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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)
  • C12N9/6435—Plasmin (3.4.21.7), i.e. fibrinolysin
• • 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)
  • C12Y304/21007—Plasmin (3.4.21.7), i.e. fibrinolysin

Definitions

• the present invention relates to a novel method of recombinantly producing, recovering and purifying Angiostatin® protein (EntreMed Inc., Rockville, MD).
• Angiostatin® is a protein which is a potent inhibitor of developing blood vessels and tumor growth. Angiostatin® is believed to play an important role in the inhibition of the development of blood vessels to new tumor metastases.
• Angiostatin® protein proteins, such as Angiostatin® protein, in high yield from biological material, such as tissue extracts, cell extracts, broth from incubation systems, and culture medium is often frought with problems in view of the numerous proteins and other undesirable molecules present in an homogenate or extract.
• biological material such as tissue extracts, cell extracts, broth from incubation systems, and culture medium.
• Angiostatin® protein proteins that will provide the large amounts of Angiostatin® protein required for clinical use, including, but not limited to, cancer therapy.
• Such methods should produce Angiostatin® protein in an efficient and convenient manner in a culture broth which is amenable to procedures designed to recover and purify Angiostatin® protein in high yields.
• Separating a specific protein of interest from potential contaminants presents a challenge in view of numerous factors, such as contamination of cellular homogenates with proteolytic enzymes that may digest the protein.
• Other undesirable cellular constituents that may be present in homogenates include but not limited to, pigments, cytochromes, lipids, free radicals, oxidases and other lysosomal enzymes, and oxides. Some of these substances may affect the protein of interest by stripping electrons, affecting disulfide bonds and changing the conformation of the protein.
• Centrifugation of cells including yeast, bacteria, insect and other cells used for recombinant production of proteins, such as Angiostatin® protein, could possibly result in damage to the cells with concomitant release of undesirable biological material. What is needed is a method for recovery and purification of protein, such as Angiostatin® protein, which does not employ centrifugation. Methods for recombinant production, recovery and purification of Angiostatin® protein on a large scale are required to produce and isolate the amounts of purified Angiostatin® protein needed for administration to patients and also for research purposes.
• Angiostatin® protein Also needed is a method for purifying recombinantly-produced Angiostatin® protein which avoids the need for centrifugation of the culture broth, thereby avoiding problems associated with cell lysis. This method should be capable of use on a large scale to recover and purify Angiostatin® protein in quantities needed for clinical administration and research.
• the present invention solves these problems inherent in the recovery and purification of proteins, particularly Angiostatin® protein, by providing new and useful methods for recombinant production, recovery and purification of proteins, especially Angiostatin® protein.
• the present invention provides new and useful methods for recombinantly producing Angiostatin® protein in large amounts.
• the present invention provides a method for recovery and purification of Angiostatin® protein.
• the present invention also provides new and useful solutions for storage of Angiostatin® protein. These methods provide the benefit of preserving the biological activity of Angiostatin® protein.
• Preservation of the biological activity of Angiostatin® protein is crucial for administration of Angiostatin® protein to humans and animals for the purpose of inhibition of undesirable angiogenesis, for other biological activities, and for research investigations or other types of biological testing.
• Angiostatin® protein is effective in treating diseases or processes that are mediated by, or involve, angiogenesis.
• the angiogenesis mediated diseases include, but are not limited to, solid tumors; blood born tumors such as solid tumors, blood borne tumors, leukemias; tumor metastases; benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; rheumatoid arthritis; psoriasis; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, colon cancer, retrolental fibroplasia, rubeosis; Osier- Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; and wound granulation.
• the present invention provides new and improved methods for recombinant production of biologically active
• Angiostatin® protein in high yield.
• the method of the present invention is useful for recovery and purification of recombinantly-produced Angiostatin® protein.
• the method of the present invention is useful for recovery and purification of Angiostatin® protein from extracts of biological fluids, cells and tissues.
• An advantage of the present invention is that higher amounts of biologically active Angiostatin® protein are recombinantly produced. Another advantage of the present invention is that greater amounts of
• Angiostatin® protein are recovered than obtained with prior art methods. Yet another advantage of the present invention is that higher yields of more purified, and biologically active Angiostatin® protein are obtained. Still another advantage of the present invention is that Angiostatin® protein may be stored in buffers for extended periods of time, and also subjected to lyophilization, while preserving biological activity. An advantage of the present invention is that it permits Angiostatin® protein to be stored in vials or other containers, either in a solution which may be liquid or frozen, or lyophilized, and optionally shipped to a recipient.
• an object of the present invention is to provide a an improved method for recombinant production of large amounts of biologically active Angiostatin® protein.
• Another object of the present invention is to provide a method for recovery and purification of recombinantly produced proteins.
• Yet another object of the present invention is to provide a method for recovery and purification of Angiostatin® protein.
• Another object of the present invention is to provide a method for recovery and purification of Angiostatin® protein, particularly recombinantly produced Angiostatin® protein.
• An advantage of the purification methods of the present invention is that undesirable proteins, lipids and pigments are efficiently separated from the desired protein, especially Angiostatin® protein. It is another object of the present invention to provide solutions which provide favorable solubility conditions for Angiostatin® protein, particularly recombinantly-produced Angiostatin® protein while retaining biological activity of Angiostatin® protein.
• Another advantage of the methods of the present invention is that centrifugation of the broth from fermentation steps in recombinant production of Angiostatin® protein is avoided, thereby preventing unwanted cellular lysis and potential contamination of Angiostatin® protein with additional proteins, pigments, enzymes and other cellular chemicals and debris.
• FIG 1 is a process overview diagram for the large scale processing of purified Angiostatin® protein.
• Figure 2 is a process flow diagram for fermentation inoculum preparation for Angiostatin® production.
• Figure 3 is a process flow diagram for fermentation and streamline SP chromatography for Angiostatin® production.
• Figure 4 is a process flow diagram for chromatography steps following streamline SP chromatography for Angiostatin® production.
• Figure 5 is a process flow diagram for chromatography, ultrafiltration, diafiltration and formulated bulk processing steps following
• Angiostatin® is a protein which is a potent inhibitor of developing blood vessels and tumor growth. Angiostatin® is believed to play an important role in the inhibition of the development of blood vessels to new tumor metastases.
• the following pages of this patent application describe new procedures and protocols for the large scale production of human recombinant Angiostatin® from Pichia pastoris fermentation of clones with nucleic acid sequences encoding for Angiostatin® protein or variants thereof. This application also provides new procedures for the large scale production, purification, characterization and storage of human recombinant Angiostatin®.
• the methods of the present invention are not limited to human recombinant Angiostatin®, and that the present methods apply to Angiostatin® from other species, as well as fragments and conservatively substituted forms thereof
• the method can serve as a large scale purification protocol for obtaining a Angiostatin® formulations which may be used in clinical human trials.
• amino terminus refers to the free alpha-amino group on the amino acid at the amino terminal of the peptide, or to the alpha-amino group (imino group when participating in a peptide bond) of an amino acid at any other location within the peptide.
• carboxy terminus refers to the free carboxyl group on the amino acid at the carboxy terminus of a peptide, or to the carboxyl group of an amino acid at any other location within the peptide.
• amino acids making up a peptide are numbered in order, starting at the amino terminal and increasing in the direction toward the carboxy terminal of the peptide. Thus, when one amino acid is said to "follow” another, that ammo acid is positioned closer to the carboxy terminal of the peptide than the preceding amino acid.
• amino acid is used herein to refer to an amino acid (D or L) or an amino acid mimetic that is incorporated into a peptide by an amide bond.
• the amino acid may be a naturally occurring amino acid or, unless otherwise limited, may encompass known analogs of natural amino acids that function in a manner similar to the naturally occurring amino acids (i.e., amino acid mimetics).
• an amide bond mimetic includes peptide backbone modifications well known to those skilled in the art.
• Angiostatin® protein refers to proteins that may be synthesized and may be isolated from biological tissues, cells, cell culture medium, and from broth and media obtained from cellular and cell-free expression systems.
• angiostatin® protein includes Angiostatin® protein produced from recombinant expression systems.
• Angiostatin® protein also includes precursor forms of the Angiostatin® protein.
• Angiostatin® protein also includes fragments of the protein, and modified proteins and peptides thereof that have a substantially similar amino acid sequence, and that are capable of inhibiting proliferation of proliferation of blood vessels. For example, silent substitutions of amino acids, wherein the replacement of an amino acid with a structurally or chemically similar amino acid does not significantly alter the structure, conformation or activity of the protein, are well known in the art. Such silent substitutions are intended to fall within the scope of the present invention.
• the term Angiostatin® protein also includes various post-translational modifications or other modifications of Angiostatin® protein, including, but not limited to, phosphorylation, glycosylation, sulfation, and disulfide bond formation or reduction.
• Angiostatin® protein includes shortened proteins or peptide fragments of Angiostatin® protein wherein one or more amino acids, preferably 1 to 10 amino acids, are removed from either or both ends of Angiostatin® protein, or from an internal region of the protein, yet the resulting molecule retains bioactivity such as inhibiting proliferation of blood vessels.
• Angiostatin® protein also includes lengthened proteins or peptides wherein one or more amino acids, preferably 1 to 10 amino acids, is added to either or both ends of Angiostatin® protein, or to an internal location in the Angiostatin® protein, yet the resulting molecule retains the ability to inhibit proliferation of blood vessels.
• Angiostatin® protein modifications of the Angiostatin® protein, its subunits and peptide fragments. Such modifications include substitutions of naturally occurring amino acids at specific sites with other molecules, including but not limited to naturally and non-naturally occurring amino acids. Such substitutions may modify the bioactivity of Angiostatin® protein and produce biological or pharmacological agonists or antagonists. Such substitutions may include conservative substitutions known to one of skill in the art, such as valine for alanine. Acceptable substitutions may also include modifications of amino acids, such as norleucine for leucine. It is to be understood that substitution of
• D amino acids for L amino acids is encompassed within the scope of the present invention. Some substitutions are described in Dictionary of Biochemistry and Molecular Biology, 2 nd ed., J. Stenesh, John Wiley & Sons, 1989, the entirety of which is incorporated herein by reference. Additional modifications include addition of an amino acid, such as a tyrosine or another amino acid at specific locations in Angiostatin® protein or fragments thereof to enhance labeling potential with radioactive and non-radioactive labels, addition of molecules such as ricin, addition of radioactive and/or non- radioactive labels. "Substantial sequence homology" means at least approximately
• Angiostatin® protein can be isolated from biological sources, including tissues, cells and biological fluids.
• Angiostatin® protein may be produced from recombinant sources, from genetically altered cells implanted into animals, from tumors, and from cell cultures, as well as other sources.
• Angiostatin® protein can be isolated from body fluids including, but not limited to, serum, urine and ascites, or synthesized by chemical or biological methods (e.g. cell culture, recombinant gene expression, cellular and cell free expression systems, peptide synthesis, and in vitro and in vivo enzymatic catalysis of precursor molecules to yield active Angiostatin® protein).
• Recombinant techniques include gene amplification from DNA sources using the polymerase chain reaction (PCR), and gene amplification from RNA sources using reverse transcriptase/PCR.
• Angiostatin® protein can be made by automated protein synthesis methodologies well known to one skilled in the art. Alternatively, Angiostatin® protein may be isolated from larger known proteins.
• Angiostatin® protein can also be produced synthetically by chemical reaction or by recombinant techniques in conjunction with expression systems.
• Angiostatin® protein can be isolated from a body fluid such as blood or urine of patients.
• Angiostatin® protein can also be produced by recombinant DNA methods or synthetic peptide chemical methods that are well known to those of ordinary skill in the art.
• Angiostatin® protein is recombinantly produced.
• a preferred method of recombinant production of Angiostatin® protein is a method employing Pichia pastoris. Novel methods of isolation and purification of Angiostatin® protein, especially recombinantly-produced Angiostatin® protein are provided in the present invention.
• expression systems may be used for recombinant production of Angiostatin® protein.
• These expression systems include, but are not limited to Pichia pastoris, yeast, E coli, insect cells, baculovirus expressions systems, expression in transgenic animals, expression in transgenic plants, mammalian systems, and other systems commonly known to one of ordinary skill in the art of expressing proteins. Some of these expression systems are described in U.S. Patent No.
• Pichia pastoris expression system was used for most of the recombinant Angiostatin® protein production presented in the present application, it is to be understood that the present invention encompasses other systems for recombinant production of Angiostatin® protein. Accordingly, modifications of the Angiostatin® protein production parameters presented herein can be made by one of ordinary skill in the art of recombinant production of proteins using specific expression systems. For example, when yeast are used for recombinant production of Angiostatin® protein, different induction methods may be used, as commonly known to one of skill in the art.
• Yeast can be induced on methanol, or a mixture of methanol and glycerol, all optionally diluted with water, at feed rates commonly known to one of ordinary skill in the use of yeast expression systems for recombinant production of molecules, including proteins.
• Tables are not limiting and may be increased or decreased in a range of 0 to 20%, preferably 0 to 10%, without altering the spirit and scope of the present invention.
• Inoculum cultures are prepared using a two stage seed process of Pichia pastoris.
• the first stage employs an enriched media (about 800mL in a 2.8L flask) and is incubated at 250 rpm and about 30°C for approximately 24 hours to a final optical density at 600 nm (OD 6 oo nm. ) o greater than 30.
• the second stage uses a similar media base (16 x 1L in 2.8L shaker flasks) and is incubated at about 250 rpm and 30°C for approximately 16 hours to a final OD 6 oonm of between about 20.0 and 30.0.
• the fermentation media consists of Calcium Sulfate, Potassium Sulfate,
• the batch glycerol phase is the beginning phase which utilizes the initial charge of Glycerol as the carbon source. This phase lasts for approximately 30 hours. The end of this phase is characterized by a sharp DO spike. The spike indicates the depletion of the carbon source.
• the fed-batch glycerol phase is initiated at a set flow (16.1 g/Kg/hr) immediately following the batch glycerol phase.
• the fed-batch glycerol phase lasts for 6 hours.
• the pH is allowed to decrease from 5.0 to 4.0.
• the temperature is also decreased from 30°C to 26°C during the last two hours of the phase.
• the methanol ramp phase is initiated immediately following the fed-batch glycerol phase.
• the methanol is used as a carbon source and as a product inducer.
• Angiostatin is produced as a secreted protein.
• the methanol flowrate to the fermentor is ramped linearly from 1.5 to 4.5 mL/Kg/hr at a rate of 1.0 mL/Kg/hr 2 .
• the final phase of the fermentation is the methanol induction phase.
• the methanol continues to be used as a carbon source and product inducer. During this phase the methanol is fed to the fermentor at a set rate of 4.5mL/Kg/hr for -83 hours.
• Harvest conditions are then set, after the conditions have been achieved the fermentation process is ready for harvest.
• the methanol and pH loops are not shutoff until the temperature is below 20°C.
• Final Angiostatin concentration is approximately 500mg/L in the supernatant.
• the final WCW is approximately 300g/L.
• KOH Potassium Hydroxide
• Glycerol Specifics 50% Glycerol Solution (by weight) with KFO 880 Antifoam (0. mL/Kg) pH Shift Start: 4 th hour of Fed Batch Glycerol pH Shift Specifics: Linear Decrease from 5.0 to 4.0 pH Shift Duration: 2 hours
• the present invention also provides a new and useful method for recovery and purification of proteins, particularly recombinantly-produced proteins.
• the methods of the present invention may be used for recovery and purification of Angiostatin® protein from biological sources, including but not limited to biological fluids, tissues, cells, culture media, and fermentation media.
• the present invention provides a new and useful method for recovery and purification of Angiostatin® protein, and more particularly, recombinantly-produced Angiostatin® protein. This method may be employed for large scale recovery and purification of recombinantly- produced Angiostatin® protein. It is to be understood that the present invention is useful for recovery and purification of Angiostatin® protein from any expression system.
• Angiostatin® protein The basic recovery process of Angiostatin® protein is accomplished using four chromatography steps and a final concentration and diafiltration step. These steps are shown schematically in Figures 1 through 5.
• FIG. 1 presents an overview of the process.
• the broth which consists of all components (cells, nutrients, and buffer) within the fermenter, is diluted with water to a conductivity that favors binding of the target protein to the first column in the process.
• the first chromatography step in the recovery and purification procedure is called the Angiostatin® protein purification capture step, and the specific resin used is called Streamline-SP (Pharmacia, Inc.).
• SP refers to the sulfopropyl functional groups that are attached to the support bead that give the resin its cationic character. It is to be understood that besides Streamline-
• SP resin other resins that act as cation exchangers may be used in the practice of the present invention.
• cation exchangers include but are not limited to carboxymethylcellulose.
• Streamline refers to a relatively new format of chromatography that is designed to capture and separate target protein from a milieu of broth, thus eliminating the need for centrifugation to separate cells from the protein-containing supernatant.
• This type of chromatography is also known as expanded bed abso ⁇ tion chromatography (EBA).
• EBA expanded bed abso ⁇ tion chromatography
• the broth is typically pumped up into a Streamline column containing about 20- 30%) by volume of settled resin and approximately 70-80%> buffer.
• the bed of resin expends and flows up, thereby accounting for the name EBA.
• protein is bound to the beads, which can only flow up a finite distance, to an equilibrium level. The cells and non-bound protein however, flow up and out of the column to waste.
• the final step in the purification procedure involved concentration and dialysis using the approach of Ultrafiltration/Diafiltration
• UF/DF UF/DF
• a membrane preferably made from polyethersulfone
• a preferred molecular cutoff for Angiostatin® protein is about 3kDa.
• Several liters of formulation buffer are run over the membrane to recover retain Angiostatin® protein, or another protein of interest remaining in the filters. This material recovered from the filters is added to the pool of Angiostatin® protein.
• parallel flow concentrators employing porous tubes may be used instead of flat membranes for concentration and dialysis.
• Streamline SP Resin expanded bed volume - 150L, expanded bed height of ⁇ 54cm at 300 cm hr
• Streamline SP Resin expanded bed volume - 150L, expanded bed height of ⁇ 54cm at 300 cm hr
• the column is rinsed with WPU until neutral conditions are met.
• the column is equilibrated with 50mM Sodium Phosphate, 24mM Citric Acid, pH 5.1 until the pH and conductivity of the column are that of the buffer.
• the Angiostatin Fermentation is loaded onto the column while performing inline dilution with WPU to maintain a load conductivity of 9-12 mS/cm.
• the column is washed with 15%> Glycerol, 15mM Sodium Phosphate, pH 6.1.
• the Angiostatin is eluted from the column using 30mM Sodium Phosphate, 200mM NaCl, pH 7.2. Collection begins when the conductivity rises sharply (to > 4 mS/cm) and the UN rises above 0.5 AU. Collection ends when UV returns to 0.2 AU.
• the volume of the eluate should be approximately 2-3CV's at an Angiostatin concentration of 3.3 g/L of eluate.
• the column is regenerated using 7 column volumes (CV's) of 2M ⁇ aCl.
• the 2M ⁇ aCl Regeneration is followed with 6M Urea. After an initial Urea Wash, Urea is recirculated for a minimum of 1 hour. The Urea dissolves the cell paste and eases removal of the cell paste from the column. The regeneration is followed with a WPU flush until the UV returns to baseline.
• the column is sanitized with 0.5M ⁇ aOH then stored in 0.1M ⁇ aOH.
• the 0.1M ⁇ aOH may be prepared inline by mixing 0.5M ⁇ aOH and WPU so that the inlet conductivity is 23 ⁇ 5mS/cm.
• the Q-Sepharose column (30cm x 15cm column, 10.6L CV) and Ceramic Hydroxyapatite (CHT) column (45cm x 37cm column, 58.8L CV) which were stored in 0.1M ⁇ aOH are rinsed with 5 CV's of lOmM Sodium Phosphate, pH 7.0.
• the maximum flowrate for this chromatography is 480LPH (300 cm/hr of CHT Column) and is performed at ambient temperature.
• the angiostatin flows through the Q Sepharose column and binds to the CHT column.
• the columns are charged with 0.5M Sodium Phosphate, pH 7.0 then equilibrated with lOmM Sodium Phosphate, pH 7.0 until the pH and conductivity are that of the equilibration buffer.
• the elution from the Streamline SP Chromatography of Angiostatin is diluted inline with
• the column is washed to baseline with lOmM Sodium Phosphate, pH 7.0.
• the Q Sepharose column is removed from the chromatography skid.
• the angiostatin is eluted from the CHT column with a 5CV linear gradient from lOmM Sodium Phosphate, pH 7.0 to 74mM Sodium Phosphate, pH 7.0.
• the 74mM Sodium Phosphate, pH 7.0 is continued until the UV returns to ⁇ 0.5 AU.
• the product is collected from peak beginning at 0.15 AU to peak ending at 0.3 AU.
• the volume of the elution should be approximately
• the CHT column is regenerated with 0.5M Sodium Phosphate, pH 7.0.
• the Q Sepharose FF column is regenerated with 2M NaCl.
• the columns are then cleaned with 0.5M NaOH and held for at least 1 hour (maximum of 24 hours).
• the columns are then stored in 0.1M NaOH which is prepared by blending 0.5M NaOH and WFI.
• the Toyopearl Phenyl 650M Column (45cm x 25cm column, 40L CV) which was stored in 0.1M NaOH is rinsed with WFI until neutral conditions have been met.
• the flowrate for this chromatography is 480LPH (300cm/hr) and is performed at ambient temperature.
• the column is equilibrated with 50mM Sodium Phosphate, 24mM Citric Acid, 1.4M Ammonium Sulfate, pH 5.1 until the pH and conductivity are that of the equilibration buffer.
• the elution from the CHT column is diluted inline with 50mM Sodium Phosphate, 24mM Citric Acid, 2.8M Ammonium Sulfate, pH 4.5 (1 part elution: 1 part buffer) and loaded onto the Toyopearl 650M column.
• the loaded column is then washed with 50mM Sodium Phosphate, 24mM Citric Acid, 1.4M Ammonium Sulfate, pH 5.1.
• the Angiostatin is eluted from the column using a 20CV linear gradient from 50mM Sodium Phosphate, 24mM Citric Acid, 1.4M Ammonium Sulfate, pH 5.1 to 50mM Sodium Phosphate,
• the UF/DF skid is rinsed with 2 x 10L flushes which are added to the diafiltered product. Due to the hold up volume of the UF/DF skid, it is necessary to perform the final concentration on a table top unit with 25 sq. feet of filter.
• the retentate is then concentrated to 20.0 mg/mL.
• the UF/DF filters are rinsed with 0.15M Sodium Chloride and the rinse is added to the concentrated product.
• the UF/DF retentate is adjusted with 0.15M Sodium
• Chloride to a final concentration of 15 mg/ml. Note: If the Toyopearl Elution was diluted the lx diafiltration may be omitted. Formulation The formulated pool is then aseptically filtered through a 0.2micron filter. The filtered Angiostatin is bulk filled into sterile bottles and then stored at -70°C.
• Angiostatin® protein of the present invention. This is the Angiostatin® protein amino acid sequence encoded by the gene sequence listed below as
• SEQ ID NO:l shows the amino acid sequence of the Angiostatin® protein produced from the production clone ENMA98 which contains the nucleotide sequence shown in SEQ ID NO:2 hASv3 protein sequence (260 aa) SEQ ID NO: 1
• Buffer 50mM Sodium Phosphate, 24mM Citric Acid, pH 5.1
• Buffer 15% Glycerol, 15mM Sodium Phosphate, pH 6.1
• Buffer 30mM Sodium Phosphate, 200mM NaCl, pH 7.2.
• Buffer 0.1M NaOH (inline dilution 0.5M NaOH / WPU)
• Buffer lOmM Sodium Phosphate, pH 7.0 Specifics: Rinse until conductivity ⁇ 3.0 mS/cm
• Buffer 0.5M Sodium Phosphate, pH 7.0
• Buffer lOmM Sodium Phosphate, pH 7.0
• Buffer lOmM Sodium Phosphate, pH 7.0
• Buffer A lOmM Sodium Phosphate, pH 7.0
• Buffer B 74mM Sodium Phosphate, pH 7.0
• Buffer 0.5M Sodium Phosphate, pH 7.0
• Rinse Specifics Perform a 3CV gradient from 0.1M NaOH to WFI then continue rinsing
• Buffer Dilution 50mM Sodium Phosphate, 24mM Citric Acid, 2.8M Ammonium
• Buffer A 50mM Sodium Phosphate, 24mM Citric Acid, 1.4M Ammonium
• Buffer B 50mM Sodium Phosphate, 24mM Citric Acid, 0.92M Ammonium Sulfate, pH 5.1
• Buffer 50mM Sodium Phosphate, 24mM Citric Acid, pH 5.1
• Citric Acid 5.04 g/L
• Citric Acid Monohydrate 5.04 g/L
• ANGIOSTATIN® protein results in one major species as well as a number of minor species which can be separated by reverse phase chromatography and SDS-PAGE.
• Western blot analysis using a polyclonal primary antibody indicated that all minor species were related to the major intact AngiostatinTM protein.
• Electrospray ionization mass spectrometry of the reduced protein detected two major components whose deconvoluted spectra indicated masses of 29788 Da and 29951 Da., consistent with an intact AngiostatinTM molecule and an intact molecule with a single hexose sugar (+

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