CN101336252A - Recombinant production of heparin binding proteins - Google Patents

Abstract

A process for recovering and purifying refolded heparin binding proteins produced in heterologous host cells includes the step of incubation of the solubilized protein with a polyanionic species such as dextran sulfate.

Description

The recombinant production of heparin-binding protein

Related application

The application requires the right of priority and the interests of following application: the U.S. Provisional Application serial number 60/807,432 that the U.S. Provisional Application serial number 60/753,615 that on December 22nd, 2005 submitted to and on July 14th, 2006 submit to, and at this complete its specification sheets of including.

Invention field

The present invention relates to be used for to obtain the method for the heparin-binding protein of producing at cell culture.The present invention includes the method that is used to reclaim with the heparin-binding protein of purifying refolding, this protein is produced in prokaryotic host cell and is present in these cells, typically is present in space in periplasmic space or the born of the same parents.The heparin-binding protein that should produce in prokaryotic host cell can also be found the mixture as soluble protein or solubility and insoluble protein.

Background of invention

Known large quantities of polypeptide naturally occurring, that biologic activity is arranged can heparin-binding.Such heparin comprises cytokine in conjunction with polypeptide, 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); Heparin binding growth factor (Burgess and Maciag, (1989) Annu.Rev.Biochem., 58:576-606; Klagsbrun, (1989) Prog.Growth Factor Res.1:207-235), such as Urogastron (EGF), Thr6 PDGF BB (PDGF), Prostatropin (bFGF), acid fibroblast growth factor (aFGF), vascular endothelial growth factor (VEGF) and pHGF (HGF) (Liu et al., (1992) Gastrointest.Liver Physiol., 26:G642-G649); And select albumen, select albumen, E-to select albumen and P-to select albumen (Norgard-Sumnicht et al., (1993) such as L- Science, 261:480-483).Also can be referring to Munoz and Linhardt, (2004) Arterioscler Thromb Vasc Biol., 24:1549-1557.

International publication number WO 95/07097 has put down in writing the heparin-binding protein preparaton that is used for the treatment of purposes, and it comprises natural heparin or other polyanionic compound of heparin binding growth factor such as VEGF and purifying.The oligosaccharides of heparin derivative and various other polyanionic compound have demonstrated activity conformation (Barzu et al., (1989) of stablizing heparin binding growth factor J.Cell.Physiol., 140:538-548; Dabora etal., (1991) J.Biol.Chem., 266:23627-23640), and heparin affinity chromatography has been used for various purification scheme (normally referring to international publication number WO 96/02562).

The heparin-binding protein of many Mammals origins is produced by recombinant technology, and is important (Munoz and Linhardt, (2004) clinically 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 effective mitogen of vascular endothelial cell.It is called vascular permeability factor (VPF) again.Referring to Dvorak et al., (1995) Am.J.Pathol., 146:1029-39.VEGF takes place to have played the part of the key player (angiogenesis) process of original vascularization neovascularity (promptly from) at vasculogenesis (vasculogenesis) (being the growth of embryo's vasculature) and blood vessel.Referring to for example Ferrara, (2004) Endocrine Reviews, 25 (4): 581-611; Risau etal., (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; Ferrara and Davis-Smyth, (1997) Endocri.Rev., 18:4-25.The clinical application of VEGF comprise the growth of those wherein new capillary beds be designated as for example promote wound healing (referring to for example international publication number WO 91/02058; Lawyer's account P2358R1 is entitled as " WoundHealing ", submit on June 16th, 2006), promote tissue growth and reparation, for example liver (referring to for example WO2003/0103581), bone (referring to for example WO2003/094617) etc.Also can be referring to Ferrara, (2004) Endocrine Reviews, 25 (4): 581-611.

Typically, important recombinant protein is produced in various host organisms in the treatment.Most protein can be expressed with their natural forms in such as Chinese hamster ovary celI at eucaryon host.Animal cell culture require usually to prolong growth time with realize maximum cell density and final obtain the cell density lower than prokaryotic cell prokaryocyte culture (Cleland, J. (1993) ACS Symposium Series 526, Protein Folding:In Vivo and In Vitro, American Chemical Society).In addition, animal cell culture often requires expensive substratum, and it comprises the cultivation component that may disturb desired protein to reclaim.The host bacterium expression system provides cost-efficient replacement scheme for production-scale recombinant protein production.The United States Patent (USP) of many conventional bacterial expressions about recombinant protein is arranged, comprise U.S. Patent number 4,565,785; 4,673,641; 4,795,706; 4,710,473.A major advantage of this production method is by the centrifugal or micro-filtration ability of separated product from cellular component easily.Referring to for example Kipriyanov and Little, (1999) Molecular Biotechnology, 12:173-201; Skerra and Pluckthun, (1988) Science, 240:1038-1040.

Reorganization heparin binding growth factor such as acid fibroblast growth factor, Prostatropin and vascular endothelial growth factor reclaim and purifying (Salter D.H.et al., (1996) from the many sources that comprise bacterium Labor.Invest., 74 (2): 546-556 (VEGF); Siemeister etal., (1996) Biochem.Biophys.Res.Commun., 222 (2): 249-55 (VEGF); Cao et al., (1996) J.Biol.Chem., 261 (6): 3154-62 (VEGF); Yang et al., (1994) Gaojishu Tongxun, 4:28-31 (VEGF); Anspach et al., (1995) J.Chromatogr.A, 711 (1): 129-139 (aFGF ﹠amp; 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) FASEB J., 9 (3): A410 (bFGF)).Yet bacterial expression system such as intestinal bacteria lack the cell mechanism that promotes the correct refolding of protein, and do not cause the larger protein secretion to enter substratum usually.It is inclusion body that the recombinant protein of expressing in bacterial host cell is often found, it is made up of with proteinic closely knit of reductive false folding partially folded.In this form, recombinant protein is non-activity normally.For example, the main activity form of VEGF is the homodimer of two 165 amino acid whose polypeptide (VEGF-165).In this structure, each subunit comprise 7 pairs of intrachain disulfide bonds and realize two subunits covalently bound other two to (Ferrara et al., (1991) J. Cell.Biochem., 47:211-218).Native conformation comprises a strong basicity structural domain, and it has demonstrated and has been easy to heparin-binding (people (1991) such as Ferrara is the same).The covalency dimerization of VEGF be effective receptors bind and biologic activity needed (

Et al., (1994) J.Biol.Chem., 269:32879-32885; Claffey et al., (1995) Biochim.et Biophys.Acta, 1246:1-9).Bacterial product comprises several false foldings and the intermediate disulphide confusion potentially.

In addition, refolding often produces the dimer that is connected with disulphide, tripolymer and polymer (Morris et al., (1990) of false folding 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 higher protein concn, and often appears as association (Cleland and Wang, (1990) that involve via the hydrophobic interaction of partially folded intermediate Biochemistry, 29:11072-11078).

False folding betides in the cell between yeast phase or separates in the rules.The protein of space reclamation must dissolve in periplasmic space or the born of the same parents, and soluble protein must refolding become native state.The in vitro method that being used for refolding protein becomes conformation correct, that biologic activity is arranged is vital for obtaining functional protein.Recovery is processed the denaturing agent that is included in high density from the proteinic typical downstream of inclusion body and is dissolved inclusion body such as urea, then dilutes denaturing agent to allow refolding taking place (referring to U.S. Patent number 4,512,922; 4,511,502; 4,511,503).Also can be referring to for example Rudolph and Lilie, (1996) FASEB J., 10:49-56; Fischer et al., (1993) Biotechnology and Bioengineering, 41:3-13.Such recovery method is considered to be in and generally is applicable to after the slight modification reclaims the recombinant protein that biologic activity is arranged from inclusion body.These methods have been applied to heparin-binding protein such as VEGF (people (1996) such as Siemeister is the same).These methods attempt to eliminate with organic disulfide Cheng Jian, and other stabilizing power by it makes recombinant protein become its biologic activity conformation afterwards, and can not eliminate incorrect folding intermediate or the correct homogeneous group who folds product is provided.

By being encapsulated in single protein in the micelle or separating them and remove denaturing agent then on resin, reverse micelle (reversed micelle) or ion exchange chromatography have been used to help refolding (Hagen et al., (1990) of denatured protein Biotechnol.Bioeng., 35:966-975; Creighton (1985) in Protein Structure Folding and Design(Oxender, D.L.Ed.) pp.249-251, New York:Alan R.Liss, Inc.).These methods have been used to prevent protein aggregation and have promoted correct refolding.For speed or the degree that changes refolding, with part and at the antibody of protein natural structure implemented the conformation specific refolding (Cleland and Wang, (1993), in Biotechnology, (RehmH.-J., and Reed G.Eds.) pp 528-555, New York, VCH).For example, creatine kinase refolding (Morris et al., (1987) when the antibody that exists at its natural structure Biochem.J., 248:53-57).Outside antibody, part and cofactor have been used to strengthen refolding.These molecules after native protein forms more likely with fold in protein interaction.Therefore, can will fold balance to native state " driving ".For example, the external part of the axial location of heme iron has improved refolding speed (Brems and Stellwagon, (1983) of ferrocytochrome c J.Biol.Chem., 258:3655-3661).Chaperone also has been used to help protein folding.Referring to for example Baneyx, (1999) Current Opinion in Biotechnology, 10:411-421.

Need from the host cell culture, fold and/or reclaim the new and more efficient methods of heparin-binding protein, for example be used for efficiently and production heparin-binding protein economically at the bacterial cell culture, thereby elimination or the minimizing and the improvement highly purified, recovery of protein that biologic activity is arranged, correct refolding of the active intermediate of abiology are provided, and generally are applicable to production-scale protein production.The present invention has satisfied these needs and other needs, will be conspicuous after reading the following discloses content.

Summary of the invention

The invention provides a kind of being used for from the method for the heparin-binding protein of cell culture recovery and purifying refolding.Particularly, the invention provides a kind of method that from prokaryotic host cell (for example bacterial cell), reclaims heparin-binding protein.For example, a kind of method comprises following steps: (a) the insoluble heparin-binding protein of spatial isolation in the periplasmic space of described bacterial cell or the born of the same parents; (b) the described isolating insoluble heparin-binding protein of dissolving in comprising first buffered soln of chaotropic agent and reductive agent; (c) the described dissolved heparin-binding protein of incubation in second buffered soln that comprises chaotropic agent and sulfation polyanion reagent, the time of incubation and condition make heparin-binding protein generation refolding; And (d) reclaim the heparin-binding protein of described refolding, wherein the protein concn that reclaims by the incubation that sulfation polyanion reagent is arranged has compared with the control increased by 2 to 10 times.In one embodiment, described second buffered soln further comprises arginine.In one embodiment, described second buffered soln further comprises halfcystine or gentle reductive agent.

In one embodiment of the invention, proteinic protein concn biologic activity, refolding that has that is reclaimed has increased for example 2-8 times, proteinic protein concn biologic activity, refolding that has that is perhaps reclaimed has increased 2-5 doubly, proteinic protein concn biologic activity, refolding that has that is perhaps reclaimed has increased 3-5 doubly, and proteinic protein concn biologic activity, refolding that has that is perhaps reclaimed has increased 2-3 doubly.In another embodiment of the invention, proteinic protein concn biologic activity, refolding that has that is reclaimed has for example increased greater than 2.0 times, 2.5 times, 2.8 times, 3.0 times, 5 times, 6 times, 7.0 times, 8 times, 9 times etc.In one embodiment of the invention, there is the protein concn of VEGF biologic activity, refolding to increase by 3 to 5 times.

Method of the present invention is widely used in heparin-binding protein, especially heparin binding growth factor, particularly vascular endothelial growth factor (VEGF).In certain embodiments of the invention, described sulfation polyanion reagent is between about 3,000 and 10,000 dalton.In one embodiment, employed described sulfation polyanion reagent is sulfuric acid dextran, sodium sulfate or heparin sulfate in the production process.In one aspect, the sulfuric acid dextran is between 3,000 dalton and 10,000 dalton.

In addition, the invention provides and be used for protein-bonded operation of purified heparin and method, or the independent or protein-bonded recovery of combined with heparin, just as described in this article.In a specific embodiment, purification process comprises heparin-binding protein contact hydroxyapatite upholder, the first hydrophobic interaction chromatography upholder, positively charged ion chromatography upholder and the second hydrophobic interaction chromatography upholder that makes described refolding, and from each upholder the selective elution heparin-binding protein.In another embodiment, purification process comprises heparin-binding protein contact cationic exchange upholder, the first hydrophobic interaction chromatography upholder and ion-exchange or the blending agent chromatography upholder that makes described refolding, and from each upholder the selective elution heparin-binding protein.Imagined each step that to implement recycling step with any order, order for example order or that change the chromatography upholder.In certain embodiments of the invention, provide from produce or plant-scale cell culture reclaim and the method for the heparin-binding protein of purifying refolding.

The accompanying drawing summary

Fig. 1 has shown that the VEGF that is produced by bacterial isolates W3110 is loaded into POROS HE2/M post (4.6 * 100mm, PerSeptive BioResearch Products, Cambridge, MA) tomographic map on.For example, POROS HE/2M post balance in containing the 10mM sodium phosphate pH7 of 0.15M sodium-chlor.Use this post of linear gradient elution of the 0.15-2M sodium-chlor among 10 minutes the 10mM sodium phosphate pH7.At 280nm monitoring eluant.The protein that reclaims in each peak is corresponding to VEGF, yet has only peak 3 corresponding to VEGF biologic activity, correct refolding is arranged.

Fig. 2 has shown and has shown the graphic representation of heparin to the stabilization of natural, correct folding VEGF.VEGF is suspended in the 50mM HEPESpH8 that contains 5mM EDTA, 0.2M NaCl and 10mM halfcystine.

Fig. 3 A-3D has shown and has been produced by bacterial isolates W3110, and with 12 μ g/ml 5,000 dalton's sulfuric acid dextran (Fig. 3 A), 12 μ g/ml 8,000 dalton's sulfuric acid dextran (Fig. 3 B), 12 μ g/ml10,000 dalton's sulfuric acid dextran (Fig. 3 C) or 25 μ g/ml 3,000 dalton's heparin (Fig. 3 D) is incubation together, and be loaded into POROS HE2/M post (4.6 * 100mm, PerSeptive BioResearchProducts, Cambridge, MA) tomographic map of the VEGF on.For example, this post balance in containing the 10mM sodium phosphate pH7 of 0.15M sodium-chlor.Use this post of linear gradient elution of the 0.15-2M sodium-chlor among 10 minutes the 10mM sodium phosphate pH7.At 280nm monitoring eluant.The protein that reclaims in each peak is corresponding to VEGF, yet has only peak 3 corresponding to VEGF biologic activity, correct refolding is arranged.

Fig. 4 has shown the influence of scale to the VEGF refolding.

Fig. 5 has shown the influence to the VEGF refolding of the heparin of lower molecular weight and high molecular (MW) and 10,000 daltonian sulfuric acid dextran.Peak 3 is corresponding to VEGF biologic activity, correct refolding is arranged.

Fig. 6 has shown the influence of sodium sulfate to the VEGF refolding.Peak 3 is corresponding to VEGF biologic activity, correct refolding is arranged.

Fig. 7 has shown the influence to the VEGF refolding of lower molecular weight and high molecular (MW) heparin and 5,000 dalton, 8,000 dalton and 10,000 daltonian sulfuric acid dextran.Peak 3 is corresponding to VEGF biologic activity, correct refolding is arranged.

Fig. 8 has shown the influence to the VEGF refolding of heparin and sulfuric acid dextran.Peak 3 is corresponding to VEGF biologic activity, correct refolding is arranged.

Fig. 9 has shown urea and the DTT influence to extracting VEGF from the bacterium inclusion body.

Figure 10 has shown the influence to the VEGF refolding of urea and DTT concentration.

Figure 11 has shown VEGF ₁₆₅Amino-acid sequence, wherein indicated disulfide linkage (SEQ ID NO:1).

Figure 12 has shown the influence of charged occurrence of amino acid.When the concentration in second buffered soln was 0.75M, arginine and Methionin both were useful, and, to compare with the buffered soln that does not contain Histidine, Histidine has a little synergistic effect.In addition, the concentration that demonstrated 0.1 to 1M of arginine has similar influence.

Figure 13 has shown the dilution effect of refolding percent efficiency, and wherein, though Zong VEGF concentration lowers with the extent of dilution increase, the refolding percent efficiency raises with the extent of dilution increase.

Detailed Description Of The Invention

Definition

" heparin " (heparin or heparinic acid) is highly Sulfated, straight chain anion mucopolysaccharide The heterogeneous group of (being called glycosaminoglycan). Although can there be other carbohydrate, the main carbohydrate in the heparin is: α-L-iduronic acid 2-sulfuric ester, 2-deoxidation-2-sulfoamino-group-phlorose 6-sulfuric ester, β-D-Glucose Aldehydic acid, 2-acetylaminohydroxyphenylarsonic acid 2-deoxidation-alpha-D-glucose and L-iduronic acid. These and optional other Carbohydrate links to each other by glycosidic bond, forms the polymer of different sizes. Because its covalently bound sulfuric ester and The existence of hydroxy-acid group, heparin are highly acid. The scope of the molecular weight of heparin is from about 3,000 to about 20,000 dalton are depended on source and assay method.

Natural heparin is the component of a plurality of tissues, especially liver and lung, and several mammalian species In mast cell. Heparin and heparinate (liquaemin) are commercially available, and mainly at various clinical settings In be used as anticoagulant.

" sulfuric acid dextran " is the sulfuric ester of dextran, and its primary structure is the poly of D-Glucose Body. Glucose links to each other by α-D (1-6) glycosidic bond with other optional carbohydrate, forms the many of different sizes Aggressiveness. Because the existence of covalently bound sulfuric ester, the sulfuric acid dextran is highly acid. Sulfur content is logical Often be no less than 10%, about 15%-20% typically is until each glucose molecule has 3 sulfate groups. The mean molecule quantity of sulfuric acid dextran is from about 1,000 to about 40,000,000 dalton. Among the present invention The example of adoptable sulfuric acid dextran comprises by microorganism such as Leuconostoc mesenteroides (Leuconostoc Mesenteroides) and the sulfuric ester of the dextran that produces of leuconostoc dextranicum bacteria (L.dextranicum).

" polyanion reagent " be meant when using within the scope of the present invention commercially available purifying natural heparin product and can with heparin-binding protein bonded compound, comprise other " polyanion reagent ", such as sodium sulfate, heparin sulfate, Suleparoid, (many) sulfuric acid piperylene, dextran, sulfuric acid dextran, hyaluronic acid, chrondroitin, chondroitin sulfate, dermatan sulfate and keratan sulfate.Useful especially in content of the present invention is " sulfation polyanion reagent ", sulfate derivative such as for example polysaccharide, such as heparin sulfate, the sulfuric acid dextran, by such as U.S. Patent number 5,314, the sulfuric ester of the cyclodextrin of the microorganisms that the bacillus macerans of putting down in writing in 872 (Bacillus macerans) is such, and the sulfuric ester of other dextran such as β-1,3 dextran sulfates (β-1,3 dextran be by the microorganisms that belongs to Alcaligenes (Alcaligenes) or Agrobacterium (Agrobacterium)), and chondroitin sulfate and sulfation heparin fragment.

Mentioned reagent normally those skilled in the art is available and approval.For example, the sulfation heparin fragment can be from by obtaining the gel permeation chromatography fractionated heparin derivative oligosaccharides library.Ishihara et al., (1993) are seen in the preparation of the oligosaccharides of affine fractionated, heparin derivative J.Biol.Chem., 268:4675-4683.These oligosaccharides with the depolymerization of nitrous acid part, are used sodium borohydride reduction from the plain preparation of commercialization pork liver, and by the gel permeation chromatography classification.Gained disaccharides, tetrose, six sugar, eight sugar and ten sugared set are applied in proper order and are covalently attached to SEPHAROSE ^TMThe affinity column of the human recombinant bFGF of 4B, and reply the sodium-chlor gradient and wash-out from this post further is classified into subclass according to them.This has produced five set, and called after Hexa-1 further assesses its structure and biologic activity to Hexa-5.The structure of Hexa-5C and 500MHz NMR spectrum thereof are seen Tyrell et al., (1993) J.Biol.Chem., Fig. 4 of 268:4684-4689.This six sugar have following structure [IdoA (2-OSO ₃) α 1-4GlcNSO ₃(6-OSO ₃) α 1-4] ₂IdoA (2-OSO ₃) α 1-4AMan _R(6-OSO ₃).All heparin derivative oligosaccharides that above discuss and other heparin sample oligosaccharides all are applicable to and can be used for the present invention.In one embodiment of the invention, use the heparin polysaccharide (for example seven sugar, eight sugar, nine sugar and ten sugar) of six sugar and higher unit-sized.In addition, use and to have big net negative charge oligosaccharides heparin derivative or the heparin sample of (for example because due to the sulfation of height) is useful.

Term " heparin-binding protein " or " HPB " when being used for this paper, be meant can heparin-binding (as hereinbefore defined) polypeptide.This definition comprises natural and the maturation of heparin-binding protein recombinant production, preceding (pre), preceding-former (pre-pro) and former (pro) form.The exemplary of heparin-binding protein is " heparin binding growth factor ", include but not limited to that Urogastron (EGF), Thr6 PDGF BB (PDGF), Prostatropin (bFGF), acid fibroblast growth factor (aFGF), vascular endothelial growth factor (VEGF), pHGF (HGF) (are called scattering factor again, SF) and nerve growth factor (NGF), IL-8 etc.

" vascular endothelial growth factor " or " VEGF " is meant initial a kind of Mammals somatomedin derived from the ox follicular cell when being used for this paper, and its amino-acid sequence is disclosed in Castor, C.W., et al., (1991) Methods in Enzymol., 198:391-405, and the functional derivatives with the qualitative biologic activity of corresponding natural VE GF include but not limited to Houck et al., (1991) Mol. Endocrin., the human VEGF aminoacid sequence of 5:1806-1814 report.Also can be referring to Leung et al., (1989) Science, 246:1306; Robinson ﹠amp; Stringer, (2001) Journal of Cell Science, 144 (5): 853-865; U.S. Patent number 5,332,671.The principal mode of VEGF is 165 amino acid whose homodimers, and it has 16 cysteine residues, and they form 7 intramolecular disulfide bonds and two intermolecular disulfide bonds.Alternative splicing involves forming of the multiple human VEGF polypeptide be made up of 121,145,165,189 and 206 amino acid, however VEGF ₁₂₁Variant lacks the heparin binding domains of other variant, therefore not in the scope of the listed heparin-binding protein definition of this paper.All isoforms (isoform) of VEGF are shared common N-terminal structural domain, but the length difference of the C-terminal of molecule part.Preferred VEGF activity form, VEGF ₁₆₅, in each monomer, between following amino-acid residue, have disulfide linkage: Cys26-Cys68; Cys57-Cys104; Cys61-Cys102; Cys117-Cys135; Cys120-Cys137; Cys139-Cys158; Cys146-Cys160.Referring to Figure 11.Also can be referring to for example Keck et al., (1997) Archives of Biochemistry and Biophysics, 344 (1): 103-113.VEGF ₁₆₅Molecule is made of two structural domains: a N-terminal receptors bind structural domain (amino acid/11-110, the homodimer that disulfide linkage links to each other) and a C-terminal heparin binding domains (residue 111-165).Referring to for example Keyt et al., (1996) J.Biol.Chem., 271 (13): 7788-7795.In certain embodiments of the invention, separate the also VEGF of purifying ₁₆₅Locate not to be glycosylated at residue 75 (Asn).Referring to for example Yang et al., (1998) Journal of Pharm.﹠amp; Experimental Therapeutics, 284:103-110.In certain embodiments of the invention, separate the also VEGF of purifying ₁₆₅At residue A sn10 place is not deamidating basically.In certain embodiments of the invention, separate the also VEGF of purifying ₁₆₅Be deamidating (at residue A sn10 place) and not deamidating proteinic mixture, typically most of protein is not deamidating.Since VEGF ₁₆₅Be homodimer, deamination can occur on one or all two polypeptide chains so.

When being used for this paper, " correct folding " or " biologic activity is arranged " VEGF or other HBP or the like refer to molecule with biologic activity conformation.Those of skill in the art can approve that false folding may have biologic activity with the intermediate disulfide linkage confusion.In this case, VEGF correct folding or that biologic activity is arranged or the HBP VEGF (mentioned above) or other HBP that are equivalent to the natural folding pattern.For example, outside two intermolecular disulfide bonds in dimer molecule, it is right that correct folding VEGF has disulphide mentioned above, yet other intermediate can be produced (Fig. 1 and 3A-3D) by the bacterial cell culture.For correct folding VEGF, described two intermolecular disulfide bonds occur in each monomeric identical residue, promptly between Cys51 and the Cys60.Referring to for example WO 98/16551 patent.The biologic activity of VEGF includes but not limited to for example promote vascular permeability, promotes vascular endothelial cell growth, combines with vegf receptor, signals (referring to for example Keyt et al., (1996) in conjunction with vegf receptor and by vegf receptor Journal ofBiological Chemistry, 271 (10): 5638-5646), induction of vascular generation etc.

Term " purifying " or " pure HBP " or the like refer to not contain usually in its recombinant production, especially the material of finding in protokaryon or bacterial cell culture of following material.So, this term refers to not contain the reorganization HBP of contaminative DNA, host cell proteins matter or other molecule relevant with its in situ environment.This term refers to about at least 75%, about at least 80%, about at least 85%, about at least 90%, about at least 95% or about at least 98% or higher purity.

Term " inclusion body " or " refractile body " refer to the interior piece of the intensive born of the same parents of accumulative polypeptide of interest, and the great part that it constitutes total cell protein matter comprises all cells component.In some situation, but not all situation, these polypeptide aggregation bodies can be identified as tangible bright spot in the cell coat under the phase microscope of reducing to 1,000 x magnification.

When being used for this paper, term " false folding " protein refers to the sedimentary or accumulative polypeptide that comprises in the refractile body.When being used for this paper, " insoluble " or " false folding " VEGF or other HBP refer to sedimentary or accumulative VEGF, it is included in the periplasmic space or the interior space of born of the same parents of prokaryotic host cell, perhaps otherwise relevant with prokaryotic host cell, and present the active conformation of abiology, have mispairing or inchoate disulfide linkage.Insoluble HBP normally but non-must being included in the refractile body, promptly it can be or can not be tangible under phase microscope.

When being used for this paper, when " chaotropic agent " refers in the aqueous solution suitable concn be arranged, thereby the sterie configuration or the conformation that can change polypeptide by the variation on polypeptide surface make polypeptide dissolve in the compound of this aqueous medium.Described variation can take place by Change Example such as hydration status, solvent environment or solvent-surface interaction.The concentration of chaotropic agent will directly influence its intensity and effectiveness.Strong sex change in solution, comprise the chaotropic agent of high density from liquor, folding with the polypeptide that exists in the solution is separated, thus eliminate this proteinic secondary structure effectively.Separating folding can be relatively widely, but reversible.Medium sex change in solution, comprise the chaotropic agent of enough concentration from liquor, allow polypeptide from any case guache conformation (this polypeptide by soluble intermediate presented solution) the such space conformation of partially folded one-tenth, wherein it finds it oneself when operating with its activity form under endogenous or homologous physiological conditions.The example of chaotropic agent comprises Guanidinium hydrochloride, urea and oxyhydroxide such as sodium hydroxide or potassium hydroxide.Chaotropic agent comprises these combination of agents, such as the mixture of oxyhydroxide and urea or Guanidinium hydrochloride.

When being used for this paper, when " reductive agent " refers in the aqueous solution suitable concn be arranged, keep free sulfhydryl groups, make intramolecularly or intermolecular disulfide bond suffer the compound of chemical depletion.The representative example of appropriate reductant comprises dithiothreitol (DTT) (DTT), dithioerythritol (DTE), beta-mercaptoethanol (BME), halfcystine, cysteamine, thioglycolate salt/ester, gsh, three [2-propyloic] phosphine (TCEP) and sodium borohydride.

When being used for this paper, " buffered soln " refers to tolerate the solution that pH changes by the effect of its acid-alkali conjugation (conjugate) component.

" bacterium " comprises eubacterium and archeobacteria for this paper.In certain embodiments of the invention, use eubacterium in methods described herein and the technology, comprise gram positive bacterium and gram negative bacterium.In one embodiment of the invention, use gram negative bacterium, for example enterobacteriaceae.The example that belongs to the bacterium of enterobacteriaceae comprises Escherichia (Escherichia), enterobacter (Enterobacter), erwinia (Erwinia), Klebsiella (Klebsiella), proteus (Proteus), salmonella (Salmonella), serratia (Serratia) and Shigella (Shigella).The suitable bacteria of other type comprises Azotobacter (Azotobacter), Rhodopseudomonas (Pseudomonas), rhizobium (Rhizobia), Vitreoscilla (Vitreoscilla) and paracoccus (Paracoccus).In one embodiment of the invention, use intestinal bacteria.Suitable escherichia coli host comprises intestinal bacteria W3110 (ATCC 27,325), intestinal bacteria 294 (ATCC 31,446), intestinal bacteria B and intestinal bacteria X1776 (ATCC 31,537).These examples are exemplary, rather than restrictive, and W3110 is an example.Also can adopt the mutant cell of any above-mentioned bacterium.Certainly, consider that when selecting cell the replicability of replicon in bacterial cell is necessary.For example, when using plasmid well known in the art such as pBR322, pBR325, pACYC177 or pKN410 that replicon is provided, intestinal bacteria, serratia or Salmonellas species can be used as the host suitably.Example about the suitable bacteria host cell also can vide infra.

When being used for this paper, statement " cell ", " clone ", " bacterial strain " and " cell culture " are used interchangeably, and all these class titles all comprise the offspring.So, word " transformant/transformant " and " transformant " comprise former generation subject cell and by its deutero-culture, no matter the number of times that shifts.Should also be understood that because have a mind to or sudden change unintentionally, all offsprings may not be accurately identical on the DNA content.The sudden change offspring with identical function or biologic activity who screens in initial transformant is included.If want to make different names, context has clearly statement.

When being used for this paper, " polypeptide " be often referred to from any cell source, have more than about ten amino acid whose peptides and protein." allogenic " polypeptide is that those are external polypeptide for employed host cell, such as the human protein who is produced by intestinal bacteria.Though allogenic polypeptide can be protokaryon or eucaryon, preferred it be eucaryon, be more preferably mammiferously, most preferably be human.In certain embodiments of the invention, it is polypeptide recombinant production or reorganization.

Heparin-binding protein

Separate heparin-binding protein

Heparin-binding protein (HBP) insoluble, false folding is separated from expressing this proteinic prokaryotic host cell by any this area standard technique.For example, separatin non-soluble HBP in suitable dissociating buffer, be about to cellular exposure in the damping fluid of appropriate ions intensity to dissolve most of host protein, but being tried protein in this solution is insoluble on substantially, and perhaps smudge cells discharges inclusion body or protein and makes that they can be by for example centrifugal the recovery with space in periplasmic space or born of the same parents.This technology is known, is recorded in for example U.S. Patent number 4,511,503.People such as Kleid disclosed by homogenate succeeded by centrifugal come the purifying refractile body (Kleid et al., (1984) in Developments in Industrial Microbiology, (Society for Industrial Microbiology, Arlington, VA) 25:217-235).Also can be referring to for example Fischer et al., (1993) Biotechnology and Bioengineering, 41:3-13.

U.S. Patent number 5,410,026 has put down in writing a kind of being used for reclaims proteinic typical method from inclusion body, and is summarized as follows.Prokaryotic cell prokaryocyte is suspended in the suitable damping fluid.Typically, damping fluid comprises and is suitable for carrying out buffered buffer reagent and salt between pH 5 to 9 or about 6 to 8.Any suitable salt comprises NaCl, is used in and keeps enough ionic strengths in this buffered soln.Typically, adopt about 0.01 to arrive the ionic strength that 2M or 0.1 arrives 0.2M.Cell uses in being suspended in this damping fluid the time technology that adopts usually to break or dissolve, such as for example method of machinery, for example homogenizer (Manton-Gaulinpress, Microfluidizer or Niro-Soavi), French press (French press), bead mill or acoustic wave oscillator perhaps pass through method chemistry or zymetology.

The example of ruptured cell method chemistry or zymetology comprises into spheroplast, wherein must use N,O-Diacetylmuramidase to come dissolution of bacteria wall (H.Neu et al., (1964) Biochem.Biophys.Res.Comm., 17:215); And osmotic shock, wherein involve viable cell is cleaned to discharge polypeptide (H.Neu et al., (1965) with the solution-treated of Gao Zhangdu and with the cold water that hangs down Zhang Du J.Biol.Chem., 240 (9): 3685-3692).Usually use supersound process to come the bacterium that is comprised in the fermenting broth of rupture Analysis scale volume.On bigger scale, typically adopt high-pressure homogenization.

After cell rupture, typically with suspension low-speed centrifugal in standard centrifuge, generally about 500 to 15, about 000xg, for example adopt in one embodiment of the invention about 12,000xg, the centrifugal time is enough to make all basically insoluble protein precipitations.This class time can be determined simply, and depend on centrifugal volume and centrifugal design.Typically, be enough to precipitate insoluble protein in about 10 minutes to 0.5 hour.In one embodiment, with suspension with 12, centrifugal 10 minutes of 000xg.

The gained throw out comprises all basically insoluble protein parts.If the cell rupture operation is incomplete, this throw out may also comprise intact cell or disruptive cell debris so.The thoroughness of cell rupture can be measured by resuspension gained throw out in a spot of identical buffered soln and with this suspension of phase-contrast microscopy.The existence of ruptured cell fragment or intact cell indicate further supersound process or other break means be remove fragment or cell and relevant non-refrangibility polypeptide necessary.So further break after, if necessary, recentrifuge suspension also reclaims throw out, resuspending, and check again.Repeating this operation has disclosed up to visual test and does not have the disruptive cell debris in the sedimentary material or fail to reduce the sedimentary volume of gained up to further processing.

No matter insoluble protein is intracellular or can adopts above-mentioned method in periplasmic space.In one embodiment of the invention, the condition that given herein being used for separates heparin-binding protein is at the inclusion body that is deposited in space in periplasmic space or the born of the same parents, and is particularly related to VEGF.Yet, think that described technology and rules are in the heparin-binding protein that totally is applicable to slightly after changing as described below.In certain embodiments of the invention, described technology and rules are applicable to production or plant-scale production, refolding and the purifying of HBP.

The refolding heparin-binding protein

With heparin-binding protein incubation in first buffered soln of isolating insoluble, false folding, this first buffered soln comprises a certain amount of chaotropic agent and reductive agent, is enough to dissolve basically this heparin-binding protein.This incubation allow some or basically all heparin-binding protein dissolving takes place and separate under the condition of folding concentration, incubation time and heated culture temperature and carry out.

The measurement of dissolution degree can be measured simply and suitably implements in the buffered soln, for example by turbidity measurement, by the classification between supernatant liquor behind the analysis centrifugal and the throw out, by reductibility SDS-PAGE, by protein determination (for example Bradford reagent protein determination (for example Pierce, Bio-Rad etc.)) or pass through HPLC.

Described first buffered soln comprises the pH scope that is suitable for damping fluid and maintains buffer reagent about at least 7.0, typical range 7.5-10.5.In one embodiment, the pH that is used for VEGF is pH8.0.Can provide the example of the suitable buffer reagent of the pH in a kind of scope in back to comprise TRIS-HCl (three [methylol] aminomethane), HEPPS (the N-[2-hydroxyethyl] piperazine-N '-[3-propane-sulfonic acid]), HEPES (the N-[2-hydroxyethyl] piperazine-N '-[2 ethane sulfonic aicd]), CAPSO (3-[cyclohexyl amino]-2-hydroxyl-1-propane sulfonic acid), AMP (2-amino-2-methyl-1-propanol), CAPS (3-[cyclohexyl amino]-the 1-propane sulfonic acid), CHES (2-[N-cyclohexyl amino] ethane sulfonic acid), glycine, and sodium acetate.In one embodiment of the invention, buffer reagent herein is the HEPPS of about pH 8.0.In another embodiment, described buffer reagent such as HEPPS, is Sulfated.

Be suitable for implementing chaotropic agent of the present invention and comprise for example salt of urea and guanidine or thiocyanic acid, for example urea, Guanidinium hydrochloride, Sodium Thiocyanate 99 etc.The amount of the chaotropic agent that must exist in the damping fluid is the amount that is enough to separate folding HBP in solution.In certain embodiments of the invention, chaotropic agent exists between about 4 and 10 moles.In one embodiment of the invention, chaotropic agent is the urea of about 5-8M or about 7M.In another example, chaotropic agent is the Guanidinium hydrochloride of about 6-8M.

The example of appropriate reductant includes but not limited to dithiothreitol (DTT) (DTT), dithioerythritol (DTE), beta-mercaptoethanol (BME), halfcystine, DTE etc.The amount of the reductive agent that exists in the damping fluid will depend primarily on the oxygen that comprises in the type of the type of reductive agent and chaotropic agent, the damping fluid that adopted and pH, the solution or introduce amount, and damping fluid in proteinic concentration.For example, 0.5-1.5mg/ml protein is arranged in the buffered soln for pH7.0-10.0, this buffered soln comprises 4-8M urea and following reductive agent, for example the halfcystine of the BME of the DTT of the about 1-15mM of concentration or the about 0.2-2mM of concentration or the about 2-10mM of concentration.In one embodiment, reductive agent is the DTT of concentration about 0.5 to about 4mM or 2-4mM.Fig. 9 has shown urea and the DTT influence to extracting VEGF.The VEGF at peak 3 has represented correct VEGF folding, that biologic activity is arranged.In one embodiment, reductive agent is the DTT of about 10mM.Damping fluid herein can use the combination of single reductive agent or reductive agent.

Protein concn in the buffered soln must make this protein of mensuration according to optical density(OD) fully dissolve.The accurate amount that adopts will depend on the concentration and the type of other composition in the buffered soln for example, the pH of particularly proteinic concentration, reductive agent and damping fluid.In one embodiment of the invention, the concentration range of heparin-binding protein is 0.5-5.5mg/ml, or 1.5-5.0mg/ml.Dissolving was typically carried out at least approximately 1-24 hour at about 0-45 ℃ or about 20-40 ℃ or about 23-37 ℃ or about 25-37 ℃ or about 25 ℃.In one embodiment, being dissolved in room temperature carried out about at least 2 hours.Typically, temperature can not be subjected to the obvious influence of salt, reductive agent and chaotropic agent level.

After the polypeptide dissolving, it is placed second buffered soln or dilute at second buffered soln, the concentration of chaotropic agent comprises aforesaid chaotropic agent and sulfation polyanion reagent in this second buffered soln, in any case will allow the heparin-binding protein refolding.

The condition of proteinic this second incubation solubility, false folding makes some or protein generation refolding basically or whole usually.Definite condition will depend on for example pH and the sulfation polyanion reagent of existence and the concentration and the type of chaotropic agent and reductive agent of damping fluid, if any.Normally about 0-40 ℃ or 10-40 ℃ of heated culture temperature, and incubation carries out about at least 1 hour usually to realize refolding.In one embodiment, this for example is reflected at approximately 15-37 ℃ or 20-30 ℃ and carries out between about at least 6 hours, about at least 10 hours or about 10 and 48 hours or between about 15 and 20 hours or between 6 and 20 hours or between 12 and 24 hours.

The following suitable mensuration of the degree of refolding, i.e. radioimmunoassay (RIA) titre by HPB or analyze by the high performance liquid chromatography (HPLC) that uses POROS HE2/M post (PerSeptive BioResearch Products) for example or other suitable heparin affinity column.RIA titre or correctly folding HBP peak size raise and directly raise corresponding to the amount that is present in the correct HPB folding, that biologic activity is arranged in the damping fluid.Carry out correct folding HPB that incubation reclaimed with the maximization ratio to false folding HPB, it is measured by RIA or HPLC.

In one embodiment, the quality of the correct VEGF that folds and quantity use the heparin binding assay to assess.The sample that will comprise the heparin-binding protein of dilution for example is loaded into POROS HE2/M post, and (Cambridge is MA) or on other suitable heparin affinity column for 4.6 * 100mm, PerSeptive BioResearch Products.For example, balance heparin affinity column in comprising the 10mM sodium phosphate pH7 of 0.15M sodium-chlor.With the flow velocity of 1ml/min or 2ml/min, use this post of 0.15-2M sodium-chlor linear gradient elution among 10 minutes the 10mM sodium phosphate pH7.At 280nm place monitoring eluant.In one embodiment, in corresponding to the simple spike that HBP biologic activity, correct refolding is arranged, reclaim protein.In one embodiment of the invention, the assay method that is used to measure the HBP of correct refolding is RPHPLC.Randomly, can confirm that disulfide linkage connects by peptide figure.Circular dichroism also can be used for measuring 2 and 3 dimensional organization/folding.

The buffer reagent that is used for second buffered soln can be any above listed about first buffered soln, HEPPS pH8.0 for example, and for example the about 50mM of concentration is used for refolding VEGF.Can dilute polypeptide, for example at least 5 times or about at least 10 times, about 20 times or about 40 times with the refolding damping fluid.Perhaps, polypeptide can be dialysed to the refolding damping fluid.

Second buffered soln comprises chaotropic agent, and the concentration of this chaotropic agent makes HPB that refolding take place.Usually, chaotropic agent exists with the concentration between about 0.5 and 2 mole.In one embodiment of the invention, chaotropic agent herein is a urea, and its concentration is about 0.5-2M, 0.5-2M or about 1M.In one embodiment, chaotropic agent is a urea, and its concentration is about 1.3M.In another embodiment of the invention, chaotropic agent is a Guanidinium hydrochloride, and its concentration is about 1M.Figure 10 has shown urea and the reductive agent DTT influence to the VEGF refolding.The VEGF at peak 3 has represented correct VEGF folding, that biologic activity is arranged.

As described, this solution randomly also comprises reductive agent.Those are above described about dissolving step suitably for reductive agent, and its concentration range is that halfcystine is about 0.5 to be 0.1-1.0mM and/or BME for less than about 0.2mM to about 10mM, DTT.In one embodiment of the invention, reductive agent is that concentration is the DTT of about 0.5-2mM.In one embodiment of the invention, reductive agent is that concentration is the DTT of about 0.5mM.The example of appropriate reductant includes but not limited to for example dithiothreitol (DTT) (DTT), beta-mercaptoethanol (BME), halfcystine, DTE etc.Although generally speaking DTT and BME can unite use with the rules that provide for heparin-binding protein herein, and be as described herein, about 0.1 is an example that is used to reclaim VEGF to about 10mM halfcystine and about 0.1 combination to about 1.0mM DTT.

The refolding step comprises sulfation polyanion reagent, and its concentration is enough to realize the proteinic complete refolding of dissolved.The example of suitable polyanion reagent is above being described, the sulfated derivative of polysaccharide for example, sulfation polyanion reagent such as heparin sulfate, sulfuric acid dextran, Suleparoid and chondroitin sulfate and sulfation heparin fragment as indicated above.For employed heparin sulfate in the content of the present invention, molecular weight is usually between about 3,000 and 10,000 dalton, perhaps between about 3,000 and 6,000 dalton.

In one embodiment of the invention, in content of the present invention, adopt the sulfuric acid dextran.The molecular weight of the sulfation polyanion that is adopted among the present invention or other reagent such as sulfuric acid dextran depends on the protein-bonded size of the heparanase specific that is reclaimed.Usually adopt the sulfuric acid dextran between about 3,000 and 10,000 dalton.In one embodiment of the invention, use the sulfuric acid dextran between about 5,000 and 10,000 dalton, for example be used for the recovery of VEGF.In another embodiment, use the sulfuric acid dextran between about 5,000 and 8,000 dalton to reclaim HBP.VEGF recovering state when Fig. 3 A-3D has shown the sulfuric acid dextran (Fig. 3 A-C) of using various concentration and molecular weight and heparin (Fig. 3 D), it is analyzed by heparin affinity chromatography.Peak 3 is corresponding to correct folding VEGF.

The concentration of the polyanionic compound that is adopted depends on the protein that reclaimed and the temperature and the pH of concentration and condition such as refolding damping fluid thereof.For sodium sulfate, typical concentration about 50 and 500mM between; For low molecular weight heparin, such as 6,000 daltonian heparin (Sigma Chemical Co.), between about 10 and 200 μ g/ml; For high molecular weight heparin, such as the plain I-A (Sigma ChemicalCo.) of pork liver, between about 10 and 200 μ g/ml; And for the sulfuric acid dextran, between the about 10 and 400 μ g/ml or between the about 10 and 200 μ g/ml.

The refolding damping fluid can randomly comprise other reagent, such as any non-ionic detergent, such as TRITON ^TMX-100, NONIDET ^TMP-40, TWEEN ^TMSeries and BRIJ ^TMSeries.Non-ionic detergent exists with big concentration between 0.01% and 1.0%.In an example, the concentration of non-ionic detergent is between about 0.025% and 0.05% or about 0.05%.

Randomly, positively charged amino acid, for example (for example L-arginine/HCl), Methionin etc. may reside in the refolding damping fluid arginine.In one embodiment of the invention, arginic concentration is for example about 0-1000mM, or about 25 to 750mM, or about 50-500mM, or about 50-250mM, or the final concentration of about 100mM etc.In certain embodiments of the invention, protein is at pH7.0-9.0 and comprises in final concentration 0.5-3M urea, 0-30mg/L sulfuric acid dextran, 0-0.2%Triton X-100,2-15mM halfcystine, 0.1-1mM DTT and the arginic buffered soln of 0-750mM.In one embodiment, used 50mM HEPPS.In one embodiment, the final concentration of refolding buffered soln is 1M urea, 50mM HEPPS, 15mg/L sulfuric acid dextran, 0.05%Triton X-100,7.5mM halfcystine, 100mM arginine, pH8.0.In one embodiment, the final concentration of refolding buffered soln is 1.3M urea, 50mM HEPPS, 15mg/L sulfuric acid dextran, 0.05%Triton X-100,7.5mM halfcystine, 0.5mM DTT, 100mM arginine, pH8.0.

The recovery of heparin-binding protein and purifying

Though from nutrient solution, reclaim and be used to separate this type of proteinic the whole bag of tricks and known rules with conjugated protein can the employing of purified heparin, the for example classification of salt and solvent, the absorption of colloidal materials, gel-filtration, ion exchange chromatography, affinity chromatography, immunoaffinity chromatography, electrophoresis and high performance liquid chromatography (HPLC), the application has described a kind of example of four step chromatography rules, it comprises the heparin-binding protein contact hydroxyapatite upholder that makes described refolding, the first hydrophobic interaction chromatography upholder, the positively charged ion chromatography upholder and the second hydrophobic interaction chromatography upholder, and from each upholder this heparin-binding protein of selective elution.Perhaps, the application has described another kind of chromatography rules, it comprises heparin-binding protein contact cationic exchange upholder, hydrophobic interaction chromatography upholder and the ion exchange chromatography upholder that makes described refolding, and from each upholder this heparin-binding protein of selective elution.Having imagined each step of arbitrary rules can implement with any order.In one embodiment of the invention, described step is that order is implemented.

Suitable first step characteristic during further recovery and purified heparin are conjugated protein provides the minimizing of concentrating of heparin-binding protein and sample volume.For example, described second incubation step can cause the volume of the heparin-binding protein that reclaims to increase greatly and protein is followed dilution in the refolding damping fluid above.The suitable first chromatography upholder provides the volume of the heparin-binding protein that reclaims to reduce, and can be favourable to a certain degree purifying proteins of interest matter is provided from undesired contaminative protein.The first suitable chromatographic step comprises can wash-out and directly be loaded into chromatography upholder on the first hydrophobic interaction chromatography upholder.For example, use can be in being suitable for being loaded on the high salt concentration of hydrophobic interaction chromatography upholder the chromatography upholder of wash-out heparin-binding protein.

The first exemplary chromatography upholder includes but not limited to the hydroxyapatite upholder, for example I type and II type CHT pottery (being called the MacroPrep pottery in the past), Bio-Gel HT, Bio-Gel HTP (Biorad, Hercules, CA) etc.; The metal chelate chromatography upholder comprises the inert plastic of immobilized metal such as copper, nickel etc.; And non-derivative tripoli gel.In one embodiment of the invention, being used for the purifying of VEGF and the first chromatography upholder of recovery is the hydroxyapatite upholder.In another embodiment of the invention, being used for the purifying of VEGF and the first chromatography upholder of recovery is the cationic exchange upholder, for example hereinafter described in more detail.

Realize according to this area standard practices from the first chromatography upholder wash-out.Suitable elution requirement and damping fluid will be convenient to wash-out HPB and directly be loaded on the first hydrophobic interaction chromatography upholder as described below.

Hydrophobic interaction chromatography is that this area is known altogether, and relies on the hydrophobic part and the interaction that is attached to the hydrophobic ligand of " chromatography upholder " of molecule.Be coupled to the hydrophobic ligand HIC of being called chromatography miscellaneous upholder, HIC gel or the HIC post etc. of matrix.Also should understand, the interactional strength between protein and the HIC post just on the protein apolar surfaces to the function of the ratio of polar surfaces, or the function of the distribution of apolar surfaces.

Many matrix can be used for preparing the HIC post.The most widely used is agarose, though tripoli and organic polymer resin also can be used.Useful hydrophobic ligand includes but not limited to have about 2 alkyl (such as butyl, propyl group or octyl group) or aryl (such as phenyl) to about 10 carbon atoms.The conventional H IC upholder that is used for gel and post can be purchased from following supplier, such as GE Healthcare, and Uppsala, Sweden, name of product are butyl-SEPHAROSE ^TM, phenyl-SEPHAROSE ^TMCL-4B, octyl group SEPHAROSE ^TMFF and phenyl SEPHAROSE ^TMFF and TosohCorporation, Tokyo, Japan, name of product are TOYOPEARL ^TMButyl 650M (FractogelTSK Butyl-650) or TSK-GEL phenyl 5PW.In one embodiment, the purifying of VEGF and the employed HIC chromatography upholder of recovery are butyl-agaroses, and the second hydrophobic chromatography upholder is a phenyl sepharose.In another embodiment, a HIC chromatography upholder is a phenyl sepharose.

Ligand density is an important parameters, because it not only influences the intensity of protein interaction, also influences the capacity of post.The ligand density of commercially available phenyl or octyl phenyl gel is the order of magnitude of 5-40 μ mol/ml gel bed.Gel capacity be the function of the concrete protein of discussing and pH, temperature and salt concn, but can estimate usually to drop in the scope of 3-20mg/ml gel.

The selection of concrete gel can be determined by those of skill in the art.Generally speaking, the intensity of protein and HIC ligand interaction increases along with the chain length of alkyl part, is suitable for the great majority separation but have about 4 parts to about 8 carbon atoms.Phenyl has the hydrophobicity roughly the same with amyl group, though selectivity can be different, this will belong to the interactional possibility with the π-π of protein aromatic base.

High salt concn helps protein adsorption to the HIC post, but actual concentrations can in very large range change, and this depends on proteinic character and selected concrete HIC part.Salt concn between common use about 1 and the 4M.

From HIC upholder wash-out, no matter be form substep or gradient, can realize in several ways,, b) pass through to change polarity of solvent by changing salt concn such as a), or c) by adding stain remover.By reducing salt concn, the protein of absorption elutes according to the order that hydrophobicity raises.The change of polarity aspect may be subjected to adding the influence of solvent such as ethylene glycol or Virahol, and this is because reduced the intensity of hydrophobic interaction.Stain remover is brought into play the function of proteinic substitute, and has been mainly used in the purifying of membranin.

Multiple anionic group can be attached to matrix to be formed for the positively charged ion upholder of chromatography.Anionic group comprises carboxymethyl, sulfydryl (sulfethyl), sulfopropyl, phosphate radical and sulfonate radical (S).Cellulose ion exchange resin such as SE52, SE53, SE92, CM32, CM52, CM92, P11, DE23, DE32, DE52, EXPRESS ION ^TMS and EXPRESS ION ^TMC can be from Whatman LTD, and Maidstone Kent U.K obtains.Also know based on SEPHADEX ^TMAnd SEPHAROSE ^TMAnd crosslinked ion-exchanger, name of product is CM SEPHADEX ^TMC-25, CM SEPHADEX ^TMC-50 and SP SEPHADEX ^TMC-25, SP SEPHADEX ^TMC-50 and SP-SEPHAROSE ^TMHigh Performance, SP-SEPHAROSE ^TMFast Flow, SP-SEPHAROSE XL, CM-SEPHAROSE ^TMFast Flow, and CM-SEPHAROSE ^TMCL-6B can obtain from GEHealthcare.The example that is used to implement ion-exchanger of the present invention includes but not limited to for example its commodity MACROPREP by name ^TMIon-exchanger, such as for example from BioRad, Hercules, the MACROPREP of CA ^TMS upholder, MACROPREP ^TMHigh S upholder and MACROPREP ^TMThe CM upholder.

Wash-out from positively charged ion chromatography upholder is realized by improving salt concn usually.Because the wash-out from ion column involves interpolation salt, and because as described, HIC is enhanced in salt concn, introduces the HIC step so choose wantonly after ion step or other salt step.In one embodiment of the invention, the cation-exchange chromatography step is before the HIC step.

The example of the method that is used for purifying VEGF has hereinafter been described, for example referring to embodiment 5 and 6.After refolding, remove insoluble material in the set by depth filtration.Then clarifying set is loaded into HEPPS/0.05%TRITON at 5mM ^TMEquilibrated pottery hydroxyapatite column among the X100/pH 8 (BioRad, Hercules, CA) on.Remove non-binding property protein by cleaning, and use 50mM HEPPS/0.05%TRITON with level pad ^TMThe step such as degree such as grade of X100/0.15M sodium phosphate/pH8 is come wash-out VEGF.The VEGF set is loaded into the HEPPS/0.05%TRITON at 50mM ^TMEquilibrated butyl SEPHAROSE among X100/0.15M sodium phosphate/pH 8 ^TMFast Flow post (GE Healthcare, Uppsala, Sweden) on.Clean pillar with level pad, and in the post effluent liquid, collect VEGF.With butyl SEPHAROSE ^TMThe set be loaded into equilibrated Macro PrepHigh S post in 50mM HEPES/pH8 (BioRad, Hercules, CA) on.Cleaning after 280nm effluent liquid absorbancy reaches baseline value, cleaning pillar with the 50mM HEPES/0.25M sodium-chlor/pH8 of two column volumes.Use 50mMHEPES/pH8 0.25-0.75M sodium-chlor gradient elution neutral line, 8 column volumes VEGF.Collect each fraction, and merge the fraction that those comprise the correct VEGF that folds, it is determined by the heparin binding assay.

Adjust (condition) MacroPrep High S set with isopyknic 50mM HEPES/0.8M Trisodium Citrate/pH7.5.Then the set that will adjust be loaded into 50mM HEPES/0.4M Trisodium Citrate/pH7.5 equilibrated phenyl 5PW TSK post (Tosoh Bioscience LLC, Montgomeryville, PA) on.With level pad non-binding property protein purge flow is crossed after the pillar, used 0.4-0M Trisodium Citrate gradient elution VEGF among the 50mM HEPESpH 7.5,10 column volumes.Analyze each fraction by the SDS-polyacrylamide gel electrophoresis, and merge the fraction that those comprise enough purity VEGF.

In host cell, express heparin-binding protein

In brief, can import host cell with respect to the expression vector of host's prokaryotic cell prokaryocyte genome self-replicating and marking protein.The structure of suitable expression vector is well known in the art, comprises the nucleotide sequence of heparin-binding protein described herein.Referring to for example Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (ColdSpring 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.Suitable prokaryotic cell prokaryocyte (comprising bacterium), expression vector can be available from for example American type culture collection (ATCC), Rockville, Maryland.The method that is used for large scale culturing prokaryotic cell prokaryocyte (especially bacterial cell) culture is well known in the art, and these methods can be used for content of the present invention.

For example, with expression vector or the cloning vector transfection of prokaryotic host cell with coding heparin-binding protein interested, and in conventional nutritional medium, cultivate, this substratum is taken the circumstances into consideration to evoked promoter, is selected the gene of transformant or the required sequence of amplification coding to improve.The nucleic acid of coding polypeptide of interest can be RNA, cDNA or the genomic dna in any source, as long as its coding polypeptide of interest.The method that selection is used at the suitable nucleic acid of microorganism host expressing heterologous polypeptide (comprising its variant) is well known in the art.Prepare the nucleic acid encoding molecule by several different methods known in the art.For example, the DNA of coding VEGF is separated and order-checking, for example by utilizing the oligonucleotide probe that can specificity be bonded to the gene of coding VEGF.

With the appropriate insertion replicable vector of heterologous nucleic acids (for example cDNA or genomic dna), be used under the control of suitable promotor, expressing in microorganism.Many carriers can be used for this purpose, and the selection of appropriate carrier will depend primarily on insert carrier nucleic acid size and will be with the concrete host cell of this carrier conversion.Every kind of carrier comprises various members, and it depends on the concrete host cell that it is compatible with it.The particular type that depends on the host, support element generally include but are not limited to next type or many types of: signal sequence, replication orgin, one or more marker gene, promotor and transcription termination sequence.

Usually, use with microorganism host and comprise derived from the replicon of the species compatible and the plasmid vector of control sequence with host cell.Carrier carries a replication site usually, and the flag sequence that Phenotypic Selection can be provided in transformant.For example, generally use the pBR322 transformed into escherichia coli, pBR322 is that plasmid derived from species Escherichia coli is (referring to for example Bolivar et al. (1977) Gene, 2:95).PBR322 comprises the gene of penbritin and tetracyclin resistance, and the easy method of identification of transformed cell so is provided.But pBR322 plasmid or other bacterial plasmid or phage also comprise usually or are modified and comprise and can be used to express the promotor of selected marker gene by the host.

(i) signal sequence

Polypeptide of the present invention is recombinant production directly not only, and can be used as with the fusion polypeptide of heterologous polypeptide and produce, and this heterologous polypeptide generally is signal sequence or has other polypeptide at the specificity cleavage site of the N-of mature protein or polypeptide end.The general allos signal sequence of selecting to be subjected to host cell identification and processing (promptly being subjected to the signal peptidase cutting).For those nonrecognition and process for the prokaryotic host cell of natural polypeptides signal sequence, the leading prokaryotic signal sequence of alkaline phosphatase, penicillinase, lpp or heat-staple enterotoxin 1 I substitutes this signal sequence with for example being selected from.

(ii) replication orgin member

Expression vector comprises the nucleotide sequence that makes that upholder can duplicate in the host cell that one or more are selected.This type of sequence of multiple microorganism is known.The replication orgin of plasmid pBR322 is applicable to most of gram negative bacteriums, such as intestinal bacteria.

(iii) select gene component

Expression vector comprises the selection gene usually, is also referred to as to select mark.The survival of the process transformed host cells that this genes encoding is cultivated in selective medium or the necessary protein of growing.In substratum, can not survive with comprising the carrier transformed host cells of selecting gene.This that select that mark utilized with the present invention and defined genetic marker are different.The typical following protein of genes encoding of selecting: (a) it gives the resistance at microbiotic or other toxin, for example penbritin, Xin Meisu, Rheumatrex or tsiklomitsin, (b) it is supplied and is not the auxotrophy that caused by the existence of genetic marker, or (c) it provides the critical nutrients that can not obtain from complex medium, for example is used for the gene of encoding D-alanine racemase of bacillus.

An example of selection scheme utilizes medicine to prevent the growth of host cell.In this case, giving chemical sproof polypeptide with nucleic acid success cell transformed interested generation also so survives from this selection scheme.The example that such dominance is selected uses medicine Xin Meisu (Southem et al., (1982) J. Molec.Appl.Genet., 1:327), mycophenolic acid (Mulligan et al., (1980) Science, 209:1422) or Totomycin (Sugden et al., (1985) Mol.Cell.Biol., 5:410-413).Above three examples of this that provides adopt bacterial gene under the eucaryon control to transmit respectively resistance at suitable drugs G418 or Xin Meisu (Geneticin), xgpt (mycophenolic acid) or Totomycin.

(iv) promotor member

The expression vector that is used to generate heparin-binding protein interested comprises the suitable promotor that is subjected to host organisms identification and can be operatively connected with the nucleic acid of coding polypeptide of interest.The promotor that is applicable to prokaryotic hosts comprises β-Nei Xiananmei and lactose promoter systems (Chang et al., (1978) Nature, 275:615; Goeddel et al., (1979) Nature, 281:544), pectinose promoter systems (Guzman et al., (1992) J.Bacteriol., 174:7716-7728), alkaline phosphatase, tryptophane (trp) promoter systems (Goeddel, (1980) Nucleic Acids Res., 8:4057 and EP 36,776) and hybrid promoter such as tac promotor (deBoer et al., (1983) Proc.Natl.Acad.Sci.USA, 80:21-25).Yet other known bacterium promotor also is suitable.Their nucleotide sequence is announced, makes the skilled work personnel can use joint or adapter to provide the restriction site of any needs it can be operatively connected to DNA (Siebenlist et al, (1980) of coding polypeptide of interest thus Cell, 20:269).Also can be referring to for example Sambrook et al., The same

The promotor of using in bacterial system also comprises DNA Shine-Dalgarno (S.D.) sequence that can be operatively connected with the coding polypeptide of interest usually.Can from bacterial origin DNA, downcut promotor and be inserted into the carrier that comprises required DNA by restriction enzyme digestion.

(the v) structure of carrier and analysis

Use standard interconnection technique makes up the suitable carrier that comprises one or more above listed members.With isolating plasmid or dna fragmentation cutting, cut out, and connect into the form that required plasmid needs that produces again.

Connect mixture transformed into escherichia coli K12 bacterial strain 294 (ATCC 31,446) or other bacterial strains in order to analyze to confirm the correct sequence in the constructed plasmid, to use, and select successful transformant by suitable penbritin or tetracyclin resistance.From this transformant, prepare plasmid, and analyze by restriction endonuclease digestion, and/or by Sanger et al. (1977) Proc.Natl.Acad.Sci.USA, 74:5463-5467 or Messing et al., (1981) Nucleic Acids Res., the method for 9:309 or by Maxam et al. (1980) Methods in Enzymology, the method order-checking of 65:499.Also can be referring to for example Sambrook et al., The sameAnd Ausubel et al.l., The same

The nucleic acid of coding heparin-binding protein interested is inserted host cell.Usually, this is by transforming this host cell with expression vector mentioned above and realizing in order to induce various promotors to take the circumstances into consideration to cultivate in the conventional nutritional medium of improvement.

(vi) cultivate host cell

The suitable prokaryotic cell prokaryocyte that is used to express heparin-binding protein interested is well known in the art.General use those with the inclusion body form or in periplasmic space or born of the same parents the host cell of great expression recombinant protein in the space.Suitable prokaryotic organism comprise bacterium, eubacterium for example, such as Gram-negative or gram-positive organism, for example intestinal bacteria (E.coli), Bacillus (Bacilli) such as Bacillus subtillis (B.subtilis), Rhodopseudomonas (Pseudomonas) species such as Pseudomonas aeruginosa (P.aeruginosa), Salmonella typhimurium (Salmonella typhimurium) or serratia marcescens (Serratia marcescens).An example of escherichia coli host is intestinal bacteria 294 (ATCC31,446).Other bacterial strain such as intestinal bacteria B, intestinal bacteria X1776 (ATCC 31,537) and intestinal bacteria W3110 (ATCC 27,325) also are suitable.These examples are exemplary rather than restrictive.The W3110 bacterial strain is a kind of typical host, because it is the host strain commonly used that is used for recombinant dna product fermentation.In one aspect of the invention, host cell can be secreted the proteolytic ferment of minimum.For example, can modify bacterial strain W3110 with place's genetic mutation in the gene of realizing coded protein, such host's example comprises intestinal bacteria W3110 bacterial strain 1A2,27A7,27B4 and 27C7, and it is recorded in the U.S. Patent number 5 of bulletin on April 25 nineteen ninety-five, 410,026.For example, a kind of bacterial strain that is used to produce VEGF is the coli strain W3110 with genotype tonA Δ ptr3 phoA Δ E15 Δ (argF-lac) 169 degP41 ilvg, is called 49B3.In another example, the bacterial strain that is used to produce VEGF is to have genotype Δ fhuA (Δ tonA) ptr3, lacI ^q, lacL8, Δ ompT Δ (nmpC-fepE) Δ degP ilvG ⁺Coli strain (62A7).Also can cross over the form of page number 23-24 referring to for example WO2004/092393.

The prokaryotic cell prokaryocyte that is used for producing heparin-binding protein interested is cultivated at substratum known in the art and that be suitable for cultivating selected host cell, comprises Sambrook et al., Molecular Cloning, A Laboratory Manual, the general substratum of describing among the Cold Spring Harbor Laboratory Press (ColdSpring Harbor, New York) (2001).The substratum that is suitable for bacterium includes but not limited to AP5 substratum, nutrient broth, Luria-Bertani (LB) meat soup, NeidhardtShi minimal medium and the C.R.A.P. limit or perfect medium, adds essential nutritional supplement.In certain embodiments, substratum also comprises selective agent, and its structure according to expression vector is selected, and allows to comprise the prokaryotic cell prokaryocyte growth of expression vector with selectivity.For example, penbritin is added into substratum to be used for the cell of culture expression ampicillin resistance gene.Can also be included in any essential fill-in outside carbon, nitrogen and the inorganic phosphate Yanyuan with suitable concentration, or introduce alone or as with the mixture of another fill-in or substratum such as compound nitrogen source.Randomly, substratum can comprise one or more reductive agents that is selected from down group: gsh, halfcystine, cystamine, thioglycolate/ester, dithioerythritol and dithiothreitol (DTT).

U.S. Patent number 5,304,472 and 5,342,763 have provided the example of suitable culture medium.C.R.A.P. phosphoric acid salt restriction substratum comprises 3.57g (NH ₄) ₂(SO ₄), 0.71g two hydration Trisodium Citrates, 1.07g KCl, 5.36g yeast extract (through what identify), 5.36g HycaseSF ^TM-Sheffield adjusts pH to 7.3 with KOH, with an amount of volume to 872 of deionized water adjustment milliliter, and autoclaving; Be cooled to 55 ℃, and additional 110ml 1M MOPS pH 7.3,11ml 50% glucose, 7ml 1M MgSO ₄Can add the concentration of Pyocianil to 50 μ g/ml then to inducing culture.

Cultivate prokaryotic host cell in suitable temperature.For colibacillary cultivation, for example temperature range is for example about 20 ℃ to about 39 ℃, perhaps about 25 ℃ to about 37 ℃, and perhaps about 30 ℃.

If use alkaline phosphatase promoter, then in suitable culture medium, cultivate the Bacillus coli cells that is used for production polypeptide of interest of the present invention, wherein can be as for example Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (Cold Spring Harbor, New York) (2001) is general describe partially or even wholly induce alkaline phosphatase promoter.Cultivation can not lack inorganic phosphate or be in the phosphate starvation level.At first, substratum comprises inorganic phosphate, and its amount is higher than the level that induced protein synthesizes and be enough to make bacterial growth.Along with cell growth and utilize phosphoric acid salt, they have reduced the phosphoric acid salt level in the substratum, cause that thus the polypeptide synthetic induces.

If but promotor is an inducible promoter, induce generation in order to allow so, general culturing cell is up to reaching certain optical density(OD), about 200 A when for example using the high-cell density operation ₅₅₀, induce (for example, grading) in this some startup, to induce the expression of gene of coding polypeptide of interest by a kind of one-tenth that exhausts substratum by adding inductor.

Can also comprise any essential fill-in of the suitable concentration that those skilled in the art will know that, or introduce alone or as with the mixture of another fill-in or substratum such as compound nitrogen source.The pH of substratum can be any pH in about 5 to 9, and this depends primarily on host organisms.For intestinal bacteria, pH is for example about 6.8 to about 7.4, perhaps about 7.0.

The preparaton of heparin-binding protein

Reclaiming the polypeptide of (for example using method described herein) can prepare in the pharmacopedics acceptable carrier, and is used for the known various diagnosis of this quasi-molecule, treatment or other purposes.For example, VEGF described herein can be used for immunoassay, such as enzyme immunoassay.Also imagined the therepic use of the heparin-binding protein of using method acquisition described herein.For example, somatomedin or hormone, for example VEGF can be used for strengthening as required growth.For example, VEGF can be used for promoting wound healing, for example acute wounds (for example burn, wound, normal wound etc.) or chronic wounds (for example diabetic ulcer, pressure ulcer, bedsore (decubitus ulcer), varicose ulcer etc.); Promote hair growth; Promote tissue growth and reparation (for example bone, liver etc.) etc.

Be prepared as follows with preparaton for the heparin-binding protein treatment of storing, mix molecule (for example polypeptide) and optional pharmacopedics acceptable carrier, vehicle or stablizer (Remington ' s with required purity Pharmaceutical Sciences 18th edition, Gennaro, A.Ed. (1995)), with the form of the freeze-dried formulation or the aqueous solution.Acceptable carrier, vehicle or stablizer are nontoxic at dosage that is adopted and concentration to the recipient, comprise buffer reagent, such as phosphoric acid salt, Citrate trianion and other organic acid; Antioxidant comprises xitix and methionine(Met); Sanitas is (such as the two methyl-benzyl ammoniums of chlorination octadecyl; Meton; Benzalkonium chloride, benzethonium chloride; Phenol, butanols or phenylcarbinol; Alkyl paraben such as methyl p-hydroxybenzoate or propyl ester; Pyrocatechol; Resorcinol; Hexalin; The 3-amylalcohol; And meta-cresol); Lower molecular weight (less than about 10 residues) polypeptide; Protein is such as serum albumin, gelatin or immunoglobulin (Ig); Hydrophilic polymer is such as polyvinylpyrrolidone; Amino acid is such as glycine, glutamine, l-asparagine, Histidine, arginine or Methionin; Monose, disaccharides and other carbohydrate comprise glucose, seminose or dextrin; Sequestrant is such as EDTA; Carbohydrate is such as sucrose, N.F,USP MANNITOL, trehalose or sorbyl alcohol; The salify gegenion is such as sodium; Metal composite (for example zinc-protein complex); And/or nonionic surface active agent, such as TWEEN ^TM, PLURONICS ^TMOr polyoxyethylene glycol (PEG).

In certain embodiments, the preparaton that is used for using in the body is aseptic.This is easy to realize by the filtration of aseptic filter membrane.HBP can lyophilized form or is stored as the aqueous solution or gel form.The pH of HBP preparation can be for example about 5 to 8, though higher or lower in some cases pH value may also be suitable.Be appreciated that the salt that uses certain vehicle, carrier or stablizer can form HBP.

When being usually used for wound healing, HBP is mixed with and is used for site-specific sending.When using outwardly, HBP is fit to combine with other composition, such as carrier and/or auxiliary agent.For the character of this type of other composition without limits, just they must be that pharmacopedics is acceptable and be effectively for their predetermined using, and can not significantly reduce the activity of the activeconstituents of preparaton.The example of suitable media comprises ointment, ointment, gelifying agent, sprays or suspension.There is or do not have the collagen of purifying.Each composition also can inject aseptic dressing, percutaneous plaster, plaster and bandage, optional liquid or semi-liquid form.

In order to obtain gel formulation, the HBP for preparing in fluid composition can mix the gel that has the suitable viscosity that is suitable for surface applications with formation with the water-soluble polysaccharide or synthetic polymer such as the polyoxyethylene glycol of significant quantity.Operable polysaccharide comprises that for example derivatived cellulose is such as the etherified cellulose derivative, comprise alkylcellulose, hydroxy alkyl cellulose and alkyl-hydroxyalkylcelluloswith, for example methylcellulose gum, Natvosol, carboxymethyl cellulose, Vltra tears and hydroxypropylcellulose; Starch and classification starch; Agar; Lalgine and alginates/ester; Sudan Gum-arabic; Pullullan; Agarose; Carrageenin; Dextran; Dextrin; Polylevulosan; Inulin; Mannosans; Xylan; Arabinan; Chitosan; Glycogen; Dextran; And synthetic biopolymer; And natural gum, such as xanthocyte gum; Guar gum; Carob gum (locust bean gum); Sudan Gum-arabic; Tragacanth gum; And karaya gum (karayagum); And derivative and mixture.In certain embodiments of the invention, jelling agent herein be for example to biology system inert, nontoxic, be easy to prepare and/or not too soft or viscosity and can not make wherein the unsettled a kind of jelling agent of HBP.

In certain embodiments, polysaccharide is the derivatived cellulose of etherificate, in another embodiment, be listed in that clearly define, purifying and the American Pharmacopeia, for example methylcellulose gum and hydroxyalkyl cellulose derivative are such as hydroxypropylcellulose, Natvosol and Vltra tears.In one embodiment, methylcellulose gum is exactly a polysaccharide.If adopt methylcellulose gum in gel, for example, it generally accounts for about 2-5% or about 3% or about 4% or about 5% of gel, and the amount of HBP is about every ml gel 300-1000mg.

The polyoxyethylene glycol that can be used for gel generally be low and the mixture of high molecular weight polyethylene glycol to obtain suitable viscosity.For example, the mixture of a kind of polyoxyethylene glycol of molecular weight 400-600 and a kind of polyoxyethylene glycol of molecular weight 1500 will be effective mixing with suitable ratio when obtaining mashed prod for this purpose.

Term " water miscible " is meant when being applied to polysaccharide and polyoxyethylene glycol and comprises colloidal solution and dispersion.Usually, the solubility of derivatived cellulose is by the decision of the replacement degree of ether group, and useful herein stable derivatives should have this type of ether group of sufficient amount on each dehydrated glucose unit of cellulose chain, so that the derivative water soluble.The ether of at least 0.35 ether group replacement degree is normally enough in each dehydrated glucose unit.In addition, derivatived cellulose can be the form of an alkali metal salt, for example lithium, sodium, potassium or cesium salt.

Activeconstituents can also be contained in the microcapsule or in the extended release preparation.Referring to for example, Remington ' s Pharmaceutical Sciences 18th edition, Gennaro, A.Ed. (1995).Also can be referring to Johnsonet 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 Productionof Single Immunization Vaccines Using Polylactide Polyglycolide MicrosphereSystems, " in Vaccine Design:The Subunit and Adjuvant Approach, Powell andNewman, eds, (Plenum Press:New York, 1995), pp.439-462; WO 97/03692, WO96/40072, and WO 96/07399; U.S. Patent number 5,654,010; DE 3,218, and 121; Epstein et al., (1985) Proc.Natl.Acad.Sci.USA, 82:3688-3692; Hwang et al., (1980) Proc. Natl.Acad.Sci.USA, 77:4030-4034; EP 52,322; EP 36,676; EP 88,046; EP143,949; EP 142,641; Japanese patent application 83-118008; U.S. Patent number 4,485,045 and 4,544,545; And EP 102,324.

Following examples are as illustration but not provide as restriction.

Embodiment

Embodiment 1: the recombinant human VEGF of expression in escherichia coli

At expression in escherichia coli recombinant human VEGF.In building-up process, protein secreting enters periplasmic space, and is accumulated into refractile body.Implement research to realize proteinic extracting and refolding for this reason.When having disclosed use standard recovery technology and do not added polyanion reagent, these researchs isolated at least 3 kinds of VEGF (Fig. 1).For studies show that of natural VE GF add heparin and improved resistance (Fig. 2) chaotropic agent and the sex change of mercaptan inductive.In addition, in small-scale refolding experiment, heparin has significantly increased the amount of the VEGF of correct refolding.In order to adjust this result to adapt to extensive technology, found the condition of allowing in sulfuric acid dextran (as the molecule of heparin structure analogue) VEGF refolding when existing.For contrast, add the sulfuric acid dextran and make the output of correct VEGF folding, that biologic activity is arranged increase 3-5 doubly.

Method

Be used for VEGF ₁₆₅The plasmid of expressing--plasmid pVEGF171 is designed for and expresses human VEGF in colibacillus periplasm ₁₆₅(referring to for example Leung et al., (1989) Science, 246:1306-1309).Transcribing under the tight control that is positioned at alkaline phosphatase (AP) promotor of VEGF encoding sequence (referring to for example Kikuchi et al., (1981) Nucleic Acids Research, 9:5671-8), provide the needed sequence of translation initiation (referring to for example Yanofsky et al., (1981) by the trpShine-Dalgamo district simultaneously Nucleic Acids Research, 9:6647-68).The VEGF encoding sequence merges downstream at the heat-staple enterotoxin 1 I of bacterium (STII) signal sequence (referring to for example Lee et al., (1983) Infect.Immun., 42:264-8; And Picken et al., (1983) Infect.Immun., 42:269-75) so that subsequent secretion enters colibacillus periplasm.Codon in the STII signal sequence is modified provides the translation skill that is adjusted, and this produces best VEGF accumulating level (referring to for example Simmons and Yansura, (1996) in pericentral siphon Nature Biotechnoloy, 14:629-34).λ _ToTranscription terminator is (referring to for example Scholtissekand Grosse, (1987) Nucleic Acids Research, 15:3185) be positioned at the downstream of VEGF Transcription Termination codon.Replication orgin, and penbritin and tetracycline resistance gene provide by plasmid pBR322.Referring to for example Bolivar et al. (1977) Gene, 2:95-113.

Cell homogenates and refractile body prepare--and the Bacillus coli cells of results is frozen in-70 ℃.Cell uses the centrifugal results of BTUX (whizzer, Alfa laval), and uses BEPEX (extensive refrigerator) freezing.Cell suspension is at the 50mM of 5 volumes HEPES/150mM NaCl/5mM EDTA pH 7.5 (5L/kg throw outs), and at 15M type laboratory homogenizer Gaulin15M (on a small scale) or M3 (on a large scale) (Gaulin Corporation, Everett, MA) middle homogenate.Use isopyknic same damping fluid diluting cells suspension then, and BTPX205 (Alfa Laval Separation AB, Tumba, Sweden) in the continuously feeding whizzer by centrifugal results refractile body.Medium-scale whizzer uses SA1.In addition, can be with cell homogenates, and can directly gather in the crops throw out and freezing and rehydration in BEPEX not.Embodiment 2: at extracting and the refolding-I of the recombinant human VEGF of expression in escherichia coli

Method

Extracting and refolding--throw out be will reflect and the extraction buffer that contains 7M urea/50mM HEPPS/pH8 (final concentration), consumption 5L damping fluid/kg throw out will be suspended in.Press the 3.7g/kg throw out then and add the solid dithiothreitol (DTT), reach the final concentration of 4mM.Referring to for example Fig. 9, it has shown that urea and DTT are to the extractive influence of VEGF.Suspension was thoroughly mixed 1-2 hour in 20 ℃.Can adjust pH to pH8.0 with 50% sodium hydroxide (w/w).Add 19 volume refolding damping fluids by each volume extraction buffer and start refolding.The refolding damping fluid contains 50mM HEPPS/1M-2M urea/2-5mM halfcystine/0.05%-0.2%TRITON ^TMX100/pH 8 (final concentration).Referring to for example Figure 10, it has shown the urea that exists in the refolding process and the influence of DTT concentration.As directed interpolation sulfuric acid dextran, heparin or sodium sulfate.Implemented the refolding incubation 4-24 hour in room temperature.Randomly, can implement incubation in room temperature and reach about 48 hours.Fold and monitor by SDS-PAGE and/or heparin HPLC.Clarify product by using Cuno 90SP filter succeeded by the depth filtration of 0.45 μ m filter.

Heparin is in conjunction with the HPLC assay method--and use the pillar that comprises the immobilization heparin to measure quality and the quantity of the VEGF of correct refolding.(4.6 * 100mm, HE2/M be available from PerSeptive BioResearch Products, Cambridge, MA) balance in containing the 10mM sodium phosphate pH 7 of 0.15M sodium-chlor for POROS HE2/M post.With the flow velocity of 1ml/min or 2ml/min, use 0.15M 10 minutes, in the level pad this post of sodium-chlor linear gradient elution to 2M.In some assay method, wash-out carried out 16 minutes.At 280nm place monitoring eluant.Usually, most of protein wash-out in void volume, and can identify 3 kinds of VEGF.High-affinity, the kind of wash-out is accredited as correct folding VEGF at the latest, is accredited as " peak 3VEGF " afterwards.

The result

Heparin is protected the sex change of VEGF opposing halfcystine mediation--and add 10mM halfcystine to the molecule that natural VE GF causes correctly folding and significantly reduce (Fig. 2).By adding the low 2 kinds of multi-form heparin of concentration, prevented this Denaturation to 20mM.

Table I a

Heparin and sulfuric acid dextran are to the influence of VEGF refolding

Numerical value in the table is the amount (unit is milligram) of the peak 3VEGF of every gram refraction throw out formation.Each concentration of adding is as directed. ^*Average control (5.6+5.0=5.3)

Table I b

Sodium sulfate is to the influence of VEGF refolding

Numerical value in the table is the amount (unit is milligram) of the peak 3VEGF of every gram refraction throw out formation.Each concentration of adding is as directed.

Table II

Heparin and sulfuric acid dextran are to the influence of VEGF refolding

Numerical value in the table is the amount (unit is milligram) of the peak 3VEGF of every gram refraction throw out formation.

Sum up

Heparin and sulfuric acid dextran improve refolding output--owing to, investigated of the influence of several multi-form sulfated polymers to refolding VEGF as mentioned above at the protectiveness characteristic of sex change.Shown in Table I a (and Fig. 5), the lower molecular weight of heparin and high molecular form all make the output of refolding VEGF improve about 3 times.Shown in Table I b (and Fig. 6), sodium sulfate makes the output of refolding VEGF improve about 2 times.The sulfuric acid dextran of 10Kd form also effectively raises refolding output; Yet the higher concentration range of being investigated causes substrate to suppress.Further investigation has proved that the sulfuric acid dextran of 5Kd, 8Kd and 10Kd form has all significantly improved the output (Table II) of refolding VEGF.See Fig. 7.Be also shown in Fig. 8.

Embodiment 3: different damping fluids and TRITON ^TMThe influence that X-100 reclaims VEGF

The result

Damping fluid VEGF (mg/g throw out)

HEPES，pH?8 13.3

HEPES, pH 8, contain TRITON ^TM14.3

HEPPS，pH?8 16.6

TrisHCl，pH?8 12.8

Damping fluid VEGF (mg/g throw out)

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 ^TM?X-100?12.4

HEPPS，pH?8+TRITON ^TM?X-100?13.9

Sum up

Embodiment 1,2 and 3 data combine have been proved by comprising that the condition of heparin sulfate or sulfuric acid dextran and recovery has realized that significantly (2-5 doubly) of output improves when refolding VEGF (a kind of heparin binding growth factor).This method is in the technical scale successful implementation.Expect that this method can be applicable to the proteinic refolding of other alkaline somatomedin and other heparin-binding.

Embodiment 4: at extracting and the refolding-II of the recombinant human VEGF of expression in escherichia coli

Method

Extracting and refolding--will reflect throw out and be suspended in extraction buffer, wherein final concentration is 7M urea, 2-30mM DTT (for example 10mM DTT), 50mM HEPPS/pH7-9 (for example pH 8), consumption 5L damping fluid/kg throw out.Suspension was thoroughly mixed 1-2 hour in room temperature.Add 19 volume refolding damping fluids by each volume extraction buffer and start refolding.The refolding damping fluid contains 1M or 1.3M urea, 2-15mM halfcystine (for example 7.5mM halfcystine), 0.5mM DTT, 0-0.75M arginine (for example 100mM arginine), 15mg/L sulfuric acid dextran, 50mM HEPPS, 0.05%TRITON ^TMX100/pH 8 (final concentration).Referring to for example Figure 12, it has shown when having charge residue the influence of refolding, adds wherein that Histidine produces and identical effect when not having the aminoacid addition thing.Implemented the refolding incubation 12-24 hour in room temperature.Randomly, can implement incubation in room temperature and reach about 48 hours.Randomly, in the refolding technological process, can add air or oxygen (0.3-1cc/min/L).Fold and monitor by SDS-PAGE and/or heparin HPLC.Clarify product by using the Cuno90SP filter succeeded by the depth filtration of 0.45 μ m filter.

The overall extent of dilution of extracting and refolding step is 1: 100.The overall extent of dilution (for example bringing up to 1: 100 to 1: 200) that improves extracting and refolding step has increased the total amount of active VEGF, though concentration reduces.Referring to Figure 13.

By measuring dimer/monomeric amount, can measure the efficient of refolding, wherein can measure monomer by C18 reversed-phase HPLC post, can measure dimer by heparin column chromatography or SP-5PW cation-exchange chromatography assay method and form.

Embodiment 5: extensive refolding

For the scalability (scalability) of checking optimizing refolding condition, implement research with the refolding kinetics of check in little (0.1L), medium (1L) and pilot plant (pilot plant) (250L is to 400L) scale.As shown in Figure 4, large-scale refolding kinetics and being difficult to are on a small scale distinguished, and the final titre of refolding VEGF increases slightly.These digital proofs utilize the scalability of the refolding of sulfuric acid dextran.Further clarify product by using Cuno 90SP filter succeeded by the depth filtration of 0.45 μ m filter.

Embodiment 6: the purifying I of rhVEGF after the refolding

MacroPrep pottery hydroxyapatite--after the refolding, remove insoluble substance in the set by depth filtration.Set after will clarifying then is loaded into the HEPPS/0.05%TRITON at 50mM ^TMEquilibrated pottery hydroxyapatite column among the X100/pH 8 (35D * 31H=30L) (Bio Rad, Hercules, CA) on.Remove debond protein by cleaning, and use 50mM HEPPS/0.05%TRITON with level pad ^TMThe VEGF such as wash-out such as step such as degree such as grade of X100/0.15M sodium phosphate/pH8.Flow velocity is 120cm/hr.Heparin HPLC by each fraction analyze to determine to merge fraction.

Butyl SEPHAROSE ^TMFast Flow chromatography--the VEGF set is loaded at 50mMHEPPS/0.05%TRITON ^TMEquilibrated butyl SEPHAROSE among X100/0.15M sodium phosphate/pH8 ^TMFast Flow post (35D * 26H=25L) (GE Healthcare, Uppsala, Sweden) on.Flow velocity is 100cm/hr.Clean this post with level pad, and in the post eluant, collect VEGF.Collect each fraction, and merge proteinaceous fraction, this measures by A280nm.

Macro Prep High S chromatography--with butyl SEPHAROSE ^TMSet be loaded into equilibrated Macro Prep High S post in 50mMHEPES/pH8 (30D * 39H=27L) (and BioRad, Hercules, CA) on.Clean to eluant after the absorbancy at 280nm place reaches baseline value, clean this post with the 50mM HEPES/0.25M sodium-chlor/pH8 of two column volumes.Use 0.25-0.75M sodium-chlor linear gradient elution VEGF among the 50mM HEPES/pH8,8 column volumes.Flow velocity is 75-200cm/hr.Collect each fraction, and merga pass heparin binding assay for example heparin HPLC be defined as containing the fraction of correct folding VEGF.

Phenyl 5PW TSK chromatography--Macro Prep High S set is regulated with isopyknic 50mMHEPES/0.8M Trisodium Citrate/pH7.5.Then the set that will regulate be loaded into equilibrated phenyl 5PW TSK post in 50mMHEPES/0.4M Trisodium Citrate/pH7.5 (18D * 43H=11L) (and Tosohaas, Montgomeryville, PA) on.Clean debond protein by after this post with level pad, use 0.4-0M Trisodium Citrate gradients among the 50mM HEPES/pH7.5,10 column volumes from this post wash-out VEGF.Analyze each fraction by the SDS-polyacrylamide gel electrophoresis, and comprise the fraction of enough purity VEGF.

Ultrafiltration/saturating filter--with the VEGF that merges at 5kD regenerated cellulose film (G30619); UnitPellicon; Ultrafiltration on the Feed Rate 17.1L/min.Regulate this film with polysorbate20.The VEGF that merges is somebody's turn to do in concentration ultrafiltration at 6g/L (UF1).With sample 7-14DV (Diavolume) diafiltration in 5mM sodium succinate/275mM trehalose/pH5.0.Final preparaton is 5mM sodium succinate/275mM trehalose/0.01% polysorbate20/ph5.0, and concentration is 5mg/ml.

Embodiment 7: the purifying II of rhVEGF after the refolding

Cationic exchange liquid phase chromatography--after the refolding, remove insoluble substance in the set by depth filtration.This refolding set is adjusted to pH5.0-7.5 and about 2-6.5mS/cm.In one embodiment, this set is adjusted to pH6.5 and 5mS millisecond/cm.This refolding set can be loaded into then sulfopropyl extreme load post (sulfopropyl extreme load column, SPXL) on, and use the cumulative gradient elution of salt concn.Can analyze to determine the fraction of merging by the heparin HPLC of each fraction.

Hydrophobic interaction post (HIC)--the set of the SPXL wash-out of VEGF can be adjusted to 50mS/cm in case be loaded into phenyl TSK chromatography column (Tosohaas, Montgomeryville, PA) on.Collect fraction, and merge proteinaceous fraction.

IEX or mixed mode--phenyl TSK set can be loaded on ion exchange chromatography (IEX) post or the mixed mode layer folding post.Collect fraction, and merga pass assay method described herein is defined as comprising the fraction of correct folding VEGF.

Ultrafiltration/diafiltration--with the VEGF that merges at 5kD regenerated cellulose film (G30619); Unit Pellicon; Ultrafiltration on the Feed Rate 17.1L/min.For example, regulate this film with polysorbate20.The VEGF that merges is somebody's turn to do in concentration ultrafiltration at 6g/L (UF1).With sample 7-14DV (Diavolume) diafiltration in 5mM sodium succinate/275mM trehalose/pH5.0.

In methods described herein and the technology, final purity and/or activity can be assessed by the following method: the mapping of peptide mapping, disulphide, SDS-PAGE (reductibility with irreducibility), circular dichroism, LAL (LAL), heparin chromatography, heparin HPLC are (for example, heparin HPLC can be used for measuring VEGF dimer concentration), anti-phase (rp) HPLC (for example, rpHPLC can be used for measuring the VEGF monomer concentration), heparin in conjunction with, receptors bind (for example for VEGF, KDR receptors bind-Bioanalytic R ﹠amp for example; D, and/or Flt1 receptors bind), SEC analysis, raji cell assay Raji, HUVEC potency assay method, the ELISA that utilizes VEGF antibody, mass spectroscopy etc.

Be to be understood that, preservation described herein, embodiment and embodiment are only used for the illustration purpose, in the multiple modification that obtains according to this paper or change was implicitly included in for those skilled in the art, they were also included within the scope of the application's spirit and scope and claims.All publications of being quoted herein, quote, patent and patent application is all complete is embodied in this as a reference, be used for all purposes.

Claims (23)

1. one kind is used for from the method for prokaryotic cell prokaryocyte culture recovery heparin-binding protein, and the method includes the steps of:

(a) from the pericentral siphon of described prokaryotic cell prokaryocyte culture, separate described heparin-binding protein;

(b) the described isolating heparin-binding protein of sex change in comprising first buffered soln of chaotropic agent and reductive agent;

(c) heparin-binding protein of the described sex change of incubation in second buffered soln that comprises chaotropic agent and sulfation polyanion reagent, the time of incubation and condition make heparin-binding protein generation refolding; With

(d) heparin-binding protein of the described refolding of recovery, the heparin-binding protein of wherein comparing the refolding of recovery with the incubation that does not have sulfation polyanion reagent has about 2 to 5 times increase.

2. the process of claim 1 wherein that described heparin-binding protein is a heparin binding growth factor.

3. the method for claim 2, wherein said heparin binding growth factor is vascular endothelial growth factor (VEGF).

4. the method for claim 3, wherein said VEGF is VEGF ₁₆₅

5. the process of claim 1 wherein that described sulfation polyanion reagent is between about 3,000 dalton and 10,000 dalton.

6. the method for claim 3, wherein said second buffered soln comprises the sulfuric acid dextran.

7. the method for claim 3, wherein said second buffered soln comprises sodium sulfate.

8. the method for claim 3, wherein said second buffered soln comprises heparin.

9. the method for claim 6, wherein said sulfuric acid dextran is between about 8,000 and 10,000 dalton.

10. the method for claim 3, wherein said first and second buffered soln comprise HEPPS pH8.0.

11. the process of claim 1 wherein that described second buffered soln further comprises reductive agent.

12. the method for claim 11, the reductive agent of wherein said second buffered soln comprises the combination of halfcystine and DTT.

13. the process of claim 1 wherein that described second buffered soln further comprises non-ionic detergent.

14. the process of claim 1 wherein that described second buffered soln further comprises arginine and/or Methionin.

15. the method for claim 1, wherein said recycling step (d) comprises the heparin-binding protein that makes described refolding and contacts hydroxyapatite upholder, the first hydrophobic interaction chromatography upholder, positively charged ion chromatography upholder and the second hydrophobic interaction chromatography upholder in proper order, and from each upholder the selective elution heparin-binding protein.

16. the method for claim 15, the wherein said first and second hydrophobic interaction chromatography upholders be selected from butyl-, propyl group, octyl group-and aryl-agarose resin.

17. the method for claim 15, the wherein said first hydrophobic interaction chromatography upholder are butyl-agarose upholders and the described second hydrophobic interaction chromatography upholder is phenyl-agarose upholder resin.

18. the method for claim 1, wherein said recycling step (d) comprises the heparin-binding protein that makes described refolding and contacts cationic exchange upholder, hydrophobic interaction chromatography upholder and ion exchange chromatography upholder in proper order, and from each upholder the selective elution heparin-binding protein.

19. a method that is used for reclaiming from the prokaryotic cell prokaryocyte culture heparin-binding protein, the method includes the steps of:

(a) from the pericentral siphon of described prokaryotic cell prokaryocyte culture, separate described heparin-binding protein;

(b) the described isolating heparin-binding protein of sex change in comprising first buffered soln of chaotropic agent and reductive agent;

(c) heparin-binding protein of the described sex change of incubation in second buffered soln that comprises chaotropic agent and sulfation polyanion reagent, the time of incubation and condition make heparin-binding protein generation refolding, and the heparin-binding protein of wherein comparing the refolding of recovery with the incubation that does not have sulfation polyanion reagent has about 2 to 5 times increase; With

(d) make the heparin-binding protein of described refolding contact hydroxyapatite upholder, the first hydrophobic interaction chromatography upholder, positively charged ion chromatography upholder and the second hydrophobic interaction chromatography upholder in proper order, and from each upholder the selective elution heparin-binding protein.

20. one kind is used for the protein-bonded method of purified heparin, the method includes the steps of: make the heparin-binding protein of refolding contact hydroxyapatite upholder, the first hydrophobic interaction chromatography upholder, positively charged ion chromatography upholder and the second hydrophobic interaction chromatography upholder in proper order, and from each upholder the selective elution heparin-binding protein.

21. a method that is used for reclaiming from the prokaryotic cell prokaryocyte culture heparin-binding protein, the method includes the steps of:

(a) from the pericentral siphon of described prokaryotic cell prokaryocyte culture, separate described heparin-binding protein;

(b) the described isolating heparin-binding protein of sex change in comprising first buffered soln of chaotropic agent and reductive agent;

(c) heparin-binding protein of the described sex change of incubation in second buffered soln that comprises chaotropic agent and sulfation polyanion reagent, the time of incubation and condition make heparin-binding protein generation refolding, and the heparin-binding protein of wherein comparing the refolding of recovery with the incubation that does not have sulfation polyanion reagent has about 2 to 3 times increase; With

(d) make the heparin-binding protein of described refolding contact cationic exchange upholder, hydrophobic interaction chromatography upholder and ion exchange chromatography upholder in proper order, and from each upholder the selective elution heparin-binding protein.

22. the method for claim 19 or 21, wherein said polyanion reagent is between about 3,000 dalton and 10,000 dalton.

23. one kind is used for the protein-bonded method of purified heparin, the method includes the steps of: make the heparin-binding protein of refolding contact cationic exchange upholder, hydrophobic interaction chromatography upholder and ion exchange chromatography upholder in proper order, and from each upholder the selective elution heparin-binding protein.

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