ES2359883T3 - RECOMBINANT PRODUCTION OF HEPARIN UNION PROTEINS. - Google Patents

Abstract

A process for recovering a heparin-binding protein from a prokaryotic cell culture, wherein said heparin-binding protein is vascular endothelial growth factor (VEGF) that binds to heparin, the process comprising the steps of : (a) isolating said heparin-binding protein from the periplasm of said prokaryotic cell culture; (b) denature said heparin binding protein in a first buffered solution comprising a chaotropic agent and a reducing agent; (c) incubating said heparin-binding protein in a second buffered solution comprising a chaotropic agent and a sulfated polyanionic agent, wherein said sulfated polyanionic agent is dextran sulfate or sodium sulfate, for a time and under conditions such that refolding of said heparin binding protein occurs; and (d) recovering said heparin-binding protein, in which there is an approximately 2 to 5-fold increase in refolded heparin-binding protein compared to incubation without sulfated polyanionic agent.

Description

Related request

[0001] This application claims the priority and benefit of the provisional US application. Serial No. 60 / 753,615, filed on December 22, 2005, and of the US provisional application. Serial No. 60 / 807,432, filed July 14, 2006.

Field of the Invention

[0002] This invention relates to methods for obtaining heparin binding proteins produced in cell culture. The invention includes methods for recovering and purifying folded heparin binding proteins that have been produced in eukaryotic 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

[0003] It is known that a wide variety of biologically active polypeptides of natural origin 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. Natl. Acad. Sci. USA, 86: 7571-7574; Talpas et al. (1991) Biochim. Biophys. Acta, 1078: 208-218; Webb et al. (1993) Proc. Natl. Acad. Sci. USA, 90: 7158-7162), factors of heparin-binding growth (Burgess and Maciag, (1989) Annu. Rev. Biochem., 58: 576-606; Klagsbrun, (1989) Prog. Growth Factor Res., 1: 207-235) such as growth factor epidermal (EGF); platelet-derived growth factor (PDGF); basic fibroblast growth factor (bFGF); acid fibroblast growth factor (aFGF); Vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) (Liu et al. (1992) Gastrointestinal. 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) Arterioscler. Thromb. Vasc Biol., 24: 1549-1557.

[0004] International Publication No. WO 95/07097 describes formulations of heparin-binding proteins that include heparin-binding growth factors such as VEGF, with purified native heparin or other polyanionic compounds for therapeutic use. Heparin-derived oligosaccharides and various other polyanionic compounds have been shown to stabilize the active conformation of heparin-binding growth factors (Barzu et al. (1989) J. Cell. Physiol. 140: 538-548; Dabora and col., (1991) J. Biol. Chem. 266: 23627-23640) and heparin affinity chromatography has been used in various purification schemes (see, in general, International Publication No. WO 96/02562).

[0005] Many of the heparin-binding proteins of mammalian origin have been produced by 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 vascular permeability factor (VPF). See Dvorak et al., (1995) Am. J. Pathol. 146: 1029-39. VEGF plays important roles both in vasculogenesis, the development of embryonic vessels, and in angiogenesis, the process of formation of new blood vessels from preexisting ones. See, for example, Ferrara, (2004) Endocrine Reviews 25 (4): 581-611; Risau et al., (1988) Dev. Biol. 125: 441-450; Zachary, (1998) Intl. J. Biochem. Cell Bio. 30: 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) Endocrin. Rev. 18: 4-25. Clinical applications for VEGF include those in which the growth of new capillary beds is indicated, for example, in the promotion of wound healing (see, for example, International Publication No. WO 91/02058 and agent file No. P2358R1, entitled "Wound healing" presented on June 16, 2006), in the promotion of tissue growth and repair, for example, liver (see, for example, document W02003 / 0103581), bone (see, for example, WO2003 / 094617), etc. See also Ferrara, (2004) Endocrine Reviews 25 (4): 581-611.

[0006] 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 achieve maximum cell density and ultimately achieves lower cell density than 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. Bacterial host expression systems provide an economical alternative for manufacturing scale production of recombinant proteins. There are numerous US patents. on the generic bacterial expression of recombinant proteins, including US Pat. No. 4,565,785, 4,673,641, 4,795,706 and 4,710,473. An important advantage of the production process 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.

[0007] Recombinant heparin-binding growth factors such as acid fibroblast growth factor, basic fibroblast growth factor and vascular endothelial growth factor have been recovered and purified from a number of sources including bacteria (Salter DH and col., (1996) Labor. Invest. 74 (2): 546-556 (VEGF); Siemeister et al., (1996) Biochem. Biophys. Res. Commun. 222 (2): 249-55 (VEGF); Scrofani et al., (2001) J. Microbiol. Biotechnol. 11 (4): 543-551 (VEGF); Cao et al. (1996) J. Biol. Chem. 261 (6): 3154-62 (VEGF ); Yang et al. (1994) Gaojishu Tongxun, 4: 28-31 (VEGF); Anspach et al. (1995) J. Chromatogr. A 711 (1): 129-139 (aFGF and bFGF); Gaulandris (1994) J. Cell. Physiol. 161 (1): 149-59 (bFGF); Estape and Rinas (1996) Biotech. Tech. 10 (7): 481-484 (bFGF); McDonald et al., (1995 ) PHASEB

J. 9 (3): A410 (bFGF)). However, bacterial expression systems such as E. coli lack the cellular machinery to facilitate the proper refolding of proteins, and generally do not result in the secretion of large proteins in the culture media. Recombinant proteins expressed in bacterial host cells are often found as inclusion bodies consisting of dense masses of reduced protein partially folded and erroneously folded. In this way, the recombinant protein is generally inactive. For example, the predominant active form of VEGF is a homodimer of two 165 amino acid polypeptides (VEGF-165). In this structure, each subunit contains 7 pairs of intracatenary disulfide bonds and two additional pairs that affect the covalent ligation of the two subunits (Ferrara et al. (1991) J. Cell. Biochem. 47: 211-218). The native conformation includes a strongly basic domain that has been shown to readily bind to heparin (Ferrara et al. (1991) supra). Covalent dimerization of VEGF is necessary for effective receptor binding and biological activity (Pötgens et al., (1994) J. Biol. Chem. 269: 32879-32885; Claffey et al., (1995) Biochim. Et Biophys. Minutes 1246: 1-9). The bacterial product potentially contains several intermediates folded wrongly and with disorganized disulfide bridges.

[0008] Additionally, refolding often produces dimers, trimers and multimers that are erroneously folded and bound by disulfide (Morris et al. (1990) Biochem. J., 268: 803-806; Toren et al. (1988) Anal Biochem., 169: 287-299). This association phenomenon is very common during protein refolding, particularly at high protein concentrations, and often seems to involve association by hydrophobic interaction of partially folded intermediates (Cleland and Wang, (1990) Biochemistry, 29: 11072-11078) .

[0009] Wrong folding occurs 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 retreated to the native state. In vitro procedures to replicate proteins in the correct biologically active conformation are essential to obtain functional proteins. Typical subsequent processing of proteins recovered from inclusion bodies includes dissolution of the inclusion body in a high concentration denaturant such as urea, followed by dilution of the denaturant to allow refolding to occur (see US Pat. No. 4,512,922, 4,511,502 and 4,511,503). See also Rudolph and Lilie, (1996) FASEB J. 10: 49-56 and Fischer et al., (1993), Biotechnology and Bioengineering, 41: 3-13. Such recovery procedures are considered to be universally applicable, with minor modifications, to the recovery of biologically active recombinant proteins from inclusion bodies. These procedures have been applied to heparin binding proteins such as VEGF (Siemeister et al. (1996) supra). These procedures seek to eliminate random disulfide bonds before achieving the recombinant protein in its biologically active conformation through its other stabilization forces, and may not eliminate improperly folded intermediates or provide homogeneous populations of properly folded product.

[0010] Inverse micelles or ion exchange chromatography have been used to aid in refolding denatured proteins including a single protein in 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.) P. 249251, New York: Alan R. Liss, Inc.). These procedures have been useful to prevent protein aggregation and facilitate proper refolding. To alter the speed or extent of refolding, a specific refolding of conformation with ligands and antibodies of the native structure of the protein (Cleland and Wang, (1993), in "Biotechnology", (Rehm H.-J. and Reed G. Eds.) P. 528-555, New York, VCH). For example, creatine kinase has been replicated in the presence of antibodies of the native structure (Morris et al. (1987) Biochem. J. 248: 53-57). In addition to antibodies, ligands and cofactors have been used to enhance refolding. These molecules would be more likely to interact with the folded protein after the formation of the native protein. Therefore, the folding equilibrium can "shift" to the native state. For example, the refolding speed of ferricitochrome c has been enhanced by the extrinsic ligand of the axial position of heme iron (Brems and Stellwagon, (1983) J. Biol. Chem.

258: 3655-3661). Chaperone proteins have also been used to aid protein folding. See, for example, Baneyx, (1999) Current Opinion in Biotechnology, 10: 411-421. Heparin and heparin sulfate have been indicated to help stabilize the heparin binding protein VEGF (Brandner et al., 2005), Biochem. Biophys Res. Commun. 340: 826-839; Gengrinovitch et al., (1999) J. Biol. Chem. 274: 10816-10822).

[0011] There is a need for new and more efficient methods of folding and / or recovering heparin-binding proteins from a host cell culture, for example, for the efficient and economical production of heparin-binding proteins in a culture of bacterial cells, which provide for the elimination or reduction of biologically inactive intermediates and an improved recovery of a properly biologically active and highly purified refolded protein which, and which are generally applicable to the production at the manufacturing scale of the proteins. The invention addresses these and other needs, as will be apparent upon review of the following disclosure.

Summary of the invention

[0012] The invention provides a method for the recovery and purification of folded heparin-binding proteins from a cell culture, in particular from prokaryotic host cells, for example bacterial cells, according to the claims. For example, the method comprises the steps of (a) isolating an insoluble heparin-binding protein from the periplasmic or intracellular space of said bacterial cells; (b) solubilizing said insoluble heparin binding protein isolated 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 a polyanionic agent sulfated for a time and conditions such that the refolding of the heparin binding protein occurs, and (d) recovering said refolded heparin binding protein, in which there is a 2 to 10 fold increase in the concentration of protein recovered by incubation with a sulfated polyanionic agent compared to a control according to the claims. In one embodiment, the second buffered solution further comprises arginine. In one embodiment, the second buffered solution further comprises cysteine or a mild reducing agent.

[0013] According to the invention, there is a 2-5 fold increase in the protein concentration of the recovered biologically active refolded protein. There may be a 3-5-fold increase in the concentration of biologically active refolded protein recovered, or a 2-3-fold increase in the concentration of biologically active refolded protein recovered. There may be an increase, for example, greater than 2.0 times, 2.5 times, 2.8 times, 3.0 times or 5 times in the concentration of biologically active refolded protein protein recovered. In one embodiment of the invention, there is a 3 to 5 fold increase in the concentration of biologically active refolded VEGF protein.

[0014] The processes of the invention are applicable to vascular endothelial growth factor (VEGF). The processes described herein are broadly applicable to heparin-binding proteins and especially to heparin-binding growth factors, and in particular, to vascular endothelial growth factor (VEGF). In certain embodiments of the invention, the sulfated polyanionic agent is between about 3,000 and 10,000 Da. The sulfated polyanionic agent used in production processes is a dextran sulfate or sodium sulfate. In one aspect, dextran sulfate is between 3,000 Da and 10,000 Da.

[0015] The invention further includes processes and methods for purification of the VEGF heparin-binding protein in relation to the recovery of the heparin-binding protein as described herein. In a particular embodiment, the purification procedures include contacting said folded 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 eluting selectively the heparin binding protein of each support. In another embodiment, a purification process comprises contacting said folded heparin binding protein with a cation exchange support, a first hydrophobic interaction chromatographic support and a mixed media ion or chromatographic exchange support, and selectively eluting the protein Heparin binding of each support. It is contemplated that the recovery steps can be carried out in any order, for example, sequentially or by altering the order of the chromatographic supports. In certain embodiments of the invention, methods are provided for recovering and purifying folded heparin binding proteins from a manufacturing or industrial scale cell culture.

Brief description of the drawings

[0016]

Figure 1 illustrates a VEGF chromatogram produced by the 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 with 10 mM sodium phosphate, pH 7, containing 0.15 M sodium chloride. The column is eluted using a linear gradient of 0.15-2 M sodium chloride, 10 mM sodium phosphate, pH 7, for 10 minutes. The eluent is controlled at 280 nm. The protein recovered in each peak corresponds to VEGF, however only peak 3 corresponds to appropriately biologically active refolded VEGF.

Figure 2 illustrates a graph depicting heparin stabilization of properly folded native VEGF. VEGF is suspended in 50 mM HEPES, pH 8, which contains 5 mM EDTA, 0.2 mM NaCl and 10 mM cysteine.

Figures 3A-3D illustrate VEGF chromatographs produced by bacterial strain W3110 and incubated with dextran sulfate of 5,000 Da 12 µg / ml (Figure 3A); dextran sulfate

8,000 Da 12 µg / ml (Figure 3B); dextran sulfate 10,000 Da 12 µg / ml (Figure 3C) or heparin

3,000 Gives 25 µg / ml (Figure 3D), and loaded onto a POROS HE2 / M column (4.6 x 100 mm, PerSeptive BioResearch Products, Cambridge, MA). For example, the column is equilibrated with 10 mM sodium phosphate, pH 7, containing 0.15 M sodium chloride. The column is eluted using a linear gradient of 0.15-2 M sodium chloride in sodium phosphate. mM, pH 7, for 10 minutes. The eluent is controlled at 280 nm. The protein recovered in each peak corresponds to VEGF, however only peak 3 corresponds to an appropriately biologically active refolded VEGF.

Figure 4 illustrates the effect of the scale on VEGF refolding.

Figure 5 illustrates the effect of low molecular weight (PM) and high PM heparin and 10,000 Da dextran sulfate on VEGF refolding. Peak 3 corresponds to a properly biologically active refolded VEGF.

Figure 6 illustrates the effect of sodium sulfate on VEGF refolding. Peak 3 corresponds to a properly biologically active refolded VEGF.

Figure 7 illustrates the effect of low molecular weight (PM) and high PM heparin and 5,000 Da, 8,000 Da and 10,000 Da dextran sulfate on VEGF refolding. Peak 3 corresponds to appropriately biologically active refolded VEGF.

Figure 8 illustrates the effect of heparin and dextran sulfate on VEGF refolding. Peak 3 corresponds to appropriately biologically active refolded VEGF.

Figure 9 illustrates the effect of urea and DTT on the extraction of VEGF from bacterial inclusion bodies.

Figure 10 illustrates the effect of urea and DTT concentration on VEGF refolding.

Figure 11 illustrates the amino acid sequence of VEGF165 with the disulfide bridges indicated (SEQ ID NO.:1).

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 arginine and lysine are beneficial, while histidine has little additive effect compared to the buffered solution without it. Additionally, arginine has been shown to have a similar effect at concentrations of 0.1 to 1 M.

Figure 13 illustrates the effect of the solution on the% of refolding efficiency, in which, although the concentration of total VEGF is lower as the dilution increases, the% of refolding efficiency is greater with more dilution. Detailed Description Definitions

[0017] "Heparin" (also referred to as heparinic acid) is a heterogeneous group of highly sulfated linear chain anionic mucopolysaccharides called glucosaminoglycans. Although others may be present, the main sugars in heparin are: α-L-iduronic acid 2-sulfate, 2-deoxy-2-sulfamino-α-glucose 6-sulfate, β-Dglucuronic acid, 2-acetamido- 2-deoxy-α-D-glucose and L-iduronic acid. These, and optionally other sugars, are linked by glycosidic bonds, forming polymers of varying sizes. Due to the presence of its covalently linked sulfate and carboxylic acid groups, heparin is strongly acidic. The molecular weight of heparin varies from about 3,000 to about 20,000 Da depending on the source and the determination procedure.

[0018] Native heparin is a constituent of various tissues, especially liver and lung, and mast cells in various mammalian species. Heparin and heparin salts (sodium heparin) are commercially available and are mainly used as anticoagulants in various clinical situations.

[0019] "Dextran sulfate" is a dextran sulfate whose main structure is a D-glucose polymer. Glucose, and optionally other sugars, are linked by αD glycosidic bonds (1-6), forming polymers of varying 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 about 15-20%, with up to 3 sulfate groups per glucose molecule. Average molecular weight of dextran sulfate is from about 1,000 to about 40,000,000 Da. Examples of dextran sulfate employed in the invention include dextran sulfate produced from microorganisms such as Leuconostoc mesenteroides and L. dextranicum.

[0020] "Polyanionic agent" as used within the scope of the invention is intended to describe commercially available purified native heparin preparations and compounds that are capable of binding to heparin-binding proteins, including other "polyanionic agents" such as sodium sulfate. , heparin sulfate, heparan sulfate, (poly) pentosan sulfate, dextran, dextran sulfate, hyaluronic acid, chondroitin, chondroitin sulfate, dermatan sulfate and queratane sulfate. Particularly useful within the context of the invention is a "sulfated polyanionic agent" such as, for example, a sulfate derivative of a polysaccharide, such as heparin sulfate, dextran sulfate, cyclodextrin sulfates produced by microorganisms such as Bacillus macerans described in U.S. Patent No. 5,314,872, as well as sulfates of other glucans such as β-1,3-glucane sulfates, producing β-1,3-glucan by microorganisms belonging to the genus Alcaligenes or Agrobacterium, and chondroitin sulfate as well as fragments of sulfated heparin.

[0021] The agents mentioned above are generally available and are recognized by the expert. For example, sulfated heparin fragments can be obtained from a collection of heparin-derived oligosaccharides that have been fractionated by molecular exclusion chromatography. The preparation of affinity fractionated heparin-derived oligosaccharides has been reported by Ishihara et al. (1993) J. Biol. Chem., 268: 4675-4683. These oligosaccharides were prepared from commercial swine heparin after partial depolymerization with nitrous acid, reduction with sodium borohydride and fractionation by molecular exclusion chromatography. The resulting sets of di-, tetra-, hexa-, octa- and decasaccharides were sequentially applied to an affinity column of human recombinant bFGF covalently bound to SEPHAROSE ™ 4B, and further fractionated into subsets based on their elution from this column in response to sodium chloride gradients. This resulted in five sets, designated Hexa-1 to Hexa-5, whose biological structures and activities were further evaluated. The structure of Hexa-5C and its 500 MHz NMR spectrum are shown in Figure 4 of Tyrell et al. (1993) J. Biol. Chem., 268: 4684-4689. This hexasaccharide has the structure [IdoA (2-OSO3) α1-4GlcNSO3 (6-OSO3) α1-4] 2IdoA (2-OSO3) α14AManR (6-OSO3). All heparin-derived oligosaccharides discussed above, as well as other heparin-type oligosaccharides, are suitable and can be used according to the invention. In one embodiment of the invention, heparin hexasaccharides and heparin polysaccharides of high unit size (eg, hepta-, octa-, nona- and decasaccharides) are used. In addition, heparin-derived or heparin-derived oligosaccharides with a large net negative charge are advantageously used, for example, due to a high degree of sulfation.

[0022] The term "heparin binding protein" or "HPB" as used herein designates a polypeptide capable of heparin binding (as defined hereinbefore). The definition includes mature, pre, prepro and pro forms of native and recombinantly produced heparin binding proteins. 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), basic fibroblast growth factor ( bFGF), acid fibroblast growth factor (aFGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) (also known as dispersion factor, SF) and nerve growth factor (NGF), IL -8, etc.

[0023] As used herein, "vascular endothelial growth factor" or "VEGF" designates a mammalian growth factor originally derived from bovine pituitary follicular cells that have the amino acid sequence disclosed in Castor, C.W. et al., (1991) Methods in Enzymol. 198: 391-405, together with functional derivatives thereof that have 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. Endocrin 5: 1806-1814. See also Leung et al. (1989) Science, 246: 1306 and Robinson and Stringer, (2001) Journal of Cell Science, 144 (5): 853-865, US Pat. No. 5,332,671. The predominant form of VEGF is a 165 amino acid homodimer that has 16 cysteine 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 VEGF121 variant lacks the heparin binding domain of the other variants and therefore so much does not fall within the definition of heparin binding protein set forth herein. All isoforms of VEGF share a common aminoterminal domain, but differ in the length of the carboxy terminal portion of the molecule. The preferred active form of VEGF, VEGF165, has disulfide bonds between the Cys26-Cys68 amino acid residues; Cys57-Cys104; Cys61-Cys102; Cys117-Cys135; Cys120-Cys137; Cys139-Cys158; Cys146-Cys160 in each monomer. See Figure 11. See also, for example, Keck et al., (1997) Archives of Biochemistry and Biophysics 344 (1): 103-113. The VEGF165 molecule is composed of two domains: an aminoterminal receptor binding domain (1-110 amino acid disulfide-linked homodimer) and a carboxy-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, the isolated and purified VEGF165 is not glycosylated in the remainder 75 (Asn). See, for example, Yang et al., (1998) Journal of Pharm. & Experimental Therapeutics, 284: 103-110. In certain embodiments of the invention, the isolated and purified VEGF165 is substantially not deamidated in the Asn10 moiety. In certain embodiments of the invention, the isolated and purified VEGF165 is a mixture of deamidated protein (in the Asn10 moiety) and non-deamidated protein, typically with most of the protein being deamidated. Since VEGF165 is a homodimer, deamination can occur in one or both polypeptide chains.

[0024] As used herein, VEGF or other BPH and the like "appropriately folded" or "biologically active" designates a molecule with a biologically active conformation. The expert will recognize that erroneously folded and disorganized disulfide intermediates may have biological activity. In that case, the VEGF or HBP properly folded or biologically active corresponds to the native folding pattern of the VEGF (described above) or other BPH. For example, properly folded VEGF has the disulfide pairs indicated above, in addition to two intermolecular disulfide bonds in the dimeric molecule; however, other intermediates can be produced by culturing bacterial cells (Figure 1 and 3A-3D). For properly folded VEGF, the two intermolecular disulfide bonds can occur between the same moieties, Cys51 and Cys60, of each monomer. See, for example, WO98 / 16551. Biological activities of VEGF include, but are not limited to, for example, promoting vascular permeability, promoting vascular endothelial cell growth, binding to a VEGF receptor, binding and signaling via a VEGF receptor (see, for example, Keyt et al., (1996) Journal of Biological Chemistry, 271 (10): 56385646), induce angiogenesis, etc.

[0025] The terms "purified" or "pure BPH" and the like designate a material free of substances that normally accompany it when it is in its recombinant production and especially in culture of prokaryotic or bacterial cells. Thus, the terms designate a recombinant BPH that is free of contaminating DNA, host cell proteins or other molecules associated with its in situ environment. The terms designate 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.

[0026] The terms "inclusion bodies" or "refractive bodies" designate dense intracellular masses of the aggregate polypeptide of interest, which constitute a significant portion of the total cellular protein, including all cellular components. In some cases, but not all, these polypeptide aggregates can be recognized as visible bright spots within the contour of the cells with a phase contrast microscope at 1000 magnifications.

[0027] As used herein, the term "erroneously folded" protein designates precipitated or aggregated polypeptides that are contained in the refractory bodies. As used herein, VEGF or other "insoluble" or "erroneously folded" BPH designates precipitated or aggregated VEGF that is contained in the periplasm or intracellular space of prokaryotic host cells, or that is otherwise associated with a host cell. prokaryotic, and adopts a biologically inactive conformation with mismatched disulfide bridges

or not formed. Insoluble BPH is generally contained, but not necessarily, in refractory bodies, specifically, it may or may not be visible with a phase contrast microscope.

[0028] As used herein, "chaotropic agent" designates a compound that, in a suitable concentration in aqueous solution, is capable of changing the spatial configuration.

or conformation of polypeptides by alterations in the surface thereof, so that the polypeptide is returned soluble in the aqueous medium. Alterations can occur, for example, by changing the hydration state, the solvent environment or the solvent-surface interaction. The concentration of chaotropic agent will directly affect its potency and effectiveness. A strongly denatured chaotropic solution contains a chaotropic agent at high concentrations that, in solution, will effectively deploy a polypeptide present in the solution, effectively eliminating the secondary structure of the proteins. The deployment will be relatively extensive, but reversible. A moderately denaturing chaotropic solution contains a chaotropic agent that, in sufficient concentrations in solution, allows partial folding of a polypeptide from any distorted conformation that the polypeptide has adopted by dissolving soluble intermediates to the spatial conformation in which it is found when it works in its active form in endogenous or homologous physiological conditions. Examples of chaotropic agents include guanidine hydrochloride, urea and hydroxides such as sodium hydroxide.

or potassium Chaotropic agents include a combination of these reagents, such as a mixture of a hydroxide with urea or guanidine hydrochloride.

[0029] As used herein, "reducing agent" designates a compound that, in suitable concentration in aqueous solution, keeps the sulfhydryl groups free so that intra-or intermolecular disulfide bridges are chemically destabilized. Representative examples of suitable reducing agents include dithiothreitol (DTT), dithioerythritol (DTE), β-mercaptoethanol (BME), cysteine, cysteamine, thioglycolate, glutathione, tris- [2-carboxyethyl] phosphine (TCEP) and sodium borohydride.

[0030] As used herein, "buffered solution" means a solution that resists changes in pH by the action of its conjugated acid / base components.

[0031] "Bacteria" for the purposes of this specification include eubacteria and archaebacteria. In certain embodiments of the invention, eubacteria, including gram-positive and gram-negative bacteria, are used in the procedures and processes described herein. In one embodiment of the invention, gram-negative bacteria are used, for example, Enterobacteriaceae. Examples of bacteria belonging to Enterobacteriaceae include Escherichia, Enterobacter; Erwinia, Klebsiella, Proteus, Salmonella, Serratia and Shigella. Other types of suitable bacteria include Azotobacter, Pseudomonas, Rhizobia, Vitreoscilla and Paracoccus. In one embodiment of the invention, E. coli is used. Suitable E. coli hosts include E. coli W3110 (ATCC 27,325), E. coli 294 (ATCC 31,446), E. coli 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 aforementioned bacteria can also be used. Of course, it is necessary to select the appropriate bacteria considering the replicability of the replicon in the cells of a bacterium. For example, E. coli, Serratia or Salmonella species can be suitably used as a host when well known plasmids such as pBR322, pBR325, pACYC177 or pKN410 are used to deliver the replicon. See below for examples of suitable bacterial host cells.

[0032] As used herein, the terms "cell", "cell line", "strain" and "cell culture" are used interchangeably and all such designations include progeny. Therefore, the words "transformants" and "transformed cells" include the primary target cell and the cultures derived therefrom without regard to the number of transfers. It is also understood that the entire progeny may not be exactly identical in the DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same biological function or activity that was screened in the originally transformed cell is included. When different denominations are intended, it will be clear from the context.

[0033] As used herein "polypeptide" generally designates peptides and proteins from any cellular source that have more than about 10 amino acids. "Heterologous" polypeptides are those polypeptides foreign to the host cell being used, such as a human protein produced by E. coli. Although the heterologous polypeptide can be prokaryotic or eukaryotic, it is preferably eukaryotic, more preferably mammalian, and most preferably human. In certain embodiments of the invention, it is a recombinant or recombinant polypeptide.

Heparin binding proteins

Isolation of heparin binding protein

[0034] Soluble heparin-binding protein (BPH) erroneously folded from prokaryotic host cells expressing the protein is isolated by any of a number of standard techniques. For example, insoluble BPH is isolated in a suitable isolation buffer by exposing the cells to a buffer of ionic strength suitable to solubilize most host proteins, but in which the subject protein is substantially insoluble, or destabilizing the cells so that they release inclusion bodies or protein from the periplasmic or intracellular space and make them available for recovery, for example, by centrifugation. This technique is well known and is described, for example, in US Pat. No. 4,511,503. Kleid et al. disclose the purification of refractive 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 also, for example, Fischer et al., (1993) Biotechnology and Bioengineering 41: 3-13.

[0035] US Pat. No. 5,410,026 describes a typical procedure for protein recovery from inclusion bodies and is summarized below. Prokaryotic cells are suspended in a suitable buffer. Typically, the buffer consists of a buffering agent suitable for buffering at between pH 5 and 9, or about 6 and 8, and a salt. Any suitable salt, including NaCl, is useful for maintaining sufficient ionic strength in the buffered solution. Typically, an ionic strength of about 0.01 to 2 M, or 0.1 to 0.2 is used

M. The cells, suspended in this buffer, are destabilized or lysed, using commonly employed techniques such as, for example, mechanical procedures, for example, homogenizer (Manton-Gaulin press, microfluidizer or Niro-Soavi), French press, mill of balls or ultrasonic oscillator, or by chemical or enzymatic procedures.

[0036] Examples of chemical or enzymatic processes of cell destabilization include spheroplasty, which involves the use of lysozyme to lyse the bacterial wall (H. Neu et al. (1964) Biochem. Biophys. Res. Comm., 17: 215 ) and osmotic shock, which involves the treatment of viable cells with a solution of high tonicity and with a wash with cold water of low tonicity to release the polypeptides (H. Neu et al., 1965 J. Biol. Chem., 240 ( 9): 3685-3692). Sonication is generally used for the destabilization of bacteria contained in analytical scale volumes of fermentation broth. At larger scales, high pressure homogenization is typically used.

[0037] After destabilizing the cells, the suspension is typically centrifuged at low speed, generally from about 500 to 15,000 xg, for example, in one embodiment of the invention about 12,000 xg is used, in a standard centrifuge, for a sufficient time to sediment substantially all insoluble protein. These times can be determined simply and depend on the volume being centrifuged as well as the design of the centrifuge. Typically, about 10 minutes to 0.5 hours is sufficient to sediment the insoluble protein. In one embodiment, the suspension is centrifuged at 12,000 x g for 10 minutes.

[0038] The resulting sediment contains substantially the entire insoluble protein fraction. If the process of cell destabilization is not complete, the sediment may also contain intact cells or broken cell fragments. The completeness of the cell destabilization can be tested by resuspending the sediment in a small amount 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 additional sonication, or other destabilization means, is necessary to remove the fragments or cells and the associated non-refractive polypeptides. After such destabilization, if necessary, the suspension is centrifuged again and the sediment is recovered, resuspended and reexamined. The process is repeated until the visual examination reveals the absence of broken cell fragments in the sedimented material or until the additional treatment fails to reduce the size of the resulting sediment.

[0039] The above process can be used if the insoluble protein is intracellular or is in the periplasmic space. The conditions given herein to isolate heparin-binding protein are directed to inclusion bodies precipitated in the periplasmic space or intracellular space, and refer in particular to VEGF according to the invention. However, it is believed that the processes and procedures are applicable to heparin-binding proteins in general with minor modifications as seen throughout the following text. In certain embodiments of the invention, the processes and procedures are applicable for manufacturing or production on an industrial scale, refolding and purification of BPH.

Replenishment of heparin binding proteins

[0040] The erroneously insoluble folded heparin binding protein isolated in a first buffered solution containing an amount of chaotropic agent and reducing agent sufficient to substantially solubilize the heparin binding protein is incubated. This incubation takes place under conditions of concentration, incubation time and incubation temperature that allow solubilization of part or substantially all of the heparin-binding protein, and for deployment to occur.

[0041] The measurement of the degree of solubilization in the buffered solution can be determined simply and carried out suitably, for example, by determining turbidimetry, analyzing the fractionation between the supernatant and the sediment after centrifugation, in gels of Reduced PAGE-SDS, by protein assay (for example, the Bradford reagent protein assay (eg, Pierce, Bio-Rad etc.)), or by HPLC.

[0042] The first buffered solution comprises a buffering agent suitable for maintaining the pH range at least about 7.0, the typical range being 7.5-10.5. In one embodiment, the pH for VEGF is pH 8.0. Examples of suitable buffers that will provide a pH in this latter range include TRIS-HCl (tris [hydroxymethyl] aminomethane), HEPPS (N- [2-hydroxyethyl] piperazin-N '- [3-propanesulfonic acid]), HEPES (acid N- [2-hydroxyethyl] piperazin-N '- [2-ethanesulfonic])), CAPSO (3- [cyclohexylamino] -2-hydroxy-1propanesulfonic acid), AMP (2-amino-2-methyl-1-propanol), CAPS (3- [cyclohexylamino] -1propanesulfonic acid), CHES (2- [N-cyclohexylamino] ethanesulfonic acid), glycine and sodium acetate. In one embodiment of the invention, the buffer herein is HEPPS at approximately pH 8.0. In a further embodiment, the buffers, for example such as HEPPS, are sulfated.

[0043] Chaotropic agents suitable for the practice of this invention include, for example, urea and guanidine or thiocyanate salts, for example, urea, guanidine hydrochloride, sodium thiocyanate, etc. The amount of chaotropic agent that is necessary to be present in the buffer is an amount sufficient to deliver the HBP in solution. In certain embodiments of the invention, a chaotropic is present at about 4 to 10 M. In one embodiment, the chaotropic agent is urea at about 5-8 M, or at about 7 M. In another example, the chaotropic agent is hydrochloride of guanidine at approximately 6-8 M.

[0044] Examples of suitable reducing agents include, but are not limited to, dithiothreitol (DTT), dithioerythritol (DTE), β-mercaptoethanol (BME), cysteine, DTE, etc. The amount of reducing agent that is 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 trapped or introduced into the solution and the concentration of protein in the buffer. tampon. For example, with 0.5-1.5 mg / ml of protein in a buffer solution at pH 7.0-10.0 containing 4-8 M urea, the reducing agent is, for example, DTT at a concentration of approximately 1-15 mM or BME at a concentration of approximately 0.2-2 mM or cysteine at a concentration of approximately 2-10 mM. In one embodiment, the reducing agent is DTT at about 0.5 to about 4 mM, or 2-4 mM. Figure 9 illustrates the effect of urea and DTT on VEGF extraction. VEGF of peak 3 designates biologically active VEGF properly folded. In one embodiment, the reducing agent is approximately 10 mM DTT. A single reducing agent or a combination of reducing agents may be used in a buffer herein.

[0045] The concentration of protein in the buffered solution should be such that the protein is substantially solubilized as determined by optical density. The exact amount to be used will depend, for example, on the concentrations and types of the other ingredients in the buffered solution, particularly the concentration of protein, reducing agent and pH of the buffer. In one embodiment of the invention, the concentration of heparin binding protein is in the range of 0.5-5.5 mg per ml, or 1.5-5.0 mg / ml. The solubilization is typically carried out at about 0-45 ° C, or at about 20-40 ° C, or at about 23-37 ° C, or at about 25-37 ° C, or at about 25 ° C for at least about 1 to 24 hours. In one embodiment, solubilization is carried out for at least about 2 hours at room temperature. Typically, the temperature is apparently not affected by the levels of salt, reducing agent and chaotropic agent.

[0046] After solubilizing the polypeptide, it is disposed or diluted in a second buffered solution containing the chaotropic agent and a sulfated polyanionic agent as described above, however, at a concentration of chaotropic agent that allows refolding of the protein from Heparin binding.

[0047] The conditions of this second incubation of the erroneously folded soluble protein will generally be such that a partial or substantial or complete refolding of the protein takes place. The exact conditions will depend, for example, on the pH of the buffer and on the types and concentrations of polyanionic sulfate agents and on the chaotropic and reducing agents present, if any. The incubation temperature is generally about 0-40 ° C

or 10-40 ° C, and incubation will generally be carried out for at least about 1 hr to effect refolding. In certain embodiments, the reaction is carried out, for example, 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 6 and 20 hours, or between 12 and 24 hours.

[0048] The degree of refolding is adequately determined by radioimmunoassay (RIA) assessment of the BPH or by high performance liquid chromatography (HPLC) analysis using, for example, a POROS HE2 / M column (PerSeptive BioResearch Products) or other appropriate heparin affinity column. The increase in the RIA value or the correctly folded HBP peak size correlates directly with the increasing amounts of biologically active folded HPB present in the buffer. Incubation is carried out to maximize the ratio of correctly folded HPB to wrongly recovered folded HPB, as determined by RIA or HPLC.

[0049] In one embodiment, the quality and quantity of properly folded VEGF is assessed using a heparin binding assay. Samples containing the diluted heparin binding protein are loaded, for example, on a POROS HE2 / M column (4.6 x 100 mm, PerSeptive BioResearch Products, Cambridge, MA) or another suitable heparin affinity column. For example, the affinity column is equilibrated by heparin with 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 using a linear gradient of 0.15-2 M sodium chloride in 10 mM sodium phosphate, pH 7, for 10 minutes. The eluent is controlled at 280 nm. In one embodiment, the protein is recovered in a single peak corresponding to the appropriately biologically active folded HBP. In one embodiment of the invention, it is an assay to determine the appropriately refolded BPH HPLC-FI. Disulfide bonds can optionally be confirmed by peptide mapping. Circular dichroism can also be used to determine the bi- and three-dimensional structure / folding.

[0050] The buffer of the second buffered solution may be any of those listed above for the first buffered solution, for example, HEPPS pH 8.0, for example, at a concentration of approximately 50 mM to refuel VEGF. The polypeptide can be diluted with the refolding buffer, for example at least 5 times or at least about 10 times, about 20 times or about 40 times. Alternatively, the polypeptide can be dialyzed against the refolding buffer.

[0051] The second buffered solution contains a chaotropic agent at a concentration such that HPB refolding occurs. Generally, a chaotropic is present at about 0.5 to 2 M. In one embodiment of the invention, the chaotropic agent herein is urea at about 0.5-2 M or about 1 M. In one embodiment, the chaotropic agent is urea at a concentration of approximately 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 DTT reducing agent on VEGF refolding. . VEGF of peak 3 designates folded appropriately biologically active VEGF.

[0052] As noted, the solution optionally also contains a reducing agent. The reducing agent is suitably selected from those described above for the solubilization step in the 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 at about 0.5-2 mM. In one embodiment of the invention, the reducing agent is DTT at approximately 0.5 mM. Examples of suitable reducing agents include, but are not limited to, for example, dithiothreitol (DTT), β-mercaptoethanol (BME), cysteine, DTE, etc. Although DTT and BME can be used with respect to the procedures provided herein for heparin-binding proteins in general, a combination of cysteine from about 0.1 to about 10 mM and DTT of about 0.1 is a VEGF recovery example. 0.1 to about 1.0 mM as described herein.

[0053] The refolding step includes a sulfated polyanionic agent at a concentration sufficient to achieve complete refolding of the solubilized protein. Examples of suitable polyanionic agents are described hereinbefore, for example, a sulfate derivative of a polysaccharide as noted above with sulfated polyanionic agents such as heparin sulfate, dextran sulfate, heparin sulfate and chondroitin sulfate, as well as Sulfate heparin fragments. For heparin sulfates, the molecular weights are generally between about 3,000 and 10,000 Da, or between about 3,000 and 6,000 Da.

[0054] In one embodiment of the invention, dextran sulfate is employed in the context of the invention. The molecular weight of the sulfated polyanionic agent or others such as dextran sulfate used in the invention depends on the size of the particular heparin binding protein being recovered. Generally, dextran sulfate of between about 3,000 and

10,000 Da. In one embodiment of the invention, dextran sulfate of between about

5,000 Da and 10,000 Da, for example, for the recovery of VEGF. In another embodiment, a dextran sulfate at between about 5,000 Da and 8,000 Da is used for the recovery of BPH. 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 folded properly.

[0055] The concentration of the polyanionic compound used depends on the protein being recovered and its concentration and on conditions such as temperature and pH of the refolding 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 Da 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 about 10 and 400 µg / ml or between about 10 and 200 µg / ml for dextran sulfates.

[0056] The refolding 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 at about 0.01% to 1.0%. In one example, the concentrations of non-ionic detergent are between about 0.025% and 0.05%, or about 0.05%.

[0057] Optionally, positively charged amino acids may be present in the refolding buffer, for example, arginine (eg, L-arginine / HCl), lysine, etc. In certain embodiments of the invention, the concentration of arginine is, for example, about 01000 mM, or about 25 to 750 mM, or about 50-500 mM, or about 50-250 mM, or about 100 mM of final concentration, etc. In certain embodiments of the invention, the protein is in a buffer solution at pH 7.0-9.0 containing 0.5-3 M urea, 0-30 mg / l dextran sulfate, 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 refolding buffer solution is 1 M urea, 50 mM HEPPS, 15 mg / ml 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 refolding buffer solution is 1.3 M urea, 50 mM HEPPS, 15 mg / l dextran sulfate, 0.05% Triton X-100, 7.5 mM cysteine, DTT 0 , 5 mM, 100 mM arginine, pH 8.0.

Recovery and purification of heparin binding proteins

[0058] Although recovery and purification of heparin-binding protein from culture media can employ various known methods for the separation of said proteins such as, for example, salt and solvent fractionation, adsorption with colloidal materials, filtration in gel, ion exchange chromatography, affinity chromatography, immunoaffinity chromatography, electrophoresis and high performance liquid chromatography (HPLC), an example of a four-stage chromatographic procedure comprising contacting said folded 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 support. Alternatively, another chromatographic method is described which comprises contacting said folded 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 each procedure can be carried out in any order. In one embodiment of the invention, the steps are performed sequentially.

[0059] A suitable first stage in the recovery and further purification of the heparin-binding protein characteristically provides the concentration of the heparin-binding protein and the reduction of the sample volume. For example, the second incubation step described above may result in a large increase in the volume of heparin binding protein recovered and a simultaneous dilution of the protein in the refolding buffer. The first suitable chromatographic supports provide a reduced volume of recovered heparin binding protein and can advantageously provide some purification of the protein from unwanted contaminating proteins. Suitable early chromatographic stages include chromatographic supports that can be eluted and loaded directly into a chromatographic carrier of hydrophobic interaction. For example, chromatographic supports are used from which heparin-binding protein can be eluted with high salt concentration suitable for loading a chromatographic carrier of hydrophobic interaction.

[0060] The first exemplary chromatographic supports include, but are not limited to, hydroxyapatite chromatographic supports, for example, CHT type I and type II ceramics (previously known as MacroPrep ceramics), Bio-Gel HT, Bio-Gel HTP, Biorad, Hercules, CA, etc .; Chelating chromatographic metal supports consisting of an inert resin of immobilized metal ions such as copper, nickel, etc .; as well as non derivatized silica gels. In one embodiment of the invention, the first chromatographic supports for purification and recovery of VEGF are chromatographic hydroxapatite supports. In another embodiment of the invention, the first chromatographic supports for purification and recovery of VEGF are cation exchange supports, for example, described in more detail below.

[0061] Elution of the first chromatographic support is achieved according to standard practices in the art. Elution conditions and suitable buffers will facilitate loading of the eluted BPH directly into the first hydrophobic interaction chromatographic support as described below.

[0062] Hydrophobic interaction chromatography is well known in the art and involves the interaction of hydrophobic portions of the molecule that interact with hydrophobic ligands attached to "chromatographic supports." A hydrophobic ligand coupled with a matrix is variously designated as a chromatographic HIC 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 proportion of non-polar to polar surfaces in the protein, but of the distribution of non-polar surfaces as well.

[0063] A series of matrices can be used in the preparation of HIC columns. The most widely used is agarose, although silica and organic polymer resins can be used. Useful hydrophobic ligands include, but are not limited to, alkyl groups having from about 2 to about 10 carbon atoms, such as butyl, propyl or octyl, or aryl groups such as phenyl. Conventional HIC supports for gels and columns can be obtained commercially from suppliers such as GE Healthcare, Uppsala, Sweden under the product names butyl-SEPHAROSE ™, phenyl-SEPHAROSE ™ CL-4B, octyl-SEPHAROSE ™ FF and phenyl- SEPHAROSE ™ FF and Tosoh Corporation, Tokyo, Japan, with the product names TOYOPEARL ™ -butyl 650M (Fractogel TSK Butyl-650) or TSK-GEL phenyl 5PW. In one embodiment, the purification and recovery of VEGF is on a first HIC chromatographic support that is butylagarose and a second hydrophobic chromatographic support that is phenylagarose. In another embodiment, the first HIC chromatographic support is phenylagarose.

[0064] Ligand density is an important parameter that not only influences the strength of the interaction of the protein, but also the capacity of the column. The ligand density of commercially available phenyl or octylphenyl gels is of the order of 5-40 µmol / ml gel bed. The gel capacity is a function of the particular protein in question, as well as the pH, temperature and saline concentration, but it can generally be expected to enter the range of 3-20 mg / ml of gel.

[0065] The choice of the particular gel can be determined by one skilled in the art. In general, the strength of the interaction of the HIC protein and ligand increases with the chain length of the alkyl ligands, but ligands having from about 4 to about 8 carbon atoms are suitable for most 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.

[0066] Adsorption of the protein to an HIC column is favored by high saline concentration, but the actual concentration may vary over a wide range depending on the nature of the protein and the particular HIC ligand chosen. In general, salt concentrations between about 1 and 4 M. are useful.

[0067] Elution of an HIC support, both in steps or in the form of a gradient, can be achieved in a variety of ways such as a) changing the salt concentration, b) changing the polarity of the solvent or c) adding detergents. By reducing salt concentrations, adsorbed proteins are eluted to increase hydrophobicity. Polarity changes can be made by additions of solvents such as ethylene glycol or isopropanol, thus reducing the strength of hydrophobic interactions. Detergents function as protein shifters and have been used primarily in relation to membrane protein purification.

[0068] Various anionic constituents can be attached to matrices to form cationic supports for chromatography. Anionic constituents include carboxymethyl, sulfoethyl groups, sulfopropyl groups, phosphate and sulphonate (S). Cellulosic ion exchange resins such as SE52, SE53, SE92, CM32, CM52, CM92, P11, DE23, DE32, DE52, EXPRESS ION ™ S and EXPRESS ION ™ C are available from Whatman LTD, Maidstone Kent R.U. The ion exchangers based on SEPHADEX ™ and SEPHAROSE ™ and cross-linked 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 ™ Fast Flow, SPSEPHAROSE XL, CM-SEPHAROSE ™ Fast Flow and CM-SEPHAROSE ™, CL-6B, all available at GE Healthcare. Examples of ion exchangers for the practice of the invention include, but are not limited to, for example, ion exchangers with the MACROPREP ™ product names such as, for example, MACROPREP ™ S support, MACROPREP ™ High S support and MACROPREP ™ CM support from BioRad, Hercules, CA.

[0069] Elution of cationic chromatographic supports is generally achieved by increasing salt concentrations. Because the elution of ionic columns involves the addition of salt and because, as mentioned, the HIC has an enhanced saline concentration, the introduction of the HIC stage after the ionic stage or other saline stage is optionally used. . In one embodiment of the invention, a cation exchange chromatographic stage precedes the HIC stage.

[0070] Examples of procedures for purifying VEGF are described herein below, for example, see Examples V and VI. After refolding, insoluble material is removed from the assembly by deep filtration. The clarified assembly is then loaded in ceramic hydroxyapatite (Bio Rad, Hercules, CA) equilibrated with 5 mM HEPPS / 0.05% TRITON ™ X100 / pH 8. The unbound protein is removed by washing with equilibration buffer and the elution is eluted. VEGF using an isocratic stage of 50 mM HEPPS / 0.05% TRITON ™ X100 / 0.15 M sodium phosphate / pH

8. The VEGF assembly is loaded on a butyl-SEPHAROSE ™ Fast Flow column (GE Healthcare, Uppsala, Sweden) equilibrated with 50 mM HEPPS / 0.05% TRITON ™ X100 / 0.15 M / pH sodium phosphate 8. The column is washed with equilibration buffer and the VEGF is collected in the column effluent. The butyl-SEPHAROSE ™ assembly is loaded on a Macro Prep High S column (BioRad, Hercules, CA) that is equilibrated with 50 mM HEPES / pH 8. After reducing the effluent absorbance by washing at 280 nm to the value of reference, the column is washed with two column volumes of 50 mM HEPES / 0.25 M sodium chloride / pH 8. The VEGF is eluted using a linear gradient of 8 column volumes of 0.25-0 sodium chloride, 75 M in 50 mM HEPES / pH 8. Fractions are collected and those combined with appropriately folded VEGF, as determined by a heparin binding assay.

[0071] The Macro Prep High S assembly is conditioned with an equal volume of 50 mM HEPES / 0.8 M sodium citrate / pH 7.5. The conditioned assembly is then loaded on a phenyl-5PW TSK column (Tosoh Bioscience LLC, Montgomeryville, PA) which is equilibrated with 50 mM HEPES / 0.4 M sodium citrate / pH 7.5. After washing the unbound protein in the column with equilibration buffer, the VEGF is eluted from the column using a 10 volume gradient of 0.4-0 M sodium citrate column in 50 mM HEPES, pH 7.5. Fractions are tested by polyacrylamide-SDS gel electrophoresis and those containing VEGF of sufficient purity are combined.

Expression of heparin binding protein in host cells

[0072] Briefly, expression vectors capable of autonomous replication and protein expression relative 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, including the nucleotide sequences of the heparin binding 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. Expression vectors are available commercially in appropriate prokaryotic cells, including bacteria, for example in the American Type Culture Collection (ATCC), Rockville, Maryland. Methods for large-scale growth of prokaryotic cells, and especially the culture of bacterial cells, are well known in the art, and these methods can be used in the context of the invention.

[0073] For example, prokaryotic host cells are transfected with expression or cloning vectors encoding the heparin-binding protein of interest and cultured in conventional nutrient media modified as appropriate to induce promoters, select transformants or amplify the genes encoding the desired sequences. The nucleic acid encoding the polypeptide of interest is suitably RNA, cDNA or genomic DNA from any source, provided that it encodes the polypeptide or polypeptides of interest. Methods for selecting the appropriate nucleic acid for the expression of heterologous polypeptides (including variants thereof) in microbial hosts are well known. Nucleic acid molecules encoding the polypeptide are prepared by a variety of procedures known in the art. For example, a DNA encoding VEGF is isolated and sequenced, for example, using oligonucleotide probes that are capable of specifically binding to the gene encoding VEGF.

[0074] The heterologous nucleic acid (eg, cDNA or genomic DNA) is suitably inserted into a replicable vector for expression in the microorganism under the control of a suitable promoter. Many vectors are available for this purpose, and the 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 to transform with the vector. Each vector contains various components depending on the particular host cell with which it is compatible. Depending on the particular type of host, the components of the vector 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 transcription termination sequence.

[0075] In general, plasmid vectors containing replicon and control sequences derived from species compatible with the host cell with respect to microbial hosts are used. The vector normally carries a replication site, as well as marker sequences that are capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from a species of

E. coli (see, for example, Bolivar et al., (1977) Gene, 2: 95). pBR322 contains genes for ampicillin and tetracycline resistance and therefore provides a simple means to identify transformed cells. Plasmid pBR322, or another 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

[0076] The polypeptides of the invention can be recombinantly produced 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 at the N-terminus of the mature protein or polypeptide. The heterologous signal sequence typically selected is one that is recognized and processed (specifically, 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 by a prokaryotic signal sequence selected, for example, from the group of leader sequences of alkaline phosphatase, penicillinase, 1pp and enterotoxin II thermostable.

(ii) Replication source component

[0077] Expression vectors contain a nucleic acid sequence that allows the vector to replicate in one or more 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 gene component

[0078] Expression vectors generally contain a selection gene, also called a selectable marker. This gene encodes a protein necessary for the survival or growth of transforming host cells grown in 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, for example, ampicillin, neomycin, methotrexate or tetracycline, (b) complement auxotrophic deficiencies other than those caused by the presence of the genetic marker or markers, or (c) provide critical nutrients not available in complex media, for example, the gene encoding bacillus D-alanine racemase.

[0079] An example of a selection scheme uses a drug to stop the growth of a host cell. In this case, those cells that are successfully transformed with the nucleic acid of interest produce a polypeptide that confers resistance to drugs and therefore survives the selection regime. 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 bacterial genes under eukaryotic control to transmit resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively.

(iv) Promoter component

[0080] The expression vector for producing the heparin binding protein of interest contains a suitable promoter that is recognized by the host organism and is operably linked with the nucleic acid encoding the polypeptide of interest. Promoters suitable for use with prokaryotic hosts include the β-lactamase and lactose promoter systems (Chang et al. (1978) Nature, 275: 615; Goeddel et al. (1979) Nature, 281: 544), the system arabinose promoter (Guzmán et al. (1992) J. Bacteriol., 174: 7716-7728), alkaline phosphatase, a 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. Natl. Acad. Sci. USA, 80: 21-25). However, other known bacterial promoters are suitable. Their nucleotide sequences have been published, thus enabling the expert to operatively link them with DNA encoding the polypeptide of interest (Siebenlist et al. (1980) Cell, 20: 269) using linkers or adapters to deliver any necessary restriction site. See also, for example, Sambrook et al., Supra and Ausubel et al., Supra.

[0081] Promoters for use in bacterial systems generally also contain a Shine-Dalgarno (S.D.) sequence operably linked with the DNA encoding the polypeptide of interest. The promoter can be removed from the bacterial source DNA by restriction enzyme digestion and inserted into the vector containing the desired DNA.

(v) Construction and vector analysis

[0082] The construction of suitable vectors containing one or more of the components listed above employs standard linking techniques. Isolated plasmids or DNA fragments are cleaved, adjusted and religed in the desired manner to generate the necessary plasmids: For analysis to confirm the correct sequences in the constructed plasmids, ligation mixtures are used to transform E. coli K12 strain 294 ( ATCC 31,446) or other strains, and successful transformants are selected by resistance to ampicillin or tetracycline when appropriate. Plasmids are prepared from the transformants, analyzed by restriction endonuclease digestion and / or sequenced by the procedure of Sanger et al., (1977) Proc. Natl Acad. Sci. USA, 74: 5463-5467 or Messing et al., (1981) Nucleic Acids Res., 9: 309, or by the procedure of Maxam et al., (1980) Methods in Enzymology, 65: 499. See also , for example, Sambrook et al., supra and Ausubel et al., supra.

[0083] 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 conventional nutrient media modified as appropriate to induce the various promoters.

Host cell culture

[0084] 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, for example, eubacteria, such as gram-negative or gram-positive organisms, for example, E. coli, bacilli such as B. subtilis, Pseudomonas species such as P. aeruginosa, Salmonella typhimurium or Serratia marcescens. An example of E. coli host is E. coli 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. Strain W3110 is a typical host because it is a common host strain for fermentation of recombinant DNA product. In one aspect of the invention, the host cell should secrete minimal amounts of proteolytic enzymes. For example, strain W3110 can be modified to effect a genetic mutation in the genes encoding proteins, including examples of said E. coli hosts W3110 strains 1A2, 27A7, 27B4 and 27C7 described in US Pat. No. 5,410,026, issued on April 25, 1995. For example, it is a strain for the production of VEGF the

E. coli strain W3110 that has the genotype tonAΔ

ptr3 phoAΔE15 Δ (argF-lac) 169 degP41 ilvg designated 49B3. In another example, it is a strain for the production of VEGF E. coli strain (62A7) that has the genotype ΔfhuA (ΔtonA) ptr3, lacIq, lacL8, ΔompT Δ (nmpC-fepE) ΔdegP ilvG +. See also, for example, the table covering pages 23-24 of document W02004 / 092393.

[0085] The prokaryotic cells used to produce the heparin binding protein of interest are cultured in media known in the art and suitable for the culture of the selected host cells, including the media generally described by Sambrook et al., "Molecular Cloning, A Laboratory Manual ”, 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), Neidhardt minimum medium and minimum or complete C.R.A.P. medium, plus the necessary nutrient supplements. In certain embodiments, the media also contains a selection agent, chosen based on 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 media for the growth of cells that express the ampicillin resistance gene. Any necessary complement may also be included in addition to sources of carbon, nitrogen and inorganic phosphate at the appropriate concentrations, introduced, alone or as a mixture with another complement 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, dithioerythritol and dithiothreitol.

[0086] Examples of suitable means are given in US Pat. No. 5,304,472 and

5,342,763. Phosphate limiting media C.R.A.P. consist of 3.57 g of (NH4) 2 (SO4), 0.71 g of sodium citrate · 2 H2O, 1.07 g of KCl, 5.36 g of yeast extract (certificate), 5.36 g HycaseSF ™ -Sheffield, pH adjusted with KOH at 7.3, cs volume adjusted to 872 ml with deionized H2O and autoclaved; cooled to 55 ° C and supplemented with 110 ml of 1 M MOPS pH 7.3, 11 ml of 50% glucose and 7 ml of 1 M MgSO4). Then carbenicillin can be added to the induction culture at a concentration of 50 µg / ml.

[0087] Prokaryotic host cells are cultured at suitable temperatures. For the growth of E. coli, for example, the temperature is in the range of, for example, about 20 ° C to about 39 ° C, or about 25 ° C to about 37 ° C, or about 30 ° C.

[0088] When the alkaline phosphatase promoter is used, the E. coli cells used to produce the polypeptide of interest of this invention are cultured in suitable means in which the alkaline phosphatase promoter can be partially or completely induced as described in general. , for example, in Sambrook et al., "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory Press (Cold Spring Harbor, New York) (2001). Cultivation never has to take place in the absence of inorganic phosphate or at phosphate deprivation levels. Initially, the medium contains inorganic phosphate in an amount greater than the level of induction of protein synthesis and sufficient for bacterial growth. As the cells grow and use phosphate, they reduce the level of phosphate in the medium, thus causing the induction of polypeptide synthesis.

[0089] If the promoter is an inducible promoter, for induction to occur, cells are typically cultured to achieve a certain optical density, for example, an A550 of about 200 using a high cell density process, at which point the induction (for example, by the addition of an inducer, the deprivation of a component of the medium, etc.) to induce the expression of the gene encoding the polypeptide of interest.

[0090] Any necessary complement may also be included at the appropriate concentrations that would be known to those skilled in the art, introduced alone or as a mixture with another complement 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. coli, the pH is, for example, from about 6.8 to about 7.4, or from about 7.0.

Formulations of heparin binding proteins

[0091] The recovered polypeptide, for example using the procedures described herein, can be formulated in a pharmaceutically acceptable carrier and is 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. The therapeutic uses for heparin binding proteins obtained using the methods described herein are also contemplated. For example, a growth factor or hormone, for example VEGF, can be used to enhance growth as desired. For example, VEGF can be used to promote wound healing, for example, of an acute wound (e.g., burn, surgical wound, normal wound, etc.) or a chronic wound (e.g., diabetic ulcer, pressure ulcer, pressure ulcer, venous ulcer, etc.), to promote hair growth, to promote tissue growth and repair (for example, bone, liver, etc.), etc.

[0092] The therapeutic formulations of heparin-binding proteins are prepared for storage by mixing a molecule, for example a polypeptide, having the desired degree of purity with optional 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 carriers, excipients or stabilizers are non-toxic to the receptors at the dosages and concentrations employed, and include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzene chloride, phenol, butyl or benzyl alcohol, alkylparabenos such as methyl- or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol and mcresol); low molecular weight polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt forming counterions such as sodium; metal complexes (for example, Zn-protein complexes) and / or non-ionic surfactants such as TWEEN ™, PLURONICS ™ or polyethylene glycol (PEG).

[0093] In certain embodiments, formulations for use in in vivo administration are sterile. This is easily achieved by filtration through sterile filtration membranes. BPH can be stored in lyophilized form or as an aqueous solution or in gel form. The pH of the HBP preparations may be, for example, from about 5 to 8, although higher or lower pH values may also be appropriate in certain cases. It will be understood that the use of certain excipients, carriers or stabilizers may result in the formation of salts of BPH.

[0094] Typically for wound healing, BPH is formulated for site specific delivery. When applied topically, BPH is properly combined with other ingredients such as carriers and / or adjuvants. There are no limitations on the nature of said other ingredients, except that they must be pharmaceutically acceptable and effective for their intended administration, and cannot significantly degrade the activity of the active ingredients of the composition. Examples of suitable vehicles include ointments, creams, gels, sprays or suspensions, with or without purified collagen. The compositions may also be impregnated in sterile dressings, transdermal patches, tape and bandages, optionally in liquid or semi-liquid form.

[0095] To obtain a gel formulation, the BPH formulated in a liquid composition can be mixed with an effective amount of water-soluble polysaccharide or synthetic polymer such as polyethylene glycol, forming a gel of the appropriate viscosity to be applied topically. The polysaccharide that can be used includes, for example, cellulose derivatives such as etherified cellulose derivatives, including alkyl celluloses, hydroxyalkyl celluloses and alkyl hydroxyalkyl celluloses, for example, methyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose, hydroxypropyl methylcellulose and hydroxypropyl cellulose; starch and fractionated starch; agar; alginic acid and alginates; gum arabic; pululane; agarose; carrageenan; dextrans; dextrins; fructans; inulin; tomorrows; xylanes; arabinanos; chitosans; glycogens; synthetic glucans and biopolymers, as well as gums such as xanthan gum, guar gum, locust bean gum, arabic gum, tragacanth gum and karaya gum and derivatives and mixtures thereof. In certain embodiments of the invention, the gelling agent herein is one that is, for example, inert to biological systems, non-toxic, simple to prepare and / or not too fluid or viscous, and will not destabilize the BPH maintained therein. .

[0096] In certain embodiments, the polysaccharide is an etherified cellulose derivative, in another embodiment, it is a well-defined, purified and listed one in the USP, for example, methylcellulose and hydroxyalkylcellulose derivatives such as hydroxypropylcellulose, hydroxyethylcellulose and hydroxypropylmethylcellulose. In one embodiment, the polysaccharide is methylcellulose. If methylcellulose is used in the gel, for example, it typically comprises about 2-5%, or about 3% or about 4% or about 5% of the gel, and BPH is present in an amount of about 300- 1000 mg / ml gel

[0097] The polyethylene glycol useful for gelation is typically a mixture of high and low molecular weight polyethylene glycols to obtain the appropriate viscosity. For example, a mixture of a polyethylene glycol of molecular weight 400-600 with one of molecular weight 1500 would be effective for this purpose when mixed in the proper ratio to obtain a paste.

[0098] The term "water soluble" as applied to polysaccharides and polyethylene glycols is intended to include colloidal solutions and dispersions. In general, the solubility of cellulose derivatives is determined by the degree of substitution of ether groups, and the stabilizing derivatives useful herein should have a sufficient amount of said ether groups per unit of anhydroglucose in the cellulose chain to return water soluble derivatives. An ether substitution degree of at least 0.35 ether groups per unit of anhydroglucose is generally sufficient. Additionally, cellulose derivatives may be in the form of alkali metal salts, for example, salts of Li, Na, K or Cs.

[0099] The active ingredients can also be trapped in microcapsules or prolonged 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), p. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; U.S. Patent No. 5,654,010; DE 3,218,121; Epstein et al., (1985) Proc. Natl Acad. Sci. USA, 82: 3688-3692; Hwang et al., (1980) Proc. Natl 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; US patents No. 4,485,045 and 4,544,545 and EP 102,324.

[0100] The following examples are offered by way of illustration and not by way of limitation.

EXAMPLES

EXAMPLE I: Recombinant human VEGF expressed in Escherichia coli

[0101] Recombinant human VEGF was expressed in Escherichia coli. During the synthesis, the protein was secreted in the periplasmic space and accumulated as refracting bodies. Studies were therefore carried out to achieve the extraction and refolding of the protein. These studies revealed that at least 3 species of VEGF were isolated (Figure 1) using standard recovery techniques without the addition of a polyanionic agent. Studies with native VEGF showed that the addition of heparin increased resistance to chaotropic and thiol-induced denaturation (Figure 2). In addition, heparin significantly increased the amount of VEGF properly retracted in small-scale refolding experiments. To adapt this result to a large-scale process, the conditions that allowed the refolding of VEGF in the presence of dextran sulfate, a molecule structurally analogous to heparin, were discovered. The addition of dextran sulfate improved the yields of biologically active VEGF properly folded 3-5 times with respect to the controls.

Procedures

[0102] Plasmid for the expression of VEGF165- Plasmid pVEGF171 was designed for the expression of human VEGF165 (see, for example, Leung et al., (1989) Science, 246: 1306-1309) in the periplasm of E. coli . Transcription of the VEGF coding sequence was arranged under close control of the alkaline phosphatase (AP) promoter (see, for example, Kikuchi et al., (1981) Nucleic Acids Research, 9: 5671-8), while the Sequences necessary for initiation of translation were provided by the Shine-Dalgarno region of trp (see, for example, Yanofsky et al., (1981) Nucleic Acids Research, 9: 6647-68). The VEGF coding sequence was fused in the 3 'direction of the bacterial thermostable enterotoxin II signal sequence (STII) (see, for example, Lee et al., (1983) Infect. Immun. 42: 264-8 and Picken and col., (1983) Infect. Immun. 42: 269-75) for subsequent secretion in the periplasm of E. coli. Codon modifications in the STII signal sequence provided an adjusted translation level, which resulted in an optimal level of VEGF accumulation in the periplasm (see, for example, Simmons and Yansura, (1996) Nature Biotechnoloy, 14: 629 -3. 4). The lambda transcriptional terminator (see, for example, Scholtissek and Grosse, (1987) Nucleic Acids Research 15: 3185) was located in the 3 ’direction of the translation termination codon. The origin of replication and both ampicillin and tetracycline resistance genes were provided by plasmid pBR322. See, for example, Bolivar et al., (1977) Gene 2: 95-113.

[0103] Cellular homogenization and preparation of refractory bodies - Cells collected from Escherichia coli were frozen and stored at -70 ° C. Cells were collected by centrifugation with BTUX (centrifuge, alpha laval) and freezing using BEPEX (large-scale freezer). The cells were suspended in 5 volumes of 50 mM HEPES / 150 mM NaCl / 5 mM EDTA, pH 7.5 (5 l / kg of sediment) and homogenized in a 15 M Gaulin 15M model laboratory homogenizer (small scale) or M3 (large scale) (Gaulin Corporation, Everett, MA). The cell suspension was then diluted with an equal volume of the same buffer and the refractory bodies were collected by centrifugation in a BTPX 205 continuous feed centrifuge (Alfa Laval Separation AB (Tumba, Sweden)). SA1 was used as an intermediate scale centrifuge. Alternatively, the cells can be homogenized and the sediment can be collected directly without freezing by BEPEX and rehydration.

EXAMPLE II: Extraction and refolding of recombinant human VEGF expressed in Escherichia coli-I

Procedures

[0104] Extraction and refolding - The refractory sediment was suspended in extraction buffer containing 7 M urea / 50 mM HEPPS / pH 8 (final concentration) at 5 l of buffer / kg of sediment. Solid dithiothreitol was then added at 3.7 g / kg sediment for a final concentration of 4 mM. See, for example, Figure 9 for the effect of urea and DTT on VEGF extraction. The suspension was thoroughly mixed for 1-2 h at 20 ° C. The pH can be adjusted with 50% sodium hydroxide (w / w) to pH 8.0. Refolding was initiated by adding 19 volumes of refolding buffer per volume of extraction buffer. The refolding buffer contained 50 mM HEPPS / 1 M-2 M urea / 2-5 mM cysteine / 0.05% -0.2% TRITON ™ X100 / pH 8, final concentration. See, for example, Figure 10 for the effect of urea and DTT concentration present during refolding. Dextran sulfate, heparin or sodium sulfate was added as indicated. The refolding incubation was performed at room temperature for 4-24 hours. Optionally, incubation can be performed at room temperature for up to about 48 hours. Folding was monitored by PAGE-SDS and / or HPLC of heparin. The product was clarified by deep filtration with a Cuno 90SP filter followed by 0.45 µm filtration.

[0105] HPLC Heparin Binding Assay - The quality and quantity of VEGF appropriately refolded was determined using a column containing immobilized heparin. The POROS HE2 / M column (4.6 x 100 mm, HE2 / M from PerSeptive BioResearch Products, Cambridge, MA) was equilibrated with 10 mM sodium phosphate, pH 7, containing 0.15 M sodium chloride. flow rate of 1 ml / min, or 2 ml / min, the columns were eluted using a linear gradient of 0.15 M to 2 M sodium chloride in equilibration buffer for 10 min. In some trials, elution was performed in 16 min. The eluent was monitored at 280 nm. Typically, most of the protein was eluted in the empty volume and three classes of VEGF could be identified. The species with the highest affinity and which eluted last as correctly folded VEGF was identified and subsequently identified as "VEGF peak 3".

Results

[0106] Heparin protects VEGF against cysteine-mediated denaturation. The addition of 10 mM cysteine to native VEGF resulted in a large reduction of the properly folded molecule (Figure 2). This denaturation was avoided by adding 2 different forms of heparin at concentrations as low as 20 mM.

TABLE Ia Effect of heparin and dextran sulfate on VEGF refolding

Addition Concentration (µg / ml)

0 10 55 100 200 400 Increase Increase in% in times

Increase None 5.3 * - Low PM Heparin (3 kDa) 12.2 14.2 14.8 14.1 179% 2.8 High PM Heparin (6 kDa) 15.3 16.6 13.9 15.3 213% 3.1 Dextran sulfate (10 kDa) 15.9 15.4 13.6 7.4 8.3 191% 2.9 The values in the table are the amount of VEGF peak 3 formed ( in mg) per g of refractory sediment. The concentration of each addition is as indicated. * Medium control (5.6 + 5.0 = 5.3)

TABLE Ib Effect of sodium sulfate on VEGF refolding

Addition Concentration (µg / ml)

0 50 98 195 293 455 Increase Increase in% in times

Increase None 5.3 - Sodium sulfate 6.9 9.1 10.4 10.9 10.4 106% 2.1

The values in the table are the amount of VEGF peak 3 formed (in mg) per g of refractory sediment. The concentration of sodium sulfate is as indicated.

TABLE II Effect of heparins and dextran sulfate on VEGF refolding

Addition

0 2.5 12.5 Concentration (µg / ml) 50 100 Increase Increase

in %

in times

None 2.2 - Dextran sulfate (5 kDa) 10.1 13.7 13.4 11.2 523% 6.2 Dextran sulfate (8 kDa) 9.9 17.2 14.0 12.9 682 % 7.8 Dextran sulfate (10 kDa) 13.8 19.2 14.6 10.1 773% 8.7 Low PM heparin (3 kDa) 10.4 16.9 14.7 668% 7.7 High PM heparin (6 kDa) 14.1 18.8 20.2 818% 9.2 The values in the table are the amount of VEGF peak 3 formed (in mg) per g of refractory sediment

10 Summary

[0107] Heparin and dextran sulfate increase refolding yields-Due to the protective properties against denaturation described above, the effect of several different forms of sulfated polymers on refolding of 15 VEGF was investigated. As seen in TABLE Ia (and in Fig. 5), both low and high molecular weight forms of heparin increased the yield of folded VEGF approximately 3 times. As seen in TABLE Ib (and in Figure 6), sodium sulfate increased the yield of refolded VEGF approximately 2 times. The 10 kDa form of dextran sulfate was also effective in increasing refolding yields; however, the highest concentration interval

20 investigated led to substrate inhibition. Additional research showed that the 5 kDa, 8 kDa and 10 kDa forms of dextran sulfate all significantly increased the yield of VEGF in refolding (TABLE II). See Figure 7. See also Figure 8.

EXAMPLE III: Effect of different buffers and TRITON ™ X- 100 on the recovery of VEGF

25

5

10

fifteen

twenty

25

30

35

40

Four. Five

Results

[0108]

VEGF buffer (mg / g of sediment) HEPES, pH 8 13.3 HEPES, pH 8 with TRITON ™ 14.3 HEPPS, pH 8 16.6 TrisHCl, pH 8 16.6 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

[0109] The combined data from Examples I, II and III demonstrate a significant improvement (2-5 times) of performance by including heparin sulfates or dextran sulfates when VEGF is replicated, a heparin-binding growth factor, as well. as of the recovery conditions. This procedure has been executed successfully on an industrial scale. This procedure is expected to be applicable in the refolding of other basic growth factors and other proteins that bind to heparin.

EXAMPLE IV: Extraction and refolding of recombinant human VEGF expressed in Escherichia coli-II

Procedures

[0110] Extraction and refolding - The refractory sediment was suspended in extraction buffer in which the final concentration was 7 M urea, 2-30 mM DTT (eg, 10 mM DTT), 50 mM HEPPS / pH 7-9 (per example, pH 8) at 5 l of buffer / kg of sediment. The suspension was thoroughly mixed for 1-2 h at room temperature. Refolding was initiated by adding 19 volumes of refolding buffer per volume of extraction buffer. The refolding buffer contained as final concentration 1 M or 1.3 M urea, 2-15 mM cysteine (for example, 7.5 mM cysteine), 0.5 mM DTT, 0-0.75 M arginine (for example, 100 mM arginine), 15 mg / l dextran sulfate, 50 mM HEPPS, 0.05% TRITON ™ X100 / pH 8. See, for example, Figure 12 for the effect on refolding in the presence of charged amino acids, in that the addition of histidine produced the same effect as without amino acid additives. The refolding incubation was performed at room temperature for 12-24 hours. Optionally, incubation can be performed at room temperature for up to about 48 hours. Optionally, air or oxygen can be added during the refolding process (0.3-1 cm3 / min / l). Folding was monitored by PAGE-SDS and / or HPLC of heparin. The product was clarified by deep filtration with a Cuno 90SP filter followed by 0.45 µm filtration.

[0111] The overall dilution of the extraction and refolding stages was 1: 100. Increasing the overall dilution of the extraction and refolding stages, for example, to 1: 100 to 1: 200, increased the total amount of active VEGF, even if the concentration is lower. See Figure 13.

[0112] The efficiency of refolding can be determined by determining the amount of dimer / monomer, in which the monomers can be determined by a C18 reverse phase HPLC column and the formation of dimers can be determined by heparin column chromatography or heparin chromatography assay. cation exchange SP-5PW.

EXAMPLE V: Large-scale deployment

[0113] To test the scalability of optimized refolding conditions, studies were conducted to examine the refolding kinetics on a small scale (0.1 L), intermediate (1 L) and pilot plant (250 L to 400 L). As shown in Figure 4, the large-scale refolding kinetics were indistinguishable from the smaller scales and the final value of retracted VEGF increased slightly. These data demonstrate the scalability of refolding 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 refolding

[0114] MacroPrep ceramic hydroxyapatite chromatography After refolding, insoluble material was removed from the assembly by deep filtration. The clarified assembly was then loaded on a ceramic hydroxyapatite column (35D x 31H = 30L) (Bio Rad, Hercules, CA) equilibrated with 50 mM HEPPS / 0.05% TRITON ™ X100 / pH 8. The non-protein was removed. bound by washing with equilibration buffer and the VEGF was eluted using an isocratic step of 50 mM HEPPS / 0.05% TRITON ™ X100 / 0.15 M sodium phosphate / pH 8. The flow rate was 120 cm / h. Combination fractions were determined by HPLC analysis of heparin fractions.

[0115] Butyl Chromatography-SEPHAROSE ™ Fast Flow- The VEGF assembly was loaded onto a butyl-SEPHAROSE ™ Fast Flow column (35 D x 26 H = 25L) (GE Healthcare, Uppsala, Sweden) balanced with 50 mM HEPPS / 0.05% TRITON ™ X100 / 0.15 M sodium phosphate / pH 8. The flow rate was 100 cm / h. The column was washed with equilibration buffer and the VEGF was collected in the column effluent. The fractions were collected and the fractions containing protein were combined, measuring the A280 nm.

[0116] Macro Prep High S Chromatography- The butyl-SEPHAROSE ™ assembly was loaded on a Macro Prep High S column (30D x 39 H = 27 L) (BioRad, Hercules, CA) that was equilibrated with 50 mM HEPES / pH 8. After washing the effluent absorbance was reduced to 280 nm to the reference value, 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 gradient of 8 column volumes of 0.25-0.75 M sodium chloride in 50 mM HEPES / pH 8. The flow rate was 75-200 cm / h. Fractions were collected and those containing appropriately folded VEGF were combined, as determined by a heparin binding assay, for example, heparin HPLC.

[0117] Phenyl-5PW Chromatography TSK- The Macro Prep High S assembly was conditioned with an equal volume of 50 mM HEPES / 0.8 M sodium citrate / pH 7.5. The conditioned assembly was then loaded on a phenyl-5PW TSK column (18D x 43 H = 11L) (Tosohaas, Montgomeryville, PA) which was equilibrated with 50 mM HEPES / 0.4 M sodium citrate / pH 7.5. After raising the unbound protein from the column with equilibration buffer, the VEGF was eluted from the column using a 10 volume gradient of 0.4-0 M sodium citrate column in 50 mM HEPES, pH 7.5. The fractions were tested by polyacrylamide-SDS gel electrophoresis and those containing VEGF of sufficient purity were combined.

[0118] Ultrafiltration / diafiltration- The combined VEGF was ultrafiltered in 5 kDa regenerated cellulose membrane (G30619); Unit Pellicon; feeding speed 17.1 l / min. The membrane was conditioned with polysorbate 20. The combined VEGF was ultrafiltered at a concentration of 6 g / l (UF1). The sample was diafiltered with 7-14 DV (diavolumes) of 5 mM sodium succinate / 275 mM trehalose / pH 5.0. The final formulation was 5 mM sodium succinate / 275 mM trehalose / 0.01% polysorbate 20 / pH 5.0, at a concentration of 5 mg / ml.

EXAMPLE VII: Purification II of rhVEGF after refolding

[0119] Cation exchange liquid chromatography- After refolding, insoluble material can be removed from the assembly by deep filtration. The refolded assembly is conditioned to pH 5.0-7.5 and approximately 2 to 6.5 mS / cm. In one embodiment, the assembly is conditioned to pH 6.5 and 5 mS / cm. The refolded assembly can then be loaded onto a sulfopropyl (SPXL) extreme loading column and eluted using a gradient of increasing saline concentration. Combination fractions can be determined by HPLC analysis of heparin fractions.

[0120] Hydrophobic interaction column (HIC) - The VEGF SPXL elution set can be conditioned at 50mS / cm to load on a phenyl-TSK chromatography column (Tosohaas, Montgomeryville, PA). Fractions are collected and protein-containing fractions are combined.

[0121] IEX or mixed mode- The phenyl-TSK assembly can be loaded onto an ion exchange chromatography (IEX) column or mixed mode chromatography. Fractions are collected and those containing appropriately folded VEGF are combined, as determined by the assays described herein.

[0122] Ultrafiltration / Diafiltration- The combined VEGF can be ultrafiltered on a regenerated cellulose membrane of 5 kDa (G30619); Unit Pellicon; feeding speed 17.1 l / min. For example, the membrane is conditioned with polysorbate 20. The combined VEGF is ultrafiltered at a concentration of 6 g / l (UF1). The sample is diafiltered with 7-14 DV (diavolumes) of 5 mM sodium succinate / 275 mM trehalose / pH 5.0.

[0123] In the procedures and processes described herein, final purity and / or activity can be assessed by peptide mapping, disulfide mapping, PAGE-SDS (both reduced and non-reduced), circular dichroism, Limulus amebocyte lysate (LAL), heparin chromatography, heparin HPLC (for example, heparin HPLC can be used to determine VEGF dimer concentration), reverse phase HPLC chromatography (fi) (for example, HPLC-FI can be used to determine the concentration of monomeric VEGF), heparin binding, receptor binding (for example, for VEGF, for example, binding to the KDR receptor of Bioanalytic R&D and / or binding to the Flt1 receptor), SEC analysis, cell assays, potency of HUVEC, ELISA with VEGF antibodies, mass spectroscopy analysis, etc.

[0124] It is understood that the deposits, examples and embodiments described herein are for illustrative purposes only and will suggest various modifications or changes in view thereof to those skilled in the art.

Claims (12)

1. A process for recovering a heparin-binding protein from a prokaryotic cell culture, wherein said heparin-binding protein is vascular endothelial growth factor (VEGF) that binds to heparin, the process comprising stages of:

(to)

 isolating said heparin-binding protein from the periplasm of said prokaryotic cell culture;

(b)

 denature said heparin-binding protein in a first buffered solution comprising a chaotropic agent and a reducing agent;

(C)

 incubating said heparin binding protein in a second buffered solution comprising a chaotropic agent and a sulfated polyanionic agent, wherein said sulfated polyanionic agent is dextran sulfate or sodium sulfate, for a time and under conditions such that refolding occurs. of said heparin binding protein; Y

(d)

 recovering said heparin-binding protein, in which there is an approximately 2 to 5-fold increase in the folded heparin-binding protein compared to incubation without sulfated polyanionic agent.

2.

The process of claim 1, wherein the VEGF is VEGF165.

3.

The process of claim 1, wherein the dextran sulfate is between about 3,000 Da and 10,000 Da.

Four.

The process of claim 1, wherein the dextran sulfate is between about 8,000 and 10,000 Da.

5.

The process of claim 1, wherein said first and second buffered solutions comprise HEPPS pH 8.0.

6.

The process of claim 1, wherein the concentration of sodium sulfate is between about 50 and 500 mM.

7.

The process of claim 1, wherein said second buffered solution further comprises

(i)

 a reducing agent;

(ii)

 a nonionic detergent; Y

(iii) arginine and / or lysine.

8.

The process of claim 7, wherein the reducing agent of the second buffered solution comprises a combination of cysteine and DTT.

9.

The process of claim 1, wherein said recovery step (d) comprises

(i)

 sequentially contacting said folded 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 support, or

(ii)

 sequentially contacting said 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.

10.

The process of claim 9 (i), wherein said first and second hydrophobic interaction chromatographic supports are selected from the group consisting of butyl-, propyl-, octyl- and arylagarose resins.

eleven.

The process of claim 9 (i), wherein said first hydrophobic interaction chromatographic support is a butylagarose support and said second hydrophobic interaction chromatographic support is a phenylagarose support resin.

12.

A method of recovering a heparin-binding protein from a prokaryotic cell culture, wherein said heparin-binding protein is the growth factor.

vascular endothelial (VEGF) that binds to heparin, the procedure comprising the steps of: (a) isolating said heparin-binding protein from the periplasm of said prokaryotic cell culture; (b) denature 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, wherein the sulfated polyanionic agent is dextran sulfate or sodium sulfate, for a time and under such conditions. that refolding of said heparin binding protein occurs, in which there is a 10 increase of approximately 2-5 times the recovered folded heparin binding protein compared to incubating without sulfated polyanionic agent; Y (d) sequentially contacting said folded heparin binding protein with (i) a hydroxyapatite chromatographic support, a first hydrophobic interaction chromatographic support and a second hydrophobic interaction chromatographic support, and selectively eluting the protein from 15 heparin binding of each support; or (ii) 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.