WO1989008117A1 - Chromatographie par bi-affinite de l'heparine et du cuivre des facteurs de croissance du fibroblaste - Google Patents

Chromatographie par bi-affinite de l'heparine et du cuivre des facteurs de croissance du fibroblaste Download PDF

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Publication number
WO1989008117A1
WO1989008117A1 PCT/US1988/000587 US8800587W WO8908117A1 WO 1989008117 A1 WO1989008117 A1 WO 1989008117A1 US 8800587 W US8800587 W US 8800587W WO 8908117 A1 WO8908117 A1 WO 8908117A1
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heparin
eluate
column
copper
contacting
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PCT/US1988/000587
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English (en)
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Yuen Shing
Judah Folkman
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Yuen Shing
Judah Folkman
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Priority to PCT/US1988/000587 priority Critical patent/WO1989008117A1/fr
Priority to PCT/US1988/001660 priority patent/WO1989008144A1/fr
Publication of WO1989008117A1 publication Critical patent/WO1989008117A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • C12N9/6462—Plasminogen activators u-Plasminogen activator (3.4.21.73), i.e. urokinase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]
    • C07K14/501—Fibroblast growth factor [FGF] acidic FGF [aFGF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]
    • C07K14/503—Fibroblast growth factor [FGF] basic FGF [bFGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21073—Serine endopeptidases (3.4.21) u-Plasminogen activator (3.4.21.73), i.e. urokinase

Definitions

  • This invention relates to methods for separating, purifying and/or identifying a compound from a sample, where the compound has an affinity for at least two ligands.
  • the method hereinafter referred to as "bi-affinity chromatography", is particularly useful for the separation, identification and purification of species of fibroblast growth factor through use of a heparin-copper bi-affinity chromatography column.
  • Affinity chromatography is a well known process which enables the efficient isolation of biological macromolecules or biopolymers by making use of a feature unique to these substances.
  • biopolymers such as enzymes and antibodies are capable" of recognizing certain chemical structures with a high degree of selectivity and then binding to them.
  • the process of affinity chromatography typically involves a selective adsorbent which is placed in contact with a solution containing several kinds of substances including the desired species, the ligate. The ligate is selectively adsorbed to the ligand, which is attached to an insoluble support or matrix. The nonbinding species are removed by washing. The ligate is then recovered by eluting with a specific desorbing agent.
  • endothelial cell locomotion and proliferation were major components of angiogenesis led to the isolation and purification of angiogenic factors.
  • endothelial cell growth factors also referred to as endothelial cell growth factors
  • FGFs fibroblast growth factor
  • bFGF basic fibroblast growth factor
  • aFGF acidic fibroblast growth factor
  • Fibroblast growth factors which have a broad spectrum of target cells in culture have been reported to induce vascular growth (angiogenesis) in vivo (Esch et al. Proc. Natl. Acad. Sci. U.S.A. 82, 6507-6511 (1985)), Thomas et al. Proc. Natl. Acad Sci. U.S.A. 82, 6409-6413 (1985)).
  • FGFs FGFs to heparin might be copper-dependent (Folkman et al., Science 235, 442-447 (1987)) .
  • ceruloplasmin and heparin become angiogenic when complexed to copper, but not when deprived of copper (Raju et al. Cancer Res. 44, 1579-1584 (1984)); and (iv) heparin can act as a copper chelator (Grushka et al. Analvt. Lett. 15, 1277-1288 (1982)) and Stivala et al. Fed. Proc. 36, 83-88, (1977)).
  • the method comprises the steps of (a) contacting the sample with a bi-affinity column comprising two ligands for which the compound of interest has affinity, (b) contacting the column with an eluate which elutes compounds which do not bind to at least one of the two ligands, and (c) contacting the column with an eluate which elutes compounds which have affinity for both of the ligands.
  • the method of the present invention has been particularly useful in the separation, identification and purification of various forms or species of fibroblast growth factors in a simple, rapid manner based on the interaction of these growth factors with heparin and copper.
  • the method for separating, identifying and purifying the various forms or species of FGFs comprises contacting a sample containing FGFs with a bi-affinity chromatography column comprising (i) heparin-Sepharose and (ii) a copper-saturated chelating-Sepharose, both commercially available from Pharmacia AB, Uppsala, Sweden.
  • the column is thereafter alternately rinsed with (A) an eluate, such as a concentrated salt solution, which will remove compounds which removably bind to heparin, but not to copper; and (B) an eluate which will remove compounds which removably bind to copper, but not to heparin, e.g.
  • the FGF proteins which have affinity to both heparin and copper, are removed from the column by elution with an eluant which will remove such proteins from both the heparin and copper binding sites.
  • the eluate contains a combination of materials, which serve to remove the proteins from both the heparin and the copper, depending on the concentration of the materials in the eluate.
  • the eluate used to remove the FGF's from the column is a linear NaCl/imidazole gradient. Fractions eluted from the column are collected, analyzed and characterized as to their molecular weight, acidic or basic nature and other properties of interest.
  • FGFs may have separate binding sites for copper and for heparin.
  • a sample containing FGFs is contacted with a bi-affinity column containing both heparin and copper on the chromatography media
  • treatment of the column singly with an eluate which would remove the FGF's from a chromatographic media containing heparin, or with an eluate which would remove the FGF's from a chromatographic media containing copper, leaves FGF's on the bi-affinity heparin/copper media.
  • the right eluate is applied, e.g. an increasing concentration gradient of both NaCl and imidazole, not only are FGF's removed from the column, but they are clearly separated in accordance with type (aFGF, bFGF) and size.
  • the present invention permits resolution of the various multiple forms of
  • FGFs based upon bi-affinity chromatography.
  • Figure 1 is a schematic illustration of a heparin/copper bi-affinity chromatography column useful in the separation, purification and identification of fibroblast growth factors.
  • FIG. 2 illustrates application of heparin/copper bi-affinity chromatography to a sample isolated from bovine hypothalmi.
  • Fig. 2A shows the presence and activity of various species of FGFs collected in fractions 6 - 20.
  • the inset to Fig. 2A is a depiction of a silver stained gel showing the protein profile prior to heparin/copper bi-affinity chromatography.
  • Fig. 2B is depiction of the silver stained fractions collected in Fig. 2A, which samples were previously subjected to SDS gel electrophoresis.
  • Fig. 2C and 2D are Western blots of the fractions probed with antibodies raised against basic FGF and acidic FGF respectively. DETAILED DESCRIPTION OF THE INVENTION
  • the method comprises contacting a sample containing the compound of interest with a bi—affinity column comprising two ligands to which the compound releasably binds.
  • the column is then contacted with a first eluate which elutes those compounds which releasably bind to one of the two ligands.
  • the column is thereafter contacted with a second eluate which elutes compounds which releasably bind to the other ligand.
  • this process is continued in an alternating fashion for a sufficient number of times until the undesirable components of the sample have been removed.
  • the compound of interest is then recovered by contacting the column with an eluate which elutes compounds which releasably bind to both ligands.
  • TPA tissue plasminogen activator
  • such compounds can be separated, purified and/or identified by (i) contacting a sample containing the compound of interest with a bi-affinity column comprising both ligands to which the compound has an affinity, (ii) alternately contacting the column with eluates which elute compounds which bind to one ligand but not the other, and (iii) contacting the column with an eluate which elutes compounds which have an affinity for both ligands.
  • the eluates which may be used in practicing the present invention will vary depending on the compound of interest, and more particularly on the ligands used to construct the bi-affinity column.
  • the eluate used should be one which releases the compound of interest from the metal without breaking the bond between the metal and the matrix used to support the metal.
  • Weak chelating agents such as imidazole may be used where the matrix is a metal saturated chelating resin such as Pharmacia's chelating-sepharose.
  • the alternate eluate used will be a strong ionic solution such as NaCl.
  • the eluates chosen will depend on the ligands used to construct the bi-affinity column as well as the relative affinity of those ligands for the compound of interest. Recovery of the compound from the column may be achieved with a gradient of the eluates used in the alternate eluting steps. Fractions are then collected and analysed for the compound of interest.
  • the method of the present invention has been particularly useful in the separation, identification and purification of FGFs.
  • the method preferably comprises the steps of: (i) contacting a sample containing FGFs with a bi-affinity column comprising heparin and copper on a suitable support; and (ii) eluting the species of FGFs with a suitable eluate such as an increasing concentration gradient of materials which are effective to remove the FGF's from the heparin and the copper.
  • a suitable eluate such as an increasing concentration gradient of materials which are effective to remove the FGF's from the heparin and the copper.
  • the column is rinsed singly with eluates for heparin-binding proteins and for copper-binding proteins prior to the elution of the FGF's themselves.
  • the method also involves collecting fractions containing the separated species of FGFs.
  • the heparin and copper components of the bi-affinity column are preferably in the form of a •mixture or blend of heparin-Sepharose (Pharmacia Fine Chemicals, Sweden) and a copper-saturated chelating-Sepharose (Pharmacia Fine Chemicals, Sweden) , although other suitable heparin-resins such as heparin-agarose (Sigma) and chelating-resins such as iminodiacetic acid-agarose (Sigma) may be used in lieu thereof.
  • the chromatographic media is preferably constituted so that the number of available heparin-binding sites is approximately equal to the number of available copper-binding sites.
  • the ratio by weight percent of heparin-Sepharose to the copper-saturated chelating-Sepharose is preferably from about 10:1 to about 1:10, and most preferably about 1:1.
  • the eluates used in connection with this invention will vary depending on the compound of interest.
  • the eluate should not contain materials which react unfavorably with the chromatography medium involved, or with the compounds sought to be recovered.
  • such eluates will normally be aqueous solutions containing physiological salt and suitable buffering materials to maintain the pH at around 7.
  • the preferred buffering composition is Tris, in a concentration of about 10 mM.
  • Preferred eluates for eluting heparin-binding proteins from the column comprise salt solutions, e.g. halides of metals of group 1 or 2 of the periodic table of the elements. NaCl is most preferred, and it can be used in increasing concentration gradients, or in varying concentrations, depending upon the usage and the materials to be eluted.
  • Other suitable eluates include sulfates such as (NH 4 ) 2 S0 4 .
  • Preferred eluates for eluting copper-binding proteins from the column comprise solutions containing materials, generally organic materials, which have a greater affinity for the metal component in the chromatographic media than the proteins to be eluted so that the materials to be eluted are displaced from their association with the metal without interfering with the bond between the metal and the supporting resin.
  • Imidazole is preferred, and can be used in increasing concentration gradients, or in varying concentrations, depending upon the usage and the materials to be eluted.
  • the bi-affinity column is pre-equilibrated with an appropriate solution in which the compound of interest is stable, for example for
  • FGF a 1 to 3M NaCl, 10 mM Tris, pH 7.
  • Pre-equilibriation helps to reduce the undesirable sticking of other components of the sample to the ligands used in the bi-affinity column.
  • Eluates, reactants and other solutions which come in contact with the compound of interest should also contain suitable buffers and other ingredients in order to avoid undesirable changes in the structure or function of the compound.
  • Samples which contain the compound of interest to be separated, identified or purified are preferably partially purified prior to contact with the bi-affinity column of the present invention by, for example, batchwise adsorption to a conventional affinity chromatography column containing a ligand known to bind to that compound. This partial purification helps to prevent overloading of the bi-affinity column with contaminants or other undesirable components of the sample.
  • the partially purified sample is applied directly to the pre-equilibrated bi-affinity column.
  • the column is thereafter consecutively rinsed with, depending on the size of the column, from about 100 to about 200 ml volumes of one or more eluates to remove materials from the column other than those which bind to both ligands.
  • the consecutive rinses comprise alternating rinses in 10 mM Tris, pH 7 of: a 2.0 to 2.5 M NaCl solution and a 10.0 to 20.0 mM solution of imidazole with intervening rinses of a 0.1 to 0.6 M NaCl solution.
  • the column is consecutively rinsed with (i) a 2 M NaCl solution, (ii) a 0.6 M NaCl solution, (iii) a 0.6 M NaCl plus a 10 mM imidazole solution, and (iv) a 0.6 M NaCl solution.
  • Rinse (i) is to remove those components of the sample which bind to heparin, but not to copper.
  • Rinse (ii) is to re-equilibrate the column to allow the protein of interest to bind back to the other ligand, i.e. heparin.
  • Rinse (iii) is to remove those components of the sample which bind to copper, but not to heparin.
  • Rinse (iv) is to again re-equilibrate the column prior to the ultimate elution of the protein of interest.
  • all of rinses (i) through (iv) would tend to remove materials or contaminants which were not bound to heparin, copper or some other component of the chromatography media.
  • the FGFs are eluted with an appropriate eluting gradient such as an NaCl/imidazole gradient.
  • the NaCl/imidazole gradient preferably comprises a 0 to 0.6 M NaCl without imidazole to a 2 to 3 M NaCl plus a 10 to 20 mM imidazole at a pH of about 7.
  • the volume of the gradient depends on the size of the column employed.
  • the NaCl/imidazole gradient comprises about 100 ml of 0.6 M NaCl without imidazole to about 100 ml of 2 M NaCl plus 10 mM imidizole at a pH of about 7.
  • imidazole is increasing in concentration from 0 to 10 mM
  • NaCl is increasing in concentration from 0.6 to 2 M.
  • the source of the sample e.g. brain vs.
  • this gradient can be optimized by changing the imidazole and NaCl concentrations to improve separation of the various species of FGF present in the particular sample.
  • the flow rate of the eluents will vary depending on a number of factors, including the size of the column. In general, for a 1 cm x 9 cm column containing 7.0 ml of the heparin and copper components, the flow rate is between about 10 ml/hr and about 40 ml/hr, preferably between about 15 ml/hr and 30 ml/hr, and most preferably about 20 ml/hr.
  • the volume of fractions collected from the column will also vary depending on a number of factors including the resolving power of the column. That is, the greater the resolving power, the greater the fraction that can be collected. In general, for the above described column, the volume of fractions collected is between about 2 and 10 ml, preferably between about 3 and 7 ml, and most preferably about 5 ml.
  • fractions eluted from the bi-affinity column in accordance with the present invention may be analyzed by a number of methods to confirm resolution of the compound of interest from the original sample.
  • analytical techniques include analysis of mitogenic activity, e.g., by measuring uptake of tritiated thymidine by quiescent confluent monolayers of BALB/c mouse 3t3 cells, as described in Klagsbrun et al., Proc. Natl. Acad. Sci. U.S.A. 82, 805-809 (1985)), the disclosure of which is incorporated herein by reference.
  • Bovine hypothalami 100 g obtained from Pel-Freez (Rogers, Arkansas) were homogenized in 300 ml of 0.15 M (NH 4 ) 2 S0 4 at pH 6 and extracted by stirring at 4°C for 2 hours.
  • the crude extract was centrifuged at 15,000 X g for 1 hour and the supernatant solution was loaded directly onto a heparin-Sepharose column (1.5 cm X 12 cm) pre-equilibrated with 0.6 M NaCl in 10 mM Tris, pH 7.
  • the column was rinsed with 300 ml of 0.6 M NaCl in 10 mM Tris, pH 7.
  • a sample of crude extract containing FGFs were substantially eluted with 40 ml of 2 M NaCl in the same buffer.
  • bovine fibroblast growth factor activity About 7 x 10 5 units of bovine fibroblast growth factor activity were extracted from bovine hypothalmi. More than 95% of this activity bound to heparin-sepharose in 0.6 M NaCl and could be eluted with 2 M NaCl as described below.
  • Copper-saturated chelating-Sepharose component was prepared by adding 100 ml of 2 mg/ml copper (II) chloride to 50 ml of chelating-Sepharose (Pharmacia) , followed by several gentle mixings over a one-hour period and decanting.
  • a heparin-copper bi-affinity column was prepared by thoroughly mixing 3.5 ml each of heparin-Sepharose (Pharmacia) and copper-Sepharose, and placing the mixture in a 7.0 ml column (1 cm x 9 cm) . The mixture was blue in appearance.
  • the sample (40 ml) of FGF which was partially purified by batchwise • adsorption to heparin-Sepharose as described above, was applied directly to this blue color bi-affinity column which had been pre-equilibrated with 2 M NaCl, 10 mM Tris, pH 7.
  • the column was rinsed consecutively with 40 ml each of the following reagents, all in 10 mM Tris, pH 7: (i) 2 M NaCl, (ii) 0.6 M NaCl, (iii) 0.6 M NaCl plus 10 mM imidazole, (iv) 0.6 M NaCl.
  • FGFs were eluted at a flow rate of 20 ml/hr with a linear NaCl/imidazole gradient from 100 ml of 0.6 M NaCl without imidazole to 100 ml of 2 M NaCl plus 10 mM imidazole in 10 mM Tris, pH 7. Fractions (10 ml) were collected and assayed for growth factor activity.
  • growth factor activity was detected starting at about fraction No. 9, which corresponded to eluate containing about 1.3 M NaCl and 5 mM imidazole. About 30% of the initial growth factor activity as recovered. FGF activity was assessed by measuring the incorporation of [ 3 H]thymidine into the DNA of quiescent, confluent monolayers of BALB/c mouse 3T3 cells in 96-well plates as previously described (Klagsbrun et al. Proc. Natl. Acad. Sci. U.S.A. 82, 805-809 (1985)).
  • One unit of activity was defined as the amount of growth factor required to stimulate half-maximal DNA synthesis in 3T3 cells (about 10,000 cells per 0.25 ml of growth medium per well) .
  • protein concentration was estimated by the method of Lowry et al. (Lowry et al. J. Biol. Chem. 193, 265-275 (1951)) .
  • Rabbits were injected at multiple dorsal intradermal sites with 500 ug each of KLH-peptide conjugate emulsified with complete Freund's adjuvant. Animals were boosted regularly at 3-6 weeks intervals with 200 ug of KLH peptide conjugated emulsified in incomplete Fruend's adjuvant. The titer of the antisera after the second booster injection was about 1:15,000 to 1:50,000 as determined in an ELISA using unconjugated peptide as the antigen.
  • nitrocellulose sheet was incubated with antiserum against either bFGF or aFGF and visualized by successive incubations with biotinylated goat anti-rabbit IgG, streptavidin-biotinylated peroxidase complex, and enzyme substrate (4-chloro-l-naphthol) until color develops (Nakane et al. J. Histoche . Cvtochem. 16, 557-560 (1968)).

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Abstract

Méthodes de séparation, d'identification et de purification de diverses formes de facteurs de croissance du fibroblaste, suivant une nouvelle technique de chromatographie par bi-affinité, basée sur l'interaction des facteurs de croissance du fibroblaste avec l'héparine et le cuivre.
PCT/US1988/000587 1988-02-24 1988-02-24 Chromatographie par bi-affinite de l'heparine et du cuivre des facteurs de croissance du fibroblaste WO1989008117A1 (fr)

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Application Number Priority Date Filing Date Title
PCT/US1988/000587 WO1989008117A1 (fr) 1988-02-24 1988-02-24 Chromatographie par bi-affinite de l'heparine et du cuivre des facteurs de croissance du fibroblaste
PCT/US1988/001660 WO1989008144A1 (fr) 1988-02-24 1988-05-17 Chromatogrophie par bi-affinite de composes presentant une affinite pour deux ou plusieurs ligands

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PCT/US1988/000587 WO1989008117A1 (fr) 1988-02-24 1988-02-24 Chromatographie par bi-affinite de l'heparine et du cuivre des facteurs de croissance du fibroblaste

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PCT/US1988/001660 WO1989008144A1 (fr) 1988-02-24 1988-05-17 Chromatogrophie par bi-affinite de composes presentant une affinite pour deux ou plusieurs ligands

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991015504A1 (fr) * 1990-04-04 1991-10-17 Sciosnova Inc. Procede de purification de facteur de croissance de fibroblaste de base
US5192312A (en) * 1991-03-05 1993-03-09 Colorado State University Research Foundation Treated tissue for implantation and methods of treatment and use
US5331095A (en) * 1993-04-12 1994-07-19 Scios Nova Inc. Process for purification of basic fibroblast growth factor
US5613982A (en) * 1994-03-14 1997-03-25 Cryolife, Inc. Method of preparing transplant tissue to reduce immunogenicity upon implantation

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BIOCHEM. BIOPHYS. RES. COMMUN., Vol. 142, No. 2, Published 31 January 1987, by ACADEMIC PRESS, INC. (NEW YORK, NEW YORK, USA), A. BAIRD et al., "Fibroblast Growth Factors are Present in the Extracellular Matrix Produced by Endothelial Cells In Vitro: Implications for a Role of Heparinase Like Enzymes in the Neovascular Response", pages 428-435. *
BIOCHEM. BIOPHYS. RES. COMMUN., Vol. 146, No. 2, Published 31 July 1987, by ACADEMIC PRESS INC. (NEW YORK, NEW YORK, USA), M. IWANE et al., "Expression of cDNA Encoding Human Basic Fibroblast Growth Factor in E. Coli", pages 470-477. *
BIOCHEMISTRY, Vol. 25, No. 21, Published 21 October 1986, by AMERICAN CHEMICAL SOCIETY (WASHINGTON, D.C.), J.M. ROWE et al., "Purification and Characterization of a Human Pituitary Growth Factor", pages 6421-6425. *
PROC. NATL. ACAD. SCI. U.S.A., Vol. 83, Published October 1986, by NATIONAL ACADEMY OF SCIENCES (WASHINGTON, D.C. USA), R. MIRA-Y-LOPEZ et al., "Identification of a Pituitary Factor Responsible for Enchancement of Plasminogen Activator Activity in Breast Tumor Cells", pages 7780-7784. *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991015504A1 (fr) * 1990-04-04 1991-10-17 Sciosnova Inc. Procede de purification de facteur de croissance de fibroblaste de base
US5136025A (en) * 1990-04-04 1992-08-04 California Biotechnology Inc. Method to purify basic fibroblast growth factor
US5192312A (en) * 1991-03-05 1993-03-09 Colorado State University Research Foundation Treated tissue for implantation and methods of treatment and use
US5772695A (en) * 1991-03-05 1998-06-30 Colorado State University Research Foundation Treated tissue for implantation and methods of treatment and use
US5855617A (en) * 1991-03-05 1999-01-05 Colorado State University Research Foundation Treated tissue for implantation and methods of treatment and use
US5863296A (en) * 1991-03-05 1999-01-26 Colorado State University Research Foundation Treated tissue for implantation and methods of treatment and use
US5331095A (en) * 1993-04-12 1994-07-19 Scios Nova Inc. Process for purification of basic fibroblast growth factor
US5613982A (en) * 1994-03-14 1997-03-25 Cryolife, Inc. Method of preparing transplant tissue to reduce immunogenicity upon implantation
US5632778A (en) * 1994-03-14 1997-05-27 Cryolife, Inc. Treated tissue for implantation and methods of preparation
US5843182A (en) * 1994-03-14 1998-12-01 Cryolife, Inc. Treated tissue for implantation and methods of preparation
US5899936A (en) * 1994-03-14 1999-05-04 Cryolife, Inc. Treated tissue for implantation and methods of preparation

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