian, and most preferably human. In certain embodiments of the invention, it is a polypeptide produced in recombinant or recombinant form. Heparin-binding proteins Isolation of the heparin-binding protein The insoluble misfolded heparin-binding protein (HBP) is isolated from the prokaryotic host cells expressing the protein by any of several standard art techniques. For example, insoluble HBP is isolated in an adequate isolation buffer by exposing the cells to a buffer of adequate ionic strength to solubilize most host proteins, but in which the subject protein is substantially insoluble, or alteration of the cells so as to release the inclusion bodies or the protein form of peripheral or intracellular space and make them available for recovery, for example, by centrifugation. This technique is well known and is described in for example, U.S. Patent No. 4,511,503. Kleid et al. Describe the purification of refractile bodies by homogenization followed by centrifugation (Kleid et al., (1984) in Developments in Industrial Microbiology, (Society for Industrial Microbiology, Arlington, VA) 25: 217-235). See lso, for ex. , Fischer et al., (1993) Biotechnology and Bioengineering 41: 3-13. U.S. Patent No. 5,410,026 describes a typical method for recovering protein from inclusion bodies and is synthesized below. The prokaryotic cells are suspended in a suitable buffer. Typically the buffer consists of a suitable buffering agent between pH 5 to 9, or approximately 6 to 8 and a salt. Any suitable salt, which includes NaCl, is useful in maintaining a sufficient ionic strength in the buffered solution. Typically an ionic strength of about 0.01 to 2 M, or 0.1 to 0.2 M is employed. The cells, while suspended in this buffer, are altered or lysate by commonly employed techniques such as, for example, methods mechanical, for ex. , Homogenizer (Manton-Gaulin press, Microfluidizer, or Niro-Soaví), a French press, a pearl mill or a sonic oscillator, or by enzymatic or chemical methods. Examples of enzymatic or chemical methods of cell disruption include the use of spheroplasts, which involves the use of a lysozyme to lyse the bacterial wall (H. Neu et al., (1964) Biochem. Biophys. Res. Comm., 17: 215 ), and osmotic shock, which includes the treatment of viable cells with a high tonicity solution and with a cold water wash and low tonicity to release the polypeptides (H. Neu et al., 1965 J. Biol. Chem., 240 (9): 3685-3692). Ultrasound treatment is generally used to alter the bacteria contained in analytical-scale volumes of the fermentation broth. High-pressure homogenization is typically used on a large scale. After the cells are disrupted, the suspension is typically centrifuged at ba at speed, generally about 500 to 15,000 x g, e.g. In one embodiment of the invention approximately 12,000 x g are used in a standard centrifuge for a sufficient time to pelletize substantially all the insoluble proteins. Such times can simply be determined and depend on the volume that is centrifuged in addition to the design of the centrifuge. Typically, about 10 minutes to 0.5 hours is sufficient to pellet the insoluble protein. In one embodiment the suspension is centrifuged at 12,000 x g for 10 minutes. The resulting pellet contains substantially all of the insoluble protein fraction. If the cell breakdown process is not complete, the pellet may also contain intact cells or broken cell fragments. The entire cell disruption can be tested by resuspending the pellet in a small proportion of the same buffer solution and examining the suspension with a phase contrast microscope. The presence of broken cell fragments or whole cells indicates that treatment with additional ultrasound or other means of rupture is necessary to remove the fragments or cells and the associated non-retractable polypeptides. After such an additional break, if necessary, the suspension is centrifuged again and the pellet is recovered and resuspended and re-examined. The process is repeated until the visual examination reveals the absence of fragments of broken cells in the sedimented material or until the additional treatment fails to reduce the size of the resulting pellet. The above process can be used if the insoluble protein is intracellular or is in the periplasmic space. In an embodiment of the invention, the conditions given herein for isolating the heparin binding protein are directed to inclusion bodies precipitated in the periplasmic or intracellular space and refer in particular to VEGF. However, it is considered that the processes and procedures are applicable to heparin-binding proteins in general with minor modifications as noted throughout the following text. In certain embodiments of the invention, the processes and methods are applicable to the manufacture or production, renaturation and purification on an industrial scale of the HBP. Renaturing of heparin-binding proteins The isolated insoluble misfolded heparin-binding protein is incubated in a first buffered solution containing an amount of a chaotropic agent and a reducing agent sufficient to substantially solubilize the heparin-binding protein. This incubation takes place under conditions of concentration, incubation time and incubation temperature that allow the solubilization of some or substantially all of the hepapin binding protein and for unfolding to occur. The measurement of the degree of solubilization of the buffered solution can be simply determined and carried out suitably, for example, by determination of the turbidity, by analysis of the fractionation between the supernatant and the pellet after centrifugation, in reduced gels of SDS-PAGEs , by two proteins (eg, protein dosing with Bradford reagent (eg, Pierce, Bio-Rad etc.)), or by HPLC. The first buffed solution comprises a buffering agent suitable for maintaining the pH range of the buffer by at least about 7.0, with the typical range in 7.5-10.5. In one embodiment, the pH for VEGF is pH 8.0. Examples of suitable buffers that will provide a pH within this latter range include TRIS-HC1 (Tris [hydroxymethyl] aminomethane), HEPPS (N- [2-Hydroxyethyl] piperazine-N '- [3-propan-sulfonic acid]), HEPES (acid N- [2-Hydroxyet piperazine-N '- [2-ethanesulfonic]), CAPSO (3- [Cyclohexylamino] -2-h? drox? -l-propanesulfonic acid), AMP (2-Am? No-2-met? L-l-propanol), CAPS (3- [Cyclohexylamino] -1-propansufonic acid), CHES (2- [N-Cyclohexylamino] -etansufonic acid), glycine and sodium acetate. In one embodiment of the invention, the buffer of the present specification is HEPPS at about pH 8.0. In a further embodiment, the buffers, e.g. , such as HEPPS, are sulfated. Suitable chaotropic agents to carry out this invention include, e.g. , urea and salts of guanidine or thiocyanate, eg. , urea, guanidine clohydrate, sodium thiocyanate, etc. The quantity of chaotropic agent necessary to be present in the buffer is an amount sufficient to deploy the HBP in solution. In certain embodiments of the invention, a chaotrope is present in a dwarfed concentration between about 4 and 10 molar. In one embodiment of the invention, the chaotropic agent is urea at about 5-8 M, or at about 7 M. In another example, the chaotropic agent is guanidine hydrochloride at about 6-8 M. Examples of suitable reducing agents include, but without limitation, dithiothreitol (DTT), dithioerythritol (DTE), β-mercaptoethanol (BME), cysteine, DTE, etc. The proportion of the reducing agent present in the buffer will depend mainly on the type of reducing agent and chaotropic agent, the type and pH of the buffer used, the amount of oxygen entered or introduced into the solution and the concentration of the protein in the buffer. For example, with 0.5-1.5 mg / ml of protein in a buffered solution at pH 7.0-10.0 containing 4-8 M urea, and the reducing agent is, e.g. , DTT with a concentration of approximately 1-15 mM, or BME with a concentration of approximately 0.2-2 mM, or cysteine with a concentration of approximately 2-10 mM. In one embodiment, the reducing agent is DTT of about 0.5 to about 4 mM, or 2-4 mM. Figure 9 illustrates the effect of urea and DTT on the extraction of VEGF. Peak 3 of VEGF refers to biologically active VEGF properly folded. In one embodiment, the reducing agent is DTT of about 10 mM. A single reducing agent or a combination of reducing agents can be used in the buffer herein. The concentration of the protein of the buffered solution must be such that the protein is substantially solubilized, which is determined by optical density. The exact amount to use will depend on, eg. , the concentrations and types of other components of the buffered solution, in particular the concentration of protein, reducing agent and buffer pH. In one embodiment of the invention, the concentration of the hepapine binding protein is in the range of 0.5-5.5 mg per ml or 1.5-5.0 mg / ml. Solubilization is typically carried out at about 0-45 ° C or about 20-40 ° C or about 23-37 ° C, or about 25-37 ° C, or about 25 ° C for at least about one to 24 hours. In one embodiment, the solubilization is carried out for at least about two hours at room temperature. Typically, the temperature is apparently not affected by the levels of salt, reducing agent and caotropic agent. After the polypeptide is solubilized, it is placed or diluted in a second buffered solution containing the chaotropic agent and a sulfated polyanionic agent as described above, with a concentration of the caotropic agent that nevertheless allows renaturation of the hepapne binding protein. . The conditions of this second incubation of the soluble misfolded protein will generally be such that partial, substantial or complete renaturation of the protein occurs. The exact conditions will depend, for example, on the pH of the buffer and the types and concentrations of the polyanionic and chaotropic agents and the reducing agents, if they were present. The incubation temperature is generally about 0-40 ° C, or 10-40 ° C and the incubation will generally be carried out for at least about 1 hour to effect renaturation. In certain embodiments, the reaction is carried out e.g. , at about 15-37 ° C or at 20-30 ° C, for at least about 6 hours, for at least about 10 hours, or between about 10 and 48 hours, or between about 15 and 20 hours, or between about 6 hours and 20 hours, or between 12 and 24 hours. The degree of renaturation is suitably determined by a radioimmunoassay (RIA) titre of the BPH or by high-resolution liquid chromatography (HPLC) analysis by e j. , a POROS HE2 / M column (PerSeptive BioResearch Products) or another appropriate hepapin affinity column. Increasing the RIA titre or peak size of properly folded HBP correlates directly with the increasing amounts of correctly folded biologically active HPB present in the buffer. Incubation is carried out to maximize the ratio of correctly folded HBP to recovered misfolded HBP, determined by RIA or HPLC.

In one embodiment, the quality and quantity of the properly folded VEGF is evaluated by a heparin binding assay. Samples containing the diluted heparin binding protein are loaded into, eg. , a POROS HE2 / M column (4.6 x 100 mm, PerSeptive BioResearch Products, Cambridge, MA) or another suitable affinity column. For example, the heparin affinity column is equilibrated in 10 mM sodium phosphate, pH 7 containing 0.15 M sodium chloride. At a flow rate of 1 ml / min or 2 ml / min, the column is eluted by a linear gradient of 0.15-2 M sodium chloride, 10 mM sodium phosphate, pH 7 for 10 minutes. The eluent is monitored at 280 nm. In one embodiment, the protein is recovered in a single peak corresponding to the biologically active HBP properly folded. In one embodiment of the invention, an assay for determining properly folded HBP is RPHPLC. The disulfide linkages can optionally be confirmed by mapping the peptide. Circular dichroism can also be used to determine the structure / folding in 2 & 3d The buffer for the second buffered solution can be any of those listed above for the first buffered solution, e. PH HEPPS. 8.0, eg. with a concentration of approximately 50 mM for the renaturation of VEGF. The polypeptide can be diluted with the renaturation buffer, e.g. , at least five times or at least approximately ten times, approximately 20 times or approximately 40 times. Alternatively, the polypeptide can be dialyzed against the renaturation buffer. The second buffered solution contains a chaotropic agent with a concentration such that the renaturation of the BPH takes place. Generally a chaotrope is present at a concentration between approximately 0.5 and 2 molar. In one embodiment of the invention, the chaotropic agent herein is urea about 0.5-2 M, 0.5-2 M, or about 1 M. In one embodiment, the chaotropic agent is urea at a concentration of about 1.3 M. In another embodiment of the invention, the chaotropic agent is guanidine hydrochloride approximately 1 M. Figure 10 illustrates the effect of urea and the reducing agent DTT on the renaturation of VEGF. Peak 3 of VEGF refers to biologically active VEGF properly folded. As indicated, the solution optionally also contains a reducing agent. The reducing agent is suitably selected from the above for the solubilization step in a concentration range of about 0.5 to about 10 mM for cysteine, 0.1-1.0 mM for DTT, and / or less than about 0 , 2 mM for BME. In one embodiment of the invention, the reducing agent is DTT about 0.5-2 mM. In one embodiment of the invention, the reducing agent is about 0.5 mM DTT. Examples of suitable reducing agents include, but are not limited to, e.g. , dithiothreitol (DTT), β-mercaptoethanol (BME), cysteine, DTE, etc. While DTT and BME can be used in connection with the methods provided herein for heparin-binding proteins in general, a cysteine combination of about 0.1 to about 10 mM and about 0.1 to about 1, 0 mM DTT as described herein is an example for the recovery of VEGF.

The renaturation step includes a sulfated polyanionic agent in a sufficient concentration to obtain a complete renaturation of the solubilized protein. Examples of suitable sulfated polyanionic agents were described hereinabove, e.g. , a sulfated derivative of a polysaccharide as indicated above with sulfated polyanionic agents such as heparin sulfate, dextran sulfate, heparin sulfate of crondroitin in addition to sulfated heparin fragments. For the heparin sulfate used in the context of the invention, the molecular weights are generally between approximately 3,000 and 10,000 daltons or between approximately 3,000 and 6,000 daltons. In one embodiment of the invention, dextran sulfate is used in the context of the invention. The molecular weight of the sulfated polyanionic agent or other agent such as the dextran sulfate employed in the invention depends on the size of the particular heparin binding protein that is recovered. Generally, the dextran sulfate employed is between about 3,000 and 10,000 dalton. In one embodiment of the invention, dextran sulfate is used between about 5,000 daltons and 10,000 daltons, e.g. , for the recovery of VEGF. In another embodiment, a dextran sulfate is used between about 5,000 and 8,000 daltons for the recovery of HBP. Figures 3A-3D show the recovery of VEGF with various concentrations and molecular weights of dextran sulfate (Figures 3A-C) and heparin (Figure 3D) analyzed by heparin affinity chromatography. Peak 3 corresponds to VEGF properly folded.

The concentration of the polyanionic compound used depends on the protein recovered and its concentration and conditions such as the temperature and pH of the renaturation buffer. Typical concentrations are between about 50 and 500 mM for sodium sulfate, between about 10 and 200 μg / ml for low molecular weight heparins such as 6,000 dalton heparin (Sigma Chemical Co.), between about 10 and 200 μg. / ml for high molecular weight heparins such as porcine heparin IA (Sigma Chemical Co.) and between approximately 10 and 400 μg / ml, or between approximately 10 and 200 μg / ml for dextran sulfates. The renaturing buffer may optionally contain additional agents such as any of a variety of non-ionic detergents such as TRITON ™ X-100, NONIDET ™ P-40, the TWEEN ™ series and the BRIJ ™ series. The non-ionic detergent is present in approximately between 0.01% and 1.0%. In one example, the concentrations for the non-ionic detergent are between about 0.025% and 0.05%, or about 0.05%. Optionally, amino acids with positive charge may be present, e.g. , arginine (eg, L-arginine / HCl), lysine, etc., in the renaturation buffer. In certain embodiments of the invention, the concentration of arginine is e.g. , approximately 0-1000 mM, or approximately 25 to 750 mM, or approximately 50-500 mM, or approximately 50-250 mM, or approximately 100 mM final concentration, etc. In certain embodiments of the invention, the protein is a buffer solution at pH 7.0-9.0 containing, urea0.5-3 M, dextran sulfate 0-30 mg / l, Triton X-100 0-0 , 2%, 2-15 mM cysteine, 0.1-1 mM DTT and 0-750 mM arginine, final concentration. In one embodiment, 50 mM HEPPS is used. In one embodiment, the final concentration of the renaturation buffer solution is 1 M urea, 50 mM HEPPS, 15 mg / L dextran sulfate, 0.05% Triton X-100, 7.5 mM cysteine, 100 mM arginine, pH 8.0 In one embodiment, the final concentration of the renaturation buffer solution is 1.3 M urea, 50 mM HEPPS, 15 mg / L dextran sulfate, 0.05% Triton X-100, 7.5 mM cysteine, argj.nine. 100 mM, pH 8.0. Recovery and purification of heparin-binding proteins While for the recovery and purification of the heparin-binding protein from the culture medium, various known methods and methods for separation of such proteins can be employed, such as, for example, fractionation of salt and solvent, adsorption with colloidal materials, filtration with gels, ion exchange chromatography, affinity chromatography, affinity immmunochromatography, electrophoresis and high resolution liquid chromatography (HPLC), a four-step chromatographic process comprising contacting said renatured heparin-binding protein with a hydroxyapatite chromatographic support, a first hydrophobic interaction chromatographic support, a cationic chromatographic support and a second hydrophobic interaction chromatographic support and selectively eluting the heparin-binding protein of each sop orte. Alternatively, another chromatographic method is described comprising contacting said renatured heparin binding protein with a cation exchange support, a hydrophobic interaction chromatographic support, and an ion exchange chromatographic support and selectively eluting the heparin binding protein. of each support. It is contemplated that the steps of any procedure can be performed in any order. In an embodiment of the invention, the steps are carried out successively.

A suitable first step for the subsequent recovery and purification of the heparin-binding protein characteristically provides the concentration of the heparin binding protein and a reduction in the volume of the sample. For example, the second step of The incubation described above produces a large increase in the volume of the recovered heparin binding protein and the concomitant dilution of the protein in the renaturation buffer. The first suitable chromatographic supports provide a reduction in volume of the recovered heparin binding protein and can advantageously provide some purification of the protein from unwanted contaminating proteins. The first chromatographic steps include chromatographic supports that can be eluted and loaded directly onto a first hydrophobic interaction chromatographic support. For example, chromatographic supports are used from which the heparin binding protein can be eluted at a high concentration of suitable salt to load a hydrophobic interaction chromatographic support. Examples of first chromatographic supports include, but are not limited to, hydroxyapatite chromatographic supports, e.g. CHT ceramic type I and type II (formally known as MacroPrep ceramic), Bio-Gel HT, Bio-Gel HTP, Biorad, Hercules, CA, etc., chelating metal supports chelating metals consisting of an inert resin with immobilized metal ions such such as copper, nickel, etc., in addition to non-derivatized silica gels. In an embodiment of the invention, the first chromatographic supports for the purification and recovery of VEGF are hydroxyapatite chromatographic supports. In another embodiment of the invention, the first chromatographic supports for the purification and recovery of VEGF are cation exchange supports, e. those that are described below in more detail. Elution of the first chromatographic support is achieved in accordance with standard art practices. The conditions and suitable elution buffer will facilitate the loading of the HPB eluted directly to the first hydrophobic interaction chromatographic support as described below. Hydrophobic interaction chromatography is well known in the art and is based on the interaction of the hydrophobic portions of the molecule interacting with the hydrophobic ligands bound to the "chromatographic supports". A hydrophobic ligand is coupled to a matrix mentioned in various forms as a HIC chromatographic support, HIC gel or HIC column and the like. It is further appreciated that the strength of the interaction between the protein and the HIC column is not only a function of the ratio of non-polar to polar surfaces of the protein but also of the distribution of the non-polar surfaces. Several matrices can be used for the preparation of the HIC columns. The most widely used is agarose, although silica and organic polymer resins can also be used. Useful hydrophobic ligands include, but are not limited to, groups having from about 2 to about 10 carbon atoms, such as butyl, propyl or octyl, or aplo groups such as femlo. Conventional HIC supports for gels and columns can be obtained from suppliers such as GE Healthcare, Uppsala, Sweden ba or the product names butyl-SEPHAROSE ™, feni 1-SEPHAROSE ™ CL-4B, octil SEPHAROSE ™ FF and phenyl SEPHAROSE ™ FF and Tosoh Corporation, Tokyo, Japan or the product names TOYOPEARL ™ butyl 650M (Fractogel TSK But? l-650) or TSK-GEL phenyl 5PW. In one embodiment, the purification and recovery of VEGF is a first HIC chromatographic support which is butyl agarose and a second hydrophobic chromatographic support which is phenyl random. In another embodiment, first HIC chromatographic support is phenyl agarose. The density of the ligand is an important parameter in the sense that it influences not only the strength of the protein interaction but also the capacity of the column. The density of the ligand of the phenyl or octyl gels available in the market is in the order of 5-40 μmol / ml of the gel bed. The capacity of the gel is a function of the particular protein in question in addition to the pH, temperature and salt concentration but it can generally be expected to be included in the range of 3-20 mg / ml gel. The choice of the particular gel can be determined by the expert professional. In general, the strength of the interaction of the protein and the HIC ligand increases with the chain length of the alkyl of the ligand but the ligands having from about 4 to about 8 carbon atoms are suitable for the majority of the separations. A phenyl group has approximately the same hydrophobicity as a pentyl group, although the selectivity may be different due to the possibility of pi-pi interaction with the aromatic groups of the protein. The adsorption of the protein to a HIC column is favored by the high salt concentration but the actual concentration can vary over a wide range according to the nature of the protein and the particular HIC ligand chosen. In general, the salt concentration between approximately 1 and 4 M is useful. The elution of a HIC support, either stepwise or in a gradient form, can be obtained in a variety of ways such as a) by changing the salt concentration, b) by changing the polarity of the solvent or c) by addition of detergents. By decreasing salt concentrations, the adsorbed proteins are eluted in order to increase hydrophobicity. Changes in polarity can be caused by additions of solvents such as ethylene glycol or isopropanol, thus decreasing the strength of hydrophobic interactions. Detergents function as displacers of proteins and have been used primarily in connection with the purification of membrane proteins. Various ammonium constituents can be attached to the matrices to form cationic supports for chromatography. The ammonium constituents include the carboxymethyl, sulfethyl, sulfopropyl, phosphate and sulfonate groups (S). Cellulose ion exchange resins such as SE52 SE53, SE92, CM32, CM52, CM92, Pll, DE23, DE32, DE52, EXPRESS ION ™ S and EXPRESS ION ™ C are available from Whatman LTD, Maidstone Kent U.K. Ion exchangers based on SEPHADEX ™ and SEPHAROSE ™ and crosslinked are also known under the product names CM SEPHADEX ™ C-25, CM SEPHADEX ™ C-50 and SP SEPHADEX ™ C-25 SP SEPHADEX ™ C-50 and SP- SEPHAROSE ™ high-performance SP-SEPHAROSE ™ rapid flow, SP-SEPHAROSE XL, CM-SEPHAROSE ™ rapid flow and CM-SEPHAROSE ™, CL-6B, all available from GE Healthcare. Examples of ion exchangers for the practice of the invention include, but are not limited to, e.g. , ion exchangers under the MACROPREP ™ product name such as, for example, MACROPREP ™ S support, MACROPREP ™ High S support and MACROPREP ™ CM support from Irad, Hercules, CA. The elution of the cationic chromatographic supports is generally obtained by increasing the salt concentrations. Since the elution of the ionic columns involves the addition of salt and because, as mentioned, in HIC the salt concentration is increased, the introduction of the HIC step after the ionic step or another salt step is optionally used. In an embodiment of the invention, a chromatographic step of cation exchange precedes the passage of the HIC.

Examples of methods for purifying VEGF herein are described below, e.g. see Example V and VI. After renaturation, the insoluble material of the mixture is removed by deep filtration. The clarified mixture is then loaded onto a hydroxyapatite ceramic (Bio Rad, Hercules, CA) equilibrated in 5 mM HEPPS / TRITON ™ 0.05% XlOO / pH 8. The non-binding protein is removed by washing with the equilibrium buffer and VEGF was eluted by an isocratic step of 50 mM HEPPS / TRITON ™ 0.05% X100 / 0.15 M sodium phosphate / pH 8. The VEGF mixture is loaded on a Butyl SEPHAROSE ™ fast flow column (GE Healthcare, Uppsala, Sweden) equilibrated in 50 mM HEPPS / TRITON ™ 0.05% XlOO / 0.15 M sodium phosphate / pH 8. The column is washed with equilibrium buffer and the VEGF is collected in the column effluent . The SEPHAROSE ™ butyl mixture is loaded onto a Macro Prep High S column (BioRad, Hercules, CA) that is balanced in 50 mM HEPES / pH 8. After washing the absorbance of the effluent is measured at 280 nm from the baseline value , the column is washed with two columns of 50 mM HEPES / 0.25 M sodium chloride / pH 8. The VEGF is eluted by a linear column 8 with a volume gradient of sodium chloride 0.25-0.75 M in 50 mM HEPES / pH 8. The fractions are collected and those containing VEGF folded appropriately, determined by a hepapin binding assay, are mixed. The Macro Prep High S mixture is conditioned with an equal volume of 50 mM HEPES / 0.8 M sodium citrate / pH 7.5. The conditioned mixture is then loaded on a 5PW TSK Feml column (Tosoh Bioscience LLC, Montgomeryville, PA) which is equilibrated with 50 mM HEPES / 0.4 M sodium citrate / pH 7.5. After washing the non-binding protein by means of the column with equilibrium buffer, the VEGF is eluted from the column using a column 10 with a volume gradient of 0.4-0 M sodium citrate in 50 mM HEPES, pH 7.5. The fractions are analyzed by SDS-polyacrylamide gel electrophoresis and those containing VEGF of sufficient purity are combined. Expression of the heparin binding protein in host cells Briefly, expression vectors capable of autonomous replication and protein expression in relation to the genome of the prokaryotic host cell are introduced into the host cell. The construction of appropriate expression vectors is well known in the art which includes the nucleotide sequences of the umon to hepapna proteins described herein. See, for example , Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (Cold Spring Harbor, New York) (2001), Ausubel et al., Short Protocols in Molecular Biology, Current Protocols John Wiley and Sons (New Jersey) ) (2002), and, Baneyx, (1999) Current Opinion in Biotechnology, 10: 411-421. Commercially available expression vectors of prokaryotic cells, including bacteria, are available through, for example, the American Type Culture Collection (ATCC), Rockville, Maryland. Methods for large-scale growth of prokaryotic cells? and especially the cultivation of bacterial cells are well known in the art and these methods can be used in the context of the invention. For example, prokaryotic host cells are transfected with the expression or cloning vectors encoding the heparin binding protein of interest and cultured in a conventional nutrient medium modified as appropriate to induce promoters, select transformants or amplify the genes that encode the desired sequences. The nucleic acid encoding the polypeptide of interest is suitably RNA, cDNA or genomic DNA from any source, as long as it encodes the polypeptide (s) of interest. The methods are well known for selecting the appropriate nucleic acid for the expression of the heterologous polypeptides (including variants thereof) in the microbial hosts. The nucleic acid molecules encoding the polypeptide are prepared by a variety of methods known in the art. For example, a DNA that encodes VEGF is isolated and sequenced, eg. , by the use of oligonucleotide probes that are capable of specifically binding to the gene encoding VEGF. The heterologous nucleic acid (eg, cDNA or genomic DNA) is suitably inserted into a replicable vector for expression in the microorganism ba or control of a suitable promoter. Many vectors are available for this purpose, and selection of the appropriate vector will depend primarily on the size of the nucleic acid to be inserted into the vector and the particular host cell transformed with the vector. Each vector contains various components that depend on the particular host cell with which it is compatible. According to the particular type of host, the vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, a promoter, and a terminator sequence of transcription. In general, plasmid vectors containing replicon and control sequences that are derived from species compatible with the host cell are used in connection with microbial hosts. The vector commonly carries a replication site, in addition to the labeling sequences that are capable of providing a phenotypic selection in the transformed cells. For example, E. col i is typically transformed by pBR322, a plasmid derived from a species of E. coli (see, for e., Bolívar et al., (1977) Gene, 2: 95). PBR322 contains genes for resistance to ampicillin and tetracycline and thus provides simple means to identify the transformed cells. Plasmid pBR322 or other bacterial plasmid or phage, also generally contains, or is modified to contain, promoters that can be used by the host for the expression of selectable marker genes. (i) Signal sequence The polypeptides of the invention can be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which is typically a signal sequence or other polypeptide having a specific cleavage site in the N-terminal end of the protein or polypeptide mature. The selected heterologous signal sequence is typically one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the signal sequence of the native polypeptide, the signal sequence is replaced with a prokaryotic signal sequence selected, for example, from the group of alkaline phosphatase, penicillinase, lpp, or thermostable enterotoxin II leaders. . (ii) Component of the origin of replication Expression vectors contain a nucleic acid sequence that allows the vector to replicate in one or more of the selected host cells. Such sequences are well known for a variety of microbes. The origin of replication of plasmid pBR322 is suitable for most Gram-negative bacteria such as E. coli (iii) Selection of the gene component Expression vectors generally contain a selection gene, also called a selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells is a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium. This selectable marker is separated from the genetic markers as used and defined by this invention. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, eg. , ampicillin, neomycin, methotrexate or tetracycline, (b) complement autotrophic deficiencies different from those caused by the presence of genetic marker (s), or (c) provide critical nutrients not available from the complex medium, e. , the gene that codes for D-alamine racemase for Bacilli. An example of a selection scheme uses a drug to stop the growth of a host cell. In this case, cells that successfully transform with the nucleic acid of interest produce a polypeptide that confers drug resistance and thus survives the selection regimen. Examples of such dominant selection use the drugs neomycin (Southern et al., (1982) J. Molec.

Appl. Genet , 1: 327), mycophenolic acid (Mulligan et al., (1980) Science 209: 1422) or hygromycin (Sugden et al., (1985) Mol Cell. Biol., 5: 410-413). The three examples given above employ ba bacteria or eukaryotic control genes to transmit resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid), or hygromycin, respectively. (iv) Promoter Component The expression vector for producing the heparin binding protein of interest contains a suitable promoter which is recognized by the host organism and is operably linked to the nucleic acid encoding the target polypeptide. Promoters suitable for use with prokaryotic hosts include the beta-lactamase and lactose promoter systems (Chang et al., (1978) Nature, 275: 615, Goeddel et al., (1979) Nature, 281: 544), arabinose promoter system (Guzman et al., (1992) ^ Bactepol., 174: 7716-7728), the alkaline phosphatase or tryptophan (trp) promoter system (Goeddel, (1980) Nucleic Acids Res., 8: 4057 and EP 36,776) and hybrid promoters such as the tac promoter (deBoer et al., (1983) Proc. Nati, Acad. Sci. USA, 80: 21-25). However, other known bacterial promoters are suitable. Their nucleotide sequences have been published, thus allowing expert practitioners to link them operably to the DNA encoding the polypeptide of interest (Siebenlist et al, (1980) Cell, 20: 269) using linkers or adapters to provide any of the required restriction sites. See also, by e. , Sambrook et al., Supra, and Ausubel et al. , supra. Promoters for use in bacterial systems also generally contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the polypeptide of interest. The promoter can be removed from the bacterial DNA source by digestion with restriction enzymes and inserted into the vector containing the desired DNA. (v) Construction and vector analysis The construction of suitable vectors containing one or more of the components listed above employs standard ligation techniques. The isolated plasmids or DNA fragments are cleaved, adapted and re-ligated in the desired form to generate the required plasmids. For the analysis to confirm the correct sequences in the constructed plasmids, the linkage mixtures are used to transform the E strain. coli K12 294 (ATCC 31,446) or other strains and successful transformants are selected for resistance to ampicillin or tetracycline as appropriate. The plasmids of the transformants are prepared, analyzed by digestion and restriction endonuclease and / or sequenced by the method of Sanger et al., (1977) Proc. Nati Acad. Sci. USA, 74: 5463-5467 or Messing et al., (1981) Nucleic Acids Res., 9: 309, or by the method of Maxam et al., (1980) Methods in Enzymology, 65: 499. See also , for ex. , Sambrook et al., Supra, and Ausubel et al., Supra. The nucleic acid encoding the heparin binding protein of interest is inserted into the host cells. Typically, this is achieved by transforming the host cells with the expression vectors described above and culturing in a modified conventional nutrient medium as appropriate to induce the various promoters. Culturing the host cells Prokaryotic cells suitable for use to express the heparin binding proteins of interest are well known in the art. Host cells that express the recombinant protein abundantly in the form of inclusion bodies or in the periplasmic or intracellular space are typically used. Suitable prokaryotes include bacteria, eg, eubacteria, such as Gram-negative or Gram-positive organisms, eg, E. col i, Ba ci lli such as B. subti lis, species of Pseudomonas s species such as P. aeruginosa, Salmonel typhimuri um, or Serra tia marcescens. An example of an E host. coli is E. col i 294 (ATCC 31, 446). Other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are also suitable. These examples are illustrative rather than limiting. The W3110 strain is a typical host because it is a common host strain for fermentations of recombinant DNA products. In one aspect of the invention, the host cell must secrete minimal amounts of proteolytic enzymes. For example, the strain W3110 can be modified to effect the genetic mutation of the genes encoding proteins, as examples of such hosts include strains of E. col i W3110 1A2, 27A7, 27B4, and 27C7 described in U.S. Patent No. 5,410,026 filed April 25, 1995. For example, one strain for the production of VEGF is the E strain. coli W3110 that has the tonA genotype? ptr3 phoA? E15? (argF-la c) 1 69 degP41 i lvg designated 49B3. In another example, a strain for the production of VEGF is the strain of E.coli (62A7) which has the genotype? FhuA (? TonA) ptr3, clg, cL8, ¿SompT? (nmpC-fepE)? degP? lvG + See also, eg. , the table covering pages 23-24 of WO2004 / 092393.

The prokaryotic cells used to produce the heparin binding protein of interest are cultured in a medium known in the art and suitable for the culture of the selected host cells, which include the means generally described by Sambrook et al., Molecular Cl Oning, A Labora Tory Manua, Cold Spring Harbor Laboratory Press (Cold Spring Harbor, New York) (2001). Means that are suitable for bacteria include, but are not limited to, AP5 medium, nutrient broth, Luria-Bertani broth (LB), minimal Neidhardt medium and complete minimum medium of C.R.A.P. More nutritional supplements needed. In certain embodiments, the media also contains a selection agent, chosen on the basis of the construction of the expression vector, to selectively allow the growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the medium for the growth of cells expressing an ampicillin-resistant gene. Any of the necessary supplements in addition to carbon, nitrogen and inorganic phosphate sources can also be included in appropriate concentrations introduced alone or as a mixture with another supplement or medium such as a complex nitrogen source. Optionally the culture medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, dithioetptipot and dithiothreitol. Examples of suitable means are given in U.S. Patent Nos. 5,304,472 and 5,342,763. The medium C.R.A.P. Phosphate-limited consists of 3.57 g of (NH4) 2 (S04), 0.71 g of Na-2H20 citrate, 1.07 g of KCl, 5.36 g of yeast extract (certified), 5, 36 g of HycaseSF ™ -Sheffield, adjusted to pH with KOH at 7.3, is volume adjusted to 872 ml with H20 desiominated and autoclaved, cooled to 55 ° C. and supplemented with 110 ml of 1 M MOPS pH 7.3, 11 ml of 50% glucose, 7 ml of 1 M MgSO4). Then, carbemzyma can be added to the induction culture at a concentration of 50 μg / ml. The prokaryotic host cells are cultured at suitable temperatures. For the growth of E. col i, for example, the temperature range varies from eg. , about 20 ° C to about 39 ° C, or about 25 ° C to about 37 ° C, or about 30 ° C. When the alkaline phosphatase promoter is employed, the cells of E. coli used to produce the polypeptide of interest of this invention are cultured in a suitable medium in which the alkaline phosphatase promoter can be partially or completely induced as generally described, e.g. , in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (Cold Spring Harbor, New York) (2001). The culture never needs to bind in the absence of inorganic phosphate or in levels of phosphate deprivation. At the beginning, the medium contains inorganic phosphate in an amount above the level of induction of protein synthesis and sufficient for the growth of the bacteria. As the cells grow and utilize phosphate, they lower the phosphate level of the medium, thereby inducing the synthesis of the polypeptide. If the promoter is an inducible promoter, for induction to occur, typically the cells are cultured until a certain density is reached, e. , an A550 of approximately 200 using a high cell density process, in which the induction point is initiated (eg, by the addition of an inducer, by reduction of a component of the medium, etc.), to induce expression of the gene encoding the polypeptide of interest. Any necessary supplements may also be included in appropriate concentrations that are known to those skilled in the art, introduced alone or as a mixture with another supplement or medium such as a complex nitrogen source. The pH of the medium can be any pH of about 5-9, depending mainly on the host organism. For E. col i, the pH is, for example. , from about 6.8 to about 7.4, or about 7.0. Formulations of heparin-binding proteins The recovered polypeptide, e.g. , by the methods disclosed herein, can be formulated into a vehicle acceptable for pharmaceutical use and used for various diagnostic, therapeutic, or other known uses for such molecules. For example, the VEGF described herein can be used in immunoassays, such as enzyme immunoassays. Therapeutic uses for the heparin-binding proteins obtained from the methods disclosed herein are also contemplated. For example, a growth factor or hormone, eg. , VEGF, can be used to increase growth when desired. For example, VEGF can be used to promote wound healing, eg. , an acute wound (eg, burn, surgical wound, normal wound, etc.) or a chronic wound (eg, diabetic ulcer, pressure ulcer, decubitus ulcer, venous ulcer, etc.), to promote capillary growth, to promote growth and repaired tissue (eg, bone, liver, etc.), etc. Therapeutic formulations of heparin-binding proteins are prepared for preservation by the mixture of a molecule, e.g. , a polypeptide, having the desired degree of purity with pharmaceutically acceptable carriers, excipients or stabilizers (Remington 's Pharmaceutical Sciences 18th edition, Gennaro, A. Ed. (1995)), in the form of lyophilized formulations or aqueous solutions. Acceptable vehicles, excipients or stabilizers are non-toxic to containers at the doses and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (such as chloride). of octadecyldimethylbenzyl amomium, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), polypeptides of low molecular weight (less than about 10 residues), proteins, such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinyl pyrrolidone, amino acids such as glycine, glutamine, asparagma, histidine, argimna, or lysine, monosaccharides, disaccharides, and other carbohydrates that include glucose, mannose, or dextpnas, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose or sorbitol, salt-forming counterions such as sodium, metal complexes (by e. Zn-protein complexes), and / or non-ionic surfactants such as TWEEN ™, PLURONICS ™ or polyethylene glycol (PEG). In certain embodiments, the formulations used for in vivo administration are sterile. This is easily accomplished by filtration through the sterile filtration membranes. BPH can be stored in lyophilized form or as an aqueous solution or gel form. The pH of the HBP preparations can be, e. , from about 5 to 8, although higher or lower pH values may also be appropriate in certain cases. It will be considered that the use of certain excipients, vehicles or stabilizers can produce the formation of salts of the HBP. Typically for wound healing, HBP is formulated for site-specific administration. When applied topically, BPH is suitably combined with other elements, such as vehicles and / or adjuvants. There are no limitations on the nature of such other elements, except that these must be acceptable for pharmaceutical use and effective for the intended administration and can not significantly degrade the active elements of the composition. Examples of suitable vehicles include ointments, creams, gels, aerosols or suspensions, with or without purified collagen. The compassions can also be impregnated in sterile bandages, transdermal patches, plasters and bandages optionally in liquid or semi-liquid form. To obtain a gel formulation, the HBP formulated in a liquid composition can be mixed with an effective amount of a water-soluble polysaccharide or synthetic polymer such as polyethylene glycol to form a gel of the appropriate viscosity to be applied. The polysaccharide that can be used includes, for example, cellulose derivatives such as etherified cellulose derivatives, which include alkyl celluloses, hydroxyalkyl celluloses, and alkyl hydroxyalkyl celluloses, for example, methyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose, hydroxypropyl methyl cellulose and hydroxypropyl. cellulose, starch and fractionated starch, agar, algic acid and alginate, gum arabic, pullulan, agarose, carrageenan, dextrans, dextrus, fructans, mulina, mannan, xylan, arabinanos, chitosan, glycogen, glycans and synthetic biopolymers, in addition to gums such as xanthe gum, guar gum, locust bean gum, gum arabic, gum tragacanth and karaya gum and derivatives and mixtures thereof. In certain embodiments of the invention, the gelling agent herein is one which, e. , is inert to biological systems, nontoxic, simple to prepare and / or not too slippery or viscous and will not destabilize the HBP retained in it. In certain embodiments, the polysaccharide is an etherified cellulose derivative, in another embodiment one that is well defined, purified and listed in USP, e.g. methylcellulose and hydroxyalkyl cellulose derivatives, such as hydroxypropyl cellulose, hydroxyethyl cellulose and hydroxypropyl methylcellulose. In one embodiment, the methylcellulose is the polysaccharide. If methylcellulose is used in the gel, eg. , typically comprises about 2-5%, or about 3% or about 4% or about 5% of the gel, and the HBP is present in a proportion of about 300-1000 mg per ml of gel. The polyethylene glycol useful for gelation is typically a mixture of low and high molecular weight polyethylene glycols to obtain the proper viscosity. For example, a mixture of a polyethylene glycol of molecular weight of 400-600 with one of molecular weight 1500 would be effective for this purpose when mixed in the proper ratio to obtain a paste. The term "water-soluble" when applied to polysaccharides and polyethylene glycols is intended to include colloidal solutions and dispersions. In general, the solubility of the cellulose derivatives is determined by the degree of substitution of the ether groups, and the stabilizing derivatives useful herein should have a sufficient amount of such ether groups per anhydrous glucose unit of the cellulose chain to turn the derivatives into hydroslubles. A degree of ether substitution of at least 0.35 anhydrous glucose unit ether groups is generally sufficient. In addition, the cellulose derivatives can be in the form of alkali metal salts, for example, Li, Na, K, or Cs salts. The active components can also be enclosed in microcapsules or sustained release preparations. See, for example , Remington's Pharmaceutical Sciences 18th edition, Gennaro, A. Ed. (1995). See also Johnson et al., Nat. Med., 2: 795-799 (1996), Yasuda, Biomed. Ther., 27: 1221-1223 (1993), Hora et al., Bio / Technology, 8: 755-758 (1990), Cleland, "Design and Production of Single Immunization Vaccines Using Polylactide Polyglycolide Microsphere Systems," in Vaccine Design : The Subunit and Adjuvant Approach, Powell and Newman, eds, (Plenum Press: New York, 1995), pp. 439-462, WO 97/03692, WO 96/40072, WO 96/07399, U.S. Patent No. 5,654,010, DE 3,218,121, Epstem et al., (1985) Proc. Nati Acad. Sci. USA, 82: 3688-3692, Hwang et al., (1980) Proc.

Nati Acad. Sci. USA, 77: 40304034, EP 52,322, EP 36,676, EP 88,046, EP 143,949, EP 142,641, Japanese Patent Application 83-118008, U.S. Patent Nos. 4,485,045 and 4,544,545, and EP 102,324. The following examples are offered by way of illustration and not by way of limitation. EXAMPLES EXAMPLE 1: Recombinant human VEGF expressed in Escheri chia coli Recombinant human VEGF was expressed in Escheri chia coli. During the synthesis, the protein was secreted in the peripasmic space and accumulated as retractable bodies. The studies were therefore carried out to obtain the extraction and renaturation of the protein. These studies revealed at least 3 species of VEGF (Figure 1) that were isolated by known recovery techniques without the addition of a polyanionic agent. Studies with native VEGF showed that the addition of heparin increased resistance to denaturing induced by the chaotrope and thiol (Figure 2). In addition, hepapna significantly increased the proportion of VEGF renatured appropriately in small-scale renaturation experiments. In order to adapt this result to a large-scale process, conditions were discovered that allowed the renaturation of VEGF in the presence of dextran sulphate, a molecule with a structure analogous to heparin. The addition of dextran sulfate improves the yields of appropriately biologically active renatured VEGF 3-5-fold relative to the controls. Methods Plamid for the expression of VEGFjßs The plasmid pVEGF171 was designed for the expression of human VEGF? 65 (see, eg, Leung et al., (1989) Science, 246: 1306-1309) in the periplasm of the E. col i. The transcription of the VEGF coding sequence was placed under close control of the alkaline phosphatase (AP) promoter (see, eg, Kikuchi et al., (1981) Nucleic Acids Research, 9: 5671-8), whereas Sequences required for translation initiation were provided by the Shine-Dalgarno trp region (see, eg, Yanofsky et al., (1981) Nucleic Acids Research, 9: 6647-68). The coding sequence of VEGF was fused downstream of the thermostable bacterial enterotoxin II signal sequence (STII) (see, eg, Lee et al., (1983) Infect. Immun. 42: 264-8, and Picken et al. al., (1983) Infect, Immun.42: 269-75) for subsequent secretion in the periplasm of E. coli. Modifications of the codon of the STII signal sequence provided an adjusted translation level, which produced an optimal level of VEGF accumulation in the periplasm der, by e. , Simmons and Yansura, (1996) Nature Biotechncloy, 14: 629-34). The lambda for the transcription terminator (see, eg, Scholtissek and Grosse, (1987) Nucleic Acids Research 15: 3185) was located downstream of the translation stop codon of VEGF. The origin of replication and the ampicillin and tetracycline resistance genes were provided by the plasmid pBR322. See, for example , Bolívar et al., (1977) Gene 2: 95-113. Cellular homogenization and preparation of refractile bodies- The collected Escherichia coli cells were frozen and stored at -70C °. Cells were harvested by centrifugation with BTUX (centrifuge, Alfa laval) and freezing by BEPEX (large-scale freezer).

Cells were suspended in 5 volumes of 50 mM HEPES / 150 mM NaCl / 5 mM EDTA pH 7.5 (5 1 / kg pellet) and homogenized in a laboratory model 15 M Gaulin 15M (small scale) or M3 homogenizer ( large scale) (Gaulm Corporation, Everett, MA). The cell suspension was then diluted with an equal volume of the same buffer and the shrinkable bodies were collected by centrifugation in a continuous feed centrifuge BTPX 205 (Alfa Laval Separation AB (Tumba, Sweden) .The intermediate scale centrifuge used was SAI. Alternatively, the cells can be homogenized and the pellet can be harvested directly without freezing in BEPEX and rehydrating EXAMPLE II: Extraction and Renaturation of the human recombinant VEGF expressed in Eschep chia col i -I Methods Extra ction and Rena turali za tion - The Retractable pellet was suspended in extraction buffer containing 7 M Urea / 50 mM HEPPS / pH 8 (final concentration) at 5 liters buffer / kg of pellet, then solid dithiothreitol was added at a rate of 3, 7 g / kg of pellet for a final concentration of 4 mM. See, for example , Figure 9 for the effect of urea and DTT on the extraction of VEGF. The suspension was mixed thoroughly for 1-2 hours at 20 ° C. The pH can be adjusted with 50% sodium hydroxide (w / w) to pH 8.0. The renaturation was initiated by the addition of 19 volumes of renaturation buffer per volume of extraction buffer. The renaturation buffer contained 50 mM HEPPS / 1 M-2M Urea / 2-5 mM / TRITON ™ 0.05% -0.2% XlOO / pH 8, final concentration. See, for example , Figure 10 for the effect of the concentration of urea and DTT present during the renaturation. Dextran sulfate, hepapna or sodium sulfate was added as indicated. The renaturation incubation was performed at room temperature for 4-24 hours. Optionally, the incubation can be carried out at room temperature for up to about 48 hours. The folding was monitored by SDS-PAGE and / or heparin HPLC. The product was clarified by deep filtration with a Cuno 90SP filter followed by 0.45 μm filtration. HPLC analysis of heparin binding - the amount and quality of appropriately renatured VEGF was determined by a column containing immobilized hepapna. The POROS HE2 / M column (4.6 x 100 mm, HE2 / M by PerSeptive BioResearch Products, Cambridge, MA) was equilibrated in 10 mM sodium phosphate, pH 7 containing 0.15 M sodium chloride. of flow of 1 ml / min or 2 ml / rnin, the columns were eluted using a linear gradient of 0.15 M to 2 M sodium chloride in equilibrium buffer for 10 min. In some trials, the elution was performed in 16 minutes. The eluent was monitored at 280 nm. Typically, most of the protein was eluted at the null volume and 3 classes of VEGF could be identified. The highest affinity species that eluted in the end was identified as correctly renatured VEGF and was later identified as "VEGF Pico 3". Results Heparin protects VEGF against stein-mediated denaturation. The addition of 10 mM cysteine to native VEGF produced a large decrease in the appropriately folded molecule (Figure 2). This denaturation was prevented by the addition of 2 different forms of heparin in concentrations as low as 20 mM. TABLE The effect of heparin and dextran sulfate on the renaturation of VEGF Addition Concentration (μg / ml) 0 10 55 100 200 400% Aum. or inc of plegam.

None 5.3 * Low PM heparin (3 kd) 12.2 14.2 14.8 14.1 179% 2.8 High PM heparin (6 kD) 15.3 16.6 13.9 15.3 213% 3.1 Dextran sulphate (10 Kd) 15.9 15.4 13.6 7.4 8.3 191% 2.9 The values in the table are the ratio of Peak 3 of the VEGF formed (in mg) per g of shrink pellet. The concentration of each addition is indicated. * average control (5.6 + 5.0 = 5.3) TABLE Ib The effect of sodium sulphate on the renaturation of VEGF Addition Concentration (μg / ml) 0 50 98 195 293 455% Aum. or inc of plegam. None 5.3 - Sodium sulphate 6.9 9.1 10.4 10.9 10.4 106% 2.1 The values in the table are the ratio of Peak 3 of VEGF formed (in mg) per g of retractable pellet The concentration of sodium sulfate is indicated TABLE II The effect of heparins and dextran sulfates on the renaturation of VEGF Addition Concentration (μg / ml) 0 2.5 12.5 50 100% Inc or inc of plegam. None 2,2-dextran sulfate (5Kd) 10, l 13.7 13.4 11.2 523% 6.2 dextran sulphate (8Kd) 9.9 17.2 14.0 12.8 682% 7 , 8 dextran sulfate (lOKd) 13.8 19.2 14.6 10.1 773% 8.7 Low PM heparin (3 kd) 10.4 16.9 14.7 668% 7.7 High heparin PM (6 kD) 14,1 18.8 20.2 818% 9.2 The values in the table are the ratio of Peak 3 of VEGF formed (in mg) per g of shrink pellet. Summary The hepapna and the dextran sulfates increase the yields of the re-turalization. Due to the protective properties against the denaturation described above, the effect of several different forms of sulaphated polymers on the renaturation of VEGF was investigated. As seen in the TABLE (and in Fig. 5), the low and high molecular weight forms of hepapna increased the yield of renatured VEGF by approximately 3-fold. As seen in TABLE Ib (and in Figure 6), sodium sulfate increased the yield of renatured VEGF by approximately 2-fold. The 10 Kd form of dextran sulfate was also effective in increasing the yields of reanturalization, however, the greater concentration range investigated produced the inhibition of the substrate. Further investigations showed that all 5 Kd, 8 Kd and 10 Kd forms of dextran sulfate significantly increased the performance of VEGF over renaturation (TABLE II). See Figure 7. See also Figure 8. EXAMPLE III: Effect of different buffer and TRITOISP X-100 on the recovery of VEGF Results Buffer VEGF (mg / g pellet, HEPES, pH 8 13.3 HEPES, pH 8 with TRITONt 14.3 HEPPS, pH 8 16, 6 TrisHCl, pH 8 12, 8 Buffer VEGF (mg / g pellet, HEPES, pH 7.2 9.1 HEPPS, pH 7.2 10.7 HEPES, pH 8 10.3 HEPPS, pH 8 12.8 HEPES, pH 8 + TRITON ™ X-100 12.4 HEPPS, pH 8 + TRITON ™ X-100 13, 9 Summary The combined data of Examples I, II and III demonstrate a significant (2-5 times) increase in yield which includes both heparin sulfates and dextran sulfates when VEGF is renatured, a hepapne-binding growth factor in addition to the conditions of recovery. This method has been implemented successfully on an industrial scale. This method is expected to be applicable in the renaturation of other basic growth factors and other proteins that bind to heparin. EXAMPLE IV: Extraction and renaturation of the human recombinant VEGF expressed in Eschep chia col i-II Methods Extra ction and Rena turali za tion - The retractable pellet was suspended in extraction buffer containing 7 M Urea, 2-30 mM DTT (e.g. ., 10 mM DTT), 50 mM HEPPS / pH 7-9 (eg, pH 8) to 5 liters of buffer / kg of pellet. The suspension was mixed thoroughly for 1-2 hours at room temperature. The renaturation was initiated by the addition of 19 volumes of renaturation buffer per volume of extraction buffer. The renaturation buffer contained as a final concentration 1 M urea or 1, 3 M, 2-15 mM cysteine (e.g., 7.5 mM cysteine), 0.5 mM DTT, 0-0.75 M argon (e.g., 100 mM arginine), dextran sulfate 15 mg / l, 50 mM HEPPS, TRITON ™ 0.05% XlOO / pH 8. See, eg. , Figure 12 for the effect of renaturation in the presence of charged amino acids, where the addition of histidine produced the same effect as without amino acid additives. The renaturation incubation was performed at room temperature for 12-24 hours. Optionally, the incubation can be carried out at room temperature for up to about 48 hours. Optionally, it can be used and oxygen during the renaturation process (0, 3-lcc / min / l). The folding was monitored by SDS-PAGE and / or heparin HPLC. The product was clarified by deep filtration with a Cuno 90SP filter followed by 0.45 μm filtration. The total dilution of the extraction and renaturation steps was 1: 100. The increase in the total dilution of the extraction and renaturation steps, eg. , at 1: 100 to 1: 200, the total proportion of active VEGF increased although the concentration is lower. See Figure 13. The efficiency of renaturation can be established by determining the proportion of dimer / monomer, where the monomers can be determined by a C18 HPLC column in reverse phase and the formation of dimers can be determined by column chromatography of heparin or an SP-5PW cation exchange chromatography assay.

EXAMPLE V: Large Scale Renaturation In order to examine the scalability of optimized renaturation conditions, studies were performed to examine the kinetics of renaturation in small scale (0.1 1), intermediate (1 1) and pilot plant (250 1 to 400 1). As shown in Figure 4, the kinetics of large-scale renaturation were indistinguishable from the smaller scales and the final titre of the renatured VEGF was slightly increased. These data demonstrate the scalability of renaturing with dextran sulfate. The product was further clarified by deep filtration with a Cuno 90SP filter followed by 0.45 μm filtration. EXAMPLE VI: Purification I of rhVEGF after Renaturing Croma tografí a in hydroxiapa ti ta cerámica Ma croPrep- After renaturation, the insoluble material of the mixture was removed by deep filtration. The clarified mixture was then loaded onto a ceramic column with hydroxyapatite (35D x 31H = 30L) (Bio Rad, Hercules, CA) equilibrated in 50 mM HEPPS / 0.05% TRITON ™ / pH 8. The unbound protein was removed by washing with equilibrium buffer and VEGF eluted using an isocratic step of HEPPS50 mM / TRITON ™ 0.0100% / 0.15 M sodium phosphate / pH 8. The flow rate was 120 cm / hr. The mixed fractions were determined by heparin HPLC analysis of the fractions. Croma tography in Bu til SEPHAROSE ™ fl uj o fast- The VEGF mixture was loaded on a Butyl SEPHAROSE ™ column Fast flow (35 D x 26 H = 25L) (GE Healthcare, Uppsala, Sweden) balanced in 50 mM / TRITON HEPPS ™ 0.05% XlOO / 0.15 M sodium phosphate / pH 8. The flow rate was 100 cm / hr. The column was washed with equilibrium buffer and the VEGF was collected in the column effluent. The fractions were collected and the fractions containing proteins were mixed, by measurement at A280nm. Croma tography Ma cro Prep High S- The Butyl SEPHAROSE ™ mixture was loaded onto a Macro Prep High S column (30D x 39 H = 27 L) (BioRad, Hercules, CA) which was equilibrated in 50 mM HEPES / pH 8. After washing the absorbance of the effluent at 280 nm was the baseline, the column was washed with two column volumes of 50 mM HEPES / 0.25 M sodium chloride / pH 8. The VEGF was eluted using a linear column 8 and a volume gradient of 0.25-0.75 M sodium chloride in 50 mM HEPES / pH 8. The flow rate was 75-200 cm / hr. The fractions were collected and those containing the renatured VEGF were mixed appropriately, determined by the heparin binding assay, e.g. , HPLC Heparin. Croma tography in phenyl 5PW TSK - The Macro Prep High S mixture was conditioned with an equal volume of 50 mM HEPES / 0.8 M sodium citrate / pH 7.5. The conditioned mixture was then loaded on a Phenyl 5PW TSK column (18D x 43 H = 11L) (Tosoh Bioscience LLC, Montgomeryville, PA) which was equilibrated with 50 mM HEPES / 0.4 M sodium citrate / pH 7, 5. After washing the non-bound protein by means of the column with equilibrium buffer, the VEGF is eluted from the column using a column 10 with a volume gradient of 0.4-0 M sodium citrate in 50 mM HEPES, pH 7.5. The fractions are analyzed by SDS-polyacrylamide gel electrophoresis and those containing VEGF of sufficient purity are mixed. Ul trafil tracion / Diaphile tracíón- The ultrafiltered VEGF mixed in a 5kD regenerated cellulose membrane (G30619), Unit Pellicon, Feed Rate 17.1 L / min. The membrane was conditioned with polysorbate 20. The mixed VEGF was ultrafiltered at a concentration of 6 g / 1 (UF1). The sample was subjected to diafiltration with 7-14 DV (Diavolume) with 5 mM sodium succinate / 275 mM Trehalose / pH 5.0. The final formulation was 5 mM sodium succinate / 275 mM Trehalose / 200.01% polysorbate / pH5.0, at a concentration of 5 mg / ml. EXAMPLE VII: Purification II of rhVEGF after Renaturation Chroma tography liquefied in terchange cationi - After renaturation, the insoluble material of the mixture can be removed by deep filtration. The renatured mixture is conditioned to pH 5.0-7.5 and approximately 2 to 6.5 mS / cm. In one embodiment, the mixture is conditioned at pH 6.5 and 5 mS / cm. The renatured mixture can then be loaded onto a sulfopropyl extreme loading column (SPXL) and eluted using a gradient of increasing salt concentration. The combination fractions can be determined by heparin HPLC analysis of the fractions. Hydrophobic interaction column (HIC): -The SPXL elution mixture of VEGF can be conditioned at 50mS / cm to load on a chromatographic column Fe l TSK (Tosohaas, Montgomeryville, PA). The fractions were collected and the fractions containing proteins were mixed. I EX or mixed mode: -The combination of Fheml TSK can be loaded on a column of ion exchange chromatography (IEX) or mixed mode chromatography. The fractions were collected and mixed fractions containing renatured VEGF appropriately, determined by the assays described herein. Ultrafiltration / Filtration Day- The ultrafiltered VEGF mixed in a 5kD regenerated cellulose membrane (G30619), Unit Pellicon, Feed Rate 17.1 L / min. For example, the membrane was conditioned with polysorbate 20. The mixed VEGF is ultrafiltered at a concentration of 6 g / 1 (UF1). The sample was subjected to diafiltration with 7-14 DV (Diavolume) with 5 mM sodium succinate / 275 mM Trehalose / pH 5.0. In the methods and processes described herein, the purity and / or final activity can be evaluated by peptide mapping, disulfide mapping, SDS-PAGE (reduced and non-reduced), circular dichroism, limulus amoebocyte lysate (LAL ), heparin chromatography, hepap HPLC (eg, Heparin HPLC can be used to determine the VEGF dimer concentration), reversed-phase HPLC (rp) chromatography (eg, rpHPLC can be used to determine the concentration of the VEGF monomer), umon to heparin, receptor binding (eg for VEGF, binding to the KDR-Bioanalytic R &; D, and / or binding to the Fltl receptor), SEC assays, cell assays, HUVEC power assays, VEGF antibody ELISA, mass spectrometry analysis, etc.

It is considered that the deposits, examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested by those skilled in the art and will be included in the spirit and scope of this application. and scope of the appended claims. All publications, citations, patents and patent applications herein are incorporated in their entirety as a reference for all purposes.

Claims (23)

1. CLAIMS Having thus specially described and determined the nature of the present invention and the manner in which it is to be put into practice, it is claimed to claim as proprietary and exclusive right 1. A process for recovering a heparin binding protein from of a prokaryotic cell culture, the process comprising the steps of (a) isolating said heparin binding protein from the periplasm of said prokaryotic cell culture, (b) denaturing said heparin binding protein in a first buffered solution comprising a chaotropic agent and a reducing agent, (c) incubating said denatured heparin-binding protein in a second buffered solution comprising a chaotropic agent and a sulfated polyanionic agent for such a period of time and under such conditions that renaturation of the protein occurs of heparin binding, and (d) recovering said renatured heparin-binding protein, where there is approximately a 2 to 5 fold increase in the recovered renatured heparin binding protein compared to incubation without a non-sulfated polyammonic agent.
2. 2. The process of claim 1, wherein the heparin binding protein is a heparin binding growth factor.
3. 3. The process of claim 2, wherein the heparin-binding growth factor is vascular endothelial growth factor (VEGF).
4. 4. The process of claim 3, wherein the VEGF is VEGFip5.
5. The process of claim 1, wherein the sulfated polyammonic agent has between about 3,000 daltons and 10,000 daltons.
6. The process of claim 3, wherein said second buffered solution comprises a dextran sulfate.
7. 7. The process of claim 3, wherein said second buffered solution comprises sodium sulfate.
8. The process of claim 3, wherein said second buffered solution comprises heparin.
9. 9. The process of claim 6, wherein the dextran sulfate has between about 8,000 and 10,000 daltons.
10. 10. The process of claim 3, wherein said first and second buffered solutions comprise HEPPS at pH 8.0.
11. The process of claim 1, wherein said second buffered solution further comprises a reducing agent.
12. The process of claim 11, wherein the reducing agent of the second buffered solution comprises a combination of cistern and DTT.
13. The process of claim 1, wherein said second buffered solution further comprises a non-ionic detergent.
14. The process of claim 1, wherein said second buffered solution further comprises argimine and / or lysine.
15. The process of claim 1, wherein said recovery step (d) comprises successively contacting said renatured heparin-binding protein with a hydroxyapatite chromatographic support, a first hydrophobic interaction chromatographic support, a cationic chromatographic support, and a second chromatographic support of hydrophobic interaction, and selectively elute the heparin binding protein of each support.
16. 16. The process of claim 15, wherein said first and second hydrophobic interaction chromatographic support is selected from the group consisting of butyl-, propyl-, octyl- and aryl-agarose resins.
17. 17. The process of claim 15, wherein said first hydrophobic interaction chromatographic support is a butyl-agarose support and said second hydrophobic interaction chromatographic support is a phenyl-agarose support resin.
18. The process of claim 1, wherein said recovery step (d) comprises successively contacting said renatured heparin binding protein with a cation exchange support, a hydrophobic interaction chromatographic support and an ion exchange chromatographic support, and selectively eluting the heparin binding protein from each support.
19. 19. A method for recovering a heparin binding protein from a culture of prokaryotic cells, the method comprising the steps of (a) isolating said heparin binding protein from the periplasm of said prokaryotic cell culture, (b) denaturing said isolated heparin binding protein in a first buffered solution comprising a chaotropic agent and a reducing agent, (c) incubating said denatured heparin-binding protein in a second buffered solution comprising a chaotropic agent and a sulfated polyanionic agent for such a period of time and under such conditions that renaturation of the heparin-binding protein occurs, where there is approximately a 2 to 5 fold increase of the renatured heparin umon protein recovered compared to the incubation without sulfated polyammonium agent and (d) successively contacting said renatured heparin binding protein with a hydroxyapatite chromatographic support , a first hydrophobic interaction chromatographic support, a support cation chromatography and a second hydrophobic interaction chromatographic support and selectively elute the heparin binding protein of each support.
20. 20. A method for purifying a hepapin binding protein, the method comprising the steps of successively contacting a renatured heparin-binding protein with a hydroxyapatite chromatographic support, a first hydrophobic interaction chromatographic support, a cation chromatographic support and a second hydrophobic interaction chromatographic support and selectively elute the heparin binding protein of each support.
21. 21. A method for recovering a heparin-binding protein from a culture of prokaryotic cells, the method comprising the steps of (a) isolating said heparin binding protein from the periplasm of said prokaryotic cell culture, (b) denaturing said isolated heparin binding protein in a first buffered solution comprising a chaotropic agent and a reducing agent, (c) incubating said denatured heparin-binding protein in a second buffered solution comprising a chaotropic agent and a sulfated polyanionic agent during such a period of time and under such conditions that renaturation of the heparin binding protein occurs, where there is approximately a 2 to 3 fold increase of the recovered renatured heparin binding protein compared to the incubation without a sulfated polyanionic agent and d) successively contacting said renatured heparin binding protein with a sop. orte of cation exchange, a hydrophobic interaction chromatographic support and an ion exchange chromatographic support and selectively elute the heparin binding protein of each support.
22. 22. The method of claim 19 or 21, wherein the polyanionic agent has between about 3,000 daltons and 10,000 daltons.
23. 23. A method for purifying a heparin-binding protein, the method comprising the steps of successively contacting a renatured heparin binding protein with a cation exchange support, a hydrophobic interaction chromatographic support, and an exchange chromatographic support. ion and selectively elute the heparin binding protein of each support.