WO1998025963A1 - Analysis and separation of platelet-derived growth factor proteins - Google Patents
Analysis and separation of platelet-derived growth factor proteins Download PDFInfo
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- WO1998025963A1 WO1998025963A1 PCT/US1997/022815 US9722815W WO9825963A1 WO 1998025963 A1 WO1998025963 A1 WO 1998025963A1 US 9722815 W US9722815 W US 9722815W WO 9825963 A1 WO9825963 A1 WO 9825963A1
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- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1858—Platelet-derived growth factor [PDGF]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0061—Use of materials characterised by their function or physical properties
- A61L26/0066—Medicaments; Biocides
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
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- 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
- C07K14/49—Platelet-derived growth factor [PDGF]
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- 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
- C07K14/575—Hormones
- C07K14/65—Insulin-like growth factors (Somatomedins), e.g. IGF-1, IGF-2
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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- A—HUMAN NECESSITIES
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0014—Skin, i.e. galenical aspects of topical compositions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
- A61L2300/414—Growth factors
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
Definitions
- the present invention relates to the field of protein analysis and separation, particularly to preparations of purified isoforms of heterologous proteins having structural heterogeneity.
- Platelet-derived growth factor (PDGF), the primary mitogen in serum for mesenchymal-derived cells, is stored in platelet alpha-granules. Injury to blood vessels activates the release of PDGF from these granules in the vicinity of the injured vessels. This mitogen acts as a potent chemoattractant for fibroblasts and smooth muscle cells, as well as monocytes and neutrophils. The mitogenic activity of the localized PDGF results in proliferation of these cells at the site of injury, contributing to the process of wound repair. Purified native platelet-derived growth factor (PDGF), a glycoprotein of about
- a and B disulfide-linked polypeptide chains. Two forms of these chains, designated A and B, have been identified.
- the native protein occurs as the homodimer AA or BB or the heterodimer AB, or a mixture thereof.
- a partial amino acid sequence for the PDGF-A chain has been identified (Johnsson et al. (1984) EMBO J. 3:921-928) and cDNAs encoding two forms of PDGF A-chain precursors have been described (U.S. Patent No. 5,219,759).
- the mature A-chain consists of a 104 amino acid polypeptide that is derived by proteolytic processing of a 211 amino acid precursor polypeptide.
- the cDNA encoding the PDGF-B chain has also been described ⁇ Nature (1985) 316:748-750).
- the mature B-chain consists of a 109 amino acid polypeptide that is derived by proteolytic processing of a 241 amino acid precursor polypeptide.
- the mature A and B chains of PDGF show sequence identity of 51%, with the eight cysteine residues being conserved in each of the chains (Johnsson et al. (1984) EMBO J. 3:921-928).
- recombinant PDGF produced in a yeast host undergoes further post-translational processing that results in a secreted mature protein having considerable structural heterogeneity. This is true for the heterodimer rPDGF-AB, and more particularly for the homodimer rPDGF-BB.
- PDGF When expressed in a yeast host for production of a bulk drug substance, PDGF undergoes endoproteolytic cleavage, or so-called clipping, of the B -chain between the Arg-32 and Thr-33 residues, resulting in a bulk drug substance having a mixture of undipped or so-called intact rPDGF and clipped rPDGF that has been cleaved in the B-chain between the Arg-32 and Thr-33 residues.
- rPDGF-BB one or both of the B -chains may be clipped, resulting in single-clipped or double-clipped rPDGF-BB, respectively.
- post-translational modifications of interest to the present invention include exoproteolytic removal of C-terminal amino acids, or so- called truncation, which may remove Arg-32 from clipped B-chains and/or Thr-109 from intact and clipped B-chains.
- These post-translational modifications lead to a number of structural forms, or so-called isoforms, of rPDGF-BB present in the secreted product.
- Methods of the present invention are directed to separation and purification of the intact, single-clipped, and double-clipped isoforms of rPDGF, more particularly rPDGF-BB.
- Proteinase-resistant mutants of recombinant PDGF-BB exhibiting improved biological activity have been identified (Cook et al. (1992) Biochem. J 281:57-65; see also European Patent Application No. 0 547 064 Bl). These mutants prevent endoproteolytic cleavage of the B-chains at Arg-32. Thus elimination of endoproteolytic cleavage in this region apparently leads to increased biological activity of the PDGF-BB produced in a yeast host during the fermentation process. This increase in biological activity is presumably associated with an increase in the relative amount of undipped, or so-called intact, PDGF-BB and a decrease in the relative amount of clipped PDGF-BB in the recombinant PDGF-BB product.
- PDGF platelet derived growth factor
- PDGF PDGF
- rPDGF PDGF
- BB PDGF-BB
- a reverse charge capillary zone electrophoresis method of the invention is useful for quantifying endoproteolytic post- translational modifications.
- compositions of the invention are substantially purified isoforms of secreted PDGF proteins having structural heterogeneity, more particularly purified intact, single-clipped, and double-clipped isoforms of PDGF-BB.
- Pharmaceutical compositions comprising at least one of these substantially purified PDGF isoforms and methods for their use in promoting wound healing are also provided.
- Figure 2 is a schematic representation of single-clipped isoforms of rhPDGF- BB with or without truncation of Arg-32 and Thr-109. Paired lines represent single- clipped rhPDGF-BB. The clip in one of the B chains is represented by a break in the line. Isoforms of single-clipped species are separated into groups B and C based on the difference in the overall charge of the protein. Truncation of Arg-32 at the clip site
- Figure 3 is a schematic representation of double-clipped isoforms of rhPDGF- BB with or without truncation of Arg-32 and Thr-109.
- Paired broken lines represent double-clipped rhPDGF-BB.
- the clip in each of the B chains is represented by a break in each line.
- the double-clipped isoform is separated into groups D, E, and F based on the difference in the overall charge of the protein. Truncation of the Arg-32 at one of the clip sites (group E) makes the isoform more acidic, while truncation of Arg-32 at both of the clip sites (group F) removes two basic amino acid residues. Truncation only of Thr-109 (group D) does not alter the molecular charge, similar to single-clipped species (group B).
- Figure 4 is a high temperature reverse phase (RP) HPLC profile of rhPDGF- BB using a linear gradient elution. Double-clipped and truncated double-clipped isoforms (A), single-clipped and truncated single-clipped isoforms (B), and intact and truncated intact isoforms (C) of rhPDGF-BB are resolved as three groups of several peaks.
- RP reverse phase
- Figure 5 is a high temperature RP HPLC profile of rhPDGF-BB using a step gradient elution. Double-clipped and truncated double-clipped isoforms (A), single- clipped and truncated single-clipped isoforms (B), intact and truncated intact isoforms (C), and a dimer/dimer (D) of rhPDGF-BB are resolved as individual peaks.
- A Double-clipped and truncated double-clipped isoforms
- B single- clipped and truncated single-clipped isoforms
- C intact and truncated intact isoforms
- D dimer/dimer
- Figure 6 is an electropherogram of reverse charge capillary zone electrophoresis (RC CZE) of carboxypeptidase B treated PDGF-BB (bottom) in comparison to non-treated rhPDGF-BB (top). Letters correspond to the groups identified in Figures 1-3. DETAILED DESCRIPTION OF THE INVENTION
- compositions having PDGF activity that are useful as therapeutic agents in the treatment of wounds. More specifically, these . compositions comprise at least one biologically active, substantially purified isoform of a recombinant PDGF (rPDGF) protein.
- rPDGF recombinant PDGF
- recombinant the protein is produced as a heterologous or foreign protein in the cells of a host organism stably transformed with a nucleotide sequence encoding the foreign PDGF protein of interest.
- Methods of the invention provide for the separation of biologically active isoforms of rPDGF from a bulk drug substance comprising a mixture of these isoforms.
- the methods are particularly useful for separating isoforms that result from post-translational endoproteolytic processing of secreted rPDGF, more particularly rPDGF-BB. Once separated, isoforms can be substantially purified and incorporated into a pharmaceutical composition for use in treatment of wounds.
- An isoform of a rPDGF protein is "biologically active" when it has the capability of performing one or more biological functions or a set of activities normally attributed to PDGF in a biological context. These biological activities include inducing chemotaxis and/or mitogenesis of responsive cell types following the binding of PDGF to a specific cell surface receptor. Other biological activities of
- PDGF may include, but are not limited to phospholipase activation, increased phosphotidylinositol turnover and prostaglandin metabolism, stimulation of both collagen and collagenase synthesis by responsive cells, indirect proliferative response of cells lacking PDGF receptors, and potent vasoconstrictor activity.
- Biological activity can be determined using any number of assays available in the art for PDGF, including but not limited to the mitogenic assay described herein.
- substantially purified isoform is intended at least about 80%, preferably at least about 90%, more preferably at least about 95% of the composition comprising rPDGF is the isoform of interest.
- substantially purified isoforms of a protein are obtained using methods of the present invention that provide for separation of the isoform of interest from a cell culture comprising a mixture of rPDGF isoforms.
- separation of the isoform of interest is intended removal of any undesirable isoforms or other components in the cell culture such that the remaining isoform is substantially purified.
- the rPDGF protein whose isoforms are separated using methods of the present invention will be a biologically active dimeric form, more particularly the homodimer rPDGF-AA and PDGF-BB or the heterodimer rPDGF-AB, as well as any substantially homologous and functionally equivalent variants thereof.
- variant is intended a protein derived from the native protein by deletion or addition of one or more amino acids to the N-terminal or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein.
- amino acid sequence variants of the PDGF protein of interest can be prepared by mutations in the DNA sequence encoding the native PDGF protein. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods Enzymol. 154:367-382; U.S. Patent No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein; herein incorporated by reference. Such variants will continue to possess the desired biological activity. Obviously, the mutations that will be made in the DNA encoding the variant PDGF protein must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. See EP Patent Application Publication No. 75,444.
- rPDGF proteins whose isoforms are separated using methods of the present invention include the naturally occurring PDGF proteins as well as variants thereof. These variants will be substantially homologous and functionally equivalent to their native PDGF protein.
- a variant of a native PDGF protein is "substantially homologous" to the native protein when at least about 80%, more preferably at least about 90%, and most preferably at least about 95% of its amino acid sequence is identical to the amino acid sequence of the native PDGF protein. Variants may differ by about 1, 2, 3, or 4 amino acids.
- “functionally equivalent” is intended that the sequence of the variant defines a chain that produces a protein having substantially the same biological activity as the native protein of interest.
- Such functionally equivalent variants that comprise substantial sequence variations are also encompassed by the invention.
- a functionally equivalent variant of the native protein will have a sufficient biological activity to be therapeutically useful, by
- therapeutically useful is intended effective in achieving a therapeutic goal, as, for example, healing a wound.
- Bioactivity can be measured using assays specifically designed for measuring activity of the native protein. Additionally, antibodies raised against the biologically active native protein can be tested for their ability to bind to the functionally equivalent analog, where effective binding is indicative of a protein having a conformation similar to that of the native protein.
- Methods of the present invention provide for improved separation and purification of isoforms of rPDGF proteins, more particularly those isoforms resulting from post-translational endoproteolytic processing of rPDGF.
- the rPDGF whose isoforms are separated may be the homodimer PDGF-BB or the heterodimer PDGF- AB.
- the isoforms of recombinant human PDGF (rhPDGF) protein are separated and purified using methods of the invention.
- the nucleotide sequence encoding the rPDGF protein whose isoforms are separated using methods of the present invention may be a genomic, cDNA, or synthetic DNA sequence.
- the nucleotide sequences encoding the hybrid precursor polypeptides comprising the A- and B-chain polypeptides may be assembled as part of one expression cassette or assembled into separate expression cassettes for cotransformation of a host cell.
- the nucleotide sequence encoding a recombinant PDGF protein of interest can be expressed in a suitable expression system for subsequent separation and purification of isoforms of interest according to methods of the present invention.
- Methods are available in the art for expressing heterologous proteins, more particularly for expressing recombinant PDGF homodimers and heterodimers in bacteria (see, for example, Hoppe et al. (1990) Eur. J. Biochem 187:207-214; Fretto et al. (1993) J Biol. Chem. 268:3625-3631), yeast (Kelly et al. ( ⁇ 9S4) EMBO J. 4:3399- 3405; Ostman et al.
- Methods of the present invention may be practiced with rPDGF produced in any host system where post-translational endoproteolytic processing leads to a secreted rPDGF product comprising a mixture of isoforms.
- yeast is intended ascosporogenous yeasts (Endomycetales), basidiosporogenous yeasts, and yeast belonging to the Fungi Imperfecti (Blastomycetes).
- the ascosporogenous yeasts are divided into two families, Spermophthoraceae and Saccharomycetaceae.
- the later is comprised of four subfamilies, Schizosaccharomycoideae (e.g., genus Schizosaccharomyces), Nadsonioideae, Lipomycoideae, and Saccharomycoideae (e.g., genera Pichia, Kluyveromyces, and Saccharomyces).
- Schizosaccharomycoideae e.g., genus Schizosaccharomyces
- Nadsonioideae e.g., genus Schizosaccharomyces
- Lipomycoideae e.g., Lipomycoideae
- Saccharomycoideae e.g., genera Pichia, Kluyveromyces, and Saccharomyces.
- the basidiosporogenous yeasts include the genera Leucosporidium, Rhodosporidium, Sporidiobolus, Filobasidium, and Filobasidiella
- Yeast belonging to the Fungi Imperfecti are divided into two families, Sporobolomycetaceae (e.g., genera Sporobolomyces, Bullera) and Cryptococcaceae (e.g., genus Candida).
- Sporobolomycetaceae e.g., genera Sporobolomyces, Bullera
- Cryptococcaceae e.g., genus Candida
- species within the genera Pichia, Kluyveromyces, Saccharomyces, Schizosaccharomyces, and Candida Of particular interest are the Saccharomyces species S. cerevisiae, S. carlsbergensis, S. diastaticus, S. douglasii, S. kluyveri, S. norbensis, and S. oviformis.
- Species of particular interest in the genus Kluyveromyces include K.
- yeast Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Skinner et al., eds. (1980) Biology and Activities of Yeast (Soc. App. Bacteriol. Symp. Series No. 9). In addition to the foregoing, those of ordinary skill in the art are presumably familiar with the biology of yeast and the manipulation of yeast genetics. See, for example, Bacila et al., eds. (1978) Biochemistry and Genetics of Yeast; Rose and Harrison, eds. (1987) The Yeasts (2 nd ed.); Strathern et al, eds. (1981) The Molecular Biology of the Yeast Saccharomyces; herein incorporated by reference.
- suitable yeast and other microorganism hosts for expression of heterologous proteins is within the skill of the art.
- suitable hosts may include those shown to have, inter alia, good secretion capacity, low proteolytic activity, and overall robustness.
- Yeast and other microorganisms are generally available from a variety of sources, including the Yeast
- Methods for expression of rPDGF in a yeast host are available in the art.
- One such method comprises transforming a yeast host cell with a plasmid expression vector comprising a nucleotide sequence encoding human PDGF-BB. This method is briefly described in Example 1 herein and is described in detail in the copending patent application filed concurrently herewith entitled “Improved Expression of Heterologous Proteins in Yeast,” Serial No. , herein incorporated by reference.
- Methods of the invention are directed to separation of these structural isoforms such that a final pharmaceutical composition comprising substantially purified isoforms of the greatest biological activity can be prepared.
- These methods are useful for separating isoforms of rPDGF-AB, and more particularly isoforms of rPDGF-BB.
- Recombinant PDGF-BB produced in a yeast host cell is secreted as a fully folded, biologically active homodimeric protein consisting of two highly twisted antiparallel pairs of B chains (Oefner et ⁇ /. (1992) EMBO J. 11 "3921-3926). Each B chain comprises 109 amino acid residues.
- truncation at Arg-32 and Thr-109, glycosylation of Ser and/or Thr residues, and oxidation of methionine.
- These post-translational modifications lead to a number of structural forms, or so-called isoforms, of rPDGF-BB present in the secreted product.
- the structural heterogeneity of the rPDGF-BB yeast product is further complicated by the presence of a rigid Pro-Pro bond at residues 41 and 42. The rotation of this rigid bond is further hindered by its proximity to a disulfide bond and the bulky side chain of Trp-40. This rotational hindrance leads to the formation of stable cis-trans isomers of rPDGF-BB at room temperature.
- Post-translational endoproteolytic processing or clipping between Arg-32 and Thr-33 leads to three different and distinct basic isoforms of rPDGF-BB: intact, single-clipped, and double-clipped.
- intact is intended both B chains remain intact and are not endoproteolytically cleaved.
- single-clipped is intended one B chain is intact and one B chain is endoproteolytically cleaved between Arg-32 and Thr-33.
- double-clipped is intended both B chains are endoproteolytically cleaved between Arg-32 and Thr-33.
- Each respective isoform may be additionally modified by C-terminal truncation of Arg-32 or Thr-109, producing a known number of derived structural species for any given basic isoform.
- Methods of the invention are directed to separation of the basic isoforms designated as intact, single-clipped, and double-clipped isomers of rPDGF-BB, each of which comprises a known number of derived structural species resulting from truncation of the Arg-32 or Thr-109 residues. It has been determined that the intact isoform has 100% mitogenic activity, the single-clipped isoform has 120% mitogenic activity, and the double-clipped isoform has 20% mitogenic activity.
- the reference sample exhibits 50% activity. Accordingly, utilizing the methods of the invention compositions can be obtained having having at least about 20% greater, preferably at least about 50% greater, more preferably at least about 100% greater PDGF activity than the unpurified cell culture.
- compositions having use as a therapeutic agent for treatment of wounds
- Methods of the invention enable preparation of such pharmaceutical compositions by providing for separation of the intact and single-clipped isoforms from the double-clipped isoform of rPDGF-BB of the bulk drug substance produced by fermentation.
- Methods of the invention utilize ion exchange enrichment, reverse phase high pressure liquid chromatography, and reverse charge capillary zone electrophoresis.
- the first of these methods is directed at separation of intact, single-clipped, and double-clipped isomers of rPDGF, particularly rPDGF-BB, where separation is to a degree of purification appropriate for therapeutic use of the rPDGF.
- this method is appropriate for separation of these isomers for use in production of a pharmaceutical composition comprising substantially purified intact, single-clipped, or intact and single-clipped rPDGF, particularly rPDGF-BB.
- the second and third methods while providing a means for separation of the intact, single-clipped, and double-clipped isomers, are primarily useful for analytical means, as described below.
- the intact and single-clipped isoforms of rPDGF-BB can be separated by ion-exchange enrichment from the double-clipped isoform to a degree of purification appropriate for therapeutic use of rPDGF-BB.
- a bulk drug comprising biologically active rPDGF-BB can be enriched in substantially purified intact and single-clipped rPDGF-BB isoforms by separating these isoforms from the double-clipped isoform using cation exchange chromatography and a TSK sulfopropyl column (TSK SP).
- TSK SP TSK sulfopropyl column
- the double-clipped isoform elutes in the earlier fractions, followed by the single-clipped and intact isoforms. Removal of the eluted fraction comprising the double-clipped isoform allows for subsequent collection of fractions eluting the single-clipped and intact isoforms. These fractions can be used separately, or together, to produce a composition comprising the isolated intact isoform, single- clipped isoform, or a mixture of the intact and single-clipped isoforms of rPDGF- BB.
- Reverse phase high pressure liquid chromatography is a method useful for separating certain isoforms of a dimeric protein preparation having multiple isoforms, where at least one isoform has two different faces.
- face is intended a term used in the context of RP HPLC, where each polypeptide chain of a dimeric protein is a “face” of the molecule.
- PDGF-BB polypeptide chain of a dimeric protein
- the two chains are identical and have no modifications that make one face different than the other, the molecule has but one face.
- rPDGF-BB expressed in the yeast Saccharomyces cerevisae exhibits multiple conformers that produce broad and heterogenous chromatographic profiles when analyzed with RP HPLC at various ambient and subambient temperatures, as disclosed in Example 3 below. Analysis and separation of rPDGF-BB by RP HPLC at these temperatures is complicated by Pro-Pro isomerizations within the molecule. Additionally, absorptive chromatography cannot simultaneously resolve clipping and truncation to opposing faces of the homodimeric protein under these conditions. When conducted at high temperature in accordance with the present invention,
- HPLC analysis is carried out at about 85 C, the heterogeneity resulting from the post- translational modifications due to clipping and truncation occurring on both faces of the dimeric molecule can be resolved.
- a combination isocratic/step gradient can also be employed in addition to the high temperature to improve separation of the isoforms, thus greatly simplifying the chromatographic profile.
- Using the high temperature RP HPLC method with the isocratic/step gradient provides for separation of the isoforms of rPDGF-BB predominantly on the basis of clipping, facilitating analysis and separation of intact, single-clipped, and double-clipped isoforms of rPDGF-BB.
- Methods for conducting an isocratic/step gradient are available in the art.
- the purified rPDGF-BB isoforms obtained in accordance with this method of the invention are useful for conducting biological activity assays to determine relative biological activity of the purified isoforms.
- a mitogenic assay to determine the biological activity of a bulk drug substance comprising at least one isoform of rPDGF-BB can be conducted as described in Example 5 disclosed herein.
- RC CZE reverse charge capillary zone electrophoresis
- truncation of the basic residue Arg-32 has a greater effect on RC CZE assay selectivity than truncation of the neutral amino acid residue Thr-109. Since truncation of the Thr-109 residue does not appreciably alter the charge or size of the protein, there is no separation between the three possible categories of isoforms within the intact isoform. The baseline separation observed between the intact isoform and the single-clipped isoform may be due to an increase in hydrodynamic size of the single-clipped isoform, which may occur through partial denaturation of the secondary structure.
- the separation of the two species is due to the decrease in charge of the species of isoform resulting from C-terminal truncation of Arg-32.
- three isoforms can be distinguished; the separation of them may be due to the charge difference resulting from either a single or a double C-terminal truncation of Arg-32.
- the RC CZE method described thus far is unable to distinguish between, and thus separate, single-clipped isoforms that have the Arg-32 truncation and double- clipped isoforms that do not have the Arg-32 truncation.
- the RC CZE method of the present invention further comprises treating the bulk drug substance comprising the various rPDGF-BB isoforms with carboxypeptidase B prior to analysis. This enzyme is specific for the basic amino acids arginine and lysine located at the carboxy terminus.
- Enzyme treatment converts single-clipped isoforms not having an Arg-32 truncation to single-clipped isoforms having the Arg-32 truncation, and converts double-clipped isoforms having no, or only one, Arg-32 truncation to isoforms having two Arg-32 truncations.
- the treatment of PDGF-BB bulk drug substance with carboxypeptidase B simplifies the RC CZE electropherogram and allows quantification of the three isoforms of rPDGF- BB as defined by the clipping between Arg-32 and Thr-33 (the isoforms referred to as intact, single-clipped, and double-clipped).
- the RC CZE method of analysis of PDGF-BB as modified in the present invention to isolate intact, single-clipped, and double-clipped by use of carboxypeptidase B, is useful, in a quality control context, for analysis of a bulk drug substance comprising the rPDGF-BB isoforms described herein.
- the advantage of use of RC CZE for analyzing PDGF-BB in a quality control context is the ease and precision achieved by this method, which makes the method an appropriate method for such routine activities of analysis as required in quality control of bulk drug substances.
- compositions comprising substantially purified isoforms of interest.
- compositions can be prepared for use as a therapeutic agent as described below.
- therapeutic agent an agent that accomplishes a therapeutic goal, as for example, healing a wound.
- compositions comprising the substantially purified intact, single-clipped, or a mixture of intact and single-clipped isoforms of rPDGF-BB are useful as therapeutic agents in the treatment of wounds, particularly those wounds that are normally repaired in response to the release of the native PDGF.
- PDGF vascular endothelial growth factor
- This growth factor has been shown to promote healing of incision wounds in rats and healing in regions of the body such as chambers or subcutaneous spaces.
- PDGF has application in healing such wounds as ulcers, burns, and skin grafts.
- PDGF can be used to heal wounds resulting from damaged connective tissue, the repair of which requires fibroblast proliferation and collagen deposition.
- wound healing is retarded by any number of factors including, but not limited to advanced age, diabetes, cancer, and treatment with anti-inflammatory drugs or anticoagulants, PDGF can be used to promote normal wound healing in mammals in situations that would otherwise delay wound healing.
- platelet-derived growth factor as a therapeutic agent for full and partial thickness burn repair has also been described by Danilenko et al. (1995) Am. J. Pathol. 147:1261-1277. In this manner, recombinant PDGF-BB induces marked extracellular matrix and granulation tissue production such that the burn defects are repaired.
- refractory diabetic wounds are the result of deficiencies in growth factors and/or cytokines that are important for the healing process.
- Some preliminary studies have shown that application of certain growth factors or cytokines can facilitate wound healing, as described in Doxey et al. (1995) Life Sciences 57(11):111-23.
- PDGF of wounded diabetic rats was produced at low levels compared to control animals. These rats had a delayed production of PDGF in cells surrounding the wounds that occurred many days after the wound was incurred, possibly explaining the delay in wound healing in the diabetic animals.
- PDGF-BB Mechanisms of action of PDGF-BB and the other PDGF dimers, including
- compositions comprising PDGF can facilitate the healing of injured or depleted bones by promoting growth of the connective tissue, growth of the bone and cementum, and by stimulation of protein and collagen synthesis in the region of the injured bone, especially as in the case, for example, of periodontal disease (see WO 88/03409).
- Compositions comprising PDGF can provide fast, effective healing for such wounds as, for example, bedsores, lacerations, and burns, as described in WO 91/15231 and US 5,019,559, wherein PDGF in a composition with insulin-like growth factor (IGF) promotes an increase in new connective tissue and an increase in the growth of epithelial tissue.
- IGF insulin-like growth factor
- Compositions comprising PDGF can be administered in a gel formulation, and such a formulation is suitable for topical administration to promote wound healing in conditions such as ulcers, superficial wounds and lacerations, abrasions, surgical wounds, burns, and bone defects, as described in WO 93/08825.
- compositions comprising the intact, single-clipped, or a mixture of intact and single-clipped isoforms of PDGF-BB described herein can be administered topically with an appropriate pharmaceutically acceptable carrier or in a substantially purified preparation of protein such that it is free from impurities and elements that would interfere with a therapeutic use.
- Such preparations that are free of toxic, antigenic, inflammatory, or other deleterious substances and that are at least about 80% pure are considered appropriate.
- the concentration of the protein can be from about 1 to about 25 ⁇ g/ml of total volume, or can be in a range as broad as about 1 ng/ml to about 50 ug/ml depending on the therapeutic use for the rPDGF-BB.
- a therapeutically effective amount sufficient to accelerate the rate of appearance and increase the number of new fibroblasts in the wound space and to stimulate DNA synthesis in and collagen deposition by those fibroblasts will be in the range of about 1 to about 5 milliliters of the preparation, depending on the characteristics of the wound.
- a pharmaceutical composition comprising substantially purified intact or single-clipped isoforms of rPDGF-BB or a mixture of such isoforms and an appropriate pharmaceutically acceptable carrier can be formulated for administration as a therapeutic agent for the treatment of wounds.
- Pharmaceutically acceptable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polygly colic acids, polymeric amino acids, amino acid copolymers, and inactive viruses in particles.
- Such carriers are well known to those of ordinary skill in the art.
- Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
- Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol, and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
- the therapeutic pharmaceutical compositions are prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
- Liposomes are included within the definition of a pharmaceutically acceptable carrier. Some liposome compositions are described in U.S. Patent No. 5,422,120; WO 95/13796; WO 94/23697; WO 91/14445; and EP 524,968 Bl.
- Administration of the PDGF-BB therapeutic pharmaceutical composition can be by any means and to any location of the body of the animal being treated as is appropriate for the wound being treated.
- the PDGF-BB can be administered, for example, parenterally or topically.
- Parenteral administration can include subcutaneous, intravenous, intra-arterial, intramuscular, or direct injection into a region or organ of the body.
- Recombinant human PDGF-BB (rhPDGF) was produced by a strain of the yeast Saccharomyces cerevisiae genetically modified with a multicopy yeast expression plasmid that includes a gene encoding the mature human PDGF-B polypeptide chain. Details of this yeast expression system are disclosed in copending application filed concurrently herewith entitled "Improved Expression of
- the S. cerevisiae strain MB2-1 was developed to contain the genotype: Mat a, ura3L ⁇ , leu2-3, leu2-112, his3-ll, his3-15,pep4 ⁇ , [cir °J. It is auxotrophic for uracil, leucine and histidine, requiring these nutritional supplements when grown in minimal medium.
- MB2-1 does not contain an endogenous 2- ⁇ plasmid, which tends to interfere with the stability of the introduced plasmids and encourages recombination between endogenous and introduced plasmids.
- the strain does not express functional protease A, the product of the PEP 4 gene, which interferes with the production of heterologous proteins.
- MB2-1 was designed to impart these favorable characteristics, which include selection for high expression of heterologous proteins.
- the vector selected for expressing rhPDGF-BB, pAB24 is a yeast-bacteria shuttle vector.
- the plasmid is a chimera of sequences from pBR322, derived from several naturally occurring bacterial plasmids, and sequences of the endogenous S. cerevisiae 2- ⁇ plasmid (Broach (1981) in Molecular Biology of the Yeast Saccharomyces (Cold Spring Harbor Press, New York), 1 :445-470). Characteristics of this vector are described in detail in the copending patent application filed concurrently herewith entitled "Improved Expression of Heterologous Proteins in
- plasmid pYL7PPB includes an expression cassette with the following features. Transcription initiation and termination are mediated by the inducible hybrid yeast promoter ADH/GAP and the Mat transcriptional terminator. The cassette further includes an open reading frame encoding a truncated yeast alpha- factor leader to mediate secretion of native human proPDGF-B. The prosequence included in the expression construct is only the native N-terminal propeptide encoding sequence; the native C-terminal propeptide sequence was not included in the construct.
- N-terminal propeptide resulted in enhanced expression of rhPDGF-BB, as disclosed in copending application Serial No. .
- Dibasic processing sites at the truncated alpha factor leader/N-terminal propeptide and N-terminal propeptide/PDGF-B junctions were included to facilitate production of correctly processed rhPDGF-B polypeptide by yeast.
- the nucleotide and predicted amino acid sequence of open reading frame from p YL7PPB encoding truncated alpha factor leader - proPDGF-B are disclosed in copending application Serial No.
- Example 2 Enrichment from rhPDGF-BB Bulk Drug Substance of Intact and Single Clipped rhPDGF-BB Isoforms Using TSK Sulfopropyl Cation Exchange Chromatography
- Example 1 Making of the rhPDGF-BB bulk drug substance is described in Example 1.
- the overall goal of this procedure was to generate milligram quantities of intact, single-clipped, and double-clipped isoforms of rhPDGF-BB using repetitive TSK SP 5PW (Tosoh-Haas) chromatography of pure rhPDGF-BB produced in a yeast host cell, then to determine the relative biological activity of the three isoforms using the mitogen assay.
- TSK SP 5PW Tosoh-Haas
- the column was prepared by packing 50 ml of TSK SP 5PW (supplied pre- swollen) into an empty Pharmacia column K26 (2.5 x 10 cm). The packed column was washed with not less than 5 column volumes (c.v.) of loading buffer (0.05M Na Acetate, pH 4.5). rhPDGF-BB produced by the method of Example 1, was applied, and the column washed with not less than 2 c.v. loading buffer, and then 2 c.v. 0.05M Na Phosphate pH 7 (with NaOH). The product was eluted with a 10 c.v. linear gradient (from 0 to 0.6 M) NaCl (buffer B) in 50 mM sodium phosphate pH 7.0. The elution peak was fractionated.
- loading buffer 0.05M Na Acetate, pH 4.5
- rhPDGF-BB produced by the method of Example 1
- the product was eluted with a 10 c.v. linear gradient (from 0 to 0.6 M) Na
- starting material was a final pool of rhPDGF-BB as described in Example 1.
- the material was in Na Phosphate at pH 6 at 9.9 mg/ml; the pH was adjusted to 4.5 with 1M NaH 3 PO 4 .
- the concentration of rh-PDGF-BB was 9 mg/ml after pH adjustment, and conductivity of the load was 1.62 mS/cm at 25°C.
- the bulk drug substance rhPDGF-BB produced in yeast as described in Example 1 was analyzed on a 5 micron, 300 Angstrom, Delta-Pak C3 (3.9 rnmID x 15 cm L) column using a Spectra Physics 8800 solvent delivery pump equipped with a WISP 712 autoinjecter and a PE 135 diode array detector. Column temperature was controlled by the use of a Nesslab glycol cooling bath of a Jones Chromatography heating block. The separation was achieved at a 1.2 ml/min flow rate using a 5 to 60% acetonitrile/0.1% TFA linear gradient over 40 minutes.
- RP HPLC analysis of nonreduced rhPDGF-BB was then performed using a Zorbax 300SB-C18 reversed-phase column (4.6 mm ID x 25 cm L) maintained at 85°C using a linear TFA/acetonitrile gradient.
- This method resolved the 21 predicted clipped and truncated isoforms of rhPDGF-BB, which are clustered into three major groups (intact, single-clipped, and double-clipped). Fractions collected across each group were analyzed using RP HPLC of reduced rhPDGF-BB. The results indicated that the multiple peaks in each group are due to different levels of C-terminal truncation of Arg-32 and Thr-109.
- Example 4 Reverse Charge Capillary Zone Electrophoresis Assay for rhPDGF-BB including Pretreatment with Carboxypeptidase B
- a sample of rhPDGF-BB recombinantly produced in yeast as described in Example 1 and a reference material having a known composition of PDGF-BB were assayed using reverse charge capillary zone electrophoresis (RC CZE).
- the samples of interest were diluted in running buffer having Tris buffer at pH 8.0 to a concentration of 1.0 mg/ml as determined by spectrophotometry. 100 ul of 25 U/ml carboxypeptidase B was added to 50 ul of test sample. After 30 minutes of incubation at room temperature, 25 ul of 0. IN HC1 was added.
- a sequence table was prepared on a Turbochrom Sequence Screen.
- Degas reagents necessary for carrying out the assay were prepared by placing 1500 ul of each of the following into a separate 1700 ul polypropylene microfuge tube: IN NaOH, eCAPTM Regenerator solution, and 50 mM Tris buffer (pH 8.0). The tubes were microfuged for 5 minutes at the highest rpm setting available.
- the assay was run on a capillary electrophoresis system (BioFocusTM 3000) configured with the following information: prep 1 : preinject NaOH, 180 sec; prep 2: preinject AMREGEN, 180 sec; prep 3: preinject AMRINSE 180 sec; pressure: 2 psi*sec; polarity: neg to pos; voltage: 15.00 kV; current limit: 50.00 uA; inlet buffer: AMRUN; outlet buffer: AMRUN; cartridge temp: 25 degrees Celsius; run time: 10 min; single wavelength programmable detector mode; and lamda(nm): 214.
- prep 1 preinject NaOH, 180 sec
- prep 2 preinject AMREGEN, 180 sec
- prep 3 preinject AMRINSE 180 sec
- pressure 2 psi*sec
- polarity neg to pos
- voltage 15.00 kV
- current limit 50.00 uA
- inlet buffer AMRUN
- outlet buffer AMRUN
- cartridge temp 25 degrees Celsius
- PDGF ability of PDGF to stimulate DNA synthesis and cell growth in culture can be tested by 3 H-Thymidine incorporation into DNA of cultured cells responsive to PDGF, and results in the demonstration of biological activity of PDGF or PDGF-like molecules.
- 3 H-Thymidine incorporation into DNA of cultured cells responsive to PDGF results in the demonstration of biological activity of PDGF or PDGF-like molecules.
- Such an assay is described in U.S. Patent No. 4,849,407, and in Cook et al. (1992) Biochem. J 281 :57-65, herein incorporated by reference. This is a standard assay for determining potential therapeutic benefit of PDGF.
- the mitogenic assay for PDGF is believed in the art to represent potential usefulness as a pharmaceutical composition. It has been determined previously that the C-terminal truncations do not have any effect on the biological activity. It has also determined that over the narrow range of peak elution, the acetonitrile and triflouroacetic acid (ACN-TFA) components of the chromatography had a consistent effect on the mitogenic assay. Purified single-clipped, double-clipped and intact forms of rhPDGF-BB were collected through repeated cycles of high temperature nonreduced RP HPLC.
- the single-clipped, double-clipped and intact forms were tested using the high temperature nonreduced and reduced RP HPLC assays, and the purities were found to be 95%o, 97%), and 95%, respectively. Samples of each form were then tested by the mitogenic activity assay. All samples were assayed in triplicate in the same assay.
- the fractions of each isoform were collected, and it was determined that the single- clipped isoform had 120% activity, the intact isoform had 100% activity, and the double clipped had 20% activity.
- the reference sample an unpurified cell culture comprising a mixture of intact, single-clipped, and double-clipped isoforms of rhPDGF-BB) exhibited 50% activity. This indicated that it may be desirable to remove the double-clipped isoform in a rhPDGF-BB bulk substance that is intended for therapeutic use.
- Example 6 PDGF-BB to Treat a Wound
- a PDGF-BB mixture of intact and single-clipped isoforms is prepared in a topical cream.
- Application of about 50 to about 100 mM of PDGF-BB mixture is made to a third degree burn wound from 1 to 3 times daily for a month, or until the wound is healed.
- Standard application procedures for dressing burn wounds are practiced for the administration of the PDGF-BB cream to the burn wound.
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ES97948629T ES2306462T3 (en) | 1996-12-13 | 1997-12-12 | ANALYSIS AND SEPARATION OF PROTEINS OF THE GROWTH FACTOR DERIVED FROM PLATES. |
JP52698598A JP4275741B2 (en) | 1996-12-13 | 1997-12-12 | Analysis and separation of platelet-derived growth factor protein |
EP97948629A EP0948538B1 (en) | 1996-12-13 | 1997-12-12 | Analysis and separation of platelet-derived growth factor proteins |
DK97948629T DK0948538T3 (en) | 1996-12-13 | 1997-12-12 | Analysis and separation of platelet-derived growth factor proteins |
AU54654/98A AU5465498A (en) | 1996-12-13 | 1997-12-12 | Analysis and separation of platelet-derived growth factor proteins |
DE69738768T DE69738768D1 (en) | 1996-12-13 | 1997-12-12 | ANALYSIS AND SEPARATION OF PDGF PROTEINS |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106496302A (en) * | 2015-09-08 | 2017-03-15 | 三生国健药业(上海)股份有限公司 | One kind uses cation exchange chromatography method of protein |
EP3206670A4 (en) * | 2014-10-14 | 2018-06-20 | Samuel Lynch | Compositions for treating wounds |
Families Citing this family (60)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ATE95236T1 (en) | 1983-04-25 | 1993-10-15 | Chiron Corp | HYBRID DNA SYNTHESIS OF MATURE INSULIN-LIKE GROWTH FACTORS. |
US5811381A (en) * | 1996-10-10 | 1998-09-22 | Mark A. Emalfarb | Cellulase compositions and methods of use |
US7883872B2 (en) * | 1996-10-10 | 2011-02-08 | Dyadic International (Usa), Inc. | Construction of highly efficient cellulase compositions for enzymatic hydrolysis of cellulose |
AU5465498A (en) * | 1996-12-13 | 1998-07-03 | Michael Kunitani | Analysis and separation of platelet-derived growth factor proteins |
ES2237159T3 (en) * | 1998-10-06 | 2005-07-16 | Mark Aaron Emalfarb | TRANSFORMATION SYSTEM IN THE FIELD OF FILAMENT MICOTIC FUNGI IN CHRYSOSPORIUM. |
US20040146918A1 (en) * | 2000-02-18 | 2004-07-29 | Weiner Michael L. | Hybrid nucleic acid assembly |
US7166712B2 (en) * | 2000-07-12 | 2007-01-23 | Philadelphia, Health And Education Corporation | Mammalian MDM2 binding proteins and uses thereof |
US20080014594A1 (en) * | 2003-01-31 | 2008-01-17 | Kevin Hestir | Lung-Expressed Polypeptides |
US7960148B2 (en) | 2003-07-02 | 2011-06-14 | Verenium Corporation | Glucanases, nucleic acids encoding them and methods for making and using them |
DE10342794A1 (en) * | 2003-09-16 | 2005-04-21 | Basf Ag | Secretion of proteins from yeasts |
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US8778880B2 (en) * | 2004-02-02 | 2014-07-15 | Ambrx, Inc. | Human growth hormone modified at position 35 |
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WO2006091231A2 (en) * | 2004-07-21 | 2006-08-31 | Ambrx, Inc. | Biosynthetic polypeptides utilizing non-naturally encoded amino acids |
US7473678B2 (en) | 2004-10-14 | 2009-01-06 | Biomimetic Therapeutics, Inc. | Platelet-derived growth factor compositions and methods of use thereof |
KR20070090023A (en) | 2004-12-22 | 2007-09-04 | 암브룩스, 인코포레이티드 | Modified human growth hormone |
AU2005322019B2 (en) | 2004-12-22 | 2010-08-26 | Ambrx, Inc. | Formulations of human growth hormone comprising a non-naturally encoded amino acid |
SG161210A1 (en) * | 2004-12-22 | 2010-05-27 | Ambrx Inc | Methods for expression and purification of recombinant human growth hormone |
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EP1893229B1 (en) * | 2005-06-03 | 2011-10-19 | Ambrx, Inc. | Improved human interferon molecules and their uses |
US20070048793A1 (en) * | 2005-07-12 | 2007-03-01 | Baynes Brian M | Compositions and methods for biocatalytic engineering |
US7632823B2 (en) | 2005-08-18 | 2009-12-15 | Ambrx, Inc. | Compositions of tRNA and uses thereof |
FR2891149B1 (en) | 2005-09-26 | 2007-11-30 | Biodex Sarl | PHARMACEUTICAL COMPOSITION WITH A HEALING ACTION COMPRISING A SOLUBLE DEXTRANE DERIVATIVE AND A PLATELET DERIVED GROWTH FACTOR. |
RS51998B (en) * | 2005-11-08 | 2012-04-30 | Ambrx Inc. | Accelerants for the modification of non-natural amino acids and non-natural amino acid polypeptides |
WO2007059312A2 (en) | 2005-11-16 | 2007-05-24 | Ambrx, Inc. | Methods and compositions comprising non-natural amino acids |
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EP1968635B1 (en) * | 2005-12-14 | 2014-09-17 | Ambrx, Inc. | Compositions containing, methods involving, and uses of non-natural amino acids and polypeptides |
EP1991282B1 (en) * | 2006-02-09 | 2013-06-19 | BioMimetic Therapeutics, LLC | Compositions and methods for treating bone |
JP5484047B2 (en) | 2006-06-30 | 2014-05-07 | バイオミメティック セラピューティクス, エルエルシー | PDGF-biomatrix composition and method for treating rotator cuff injury |
US9161967B2 (en) | 2006-06-30 | 2015-10-20 | Biomimetic Therapeutics, Llc | Compositions and methods for treating the vertebral column |
AU2007292893B2 (en) * | 2006-09-08 | 2012-03-01 | Ambrx, Inc. | Suppressor tRNA transcription in vertebrate cells |
KR20090051227A (en) | 2006-09-08 | 2009-05-21 | 암브룩스, 인코포레이티드 | Hybrid suppressor trna for vertebrate cells |
JP5840345B2 (en) * | 2006-09-08 | 2016-01-06 | アンブルックス, インコーポレイテッドAmbrx, Inc. | Modified human plasma polypeptides or modified human Fc scaffold proteins and uses thereof |
EP2462895B1 (en) | 2006-11-03 | 2016-11-02 | BioMimetic Therapeutics, LLC | Compositions and methods for arthrodetic procedures |
US8680252B2 (en) | 2006-12-10 | 2014-03-25 | Dyadic International (Usa), Inc. | Expression and high-throughput screening of complex expressed DNA libraries in filamentous fungi |
US9862956B2 (en) | 2006-12-10 | 2018-01-09 | Danisco Us Inc. | Expression and high-throughput screening of complex expressed DNA libraries in filamentous fungi |
US8012931B2 (en) | 2007-03-30 | 2011-09-06 | Ambrx, Inc. | Modified FGF-21 polypeptides and their uses |
CA2685596A1 (en) | 2007-05-02 | 2008-11-13 | Ambrx, Inc. | Modified interferon beta polypeptides and their uses |
US8551751B2 (en) * | 2007-09-07 | 2013-10-08 | Dyadic International, Inc. | BX11 enzymes having xylosidase activity |
JP5547083B2 (en) | 2007-11-20 | 2014-07-09 | アンブルックス,インコーポレイテッド | Modified insulin polypeptides and their use |
CA2715254A1 (en) | 2008-02-07 | 2009-08-13 | Biomimetic Therapeutics, Inc. | Compositions and methods for distraction osteogenesis |
NZ620606A (en) | 2008-02-08 | 2015-08-28 | Ambrx Inc | Modified leptin polypeptides and their uses |
PE20110426A1 (en) | 2008-07-23 | 2011-07-01 | Ambrx Inc | MODIFIED BOVINE G-CSF POLYPEPTIDES |
WO2010030714A2 (en) | 2008-09-09 | 2010-03-18 | Biomimetic Therapeutics, Inc. | Platelet-derived growth factor compositions and methods for the treatment of tendon and ligament injuries |
KR101647164B1 (en) | 2008-09-26 | 2016-08-09 | 암브룩스, 인코포레이티드 | Modified animal erythropoietin polypeptides and their uses |
MX357314B (en) | 2008-09-26 | 2018-07-04 | Ambrx Inc | Non-natural amino acid replication-dependent microorganisms and vaccines. |
US8183346B2 (en) * | 2008-12-05 | 2012-05-22 | Eli Lilly And Company | Anti-ferroportin 1 monoclonal antibodies and uses thereof |
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SG181769A1 (en) | 2009-12-21 | 2012-07-30 | Ambrx Inc | Modified porcine somatotropin polypeptides and their uses |
NZ601559A (en) | 2010-02-22 | 2014-10-31 | Biomimetic Therapeutics Llc | Platelet-derived growth factor compositions and methods for the treatment of tendinopathies |
EP2605789B1 (en) | 2010-08-17 | 2019-06-05 | Ambrx, Inc. | Modified relaxin polypeptides and their uses |
US9567386B2 (en) | 2010-08-17 | 2017-02-14 | Ambrx, Inc. | Therapeutic uses of modified relaxin polypeptides |
AR083006A1 (en) | 2010-09-23 | 2013-01-23 | Lilly Co Eli | FORMULATIONS FOR THE STIMULATING FACTOR OF COLONIES OF GRANULOCITS (G-CSF) BOVINE AND VARIANTS OF THE SAME |
CN105085652B (en) * | 2014-05-16 | 2021-06-22 | 中国人民解放军军事医学科学院生物工程研究所 | Platelet-derived growth factor B mutant, preparation method and application thereof |
CN114805532A (en) | 2014-10-24 | 2022-07-29 | 百时美施贵宝公司 | Modified FGF-21 polypeptides and uses thereof |
CN108239648B (en) * | 2016-12-26 | 2023-04-07 | 丰益(上海)生物技术研发中心有限公司 | Method for efficiently expressing rhizomucor miehei lipase |
AU2018219283B2 (en) | 2017-02-08 | 2022-05-19 | Bristol-Myers Squibb Company | Modified relaxin polypeptides comprising a pharmacokinetic enhancer and uses thereof |
CN110590930B (en) * | 2018-06-13 | 2023-03-31 | 四川好医生攀西药业有限责任公司 | Application of periplaneta americana growth factor PDGF in preparation of injury repair drugs |
EP3884027A1 (en) * | 2018-11-19 | 2021-09-29 | Basf Se | Leader sequence for yeast |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0259632A1 (en) * | 1986-08-13 | 1988-03-16 | Zymogenetics, Inc. | Expression of biologically active PDGF analogs in eucaryotic cells |
WO1990008163A1 (en) * | 1989-01-12 | 1990-07-26 | Hoppe Juergen | Pdgf-a, pdgf-aa, pdgf-ab, process for producing them and drugs containing them |
WO1992001716A1 (en) * | 1990-07-23 | 1992-02-06 | Zymogenetics, Inc. | Protease resistant pdgf and methods of use |
Family Cites Families (46)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US2359180A (en) * | 1942-08-11 | 1944-09-26 | Gen Motors Corp | Dynamic balancer |
NZ201918A (en) | 1981-09-18 | 1987-04-30 | Genentech Inc | N-terminal methionyl analogues of bovine growth hormone |
US5010003A (en) * | 1983-04-25 | 1991-04-23 | Genentech, Inc. | Use of yeast homologous signals to secrete heterologous proteins |
EP0487116B1 (en) * | 1984-10-12 | 1999-12-29 | ZymoGenetics, Inc. | Biologically active PDGF analogs in eucaryotic cells |
US4801542A (en) | 1984-10-12 | 1989-01-31 | Zymogenetics, Inc. | Expression of biologically active PDGF analogs in eucaryotic cells |
US4889919A (en) * | 1986-08-13 | 1989-12-26 | Zymogenetics, Inc. | Biologically active PDGF derived A-chain homodimers |
US4769328A (en) | 1984-10-12 | 1988-09-06 | Zymogenetics Inc. | Expression of biologically active PDGF analogs in yeast |
US4766073A (en) | 1985-02-25 | 1988-08-23 | Zymogenetics Inc. | Expression of biologically active PDGF analogs in eucaryotic cells |
US5187263A (en) | 1984-10-12 | 1993-02-16 | Zymogenetics, Inc. | Expression of biologically active PDGE analogs in eucaryotic cells |
US5045633A (en) | 1985-02-25 | 1991-09-03 | Zymogenetics, Inc. | Expression of biologically active PDGF analogs in eucaryotic cells |
US5516896A (en) * | 1985-02-25 | 1996-05-14 | Zymogenetics, Inc. | Biologically active B-chain homodimers |
JPS6236183A (en) * | 1985-06-20 | 1987-02-17 | ザ・サルク・インステイチユ−ト・バイオテクノロジ−/インダストリアル・アソシエイツ・インコ−ポレ−テツド | Ugenoyobibunpi |
KR960005708B1 (en) | 1986-11-14 | 1996-05-01 | 인스티튜트 오브 몰레큘러 바이올로지, 인코오포레이티드 | Composition for wound healing and for bone regenerating |
US5019559A (en) | 1986-11-14 | 1991-05-28 | President And Fellows Of Harvard College | Wound healing using PDGF and IGF-II |
US4873192A (en) | 1987-02-17 | 1989-10-10 | The United States Of America As Represented By The Department Of Health And Human Services | Process for site specific mutagenesis without phenotypic selection |
US5219759A (en) | 1987-04-22 | 1993-06-15 | Chiron Corporation | Recombinant DNA encoding PDGF A-chain polypeptide and expression vectors |
CA1340772C (en) * | 1987-12-30 | 1999-09-28 | Patricia Tekamp-Olson | Expression and secretion of heterologous protiens in yeast employing truncated alpha-factor leader sequences |
US5094941A (en) * | 1987-12-31 | 1992-03-10 | Zymogenetics, Inc. | Monoclonal antibodies to PDGF |
US5422120A (en) | 1988-05-30 | 1995-06-06 | Depotech Corporation | Heterovesicular liposomes |
DK105489D0 (en) * | 1989-03-03 | 1989-03-03 | Novo Nordisk As | POLYPEPTIDE |
US5968778A (en) * | 1989-01-12 | 1999-10-19 | Jurgen Hoppe | PDGF-AB, preparation process and pharmaceuticals containing them |
NZ237464A (en) | 1990-03-21 | 1995-02-24 | Depotech Corp | Liposomes with at least two separate chambers encapsulating two separate biologically active substances |
ATE156363T1 (en) | 1990-04-10 | 1997-08-15 | Inst Molecular Biology Inc | WOUND HEALING |
US5962219A (en) * | 1990-06-11 | 1999-10-05 | Nexstar Pharmaceuticals, Inc. | Systematic evolution of ligands by exponential enrichment: chemi-selex |
CA2093664C (en) * | 1990-10-12 | 2003-07-29 | Fritz Eckstein | Modified ribozymes |
DK300090D0 (en) * | 1990-12-19 | 1990-12-19 | Novo Nordisk As | PROCEDURE FOR PREPARING LEADER SEQUENCES |
DE4216134A1 (en) * | 1991-06-20 | 1992-12-24 | Europ Lab Molekularbiolog | SYNTHETIC CATALYTIC OLIGONUCLEOTIDE STRUCTURES |
WO1993008825A1 (en) | 1991-11-04 | 1993-05-13 | Zymogenetics, Inc. | Pdgf gel formulation |
JPH08505872A (en) * | 1993-01-22 | 1996-06-25 | ユニバーシティ・リサーチ・コーポレイション | Localization of therapeutic agents |
EP0695169B1 (en) | 1993-04-22 | 2002-11-20 | SkyePharma Inc. | Multivesicular cyclodextrin liposomes encapsulating pharmacologic compounds and methods for their use |
DK82893D0 (en) * | 1993-07-08 | 1993-07-08 | Novo Nordisk As | PEPTIDE |
US5624803A (en) * | 1993-10-14 | 1997-04-29 | The Regents Of The University Of California | In vivo oligonucleotide generator, and methods of testing the binding affinity of triplex forming oligonucleotides derived therefrom |
ES2149955T3 (en) | 1993-11-16 | 2000-11-16 | Skyepharma Inc | VESICLES WITH CONTROLLED RELEASE OF ACTIVE SUBSTANCES. |
US5902880A (en) * | 1994-08-19 | 1999-05-11 | Ribozyme Pharmaceuticals, Inc. | RNA polymerase III-based expression of therapeutic RNAs |
US5627053A (en) * | 1994-03-29 | 1997-05-06 | Ribozyme Pharmaceuticals, Inc. | 2'deoxy-2'-alkylnucleotide containing nucleic acid |
CA2187626C (en) * | 1994-04-13 | 2009-11-03 | Michael G. Kaplitt | Aav-mediated delivery of dna to cells of the nervous system |
US5639642A (en) * | 1994-06-16 | 1997-06-17 | Novo Nordisk A/S | Synthetic leader peptide sequences |
ZA954983B (en) * | 1994-06-17 | 1996-02-14 | Novo Nordisk As | N-terminally extended proteins expressed in yeast |
US6146886A (en) * | 1994-08-19 | 2000-11-14 | Ribozyme Pharmaceuticals, Inc. | RNA polymerase III-based expression of therapeutic RNAs |
US5716824A (en) * | 1995-04-20 | 1998-02-10 | Ribozyme Pharmaceuticals, Inc. | 2'-O-alkylthioalkyl and 2-C-alkylthioalkyl-containing enzymatic nucleic acids (ribozymes) |
ZA9610456B (en) * | 1995-12-20 | 1997-06-20 | Novo Nordisk As | N-terminally extended proteins expressed in yeast |
US5998203A (en) * | 1996-04-16 | 1999-12-07 | Ribozyme Pharmaceuticals, Inc. | Enzymatic nucleic acids containing 5'-and/or 3'-cap structures |
AU5465498A (en) * | 1996-12-13 | 1998-07-03 | Michael Kunitani | Analysis and separation of platelet-derived growth factor proteins |
US6248878B1 (en) * | 1996-12-24 | 2001-06-19 | Ribozyme Pharmaceuticals, Inc. | Nucleoside analogs |
US6001311A (en) * | 1997-02-05 | 1999-12-14 | Protogene Laboratories, Inc. | Apparatus for diverse chemical synthesis using two-dimensional array |
US6395713B1 (en) * | 1997-07-23 | 2002-05-28 | Ribozyme Pharmaceuticals, Inc. | Compositions for the delivery of negatively charged molecules |
-
1997
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- 1997-12-12 EP EP03012465A patent/EP1365028B1/en not_active Expired - Lifetime
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- 1997-12-12 WO PCT/US1997/022815 patent/WO1998025963A1/en active Application Filing
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-
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- 1999-07-01 US US09/340,250 patent/US6083723A/en not_active Expired - Fee Related
-
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- 2000-03-17 US US09/528,108 patent/US6312923B1/en not_active Expired - Fee Related
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-
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- 2001-08-02 US US09/921,398 patent/US6706496B2/en not_active Expired - Fee Related
-
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- 2002-07-26 US US10/205,693 patent/US7084262B2/en not_active Expired - Fee Related
- 2002-10-25 US US10/280,826 patent/US6897043B2/en not_active Expired - Fee Related
-
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- 2005-01-28 US US11/046,099 patent/US7166446B2/en not_active Expired - Fee Related
-
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- 2006-07-07 US US11/483,033 patent/US20060252695A1/en not_active Abandoned
-
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- 2007-08-09 JP JP2007208669A patent/JP2008031173A/en not_active Withdrawn
- 2007-12-27 JP JP2007338286A patent/JP2008119008A/en not_active Withdrawn
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0259632A1 (en) * | 1986-08-13 | 1988-03-16 | Zymogenetics, Inc. | Expression of biologically active PDGF analogs in eucaryotic cells |
WO1990008163A1 (en) * | 1989-01-12 | 1990-07-26 | Hoppe Juergen | Pdgf-a, pdgf-aa, pdgf-ab, process for producing them and drugs containing them |
WO1992001716A1 (en) * | 1990-07-23 | 1992-02-06 | Zymogenetics, Inc. | Protease resistant pdgf and methods of use |
Non-Patent Citations (1)
Title |
---|
COOK, A.L. ET AL.: "Purification and analysis of proteinase-resistant mutants ...", BIOCHEM. J., vol. 281, 1992, pages 57 - 65, XP000400719 * |
Cited By (3)
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EP3206670A4 (en) * | 2014-10-14 | 2018-06-20 | Samuel Lynch | Compositions for treating wounds |
CN106496302A (en) * | 2015-09-08 | 2017-03-15 | 三生国健药业(上海)股份有限公司 | One kind uses cation exchange chromatography method of protein |
CN106496302B (en) * | 2015-09-08 | 2021-12-10 | 三生国健药业(上海)股份有限公司 | Method for purifying protein by ion exchange chromatography |
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