Abstract: The present invention provides methods for determining a ratio of an amount of a glycated form of a protein to a total amount of the protein in a sample containing the glycated protein, the glycosylated protein, or the glycoprotein. The method incorporates lateral flow test strip or vertical flow test strip devices having negatively charged carboxyl or carboxylate groups and hydroxyboryl groups immobilized and interspersed on a solid support matrix. The solid support matrix may include derivatives of cellulose (e.g., carboxy cellulose) derivatized with carboxylic acid (e.g., carboxylate, carboxyl) groups and hydroxyboryl compounds including phenylboronic acid (e.g., phenylborate), aminophenylboronic acid, boric acid (e.g., borate), or other boronic acid (e.g., boronate) compounds. The present invention is usefi.il for monitoring glycation or glycosylation of hemoglobin or albumin for monitoring glycemic control (e.g., glycemia in diabetes).

Abstract: Toxin derivatives are prepared by proteolytic treatment of holotoxin, and their toxicity is reduced by contacting the preparation with a ligand, which can be a metal or an antibody or another ligand. This ligand selectively binds to the toxin but not to the toxin derivative. Removing the ligand and toxin bound to the ligand further reduces toxicity. A second ligand is used to remove conjugates of the toxin and the first ligand. Compositions contain the purified derivative, optionally plus the toxin and the ligand.

Abstract: A method for separating one or more components of a biomolecule mixture by means of an ion exchange chromatographic system operated in the displacement mode includes sequentially perfusing the system with a first solution including the biomolecule mixture, and a second solution including a displacer having a structure selected from formula I and formula II:

Abstract: The present invention is related to an osteogenic device and its preparation. Said device comprises a bone morphogenetic protein (BMP), preferably a modified BMP complex obtainable by a modification of the conventional guanidum hydrochloride extraction method and collagens, preferably collagen I or collagen IV, impregnated in and/or adsorbed on a bioceramic carrier, preferably a shapable body (block) originating from a coral skeleton. The method of isolating said modified BMP complex which lacks an immunogenic component and consists essentially of a 100–700 kD and a 15–25 kD protein with osteoinductive properties and preferably of the 15–25 kD protein which has improved storage properties as well as its use in the osteogenic device with improved osteoinductive properties is also disclosed.

Abstract: The invention encompasses an improved, rapid, size-exclusive method for screening for small molecular weight ligands that bind specifically to a protein target, using size-exclusion separation, ultrafiltration, and mass spectrometry.

Abstract: The invention provides methods of increasing yields of desired conformation of proteins. In particular embodiments, the invention includes contacting preparations of a recombinant protein with a reduction/oxidation coupling reagent for a time sufficient to increase the relative proportion of a desired configurational isomer.

Abstract: A method for predicting the elution time of a peptide in chromatographic and electrophoretic separations by first providing a data set of known elution times of known peptides, then creating a plurality of vectors, each vector having a plurality of dimensions, and each dimension representing the elution time of amino acids present in each of these known peptides from the data set. The elution time of any protein is then be predicted by first creating a vector by assigning dimensional values for the elution time of amino acids of at least one hypothetical peptide and then calculating a predicted elution time for the vector by performing a multivariate regression of the dimensional values of the hypothetical peptide using the dimensional values of the known peptides. Preferably, the multivariate regression is accomplished by the use of an artificial neural network and the elution times are first normalized using a transfer function.

Abstract: The present invention relates generally to the production of recombinant human uteroglobin (rhUG) for use as a therapeutic in the treatment of inflammation and fibrotic diseases. More particularly, the invention provides processes, including broadly the steps of bacterial expression and protein purification, for the scaled-up production of rhUG according to current Good Manufacturing Practices (cGMP). The invention further provides analytical assays for evaluating the relative strength of in vivo biological activity of rhUG produced via the scaled-up cGMP processes.

Abstract: A universal folding method that has been demonstrated to be effective in refolding a variety of very different proteins expressed in bacteria as inclusion bodies has been developed. Representative proteins that can be dissolved and refolded in biologically active form, with the native structure, are shown in Table I. The method has two key steps to unfold and then refold the proteins expressed in the inclusion bodies. The first step is to raise the pH of the protein solution in the presence of denaturing agents to pH greater than 9, preferably 10. The protein solution may be maintained at the elevated pH for a period of up to about 24 hours, or the pH immediately decreased slowly, in increments of about 0.2 pH units/24 hours, until the solution reaches a pH of about 8.0, or both steps used. In the preferred embodiment, purified inclusion bodies are dissolved in 8 M urea, 0.1 M Tris, 1 mM glycine, 1 mM EDTA, 10 mM beta-mercaptoethanol, 10 mM dithiothreitol (DTT), 1 mM reduced glutathione (GSH), 0.

Abstract: Disclosed are a novel hedgehog protein, i.e., a Desert hedgehog protein of human origin including mature and precursor forms, a DNA encoding the protein, a monoclonal antibody recognizing the protein, a process for producing the protein, and a method for detecting the protein. The hedgehog protein is useful in establishment of hybridomas which produce antibodies recognizing the protein, and the monoclonal antibody is useful in detection and purification of the protein. The hedgehog protein, DNA, and monoclonal antibody of this invention have efficacy in elucidation of hereditary morphological abnormalities in humans to establish their treatments and diagnoses.

Abstract: The present invention relates to the purification and production of human albumin from various sources through crystallization and repeated crystallization. Basic features of the invented process include providing specific reaction conditions and precipitating reagents to maximize albumin crystallization. Solubility diagrams are utilized as the basis for process control of the invented method. The current invention specifically controls phosphate concentration, pH and temperature to precisely guide crystallization kinetics and crystal yield.

Abstract: Methods for purifying authentic, properly folded IGF polypeptides from yeast transformants are disclosed. The methods include a refolding step and provide for high yields of authentic IGF from a variety of yeast strains.

Abstract: In a method for reconstituting a recombinant protein from a denatured state to its active form, a feed solution containing the recombinant protein in its denatured and/or in biologically inactive intermediate forms is subjected to a chromatographic separation process, in which the protein is reconstituted under conditions that promote refolding of the protein and the intermediate forms are separated from the refolded protein. The denatured form and/or the inactive intermediate forms of the protein are separated from the refolded protein in a continuous or quasi-continuous manner and optionally recycled to the feed solution.

Abstract: A method for extracting proteins from the intercellular space of plants is provided. The method is applicable to the large scale isolation of many active proteins of interest synthesized by plant cells. The method may be used commercially to recover recombinantly produced proteins from plant hosts thereby making the large scale use of plants as sources for recombinant protein production feasible.

Abstract: Purification process of humanPurification process of human urinary gonadotropins of high biological activity and chemical purity absolutely free of foreign contaminating materials derived from the use of biological reagents or chromatography dyes, from crude of gonadotropins. The high biological activity and chemically pure composition of human gonadotropins obtained by this process, are used for the treatment of infertility and are selected from the group of follitropin or menotropins, having a bioactivity greater than 2500 IU/mg protein as tested by biological assay in rats, for both FSH and LH hormones for menotropins and greater than 5000 IU/mg protein for follitropin having an FSH:LH ratio about 75:1. Pharmaceutical preparations of said gonadotropins free of these contaminating materials are also comprised within the present invention.

Abstract: The present invention relates to a method of isolating and/or purifying hypothalamic inhibitory factor (HIF) from a sample (e.g., tissue fluid) containing HIF. The present invention provides for isolation of large amounts of HIF using diafiltration, solid phase extraction and immunoaffinity techniques. In one embodiment, the invention relates to a method of purifying hypothalamic inhibitory factor from a sample containing hypothalamic inhibitory factor comprising subjecting the sample to diafiltration, solid phase extraction and immunoaffintiy chromatography.

Abstract: Method for preparing a factor VIII solution that is essentially free of viruses and essentially devoid of vWF (von Willebrand factor) and factor VIII-vWF complexes by (a) obtaining a starting factor VIII solution devoid of factor VIII-vWF complexes; and (b) filtering the solution through a hydrophilic virus filter.

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 producing a protein suitable for X-ray crystallographic analysis, in a cell-free protein synthesis system comprising a cell-free extract, a nucleic acid coding for said protein, and amino acids for the substrate of said protein, wherein said amino acids comprises at least one amino acid comprising a heavy atom, and wherein the introduced rate of said amino acid comprising the heavy atom into the synthesized protein is at least 80%.

Abstract: A process and apparatus for purifying one or more target substances from a source liquid, employing one or more cross-flow filter elements, and one or more types of chromatography resins, in combination, to provide purification with advantageous yield, product purity, and cost- and time-efficiency.

Abstract: A method for separating one or more components of a biomolecule mixture by means of an ion exchange chromatographic system operated in the displacement mode includes sequentially perfusing the system with a first solution including the biomolecule mixture, and a second solution including a displacer having a structure selected from formula I and formula II:

Abstract: The present invention comprises a method of protecting organs or tissue susceptible to reperfusion-induced dysfunction after ischemia. The method comprises parenterally administering to a patient a therapeutical composition containing natural alpha-1 acid glycoprotein, natural alpha-1 antitrypsin or a mixture thereof. Alternatively, organ or tissue transplants can be contacted with natural alpha-1 acid glycoprotein, natural alpha-antitrypsin or mixtures by perfusing or flushing them with a solution containing natural alpha-1 acid glycoprotein, natural alpha-1 antitrypsin or mixtures thereof in a concentration of 0.1 to 5 g/l.

Abstract: The present invention relates to a novel method for the isolation or purification of immunoglobulins (a special class of proteins) from a solution containing immunoglobulins, e.g. hybridoma cell culture supernatants, animal plasma or sera, or colostrum. The method includes the use of a minimum of salts, such as lyotropic salts, in the binding process and preferably also the use of small amounts of organic solvents in the elution process. The solid phase matrices, preferably epichlorohydrin activiated agarose matricees, are functionalised with mono- or bicyclic aromatic or heteroaromatic ligands (molecular weight: at the most 500 Dalton) which, preferably, comprises an acidic substituent, e.g. a carboxylic acid. The matrices utilised show excellent properties in a “Standard Immunoglobulin Binding Test” and in a “Monoclonal Antibody Array Binding Test” with respect to binding efficiency and purity, and are stable in 1M NaOH.

Abstract: Methods and apparatus for qualitative and quantitative proteome analysis are provided. The methods and apparatus allow for the isolation of a subset of peptides out of complex mixtures of peptides. The isolation is based on a specific chemical and/or enzymatic alteration of one or more types of peptides. This alteration modifies the biophysical, chemical or any other biochemical property of the affected types of peptides (e.g., net electrical charge and/or hydrophobicity) in such way that the altered peptides can be separated from the unaltered peptides.

Abstract: Human heat-shock protein-binding proteins (HspBP-1 and HspBP-2) and fragments thereof are disclosed with the polynucleotides which identify and encode them. Genetically engineered expression vectors and host cells comprising the nucleic acid sequences encoding heat-shock protein-binding proteins (HspBP) are also disclosed and a method for producing HspBP polypeptides.

Abstract: A process for the partial or complete separation of glycosylated and nonglycosylated proteins is described, in which: a) a triazine dye immobilized on a matrix is incubated with a mixture of glycosylated and nonglycosylated proteins, b) the matrix is then washed to remove the unbound proteins, and c) the proteins are eluted by means of a stepwise or continuous increase in the ionic strength or in the pH, nonglycosylated proteins and proteins having an increasing degree of glycosylation being collected separately from one another in the eluate fractions obtained. By means of the use of this process it is possible, for example, to prepare a human albumin which is free of glycoside bonds and is expressed in yeast cells.

Abstract: Human heat-shock protein-binding proteins (HspBP-1 and HspBP-2) are disclosed with the polynucleotides which identify and encode them. Genetically engineered expression vectors and host cells comprising nucleic acid sequences encoding heat-shock protein-binding proteins (HspBP) are also disclosed, as well as methods for producing HspBP polypeptides and for detecting polynucleotide sequences that encode HspBP polypeptides.

Abstract: The invention relates to a method for solubilising and purifying recombinant proteins which are expressed in bacterial host cells and are deposited as insoluble aggregates (inclusion bodies). The purification is based on conversion of the inclusion bodies into soluble forms using organic denaturing reagents and using chromatographic methods. Inorganic, alkaline, salt-containing eluents are selected here which, after purification is complete, enable the recombinant proteins, after neutralisation, to be made available in a form which can be employed directly for medical use and is physiologically acceptable. The method is particularly suitable for purifying allergens and allergen fragments.

Abstract: The present invention relates to a method for purifying a calcium ion-binding protein by cation exchange chromatography. The present invention provide a method for isolating and purifying a calcium ion-binding protein in a simple and efficient manner from a liquid sample containing a calcium ion-binding protein and contaminants without any pretreatment such as addition of a chelating agent. More specifically, the present invention relates to a method for purifying a calcium ion-binding protein which comprises contacting said protein with a cation exchange carrier in the presence of calcium ions to let the said protein be adsorbed to the carrier, and after washing, eluting said protein, and to a calcium ion-binding protein having substantially no contaminants obtained by the method of the present invention.

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, gel filter, or size exclusion device. The isolated plasminogen solution is then concentrated by a factor of from 2 to 10. The separated blood component, e.g.

Abstract: Method for desalting an aqueous liquid containing a charged substance comprising the steps of: (i) contacting liquid (I) with an ion-exchanger (1) under conditions permitting binding between ion-exchanger and substance, said ion-exchanger comprising a base matrix carrying an ion-exchange ligand (Ligand 1) with the opposite charge compared to the substance, (ii) desorbing said substance from said ion-exchanger by the use of a liquid (liquid (II)). Wherein: (A) ion-exchanger (1) is an ion-exchanger: (a) that can bind the substance in an aqueous reference liquid at ionic strength corresponding to 0.1 M NaCl, preferably 0.25 M NaCl; and (b) permits a breakthrough capacity at the pH provided by liquid (I) which is more than 2 mg/ml of gel, at a breakthrough of 10% and linear flow velocity of 300 cm/h; and (B) in step (ii) the pH of liquid (11) is adjusted to a pH value where the charge difference between the substance, the ligand and/or the ion-exchanger is lowered.

Abstract: Compositions of purified biologically active Wnt proteins are provided. Wnt proteins are found to be hydrophobic and post-translationally modified by addition of a lipid moiety at a conserved cysteine residue. Methods for isolation of Wnt utilize detergents that maintain the solubility of the modified protein.

Abstract: The invention relates to a process for the isolation and/or purification of a proteinaceous material, wherein a solid phase comprising a mixture of hydrophobic and hydrophilic groups is used.

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: This invention provides methods of retentate chromatography for resolving analytes in a sample. The methods involve adsorbing the analytes to a substrate under a plurality of different selectivity conditions, and detecting the analytes retained on the substrate by desorption spectrometry. The methods are useful in biology and medicine, including clinical diagnostics and drug discovery.

Abstract: The present invention relates to fractions of sources and or preparations of human chorionic gonadotropin, such as fractions of human early pregnancy urine, which fractions have anti-HIV activity. The present invention further relates to pharmaceutical compositions comprising such fractions for treating the effects of HIV infection.

Abstract: A cell culture preparation and methods are provided which are suitable for production in cells of a first protein in a class of proteins where the medium is deficient in a second protein in a related class, where the second protein is normally present in the serum and capable of interfering with the purification of the first compound.

Abstract: The present invention refers to the use of novel molecules able to bind tenaciously to silica, borosilicate and silicate surfaces, and thus to modify their properties and characteristics. When applied to silica-based chromatography, it offers important advantages in all cases in which it is necessary to modulate the interaction of analytes with the stationary phase. In capillary zone electrophoresis (CZE), such compounds will eliminate or invert the electroendoosmostic (EEO) flow, greatly simplifying the analysis of negatively-charged compounds and permitting the analysis of bio(macro)molecules via the direct use of naked capillaries.

Abstract: A 24 kd protein capable of binding the E2 envelope protein of hepatitis C virus (HCV), and functionally equivalent variants or fragments of the 24 kd protein, are disclosed. Processes for production and purification of the 24 kd protein, and functionally equivalent variants or fragments thereof, are also disclosed.

Abstract: NTNR&agr;, NTNR&agr; extracellular domain (ECD), NTNR&agr; variants, chimeric NTNR&agr; (e.g., NTNR&agr; immunoadhesion), and antibodies which bind thereto (including agonist and neutralizing antibodies) are disclosed. Various uses for these molecules are described, including methods to modulate cell activity and survival by response to NTNR&agr;-ligands, for example NTN, by providing NTNR&agr; to the cell.

Abstract: An affinity ligand-matrix conjugate comprises the matrix and, conjugated thereto by the group Z, a ligand having general formula (I) wherein on X is N and the other X is N, CCL or CCn; A1 and A2 are each independently O, S or N—R1 and R1 is H, C1-6alkyl, C1-6 hydroxyalkyl, benzyl or &bgr;-phenylethyl; B1 and B2 are each independently an optionally substituted hydrocarbon lingkage containing from 1 to 10 carbon atoms; D1 is H or a primary amino, secondary amino, tertiary amino, quaternary ammonium, imidazole, guanidino or amidino group; and D2 is a secondary amino, tertiary amino, quaternary ammonium, imadazole, guanidino or amidino group; of B2-D2 is —CHCOOH—(CH2)3-4—NH2; AND p is 0 or 1. Such conjugates are useful for the separation, isolation, purification, characterisation, identification or quantification of an endotoxin.

Abstract: The present invention provides porous monoliths with high flow permeability that can be produced by polymerising and divinylbenzene in the presence of an initiator, a carboxy-functionalized nitroxide stable free radical and polymeric porogens. The monoliths produced with these stable free radicals functionalized with carboxylic groups feature porosities very different from those produced by polymerizations involving other SFRs, characterized by very large surface areas in combination with relatively large through-pores. The invention also provides a method for producing the monoliths, a column containing the monoliths and an assay method using the column.

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: The invention concerns the use of an adsorptive size exclusion chromatography gel, said gel essentially consisting of a polysaccharide matrix whereon is grafted a polymer coupled with an affinity ligand and having a cleavage threshold ranging between 2 kDa and 60 kDa for eliminating a purifying biomolecules.

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: A 24 kd percent capable of binding the E2 envelope protein of hepatitis C virus (HCV), and functionally equivalent variants or fragments of the 24 kd protein, are disclosed. Processes for production and purication of the 24 kd protein, and functionally equivalent variants or fragments thereof, are also disclosed.

Abstract: The present invention relates to cartilage extracts and to a method of producing the same. Shark cartilage extracts having anti-angiogenic, anti-tumor, anti-inflammatory and anti-collagenolytic activities have been obtained by an improved process. The process comprises the steps of obtaining a crude cartilage extract in an aqueous solution, this crude extract being fractionated to recover molecules of a molecular weight less than about 500 kDa. Some of the biologically active components of the extract are prepared by further fractionation. The cartilage extract can be used for treating diseases or conditions having etiological components selected from the group consisting of tumor proliferation, angiogenesis, inflammation, metalloprotease activity and collagenolysis. Several cosmetic applications based on the capacity of the liquid extract to improve skin conditions are also disclosed. A simple and efficient process for the preparation of cartilage extracts is also disclosed.

Abstract: A web contactor for the purposes of continuous separation of specific proteins from a mixture of proteins comprises an endless, inert, non-porous, flexible web is coated with an activated matrix material which is, in turn, bound to a plurality of ligand molecules. The ligand molecules are chosen to correspond to a desired biological molecule or class of molecules, typically a desired protein. The web is translated over a series of rolls such that it contacts a feed solution of a mixture of biological molecules in a countercurrent manner. During contact of the web and solution, the ligand molecules which are attached to the web matrix, selectively bind to the protein or other biological molecule which are to be separated from the feed solution mixture.

Abstract: The invention concerns a method for purifying the Helicobacter adhesin-like protein A (AlpA) which consists in: (i) contacting an AlpA preparation and 2.5 to 3.5 M of guanidine with a hydrophobic interaction chromatography material, so that the AlpA is adsorbed on the material; and (ii) eluting the AlpA with a solution containing 3.5 to 4.5 M of guanidine. The AlpA preparation to be purified can be in particular derived from an E. coli culture capable of expressing AlpA in a high-level recombinant form, rAlpA being in the form of inclusion bodies, the latter being recovered and solubilized in the presence of guanidine, and optionally ammonium sulphate-precipitated for the purpose of preliminary purification. The hydrophobic interaction chromatography can be followed up by an anion exchange chromatography in the presence of 8 M of urea.

Abstract: Object of the invention is a process for the chromatographic purification of cyclosporin A from a crude product containing cyclosporin complex by using a column filled with silica gel and by the application of multistep chromatography with a column filled with normal phase silica gel and by a solvent mixture containing toluene as the major component.