Abstract: Methods are provided for large scale purification of neurotrophins, including mature NGF, suitable for clinical use. The methods provide means to separate neurotrophins from various less desirable misprocessed, misfolded, size, glycosylated, or charge forms. Compositions of neurotrophins, including mature NGF, substantially free of these variants are also provided.

Abstract: A method for purifying a polypeptide by ion exchange chromatography is described which involves changing the conductivity and/or pH of buffers in order to resolve a polypeptide of interest from one or more contaminants.

Abstract: A method of purifying antithrombin III (AT III) from a starting material containing an AT III/heparin complex or an AT III/heparinoid complex is disclosed. First, the method comprises adsorbing the AT III/heparin complex or the AT III/heparinoid complex on an anion exchanger material. Second, the method involves separating the AT III from the adsorbed AT III/heparin complex or an AT III/heparinoid complex by elution with a buffer having a pH ranging from 8.5 to 10.5 and a conductivity between 10 and 60 mS.

Abstract: An adsorbent or bradykinin comprising styrene-divinylbenzene copolymer having sulfonic acid groups and a method for adsorbing and removing bradykinin comprising contacting the adsorbent with a fluid containing bradykinin are provided. An adsorber for adsorbing bradykinin in which the adsorbent is charged in a vessel having an inlet and an outlet for a fluid is also provided.

Abstract: The invention relates to a method of removing endotoxin from preparations of alpha-1-acid glycoprotein (orosomucoid) by contact with a finely divided non-toxic resin such as fumed silica. The invention also relates to a purification process for alpha-1-acid glycoprotein which includes this deprogenation step, and to the depyrogenated product and its clinical uses.

Abstract: Endotoxin binding/neutralizing proteins capable of binding endotoxin in vivo, thereby neutralizing the toxic effect or bioactivity of endotoxin which are isolated from a horseshoe crab such as Limulus polyphemus, pharmaceutical compositions and pharmaceutical uses of the proteins, a method of purifying the proteins and an assay for endotoxin based on the proteins, are disclosed.

Abstract: Expression systems are disclosed for the direct expression of peptide products into the culture media where genetically engineered host cells are grown. High yield was achieved with novel vectors, a special selection of hosts, and/or fermentation processes which include careful control of cell growth rate, and use of an inducer during growth phase. Special vectors are provided which include control regions having multiple promoters linked operably with coding regions encoding a signal peptide upstream from a coding region encoding the peptide of interest. Multiple transcription cassettes are also used to increase yield. The production of amidated peptides using the expression systems is also disclosed.

Abstract: Fibrinogen is obtained from milk by contact with a cation exchange chromatography substrate under conditions where the fibrinogen binds to the substrate, followed by optional washing of the substrate. The bound fibrinogen is removed from the substrate by irrigation under increased ionic strength or increased pH conditions. The obtained fibrinogen may be naturally produced or transgenic.

Abstract: This invention relates to methods for isolating highly-purified mixtures of natural type I interferons from white blood cells. The invention also relates to highly-purified mixtures of natural type I interferons which resemble natural type I interferon in that it includes 9 subtypes, i.e., alpha-1, alpha-2, alpha-5, alpha-7, alpha-8, alpha-10, alpha-14, alpha-21 and omega, giving rise to possibly 20 molecular species, including alpha-1a, alpha-1new, alpha-2a, alpha-2b, alpha-2c, alpha-5, alpha-5LG, alpha-7, alpha-8a, alpha-8c, alpha-10a, alpha-14a, alpha14-b, alpha 14-c, alpha-14LG, alpha-21a, alpha-21b, alpha-21c, omega and omega LG.

Abstract: The present invention relates to a process for the controlled enzymatic cleavage of purified and depigmented active allergenic proteins from indoor and outdoor source materials, which process produces fragments of allergens that retain the the natural T-lymphocyte stimulating epitopes, but are depleted of IgE-binding B-cell epitopes and complement-activating agents. The invention also relates to the new pharmaceutical products. These allergen fragments do not exhibit the disadvantages of conventional allergenic extracts for immunotherapy and can be safely used to induce a state of specific T-cell anergy and immunological tolerance in allergic human beings.

Abstract: A procedure is described for obtaining valuable endoproteases (or zonases) from hatchery-produced Atlantic salmon eggs. Synchronized hatching by for instance elevated temperature, is followed by filtration through cheese cloth. The filtrate (hatching fluid) may be stored for months or year (depending on conditions) without loss of activity. Extraneous matters are removed by centrifugation (16,000 g, 2×15 min) after addition of urea (2 or 4 M, or more). High purity zonases are obtained by simple chromatographic procedures (gel filtration, affinity chromatography, isoelectric focusing), yielding sequence-grade purity after all three steps are performed in sequence. All preparations of salmon zonases exhibit valuable enzymatic properties with regard to proteolysis, both in terms of catalysis and stability.

Abstract: Disclosed is an economical method for the preparation of chondroitin sulfates A and C useful as an effective ingredient of medicaments from fish scales as a waste material discharged from fishery in large quantities. Fish scales are enzymatically decomposed in an aqueous medium in the presence of a protease to isolate the chondroitin sulfate compounds and by-product polypeptides followed by removal of the by-product polypeptides from the aqueous solution by a cation-exchange treatment and then the aqueous solution of the chondroitin sulfate compounds is subjected to fractional precipitation by the addition of ethyl alcohol as the precipitant.

Abstract: The present invention relates to pharmaceutical compositions of transferrin and to the manufacture of such preparations. The present invention concerns specifically a pharmaceutical composition of apotransferrin, the iron-free form of transferrin, wherein the composition has at least 90% of the theoretical iron binding capacity of iron-free transferrin, contains less than 3% transferrin dimers and no detectable transferrin polymers or aggregates, and has an iron-saturation of less than 5%. The pharmaceutical apotransferrin composition of this invention effectively binds non-transferrin-bound-iron to a harmless form when administered as an intravenous injection or infusion to patients. The pharmaceutical composition of this invention does not contain viruses, denatured forms of transferrin or other harmful components.

Abstract: A method of modifying protein solubility employs polyionic polymers. These facilitate the solubilization, formulation, purification and refolding of proteins especially incorrectly folded proteins and aggregated proteins. Compositions are described that are suitable for formulating TFPI. The compositions allow preparation of pharmaceutically acceptable compositions of TFPI at concentrations above 0.2 mg/mL and above 10 mg/mL.

Abstract: Purified enzymatic compositions are provided having alpha-amidating enzymes capable of catalyzing the conversion of a peptidyl compound having a C-terminal glycine residue to a corresponding peptidyl amide having an amino group in place of the C-terminal glycine. The purified compositions have specific activities above 25 mU per mg protein and are sufficiently free of proteases to allow effective catalysis of even peptidyl compounds having L-amino acids. Biologically important alpha-amidated products such as calcitonin and other regulatory hormones are efficiently produced using the alpha-amidation reaction catalyzed by the enzymes. Purification by size exclusion chromatography in combination with strong anion exchange chromatography results in homogeneous enzyme species which are used to prepare antibodies specific for the alpha-amidating enzyme. A gene capable of expressing the alpha-amidating enzyme is ligated into an expression vector and transformed into a host cell capable of expressing the gene.

Abstract: The invention relates to pharmaceutical or veterinary compositions for the treatment of surface wounds; pharmaceutical or veterinary compositions for the treatment of gastrointestinal injuries, diseases or ulcers; methods of treating surface wounds in animals, including humans; and methods for the treatment of gastrointestinal injuries, diseases or ulcers which compositions and methods include compositions of milk product extracts including growth factors with basic to approximately neutral isoelectric points.

Abstract: A method of modifying protein solubility employs polyionic polymers. These facilitate the solubilization, formulation, purification and refolding of proteins especially incorrectly folded proteins and aggregated proteins. Compositions are described that are suitable for formulating TFPI. The compositions allow preparation of pharmaceutically acceptable compositions of TFPI at concentrations above 0.2 mg/mL and above 10 mg/mL.

Abstract: The present invention relates to the C3b/C4b receptor (CR1) gene and its encoded protein. The invention also relates to CR1 nucleic acid sequences and fragments thereof comprising 70 nucleotides and their encoded peptides or proteins comprising 24 amino acids. The invention further provides for the expression of the CR1 protein and fragments thereof. The genes and proteins of the invention have uses in diagnosis and therapy of disorders involving complement activity, and various immune system or inflammatory disorders. In specific embodiments of the present invention detailed in the examples sections infra, the cloning, nucleotide sequence, and deduced amino acid sequence of a full-length CR1 cDNA and fragments thereof are described. The expression of the CR1 protein and fragments thereof is also described. Also described is the expression of a secreted CR1 molecule lacking a transmembrane region.

Abstract: The fusion (F) protein, attachment (G) protein and matrix (M) protein of respiratory syncytial virus (RSV) are isolated and purified from respiratory syncytial virus by mild detergent extraction of the proteins from concentrated virus, loading the protein onto a hydroxyapatite or other ion-exchange matrix column and eluting the protein using mild salt treatment. The F, G and M proteins, formulated as immunogenic compositions, are safe and highly immunogenic and protect relevant animal models against decreased caused by respiratory syncytial virus infection.

Abstract: PROTEIN PURIFICATION I

Abstract: An improved process for the purification of antibodies from human plasma or other sources is disclosed. The process involves suspension of the antibodies at pH 3.8 to 4.5 followed by addition of caprylic acid and a pH shift to pH 5.0 to 5.2. A precipitate of contaminating proteins, lipids and caprylate forms and is removed, while the majority of the antibodies remain in solution. Sodium caprylate is again added to a final concentration of not less than about 15 mM. This solution is incubated for 1 hour at 25° C. to effect viral inactivation. A precipitate (mainly caprylate) is removed and the clear solution is diluted with purified water to reduce ionic strength. Anion exchange chromatography using two different resins is utilized to obtain an exceptionally pure IgG with subclass distribution similar to the starting distribution. The method maximizes yield and produces a gamma globulin with greater than 99% purity. The resin columns used to obtain a high yield of IgG, retain IgM and IgA, respectively.

Abstract: A peptide containing a sequence for the first fifteen amino acids from the N-terminal of Asn-Leu-Val-Glu-Phe-Gly-Lys-Met-Ile-Glu-Cys-Ala-Ile-Arg-Asn is used in a cell culture medium to promote cell growth in vitro and in the treatment of wounds. A method of preparation of the peptide is also claimed.

Abstract: Removing free protein from a liquid mixture containing the protein and a protein-polysaccharide conjugate can be a technologically difficult, expensive, and time consuming process. The procedure described herein for removing the protein from such a mixture simplifies this removal. The procedure includes contacting the liquid mixture, including the protein and the protein-polysaccharide conjugate, with a solid phase, restricted-access media material. This material at least partially binds with the protein and separates it from the liquid mixture, thereby providing a purified liquid containing at least a portion of the protein-polysaccharide conjugate and a reduced amount of the protein. The purified liquid can be collected for further processing or use. As one example, the restricted-access media material can include porous silica particles that generally allow the protein to enter, but restrict or limit access to the protein-polysaccharide conjugate.

Abstract: The present invention is directed to a process for purifying &agr;1-proteinase inhibitor. The process comprises providing an impure protein fraction which comprises &agr;1-proteinase inhibitor. The impure protein fraction is precipitated with a precipitant comprising PEG. The supernatant from the PEG precipitation, which comprises &agr;1-proteinase inhibitor, is collected and applied to an anion-exchange medium. A fraction comprising &agr;1-proteinase inhibitor is recovered from the anion-exchange medium and applied to a metal chelate medium. A fraction comprising &agr;1-proteinase inhibitor is then recovered from the metal chelate medium. Alpha1-proteinase inhibitor purified by the process has a specific activity greater than 0.6 units/mg.

Abstract: The present invention relates to a process for purifying immunoglobulin G from a crude immunoglobulin-containing plasma protein fraction. Said process includes a number of steps of which the anion exchange chromatography and the cation exchange chromatography are preferably connected in series. An acetate buffer having a pH of about 5.0-6.0 and having a molarity of about 5-25 mM is preferably used throughout the purification process. The invention further comprises an immunoglobulin product which is obtainable by this process. The invention also relates to an immunoglobulin product which has a purity of more than 98%, has a content of IgG monomers and dimers of more than 98.5%, has a content of IgA less than 4 mg of IgA/l, and contains less than 0.5% polymers and aggregates. Said product does not comprise detergent, PEG or albumin as a stabilizer. The product is stable, virus-safe, liquid and ready for instant intravenous administration.

Abstract: A method of separating a soluble milk component from milk is disclosed. The method involves the use of tangential flow filtration across a membrane to form a retentate and a permeate, combining the permeate with the original milk sample, and repeating this procedure until the milk has been sufficiently purified. Preferably, the milk is combined with a chelating agent, such as EDTA, to improve the purification efficiency. This procedure is advantageously employed with milk from transgenic animals which have been genetically altered to express exogenous proteins, such as therapeutic proteins, in their milk.

Abstract: A process for purifying molecules from contaminants is provided. In this process a mixture containing the molecule (peptide, polypeptide, or biologically active non-peptidyl compound) and its contaminants is loaded onto a reversed-phase liquid chromatography column and the molecule is eluted from the column with a buffer containing hexylene glycol.

Abstract: The present invention relates to a product and process for suppressing an immune response using a T lymphocyte veto molecule capable of blocking cell surface molecules responsible for T cell activation. Disclosed is a CD4 or CD2 molecule, associated with an immunoglobulin molecule capable of binding to a major histocompatibility antigen. Also disclosed is a method to produce a T lymphocyte veto molecule, a therapeutic composition comprising a T lymphocyte veto molecule and methods to use T lymphocyte veto molecules in therapeutic processes requiring suppression of an immune response.

Abstract: The present invention relates to a process for preparing functional recombinant tissue factor in a prokaryotic host organism.

Abstract: The present invention provides isolated DNA molecules encoding human and murine LIF and methods of producing LIF by the expression of the isolated DNA and suitable host cells. The invention further provides human and murine LIF polypeptides, pharmaceutical compositions containing LIF, and methods of use thereof.

Abstract: The present invention relates to a process for the one-stage resalting and purification of oligopeptides. Oligopeptides are often not formed directly as acetates when synthesised. Acetate salts of oligopeptides are however desirable as bulk-active material for medical and formulation reasons. Processes known from the prior art have hitherto involved two separate steps or pyridine-containing solvents. The resalting and purification can be combined in one step and the use of pyridine as solvent can be avoided, if the oligopeptide in the form of its chloride salt is purified with an acetate-containing solvent by liquid chromatography methods.

Abstract: The present invention provides formulations that prevent loss and damage of PEG-interferon alpha conjugates during and following lyophilization. The formulations of the present invention protect PEG-interferon alpha conjugates from loss and degradation during the lyophilization process, as well as degradation during subsequent storage. The formulations of the present invention are suitable for protection of PEG-interferon alpha conjugates from various types of degradation, including, but not limited to loss of biological activity and changes in the degree and/or nature of conjugation. A preferred PEG-interferon alpha conjugate protectable in the formulations of the present invention is an interferon alpha-2b-polyethylene glycol (12,000) conjugate.

Abstract: The present invention relates to pharmaceutical compositions of transferrin and to the manufacture of such preparations. The present invention concerns specifically a pharmaceutical composition of apotransferrin, the iron-free form of transferrin, wherein the composition has at least 90% of the theoretical iron binding capacity of iron-free transferrin, contains less than 3% transferrin dimers and no detectable transferrin polymers or aggregates, and has an iron-saturation of less than 5%. The pharmaceutical apotransferrin composition of this invention effectively binds non-transferrin-bound-iron to a harmless form when administered as an intravenous injection or infusion to patients. The pharmaceutical composition of this invention does not contain viruses, denatured forms of transferrin or other harmful components.

Abstract: Invaplex, a novel composition comprising invasin proteins and LPS from gram-negative bacteria is described as well as methods of using the novel composition as an adjuvant and a diagnostic tool.

Abstract: Disclosed are amino acid sequences of the late 64 kilodalton protein of human cytomegalovirus (HCMVgp64), useful in diagnosing and preventing HCMV infections.

Abstract: A process for the preparation of a concentrate of von Willebrand factor is described, entailing a solution of a complex of this factor with factor VIII:C being optionally pasteurized and treated with an anion exchanger, there being no binding of the von Willebrand factor.

Abstract: A method for purification of proteins by displacement chromatography in hydrophobic interaction and reversed phase chromatographic systems uses low molecular weight (less than about 10,000) surface-active compounds as displacers. Examples of effective displacers are benzethonium chloride, benzyltributylammonium chloride, and tetrahexylammonium chloride.

Abstract: There are disclosed a stable factor VIII/vWF-complex, particularly comprising high-molecular vWF multimers, being free from low-molecular vWF molecules and from proteolytic vWF degradation products, as well as a method of producing this complex.

Abstract: Expression systems are disclosed for the direct expression of peptide products into the culture media where genetically engineered host cells are grown. High yield was achieved with novel vectors, a special selection of hosts, and/or fermentation processes which include careful control of cell growth rate, and use of an inducer during growth phase. Special vectors are provided which include control regions having multiple promoters linked operably with coding regions encoding a signal peptide upstream from a coding region encoding the peptide of interest. Multiple transcription cassettes are also used to increase yield. The production of amidated peptides using the expression systems is also disclosed.

Abstract: Disclosed is an improved process for obtaining purified, monomeric, intact, correctly-folded insulin-like growth factor-I (also known as somatomedin-C). The improvements, consisting primarily of the addition of an IGF-I unfolding/refolding step and the substitution of a reverse phase chromatography step for a gel filtration chromatography step result in a three-fold increase in final yield. The process includes the following steps, in order: first cation exchange, unfolding/refolding, hydrophobic interaction chromatography, second cation exchange, and reverse phase chromatography.

Abstract: An isolated and purified polypeptide having heparin binding properties with the amino acid sequence of SQ ID No: 1 and pharmaceutical compositions for treating skin wounds.

Abstract: A method for purifying human or other alpha-1 proteinase inhibitor (&agr;1-PI) from a solution (which may be derived from the milk of a transgenic animal expressing the &agr;1-PI) which comprises contacting the solution with a cation exchange substrate under conditions sufficient to bind non-tg-&agr;1-PI contaminants to the substrate while not substantially binding tg &agr;1-PI to the substrate. Using the preferred embodiment, the purified tg &agr;1-PI contains as little as 40 pg non-&agr;1-PI-whey protein per mg total protein.

Abstract: A method for rapid purification of a blood component from blood is described in which the blood plasma is first separated from the cellular blood elements by any conventional means, such as centrifugation. An affinity cartridge is then activated with a molecule, such as an amino acid, which binds with a blood component such as plasminogen. The separated blood plasma is then passed through the affinity cartridge such that the blood component is retained by the affinity cartridge. Thereafter, the blood component is eluted from the affinity cartridge by passing a buffer solution containing a releasing agent through the affinity cartridge. This releasing agent disengages the blood component from the affinity cartridge. The releasing agent is then separated from the eluted solution by passing the eluted solution through a device, such as an ion exchange or size exclusion device. The separated blood component, e.g.

Abstract: Methods are provided for large scale purification of neurotrophins, including mature NGF, suitable for clinical use. The methods provide means to separate neurotrophins from various less desirable misprocessed, misfolded, size, glycosylated, or charge forms. Compositions of neurotrophins, including mature NGF, substantially free of these variants are also provided.

Abstract: The invention concerns a process of chromatographically separating glycosylated proteins from non-glycosylated proteins by subjecting a solution comprising glycosylated and non-glycosylated proteins to chromatography using a Ca++ containing eluant. By using this process a fraction comprising non-glycosylated proteins substantially free from glycosylated proteins is obtained. The process may be applied to the separation of proteins used in the medical industry, such as insulin.

Abstract: A soluble form of DTctGMCSF is produced with high yield in insect cells, preferably using the baculovirus expression vector system (BEVS).

Abstract: A process for producing an intravenously-administrable gamma globulin solution substantially free of contaminating viruses by heat treating for viral inactivation and fractionating an impure gamma globulin solution and then treating the purified gamma globulin with a solvent-detergent for further viral inactivation. In a continuous process disclosed herein, partially purified gamma globulin solids is not recovered as an intermediate product during the disclosed process. In the continuous process, the fractionation to obtain a purified gamma globulin solution is carried out without precipitation of the desired gamma globulin.

Abstract: A new process, particularly simple and economical, for FSH and LH separation and purification starting from crude HMG preferably urinary, comprising the following steps:1) optional exhaustion of crude HMG viral charge in aqueous EtOH2) ion-exchange chromatography on weakly basic anionic resins of DEAE type;3) affinity chromatography on resin having an antraquinone derivative as a ligand;4) optional ion-exchange chromatography on strongly basic anionic resins;Hormones obtained thereby, in particularly pure form and having high specific activity, may subsequently undergo a depyrogenation step.

Abstract: A method for producing an immunoglobulin preparation for intravenous injection, which comprises the steps of: fractionating an immunoglobulin-containing solution with 4 to 10 w/v % of polyethylene glycol having a molecular weight of from 1,000 to 10,000, at a pH value of from 4.5 to 6.5, an ionic strength of from 0.0001 to 0.1 M and a temperature of from 0 to 4.degree. C. to recover a supernatant fraction; and concentrating the supernatant fraction at a pH of from 3.5 to 5.0.

Abstract: Recombinantly produced serum albumin is purified in a series of steps, optionally by incubation with an anion-exchange adsorbent, followed by affinity chromatography employing a hydrophobic solid phase and using a water-soluble lipid anion as desorbens in the aqueous phase. The immobile phase comprises a carrier coupled to a 2-mercapto or 2-hydroxy alkanoic acid.