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Небесная энциклопедия

Космические корабли и станции, автоматические КА и методы их проектирования, бортовые комплексы управления, системы и средства жизнеобеспечения, особенности технологии производства ракетно-космических систем

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Мониторинг СМИ

Мониторинг СМИ и социальных сетей. Сканирование интернета, новостных сайтов, специализированных контентных площадок на базе мессенджеров. Гибкие настройки фильтров и первоначальных источников.

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Форма поиска

Поддерживает ввод нескольких поисковых фраз (по одной на строку). При поиске обеспечивает поддержку морфологии русского и английского языка
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Применить Всего найдено 5447. Отображено 100.
26-01-2012 дата публикации

Preparative chromatography column and methods

Номер: US20120018380A1
Автор: Sarfaraz K. Niazi
Принадлежит: Therapeutic Proteins Inc

A chromatography column that captures components in a process liquid in a free flow state and allows elution in steps is described.

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Номер записи: 1
12-07-2012 дата публикации

Olefin metathesis reactions of amino acids, peptides and proteins containing allyl sulfide groups

Номер: US20120178913A1
Автор: Benjamin Guy Davis, Justin Mark Chalker, Yuya Angel Lin
Принадлежит: Oxford University Innovation Ltd

A method for the modification of an amino acid, protein or peptide is disclosed. The method comprises reacting a carbon-carbon double bond-containing compound with an amino acid, a protein or a peptide containing an allyl sulfide group in the presence of a catalyst which promotes olefin metathesis, to form a modified amino acid, protein or peptide. Preferred carbon-carbon double bond-containing compounds include carbohydrates.

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Номер записи: 2
14-02-2013 дата публикации

Chemical preparation of ubiquitin thioesters and modifications thereof

Номер: US20130041132A1
Автор: Ajish Kumar, Ashraf Brik, Leslie Erlich, Liat Spasser, Mahmood Haj-Yahya
Принадлежит: Ben Gurion University of the Negev Research and Development Authority Ltd

The present invention discloses latent thioester functionalities attached to the C-terminus of a first polypeptide, or a first fragment thereof having a Cys residue at its N-terminus, and a process using this functionality for the preparation of polypeptide thioesters, in particular of ubiquitin thioesters, this process comprising preparing a polypeptide or a fragment thereof, being attached to a latent thioester functionality, which can then be ligated with a second polypeptide fragment, followed by selective activation of the latent thioester functionality group, to provide the requested polypeptide thioester. There are also provided the polypeptides obtained by this method, specific unnatural amino acids useful to be incorporated within the polypeptide thioesters, and kits for preparing them.

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Номер записи: 3
14-03-2013 дата публикации

Synthesis of Site Specifically-Linked Ubiquitin

Номер: US20130065272A1
Автор: Chin Jason, Nguyen Duy, Virdee Satpal
Принадлежит: Medical Research Council

The invention relates to a method of modifying a specific lysine residue in a polypeptide comprising at least two lysine residues, said method comprising (a) providing a polypeptide comprising a target lysine residue protected by a first protecting group, and at least one further lysine residue; (b) treating the polypeptide to protect said further lysine residue(s), wherein the protecting group for said further lysine residues is different to the protecting group for the target lysine residue; (c) selectively deprotecting the target lysine residue; and (d) modifying the deprotected lysine residue of (c). 1. A method of modifying a specific lysine residue in a polypeptide comprising at least two lysine residues , said method comprising:a. providing a polypeptide comprising a target lysine residue protected by a first protecting group, and af least one further lysine residue;b. treating the polypeptide to protect said further lysine residue(s), wherein the protecting group for said further lysine residues is different to the protecting group for the target lysine residue;c. selectively deprotecting the target lysine residue; andd. modifying the deprotected lysine residue of (c).2. A method according to wherein producing the polypeptide comprisesa. providing a nucleic acid encoding the polypeptide which nucleic acid comprises an orthogonal codon encoding the target lysine;b. translating said nucleic acid in the presence of an orthogonal tRNA synthetase/tRNA pair capable of recognising said orthogonal codon and incorporating said target lysine residue protected by a first protecting group into the polypeptide chain.3. A method according to wherein said orthogonal codon comprises TAG claim 2 , said tRNA comprises MDtRNACUA and said tRNA synthetase comprises MbPylRS.4. A method according to claim 1 , wherein the target lysine residue protected by a first protecting group is Nε-(1-butyloxycarbonyl)-L-lysine.5. A method according to claim 1 , wherein the protecting group ...

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Номер записи: 4
28-03-2013 дата публикации

DETECTION COMPOSITION

Номер: US20130078621A1
Автор: McGarrigle Philip, Nolan Garry P.
Принадлежит: NODALITY, INC.

The invention provides methods, kits, devices and compositions for detecting one or more target analytes. In some embodiments, the invention provides binding elements and labeling elements capable of being joined through a plurality of joining elements. 1. A composition comprising:a binding element directed against an activatable element, wherein said binding element comprises a first joining element, wherein said first joining element comprises a first oligonucleotide attached to said binding element, and wherein said first oligonucleotide comprises a first complementary oligonucleotide region;a second joining element comprising a second oligonucleotide, wherein said second oligonucleotide comprises a labeling element, and wherein said second oligonucleotide comprises a second complementary oligonucleotide region; anda third joining element comprising a third oligonucleotide, wherein said third oligonucleotide comprises a third complementary oligonucleotide region and a fourth complementary oligonucleotide region, wherein said third complementary oligonucleotide region is complementary to said first complementary oligonucleotide region in said first joining element and wherein said fourth complementary oligonucleotide region is complementary to said second complementary oligonucleotide region in said second joining element.2. A composition comprising:a binding element directed against an activatable element, wherein said binding element comprises a first joining element, wherein said joining element comprises a first oligonucleotide attached to said binding element, and wherein said first oligonucleotide comprises a first complementary oligonucleotide region;a second joining element comprising a second oligonucleotide, wherein said second oligonucleotide comprises a labeling element, and wherein said second oligonucleotide comprises a second complementary oligonucleotide region, wherein said second complementary oligonucleotide region is complementary to said first ...

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Номер записи: 5
28-03-2013 дата публикации

METHODS OF PURIFYING POLYPEPTIDES

Номер: US20130079272A1
Автор: Kelley Brian David, Liu Hui F., McCooey Beth, Myers Deanna E., Petty Krista Marie
Принадлежит: Genentech, Inc.

The present invention provides methods for purifying a polypeptide from a composition comprising the polypeptide and at least one contaminant and formulations comprising the polypeptide purified by the methods. The methods for purifying include cation exchange material and/or mixed mode material. 1. A method for purifying a polypeptide from a composition comprising the polypeptide and at least one contaminant , wherein the method comprises either (i) or (ii):(i) sequential steps of (a) loading the composition onto a cation exchange material at a loading density of greater than about 150 g/L of cation exchange material; and (b) loading a composition recovered from the cation exchange material onto a mixed mode material; or(ii) sequential steps of (a) loading the composition onto a mixed mode material; and (b) loading a composition recovered from mixed mode material onto a cation exchange material at a loading density of greater than about 150 g/L of cation exchange material.2. The method of claim 1 , wherein the polypeptide has a pI of between about 6 and about 10.3. The method of claim 2 , wherein the polypeptide has a pI of between about 7 and about 9.4. The method of claim 1 , wherein the polypeptide is an antibody or an immunoadhesin.5. The method of claim 4 , wherein the polypeptide is an immunoadhesin.6. The method of claim 4 , wherein the polypeptide is an antibody.7. The method of claim 6 , wherein the antibody is a monoclonal antibody.8. The method of claim 7 , wherein the monoclonal antibody is a chimeric antibody claim 7 , humanized antibody claim 7 , or human antibody.9. The method of claim 7 , wherein the monoclonal antibody is an IgG monoclonal antibody.10. The method of claim 6 , wherein the antibody is an antigen binding fragment.11. The method of claim 10 , wherein the antigen binding fragment is selected from the group consisting of a Fab fragment claim 10 , a Fab′ fragment claim 10 , a F(ab′)fragment claim 10 , a scFv claim 10 , a Fv claim 10 , and ...

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Номер записи: 6
18-04-2013 дата публикации

PROCESS FOR THE PURIFICATION OF A GROWTH FACTOR PROTEIN

Номер: US20130096279A1
Автор: Gilljam Gustav, Tiemeyer Maya, Winge Stefan
Принадлежит: Octapharma AG

A process of purifying a Growth Factor Protein in a purification sequence employing chromatography characterized in that 1. A process of purifying a Growth Factor Protein selected from the group consisting of Colony Stimulating Factor (CSF) , interleukin 3 (IL-3) , Hepatocyte growth factor , Epidermal growth factor and fibroblast growth factor (acid) in a purification sequence employing chromatography comprisingbinding the Growth Factor Protein to a multimodal resin at a pH between 4 to 6.2, andeluting the Growth Factor Protein at a pH>6.3.2. The process of wherein the multimodal resin comprises moieties bound to a matrix and the moieties are able to interact with the Growth Factor Protein in a mixture by ionic interactions claim 1 , hydrogen bonding claim 1 , and/or hydrophobic interaction.3. The process of wherein the mixture comprising the Growth Factor Protein is a solution.4. The process of wherein the Growth Factor Protein is a recombinant Growth Factor Protein.5. The process of wherein the Growth Factor Protein is eluted by a pH change>pH 6.3.6. The process of wherein the elution is performed with a change of the pH or with an elution agent comprising an amino acid having a basic side chain and/or high ionic strength in an elution buffer.7. The process according to wherein the concentration of the elution agent is in the range of from about 0.1 M to about 2.0 M.8. The process of wherein the Growth Factor Protein binds to the multimodal resin at pH about 4.0 to pH about 6.0 claim 1 , and the Growth Factor Protein is eluted from the multimodal resin at pH 6.5 or higher.9. The process of comprising using a buffering substance comprising sodium citrate claim 1 , sodium acetate claim 1 , or HEPES.10. The process of wherein a non-ionic detergent is present in any of the buffers used.11. The process of wherein the amino acid having a basic side chain is arginine claim 8 , lysine and/or histidine.12. The process of wherein buffers comprising about 0.1 M-2 M sodium ...

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Номер записи: 7
18-04-2013 дата публикации

METHOD FOR PURIFYING PROTEIN USING AMINO ACID

Номер: US20130096284A1
Автор: ISHIHARA Takashi
Принадлежит: KYOWA HAKKO KIRIN CO., LTD.

In large-scale purification of proteins such as antibodies, an economic high-purity purification method is required. The present invention relates to a method for purifying a protein, including one or more chromatographic processes, in which an amino acid; or a dipeptide, an oligopeptide, or a polyamino acid thereof is included in a buffer solution used in at least one chromatographic processes (equilibration buffer, wash buffer, and elution buffer), thereby purifying a high-purity protein with a very small quantity of the impurity (e.g., polymers or host cell proteins). 1. A method for purifying a protein , comprising one or more chromatographic processes , wherein an amino acid is included as an ingredient of a buffer solution or a part thereof used in at least one chromatographic process.2. The purification method according to claim 1 , wherein an amino acid is included as an ingredient of a buffer solution or a part thereof used in a cation exchange chromatographic process.3. The purification method according to claim 2 , which further comprises claim 2 , as the chromatographic process claim 2 , a Protein A affinity chromatographic process and one or more chromatographic processes selected from an anion exchange chromatographic process claim 2 , a gel filtration chromatographic process claim 2 , a hydrophobic chromatographic process claim 2 , a hydroxyapatite chromatographic process claim 2 , and a mixed mode chromatographic process.4. The purification method according to claim 2 , wherein the cation exchange chromatographic process is carried out subsequent to the Protein A affinity chromatographic process.5. The purification method according to claim 4 , wherein the anion exchange chromatographic process is further subsequently carried out.6. The purification method according to claim 3 , wherein the cation exchange chromatographic process carried out subsequent to the Protein A affinity chromatographic process and the anion exchange chromatographic process.7. ...

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Номер записи: 8
25-04-2013 дата публикации

METHOD TO PRODUCE AN IMMUNOGLOBULIN PREPARATION WITH IMPROVED YIELD

Номер: US20130101579A1
Автор: Bruckschwaiger Leopold, Butterweck Harald Arno, Grausenburger Reinhard, Gundinger Thomas, Koelbl Bernhard, Nuernberger Julia, Pljevljakovic Azra, Schwarz Hans-Peter, Svatos Sonja, Teschner Wolfgang
Принадлежит:

The present invention provides improved methods for the manufacturing of IVIG products. These methods offer various advantages such as reduced loss of IgG during purification and improved quality of final products. In other aspects, the present invention provides aqueous and pharmaceutical compositions suitable for intravenous, subcutaneous, and/or intramuscular administration. In yet other embodiments, the present invention provides methods of treating a disease or condition comprising administration of an IgG composition provided herein. 1. A method for preparing an enriched IgG composition from plasma , the method comprising the steps of:(a) precipitating a cryo-poor plasma fraction, in a first precipitation step, with from about 6% to about 10% alcohol at a pH of from about 7.0 to about 7.5 to obtain a first precipitate and a first supernatant;(b) precipitating IgG from the first supernatant, in a second precipitation step, with from about 23% to about 27% alcohol at a pH of from about 6.7 to about 7.1 at a temperature of from about −7° C., to about −9° C. to form a second precipitate;(c) suspending the second precipitate to form a suspension;(d) precipitating IgG from the suspension formed in step (c), in a third precipitation step, with from about 22% to about 28% alcohol at a pH of from about 6.7 to about 7.3 to form a third precipitate;(e) re-suspending the third precipitate to form a suspension; and(f) separating the soluble fraction from the suspension formed in step (e), thereby forming an enriched IgG composition.258-. (canceled)59. The method of claim 1 , wherein the temperature of precipitation step (b) is about −7° C.60. The method of claim 1 , wherein the alcohol concentration of precipitation step (b) is about 25%.61. The method of claim 59 , wherein the alcohol concentration of precipitation step (b) is about 25%.62. The method of claim 1 , wherein at least one of the first precipitation step claim 1 , second precipitation step claim 1 , or third ...

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Номер записи: 9
25-04-2013 дата публикации

IRON (III) CASEINSUCCINYLATE AND METHOD FOR THE PREPARATION THEREOF

Номер: US20130102529A1
Автор: De Ferra Lorenzo, Pinto Barbara, Stevenazzi Andrea
Принадлежит: ITALFARMACO S.P.A.

The present invention refers to an iron (III) caseinsuccinylate characterised by a content of iron comprised between 4.5% and 7% by weight, and by a solubility in water approximately greater than 92%. 118-. (canceled)19. Iron (III) caseinsuccinylate characterised by a content of iron comprised between 4.5 and 7% by weight , by a solubility greater than about 92% and by having a phosphorous/nitrogen ratio greater than about 5% by weight.20. Iron (III) caseinsuccinylate according to claim 19 , containing an amount of protein impurities lower than about 15% by weight.21. Method for preparing iron (III) caseinsuccinylate comprising the following steps:a. reacting casein and succinic anhydride in water to obtain succinylated casein; andb. reacting said succinylated casein with iron (III) chloride to obtain iron (III) caseinsuccinylate,wherein said casein according to step a) is purified casein, having a protein impurities content lower than 15% by weight and/or inorganic impurities content lower than 1% by weight and/or a phosphorous/nitrogen ratio greater than about 5%.22. Method according to claim 21 , wherein said protein impurities content is lower than about 10% by weight and/or said inorganic impurities content is lower than about 0.4% by weight.23. Method according to claim 21 , wherein said protein impurities are proteins different from alpha-S1 casein claim 21 , alpha-S2 casein claim 21 , beta casein claim 21 , kappa casein claim 21 , pseudo-kappa casein.24. Method according to claim 21 , wherein said purified casein has a phosphorous/nitrogen ratio greater than about 5% by weight.25. Method according to claim 21 , wherein said purified casein is obtained from acid casein or presamic casein.26. Method according to claim 21 , comprising an additional step of microfiltration of the aqueous solution of iron (III) caseinsuccinylate.27. Iron caseinsuccinylate claim 21 , obtainable through the method according to .28. Pharmaceutical composition containing iron (III) ...

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Номер записи: 10
25-04-2013 дата публикации

MANUFACTURE OF FACTOR H (FH) AND FH-DERIVATIVES FROM PLASMA

Номер: US20130102537A1
Автор: Bairstow Shawn F., JOHNSON Richard, Madlener Ruth, Ramachandran Sindhu, Schwarz Hans-Peter, Teschner Wolfgang
Принадлежит:

The present invention provides compositions and pharmaceutical formulations of Factor H derived from plasma. Also provided are methods for the manufacture of the Factor H compositions and formulations, as well as methods for the treatment of diseases associated with Factor H dysfunction. 114-. (canceled)15. A method for preparing an enriched Factor H composition from plasma , the method comprising the steps of:(a) extracting Factor H from a Fraction I precipitate and/or a Fraction II+III precipitate, to form a Factor H extract; and(b) precipitating an impurity from the Factor H extract to form a supernatant containing Factor H, thereby preparing an enriched Factor H composition.1627-. (canceled)28. The method of claim 15 , wherein extraction step (a) comprises re-suspending the Fraction II+III precipitate with a re-suspension buffer having a pH of from 4.0 to 5.5.29. The method of claim 15 , wherein precipitation step (b) comprises precipitating an impurity from the Factor extract using a final alcohol concentration of from 10% to 20% at a pH of from 7.0 to 9.0.30. The method of claim 15 , wherein precipitation step (b) comprises precipitating an impurity from the Factor H extract using a final alcohol concentration of from 14% to 16% at a pH of from 7.5 to 8.5.31. The method of claim 15 , wherein the method further comprises the step of:(c) precipitating Factor H from the supernatant formed in step (h) using a final alcohol concentration of from 20% to 30% at a pH of from 5.0 to 7.0.32. The method of claim 15 , wherein the method further comprises the step of:(c) precipitating Factor H from the supernatant formed in step (b) using a final alcohol concentration of from 24% to 26% at a pH of from 5.8 to 6.2.33. The method of claim 28 , wherein the method further comprises the step of:(c) precipitating Factor H from the supernatant formed in step (b) using a final alcohol concentration of from 20% to 30% at a pH of from 5.0 to 7.0.34. The method of claim 28 , wherein ...

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Номер записи: 11
02-05-2013 дата публикации

DIFFERENTIATION OF ISOBARIC AMINO ACIDS AND OTHER SPECIES

Номер: US20130109578A1
Автор: Cha Junhoe, Heath James R., Lee Su Seong, Lim Jaehong, Tan Sylvia, Yeo Shi Yun
Принадлежит: AGENCY FOR SCIENCE, TECHNOLOGY AND RESEARCH

Techniques for differentiating isobaric species are described. An isobaric species may be substituted with a tagging species identified using mass spectrometry. The isobaric species may be a subunit of a first polymer having a defined sequence, e.g., the isobaric species may be an amino acid in a protein or a peptide sequence. A tagging species may be substituted for the isobaric species in a second polymer having an otherwise identical sequence as the first polymer. The second polymer may have the same number of sequences as the first polymer, and substantially the same sequence of subunits, with a few exceptions such as the tagging species for the isobaric species. The first polymer and the second polymer may be prepared in the same reaction vessel. A polymer/protein of defined subunit sequence containing an isobaric species or a tagging species may be analyzed by mass spectrometry to determine the sequence. 1. A method , comprising:growing amino acid sequences on a surface, wherein at least one of the amino acids within the amino acid sequence is an isobaric amino acid, and wherein, when the isobaric amino acid is added to a medium exposed to the sequences for addition thereto, the medium containing the isobaric amino acid further contains an entity, incorporatable in an amino acid sequence, that has a molecular weight different than the isobaric amino acid.2. The method of claim 1 , wherein at least some of the amino acid sequences are attached to the surface by a cleavable linker.3. The method of claim 1 , further comprising analyzing the amino acid sequence by mass spectrometry.4. The method of claim 3 , wherein the mass spectrometry comprises MALDI-TOF/TOF.5. The method of claim 3 , wherein analyzing the amino acid sequences comprises sequencing the amino acid sequences.6. The method of claim 1 , wherein isobaric amino acid is an unnatural amino acid.7. The method of claim 1 , wherein the entity comprises an unnatural amino acid.8. A composition claim 1 , ...

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Номер записи: 12
02-05-2013 дата публикации

Method for processing protein-containing materials to obtain mixtures of natural amino acids, low-molecular weight peptides and oligopeptides

Номер: US20130109835A1
Автор: Ermilov Valeriy V., Fedorova Tatiyana Y., Kolotilin Dmitriy V., Polyakov Victor S.
Принадлежит: P.M.K. TECHNOLOGICAL TREATMENT LTD.

A method for processing protein-containing materials and utilization of protein-containing waste for production of mixtures of natural amino acids, mixtures of low-molecular weight peptides, and mixtures of oligopeptides, which can be used in the manufacture of reagents for the detoxification and bactericidal treatment of sewage sludge, surfactants, wetting agents, foaming agents, detergents and cleaners. The method includes processing protein-containing waste by hydrolysis with aqueous solutions of alkalis with additives in the form of ammonium salts of organic and mineral acids introduced into the reaction mixture of these components, and the wastes are processed in the mixture with water, alkali and additive, in the ratio of 1:(0.3-1.5):(0.03-0.45):(0.01-0.2), at 100-110° C. for 20-60 min. 1. A method for processing protein-containing waste by hydrolysis with aqueous solutions of alkalis , comprising:introducing additives in a form of ammonium salts of organic and mineral acids into a reaction mixture of the protein-containing waste;agitating the waste in the mixture with water, an alkaline agent and the additive, in a ratio of 1:(0.3-1.5):(0.03-0.45):(0.01-0.2) at a temperature of 100-110° C. for 20-60 min.2. The method of claim 1 , wherein the alkaline agent is sodium hydroxide.3. The method of claim 1 , wherein the alkaline agent is potassium hydroxide.4. The method of claim 1 , wherein the processing of the protein-containing waste mixed with the water claim 1 , the alkaline agent and the additive claim 1 , in a ratio of 1:(1.0-1.5):(0.30-0.45):(0.01-0.2) claim 1 , is carried out at 100-110° C. for 20-45 min yielding a mixture of alkali metal salts of natural amino acids.5. The method of claim 1 , wherein the processing of the protein-containing waste mixed with the water claim 1 , the alkaline agent and the additive claim 1 , in a ratio of 1:(0.6-1.3):(0.16-0.29):(0.01-0.14) claim 1 , is carried out at 100-110° C. for 25-50 min claim 1 , yielding the ...

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Номер записи: 13
09-05-2013 дата публикации

PURIFICATION OF PROTEINS

Номер: US20130116413A1
Автор: Kardekar Prashant, Kulkarni Samir, Raghvendra Y., Subrahmanyam Satyam
Принадлежит:

The invention describes a method for protein purification. More particularly, the invention relates to a purification process comprising protein A chromatography and anion exchange chromatography wherein protein A chromatography eluate is further purified by anion exchange chromatography at similar pH or at a pH less than or equal to 6. 1. A process for purifying an antibody , comprising:a) purifying using protein A chromatography, wherein the antibody is eluted at low pH; andb) purifying using anion exchange chromatography performed in the flow-through mode, wherein eluate obtained from step a) is loaded onto an anion exchange resin at pH values less than or equal to 6.2. A process according to claim 1 ,wherein the antibody is eluted in step a) at pH values about 3.3 to about 4.5 and loaded onto the anion exchange resin at pH values about 3.3 to about 6.3. A process according to claim 1 , wherein the antibody is eluted in step a) at pH values about 3.5 and loaded onto the anion exchange resin at pH values about 3.5 to about 6.4. A process according to claim 1 , wherein the antibody is loaded onto the anion exchange resin at pH values about 4.5. A process according to claim 1 , wherein the antibody is loaded onto the anion exchange resin at pH values about 6.6. A process according to claim 1 , wherein the anion exchange chromatography is followed by a cation exchange chromatography step in a bind-elute mode claim 1 , and wherein the flow-through from the anion exchange chromatography step is loaded onto a cation exchange resin at pH values less than or equal to 6.7. A process for purifying an antibody claim 1 , comprising:a) purifying using protein A chromatography, wherein the antibody is eluted at low pH values; andb) purifying using anion exchange chromatography, performed in the flow-through mode, wherein eluate obtained from step a) is loaded on to the anion exchange resin without substantial adjustment of pH. Aspects of this application relate to purification ...

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Номер записи: 14
16-05-2013 дата публикации

Simultaneous synthesis of temperature-tunable peptide and gold nanoparticle hybrid spheres

Номер: US20130123466A1
Автор: Hor-Gil Hur, Jungok Kim
Принадлежит: GWANGJU INSTITUTE OF SCIENCE AND TECHNOLOGY

The present invention relates to a novel synthesis of peptide-gold nanoparticle hybrid spheres comprising a step of forming a hybrid structure by inducing self-assembly of a gold-binding peptide, and forming a gold nanoparticle in the structure at the same time. According to the present invention, size of the structure can be controlled according to temperature, and it can be used for various biomedical and electronic applications using the structure.

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Номер записи: 15
16-05-2013 дата публикации

METHOD FOR EXTRACTING UNDENATURED TYPE II COLLAGEN HAVING ACTIVE EPITOPE

Номер: US20130123468A1
Автор: Moriyama Hiroyoshi, Shiojima Yoshiaki, Yoshinari Orie
Принадлежит:

A method for extracting water-soluble undenatured type II collagen having an active epitope comprises a first step for extracting animal-derived cartilage using an acidic solution at 50° C. or less, and a second step for adding pepsin to the acidic solution and performing extraction at 40° C. or less. This allows the epitope of undenatured type II collagen to be extracted in large quantities and with high efficiency without any loss of activity, and makes it possible to provide a food or beverage product obtained using the water-soluble undenatured type II collagen extracted by the extraction method. 111-. (canceled)12. An extraction method wherein water-soluble undenatured type II collagen is extracted and an active epitope contained in the water-soluble undenatured type II collagen is extracted , comprising the steps of: (1) temperature: 5 to 35° C.,', '(2) pH: 3.4 or less, and', '(3) extraction time period: 12 to 36 hours; and, 'extracting animal-derived cartilage with an acidic solution under the following conditions (1) temperature: 5 to 35° C.,', '(2) pH: 3.4 or less, and', '(3) extraction time period: 12 to 36 hours., 'adding pepsin to the acidic solution and extracting an active epitope with the acidic solution under the following conditions13. The extraction method according to claim 12 , wherein the step of extracting animal-derived cartilage with the acidic solution is a first step and the step of adding pepsin to the acidic solution and extracting an active epitope with the acidic solution is a second step claim 12 , and the total extraction time for both the first step and the second step is 48 hours or less.14. The extraction method according to claim 13 , wherein the second step is performed at lower temperature than the first step.15. The extraction method according to claim 13 , wherein elastase is further added in the second step of extraction.16. The extraction method according to claim 14 , wherein elastase is further added in the second step of ...

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Номер записи: 16
16-05-2013 дата публикации

FRACTIONATION OF PROTEINS AND LIPIDS FROM MICROALGAE

Номер: US20130123469A1
Автор: Hatcher Patrick G., Kumar Sandeep
Принадлежит: Old Dominion University Research Foundation

Methods of fractionating proteins and lipids in algae are provided. The methods can extract peptides and amino acids from algae, can produce components used in food or fertilizer, and can improve algal biomass feed for biofuel production, as non-limiting examples. One embodiment of a method as disclosed herein comprises providing a feed material of algae saturated with water to a reactor, bringing the water saturating the algae to a subcritical temperature within the reactor and separating a reactor effluent into solids and liquid. 1. A method of fractionating proteins from algae comprising:feeding to a reactor a biomass of algae saturated with water;bringing the water saturating the algae to a subcritical temperature within the reactor; andseparating a reactor effluent into solids and liquid.2. The method of claim 1 , wherein feeding the biomass comprises:providing algae from an algae source;providing water from a water source; andmixing the algae and water prior to feeding the reactor.3. The method of claim 1 , wherein the subcritical temperature is between 200° C. and 350° C.4. The method of claim 1 , wherein the reactor is a continuous feed reactor and a residence time of within the reactor is less than two minutes.5. The method of claim 4 , wherein the residence time is less than or equal to ten seconds.6. The method of claim 1 , wherein the algae saturated with water has a solids load of between 0.01 and 7.5 dry weight percent.7. The method of further comprising recycling the liquid for use in algae cultivation.8. The method of further comprising:selecting the subcritical temperature in combination with a residence time to extract at least 60 percent of a total nitrogen content in the algae from the solids into the liquid.9. The method of claim 8 , wherein the nitrogen content of the liquid is in the form of proteins and peptides.10. The method of claim 9 , wherein the proteins and peptides are amino acids.11. The method of claim 8 , wherein the subcritical ...

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Номер записи: 17
23-05-2013 дата публикации

COMPOUND FOR USE IN PEPTIDE SYNTHESIS

Номер: US20130131286A1
Автор: Liu Chuan Fa
Принадлежит: NANYANG TECHNOLOGICAL UNIVERSITY

The present invention generally relates to processes and methods of peptide and protein synthesis. The present invention also relates to specific compounds for use in such processes and methods. It is shown herein that peptides with a C-terminal tertiary N,N-bis(2-mercaptoethyl)-amide (BMEA) undergo N-to-S acyl transfer at weakly acidic pH to form a transient thioester which can be captured for direct ligation with a cysteinyl peptide. These C-terminal BMEA peptides are easily prepared with standard Fmoc solid-phase synthesis protocols, thus giving a very convenient access to the thioester components for native chemical ligation. 1. A method of peptide synthesis comprising a compound as set forth in or , or a resin as set forth in .2. A process for the production of a peptide comprising a C-terminal tertiary N claim 11 ,N-bis(2-mercaptoethyl)-amide group; N-(2-mercaptoethyl)-N-(3-aminopropyl)-amide group or N claim 11 ,N-bis(3-mercaptopropyl)-amide claim 11 , the process comprising the step of providing a dialkylamine resin as set forth in .3. A method according to or a process according to wherein the method or process respectively comprises native chemical ligation (NCL).4. A method or process according to wherein the method or process respectively further comprises: (i) converting the ligation product to a thioester and performing NCL between the thioester and a cys-peptide; or (ii) allowing the ligation product to directly react with a cys-peptide in situ;and wherein one or more further ligation steps may optionally follow.5. A method or process according to any one of the preceding claims wherein the process comprises the addition of a thiol additive and wherein the thiol additive is optionally selected from the group consisting of: alkyl thiol claim 3 , benzylmercaptan claim 3 , MESNa and methyl mercaptoacetate.6. A method or process according to any one of the preceding claims wherein the method or process comprises Fmoc solid-phase peptide synthesis.7. A ...

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Номер записи: 18
23-05-2013 дата публикации

PREPARATION OF CANOLA PROTEIN ISOLATE FROM CANOLA OIL SEEDS ("BLENDERTEIN")

Номер: US20130131316A1
Автор: Green Brent E., Schweizer Martin, Segall Kevin I.
Принадлежит:

Canola protein isolate is recovered from canola oil seeds by crushing the oil seeds and extracting the crushed canola oil seeds. Fat co-extracted from the crushed oil seeds is removed from the aqueous canola protein solution which then is processed by the micellar route to obtain the canola protein isolate. 1. A process for the preparation of a canola protein isolate , which comprises:grinding canola oil seeds,extracting the ground canola oil seeds with an aqueous extracting medium to solubilize canola protein and fats in the ground canola oil seeds to form an aqueous canola protein solution,separating the aqueous canola protein solution from residual ground canola oil seeds,defatting the aqueous canola protein solution,clarifying the defatted aqueous canola protein solution,concentrating the clarified aqueous canola protein solution while maintaining the ionic strength substantially constant to form a concentrated canola protein solution,diluting the concentrated protein solution into chilled water to cause the formation of canola protein micelles,collecting the canola protein micelles as a protein micellar mass, anddrying the protein micellar mass to form a canola protein isolate having a protein content of at least about 90 wt % (N×6.25) d.b.2. The process of claim 1 , wherein said aqueous extracting medium is an aqueous salt solution having an ionic strength of at least about 0.05 M with a pH of about 5 to about 6.8 to form a canola protein solution having a concentration of about 3 to about 40 g/L.3. The process of wherein an antioxidant is present in the aqueous extracting medium.4. The process of wherein said defatting step is effected by chilling the canola protein solution to a temperature of about 3° to about 7° C. and removing fat that separates from the canola protein solution.5. The process of wherein claim 4 , following the defatting step claim 4 , the separated aqueous canola protein solution is subjected to a colour removal step.6. The process of ...

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Номер записи: 19
06-06-2013 дата публикации

METHOD FOR PRODUCING PEPTIDE

Номер: US20130143262A1
Автор: FUKE Ichiro, Furukawa Shinya, Hasegawa Kazuhiro
Принадлежит: AJINOMOTO CO., INC.

Peptides may be produced by allowing (A) a first amino acid or peptide, which is converted into its ionic liquid form, (B) a second amino acid or peptide, and (C) a peptide hydrolase to simultaneously exist in a single reaction system, wherein the first amino acid or peptide, which is converted into its ionic liquid form, is used as both a reaction solvent and a reaction starting material; and forming a peptide bond between the first amino acid or peptide and the second amino acid or peptide. By such a process, it is possible to synthesize a peptide at a high concentration and at a high yield, and the method is excellent for producing peptides on an industrial scale. 1. A method for producing a peptide , which comprises:(a) allowing (A) a first amino acid or peptide, which is converted into its ionic liquid form, (B) a second amino acid or peptide, and (C) a peptide hydrolase to simultaneously exist in a single reaction system, wherein the first amino acid or peptide, which is converted into its ionic liquid form, is used as both a reaction solvent and a reaction starting material; and(b) forming a peptide bond between said first amino acid or peptide and said second amino acid or peptide.2. A method according to claim 1 , wherein said second amino acid or peptide (B) is converted into its ionic liquid form.3. A method according to claim 2 , wherein water is present in said reaction system.4. A method according to claim 1 , wherein said first amino acid or peptide of (A) is protected at its amino group or carboxyl group.5. A method according to claim 2 , wherein said first amino acid or peptide of (A) is protected at its amino group or carboxyl group.6. A method according to claim 3 , wherein said first amino acid or peptide of (A) is protected at its amino group or carboxyl group.7. A method according to claim 1 , wherein (A) said first amino acid or peptide claim 1 , which is converted into its ionic liquid form claim 1 , is a carboxylate.8. A method according to ...

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Номер записи: 20
13-06-2013 дата публикации

99mTc-LABELED TRIPHENYLPHOSPHONIUM DERIVATIVE CONTRASTING AGENTS AND MOLECULAR PROBES FOR EARLY DETECTION AND IMAGING OF BREAST TUMORS

Номер: US20130149240A1
Автор: Hardy Micael Joel, Kalyanaraman Balaraman, Lopez Marcos, Zhao Ming
Принадлежит:

99mTc-labeled triphenylphosphonium contrasting agents that target the mitochondria and are useful for early detection of breast tumors using scintimammographic imaging. 99mTc-Mito10-MAG3 possesses advantageous radiopharmaceutical properties. The uptake in the myocardium is reduced by one to two orders of magnitude compared to 99mTc-MIBI. 99mTc-Mito10-MAG3 exhibits fast blood clearance, with a blood half-life of less than 2 minutes in rats. A diminished myocardial uptake combined with a prompt reduction of cardiovascular blood pool signal to facilitate improved signal-to-background ratios. 2. The compound of claim 1 , wherein X— is Cl— claim 1 , I— or F—.3. The compound of claim 1 , wherein the salt-forming counterion is acetate claim 1 , benzenesulfonate claim 1 , benzoate claim 1 , bicarbonate claim 1 , bitartrate claim 1 , bromide claim 1 , calcium edetate claim 1 , camsylate claim 1 , carbonate claim 1 , chloride claim 1 , citrate claim 1 , dihydrochloride claim 1 , edetate claim 1 , edisylate claim 1 , estolate claim 1 , esylate claim 1 , fumarate claim 1 , gluceptate claim 1 , gluconate claim 1 , glutamate claim 1 , glycollylarsanilate claim 1 , hexylresorcinate claim 1 , hydrabamine claim 1 , hydrobromide claim 1 , hydrochloride claim 1 , hydroxynaphthoate claim 1 , iodide claim 1 , isethionate claim 1 , lactate claim 1 , lactobionate claim 1 , malate claim 1 , maleate claim 1 , mandelate claim 1 , mesylate claim 1 , methylbromide claim 1 , methylnitrate claim 1 , methylsulfate claim 1 , mucate claim 1 , napsylate claim 1 , mitrate claim 1 , pamoate claim 1 , pantothenate claim 1 , phosphate claim 1 , diphosphate claim 1 , polygalacturonate claim 1 , salicylate claim 1 , stearate claim 1 , subacetate claim 1 , succinate claim 1 , sulfate claim 1 , tannate claim 1 , tartrate claim 1 , teoclate or triethiodide.4. The compound of claim 1 , wherein R2 is a branched or straight chain claim 1 , saturated or unsaturated claim 1 , substituted or unsubstituted C4-15 ...

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Номер записи: 21
13-06-2013 дата публикации

METHOD FOR THE PURIFICATION OF RECOMBINANT BLOOD COAGULATION FACTOR IX ENRICHED IN SULFATED AND/OR PHOSPHORYLATED MOLECULES

Номер: US20130150557A1
Автор: Fielder Christian, Hasslacher Meinhard, Mitterer Artur
Принадлежит:

The present invention relates to a method for the purification of rFIX using anion exchange chromatography in the pseudo-affinity mode, wherein said method comprises a wash step with a wash buffer having a salt concentration of more than 200 mM. The purification according to the invention provides a method to enrich rFIX molecules which have been posttranslationally modified by sulfation and/or phosphorylation. The present invention further relates to purified rFIX compositions enriched in monosulfated and/or monophosphorylated rFIX molecules. 17-. (canceled)8. A purified composition comprising rFIX obtained by a method , the method for obtaining the composition comprising the steps:a) loading a starting composition comprising rFIX onto an anion exchange column.,b) washing the anion exchange material using a wash buffer which has a salt concentration of more than 200 mM (High Salt Wash Buffer), andc) eluting the rFIX from the anion exchange material using an elution buffer comprising divalent cations, andd) collecting the eluate.9. The purified composition according to claim 8 , wherein said purified composition comprises an increased relative amount of monosulfated and/or monophosphorylated rFIX molecules compared to the relative amount of monosulfated and/or monophosphorylated rFIX present in the High Salt Wash Buffer fraction.10. The purified composition according to claim 8 , wherein said purified composition comprises a relative amount of monosulfated and monophosphorylated rFIX molecules which is increased at least 3 fold compared to the relative amount of monosulfated and monophosphorylated rFIX present in the High Salt Wash Buffer fraction.11. The purified composition according to claim 8 , wherein said purified composition comprises a relative amount of at least 30% of monosulfated and/or monophosphorylated rFIX molecules.12. The purified composition according to claim 8 , wherein said purified composition comprises a relative amount of at least 50% of ...

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Номер записи: 22
20-06-2013 дата публикации

METHODS AND COMPOSITIONS FOR CONTROLLING ASSEMBLY OF VIRAL PROTEINS

Номер: US20130156818A1
Автор: de los Rios Miguel, de los Rios Stephanie, Oh Kenneth J., Ostrowski Jacek, Zipkin Ilan
Принадлежит:

Provided herein are methods and compositions for controlling assembly of modified viral core proteins, for example, into a viral capsid or a nanocage. In some embodiments, the disclosed modified viral core proteins comprise at least one mutation or modification that can substantially prevent assembly of the viral core proteins until assembly is desired. In some embodiments, assembly of the viral core proteins may be triggered, for example, by contacting the viral core proteins with a reducing agent and/or by reducing the concentration of a denaturant. The viral core proteins may self-assemble to form a viral capsid or nanocage. 1. A method for assembling a modified Hepatitis B Virus (HBV) core protein into a capsid structure , the method comprising:providing a solution comprising a modified HBV core protein and a first concentration of a denaturing agent, wherein the spike region of the modified HBV core protein comprises a cysteine residue; andadding a reducing agent to the solution,thereby to form an assembled capsid structure.2. The method of claim 1 , wherein the modified HBV core protein comprises one or more modifications of as least one of amino acid sequence SEQ ID NO: 1 and SEQ ID NO: 2.3. The method of claim 1 , wherein the spike region comprises amino acids 74 to 84 of at least one of SEQ ID NO: 1 and SEQ ID NO: 2.4. The method of claim 2 , wherein the modified HBV core protein comprises a cysteine at amino acid position 77 claim 2 , 79 or 80 of at least one of SEQ ID NO: 1 and SEQ ID NO: 2.5. The method of claim 2 , wherein the modified HBV core protein comprises a cysteine at amino acid position 77 of at least one of SEQ ID NO: 1 and SEQ ID NO: 2.6. The method of claim 1 , wherein the reducing agent is at least one of beta-mercaptoethanol (BME) claim 1 , tris(2-carboxyethyl)phosphine (TCEP) claim 1 , glutathione (GSH) claim 1 , dithiothreitol (DTT) claim 1 , 2-mercaptoyethylamine (BMA) and free cysteine.7. The method of claim 1 , wherein the ...

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Номер записи: 23
20-06-2013 дата публикации

Extracellular Matrix-Derived Gels and Related Methods

Номер: US20130156862A1
Автор: Badylak Stephen F., Freytes Donald
Принадлежит: UNIVERSITY OF PITTSBURGH-OF THE COMMONWEALTH SYSTEM OF HIGHER EDUCATION

Provided are methods for preparing gelled, solubilized extracellular matrix (ECM) compositions useful as cell growth scaffolds. Also provided are compositions prepared according to the methods as well as uses for the compositions. In one embodiment a device, such as a prosthesis, is provided which comprises an inorganic matrix into which the gelled, solubilized ECM is dispersed to facilitate in-growth of cells into the ECM and thus adaptation and/or attachment of the device to a patient. 196-. (canceled)97. A method of preparing an extracellular matrix-derived gel comprising: (a) solubilizing decellularized extracellular matrix (ECM) derived from cardiac tissue by digestion with an acid protease in an acidic solution to produce digested cardiac ECM; and (b) raising the pH of the digested cardiac ECM to a pH between 7.2 and 7.8 to produce a neutralized digest solution , and (c) gelling the neutralized digest solution at a temperature greater than 25° C.98. The method of claim 97 , wherein the ECM is not dialyzed or subjected to a cross-linking process prior to the gelling step (c).99. The method of claim 97 , wherein the ECM is intact ECM.100. The method of claim 97 , wherein the neutralized digest solution is maintained at or below 25° C. before the gelling step (c).101. The method of claim 97 , wherein the protease is pepsin claim 97 , trypsin or a combination thereof.102. The method of claim 97 , wherein in the step of raising the pH of the digested cardiac ECM (b) claim 97 , a base or an isotonic buffer is added to raise the pH of the digested cardiac ECM.103. The method of claim 102 , wherein the base or isotonic buffer is NaOH or phosphate buffered saline.104. The method of claim 97 , wherein the pH is raised to 7.4 in the step of raising the pH of the digested cardiac ECM (b).105. The method of claim 97 , wherein the digest solution is gelled at 30° C. or higher.106. The method of claim 97 , wherein the digest solution is gelled at 37° C.107. The method of ...

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Номер записи: 24
27-06-2013 дата публикации

CELL-PENETRATING PEPTIDES AND USES THEREOF

Номер: US20130164219A1
Автор: Brinkmann Ulrich, Haas Alexander, Maisel Daniela
Принадлежит: Hoffmann-La Roche Inc.

The present invention relates to the identification and functional characterization of human cell-penetrating peptides (CPPs) and their use; in particular as transfection vehicles. 1. A peptide molecule capable of being internalized into a cell , wherein the peptide molecule: GAAEAAARVYDLGLRRLRQRRRLRRERVRA (SEQ ID NO: 2);', 'IREIMEKFGKQPVSLPARRLKLRGRKRRQR (SEQ ID NO: 3);', 'YLKVVRKHHRVIAGQFFGHHHTDSFRMLYD (SEQ ID NO: 4); and', 'an amino acid sequence having over its total length at least 70%, preferably at least 80% overall sequence identity with any one of SEQ ID NO: 2 to SEQ ID NO: 4; and, '(a) has an amino acid sequence selected from the group consisting of(b) is internalized into a cell with an efficacy being at least 200% of the internalization efficacy of the TAT peptide having the amino acid sequence GRKKRRQRRRPPQ (SEQ ID NO: 1).2. The peptide of claim 1 , wherein at least a part of the peptide forms an alpha-helical secondary structure.3. The peptide of claim 1 , wherein the peptide is of mammalian claim 1 , preferably of human origin.45-. (canceled)6. A nucleic acid molecule encoding the peptide of any one of to .7. A vector comprising the nucleic acid molecule of .8. A host cell comprising the vector of .9. A method of producing the peptide of any one of to claim 7 , comprising:{'claim-ref': {'@idref': 'CLM-00008', 'claim 8'}, '(a) culturing the host cell of under suitable conditions; and'}(b) isolating the peptide produced.10. A composition comprising at least one peptide of any one of to being attached to any one of the group consisting of one or more nucleic acid molecules claim 7 , one or more peptides or proteins claim 7 , one or more small molecules claim 7 , and one or more nanoparticles claim 7 , wherein the attachment is accomplished by a linkage selected from the group consisting of a covalent linkage and a non-covalent linkage.11. The composition of claim 10 , wherein the at least one peptide is attached to one or more other peptides.12. The ...

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Номер записи: 25
27-06-2013 дата публикации

PROTEIN SEPARATION VIA ION-EXCHANGE CHROMATOGRAPHY AND ASSOCIATED METHODS, SYSTEMS, AND DEVICES

Номер: US20130164827A1
Автор: Torres Anthony R.
Принадлежит:

The present invention provides methods for separating proteins from a protein mixture. In one aspect, a method for separating a high concentration protein mixture into a bound protein fraction and a flow-through protein fraction can include delivering a protein mixture through an ion exchange column at a fixed pH and a fixed salt concentration. The fixed pH and the fixed salt concentration have been preselected to cause separation of the protein mixture into a bound protein fraction and a flow-through protein fraction. In this case, the bound protein fraction binds to the ion exchange column and the flow-though protein fraction flows though the ion exchange column. The method can further include receiving the flow-through protein fraction from the ion exchange column separate from the bound protein fraction, wherein either the bound protein fraction or the flow-through fraction contains a protein of interest. 1. A method for separating a high concentration protein mixture into a bound protein fraction and a flow-through protein fraction , comprising:delivering a high concentration protein mixture through an ion exchange column at a fixed pH and a fixed salt concentration, wherein the fixed pH and the fixed salt concentration have been preselected to cause separation of the high concentration protein mixture into a bound protein fraction and a flow-through protein fraction, wherein the bound protein fraction binds to the ion exchange column and the flow-though protein fraction flows though the ion exchange column; andreceiving the flow-through protein fraction from the ion exchange column separate from the bound protein fraction, and wherein either the bound protein fraction or the flow-through fraction contains a protein of interest.2. The method of claim 1 , wherein the protein of interest is in the flow-through protein fraction.3. The method of claim 1 , wherein the protein of interest is in the bound protein fraction.4. The method of claim 1 , wherein the high ...

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Номер записи: 26
27-06-2013 дата публикации

CROSSLINKING OF PROTEINS AND OTHER ENTITIES VIA CONJUGATES OF ALPHA-HALOACETOPHENONES, BENZYL HALIDES, QUINONES, AND THEIR DERIVATIVES

Номер: US20130165382A1
Автор: Krantz Alexander, Yu Peng
Принадлежит: ADVANCED PROTEOME THERAPEUTICS INC.

The present invention relates to the formation of conjugates (e.g., protein-protein dimers) using a-halo-acetophenones, benzylic halides, quinones, and related compounds as a conjugating system. The invention also features compositions that include the conjugates described herein, as well as uses of these conjugates in methods of medical treatment. 23.-. (canceled)4. The method of claim 1 , wherein two claim 1 , three claim 1 , or four of R claim 1 , R claim 1 , R claim 1 , and Rare H.58.-. (canceled)1012.-. (canceled)1418.-. (canceled)19. The method of claim 1 , wherein R″ is a protein claim 1 , or R″ is a polymer claim 1 , nucleic acid claim 1 , carbohydrate claim 1 , small molecule therapeutic agent claim 1 , imaging agent claim 1 , or diagnostic agent.2023.-. (canceled)24. A conjugate prepared according to the method of .25. (canceled)26. A pharmaceutical composition comprising the conjugate of and a pharmaceutically acceptable excipient.27. A method of delivering a therapeutic agent to a cell undergoing necrosis or apoptosis claim 24 , said method comprising contacting said cell or tissue with an agent that is the conjugate of claim 24 , wherein one of R and R″ comprises a protein or biologically active agent claim 24 , optionally one of R and R″ is an annexin protein.2829.-. (canceled)31. The conjugate of claim 30 , wherein{'sup': '1', 'Lis a covalent bond;'}{'sup': '2', 'Lis a covalent bond;'}n1 is 1;n2 is 1;{'sup': '1', 'sub': 2', '2, 'Qis C(═O), S(═O), CHOH, or CH; and/or'}{'sup': '2', 'sub': 2', '2, 'Qis C(═O), S(═O), CHOH, or CH.'}3236-. (canceled)37. The conjugate of claim 30 , whereinR is a protein or a biologically active or biologically compatible agent; and/orR″ is a protein or a biologically active or biologically compatible agent.3840.-. (canceled)41. The conjugate of claim 30 , wherein one or both of R and R″ is a protein that is an annexin protein claim 30 , an antibody claim 30 , a cytokine claim 30 , a wild-type protein that includes a free ...

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Номер записи: 27
04-07-2013 дата публикации

Intravenous Cytomegalovirus Human Immune Globulin and Manufacturing Method Thereof

Номер: US20130172536A1
Автор: CHEN Yuqin, Dai Meilan, Ding Yujiang, Guo Caiping, Huang Qiaoyun, Huang Weirong, Li Hui, Liu Yongdi, Luo Shan, Song Qingshuang, WANG CHUNHUA, WANG Jincai, Wu Enying, Wu Kaiyong, Zhang Pei, Zhang Xin, ZHANG Yanpeng, Zhang Yunjia
Принадлежит: SHENZHEN WEIGUANG BIOLOGICAL PRODUCTS CO.,LTD.

The present invention discloses an intravenous cytomegalovirus human immune globulin and a manufacturing method thereof, wherein the technical problem to be solved is to improve the purity, yield, and safety of the product. The intravenous cytomegalovirus human immune globulin of the present invention has a specific activity of no less than 2.5 PEI-U/mg, an anti-CMV titer of no less than 100 PEI-U/ml, a purity of greater than 98.2%, and a protein content of 51˜55 mg/ml. The present invention employs caprylic acid precipitation and anion exchange chromatography for replacing the step of ethanol precipitation in the conventional cold ethanol method to keep IgG in the supernatant and maintain the activity of the IgG; the present invention employing processes of caprylic acid inactivation of virus and nanometer film virus removal can effectively protect the safety of the product, and studies show that the preparing method of the present invention not only improves the purity, yield, and safety of the product; but also saves energy and reduces the cost of production. 1. An intravenous cytomegalovirus human immune globulin having a specific activity of no less than 2.5 PEI-U/mg , an anti-CMV titer of no less than 100 PEI-U/ml , a purity of greater than 98.2% , and a protein content of 51˜55 mg/ml.2. A method for preparing intravenous cytomegalovirus human immune globulin , comprising the steps of:(a) preparing FI+II+III and FII+III deposits:preparing human plasma measured by enzyme linked immunosorbent assay, dissolving said human plasma at 2˜30° C., and mixing said human plasma, wherein said human plasma has a high titer of anti-CMV;preparing FI+II+III deposit;adjusting the protein content of said human plasma to 45˜55 mg/ml with saline, adjusting the pH of said human plasma to 6.0˜6.5 with glacial acetic acid, and adding 95% ethanol or ethanol to adjust the concentration of ethanol of said human plasma to 20˜25%, wherein the reaction temperature is −5.5˜−4.5° C., and ...

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Номер записи: 28
11-07-2013 дата публикации

C-TERMINAL MODIFICATION OF POLYPEPTIDES

Номер: US20130177940A1
Автор: Bordusa Frank, Hermann Rupert, Hoess Eva, Jakubke Hans-Dieter, Tischer Wilhelm
Принадлежит:

The invention relates to a mutated trypsin comprising an amino acid substitution both at position K60 and D189, and at least one more amino acid substitution by histidine at position N143 or position E151. Such trypsin mutant has a preferred cleavage site comprising the amino acids Xaa-Xaa-His, wherein Xaais L, Y or F and Xaais R or K. The invention also relates to a man-made polypeptide comprising a target peptide and the above cleavage site as well as to a method of producing C-terminally modified target peptides by using this mutated trypsin. 1. A mutated trypsin comprising an amino acid substitution at position K60 and at position D189 , and an amino acid substitution by histidine at position N143 or position E151.2. The mutated trypsin of wherein K60 is substituted by E or D.3. The mutated trypsin of wherein D189 is substituted by K claim 1 , H or R.4. (canceled)5. A method of producing a C-terminally transacylated target peptide comprising the steps of:{'sub': 1', '2', '1', '2', '1, 'providing a polypeptide comprising a target peptide and a restriction site peptide comprising the cleavage site Xaa-Xaa-His, wherein Xaais L, Y or F, and Xaais R or K, wherein said restriction site peptide overlaps with the target peptide by the amino acid Xaaat the C-terminal end of said target peptide,'}{'claim-ref': {'@idref': 'CLM-00001', 'claim 1'}, 'sub': '1', 'bringing said peptide into contact with a trypsin mutant according to under conditions allowing for endoproteolytic cleavage after Xaa, thereby forming an endoprotease target peptide peptide-acyl-intermediate,'}adding an appropriate nucleophile, and,upon nucleophilic attack and binding of said nucleophile to the C-terminus of the target peptide, releasing the mutated trypsin from the endoprotease target peptide-acyl-intermediate.6. The method of wherein said nucleophile is selected from the group consisting of a primary amine group claim 5 , an imine group claim 5 , a secondary amine group claim 5 , a thiol group and ...

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Номер записи: 29
11-07-2013 дата публикации

PROTEIN PURIFICATION BY ION EXCHANGE

Номер: US20130178608A1
Автор: Kulkarni Samir, Subrahmanyam Satyam
Принадлежит:

The application describes an antibody purification method comprising multiple chromatographic steps wherein the low pH eluate from a protein A chromatography is further purified without the need of substantial pH adjustment. 1. A process for purifying antibodies comprising:a) purifying using protein A chromatography, wherein the antibody is eluted at a particular pH value; andb) purifying using cation exchange chromatography in the bind-elute mode, wherein an eluate from step a) is loaded onto a cation exchange resin without substantial adjustment of pH.2. A process according to claim 1 , wherein the antibody is eluted in step a) at pH values about 3.3 to about 4.5.3. A process according to claim 1 , wherein the antibody is eluted in step a) at a pH value about 3.5.4. A process for purifying antibodies claim 1 , comprising:a) purifying using protein A chromatography, wherein an antibody is eluted at a first pH value;b) purifying using cation exchange chromatography performed in the bind-elute mode, wherein eluate obtained from step a) is loaded onto a cation exchange resin at a second pH value, and elution is carried out at a third pH value; andc) purifying using anion exchange chromatography performed in the flow-through mode, wherein eluate from step b) is loaded on to an anion exchange resin without substantial adjustment of pH.5. A process according to claim 4 , wherein the first and second pH values are similar.6. A process according to claim 4 , wherein the first and second values are different.7. A process according to claim 4 , wherein the second and third pH values are similar.8. A process according to claim 4 , wherein the first pH value is about 3.59. A process according to claim 4 , wherein the second pH value is about 6 to about 8.10. A process according to claim 4 , wherein the second pH value is about 6.11. A process according to claim 4 , wherein the third pH value is about 6 to about 8.12. A process according to claim 4 , wherein the third pH value ...

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Номер записи: 30
18-07-2013 дата публикации

METHOD FOR ISOLATING A CYCLOHEXAPEPTIDE

Номер: US20130184433A1
Автор: De Pater Robertus Mattheus, Jagesar Dhiredj Chandre
Принадлежит: DSM Sinochem Pharmaceuticals

The present invention relates to a method for isolating acyclohexapeptide and to a novel crystalline form of caspofungin diacetate thus obtained. 1. Method for isolating a cyclohexapeptide comprising the subsequent steps of:(a) Contacting a solution comprising a cyclohexapeptide and water with a resin;(b) Removing the liquid phase from the mixture obtained in step (a);(c) Eluting said cyclohexapeptide by contacting the material retained after removal of the liquid phase in step (b) by addition of an organic solvent;(d) Separating resin from the mixture obtained in step (c);(e) Crystallizing said cyclohexapeptide from the solution retained after removal of said resin(f) Isolating said cyclohexapeptide from the mixture obtained in step (e)2. Method according to wherein said resin is contained in a chromatography column claim 1 , wherein step (a) is carried out by introducing said solution comprising a cyclohexapeptide and water onto said chromatography column and wherein step (b) is carried out by removing said liquid phase from said chromatography column.3. Method according to wherein said resin is washed with an aqueous mixture after step (a) and before step (c).4. Method according to wherein said resin is hydrophobic interaction chromatography material claim 1 , reversed phase chromatography material claim 1 , ion-exchange chromatography material or a mixture of two or more of these materials.5. Method according to wherein said organic solvent in step (c) is an alcohol.6. Method according to wherein said cyclohexapeptide is anidulafungin or a salt thereof or caspofungin or a salt thereof or micafungin or a salt thereof.7. Method according to wherein the concentration of said cyclohexapeptide in said solution of step (a) is from 2 to 50 times lower than the concentration of said cyclohexapeptide in the solution retained after removal of said resin in step (d).8. Method according to wherein the concentration of said cyclohexapeptide in said solution of step (a) is ...

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Номер записи: 31
18-07-2013 дата публикации

PROTEIN PURIFICATION

Номер: US20130184439A1
Автор: Pearce-Higgins Mark Robert, Spitali Mariangela, Symmons Jonathan, Whitcombe Richard
Принадлежит: UCB PHARMA, S.A.

The present invention relates to a process for the purification of an antibody fragment from a periplasmic cell extract comprising a first cation exchange chromatography step and a second anion exchange chromatography step. 120-. (canceled)21. A process for the purification of an antibody fragment from periplasmic cell extract comprising:a) a first chromatography step to capture the antibody fragment wherein a mixture containing an antibody fragment at a concentration of at least 1.5 g/L is subjected to cation exchange chromatography and subsequently eluted to produce a first eluate containing the antibody fragment; andb) a second chromatography step wherein the first eluate is subjected to anion exchange chromatography to capture impurities and produce a flow through containing the antibody fragment.22. The process according to claim 21 , wherein the process comprises not more than two chromatography steps.23. The process according to claim 21 , wherein all chromatography steps are performed on a chromatography column.24. The process according to claim 21 , wherein the cation exchange chromatography is performed in elution mode.25. The process according to claim 21 , wherein the first cation chromatography step comprises the following steps in sequential order:a) loading a mixture containing an antibody fragment onto the cation exchange column,b) washing the cation exchange column with a wash buffer wherein during the washing the conductivity, pH and salt concentration of the buffer remains essentially unchanged, andc) eluting the antibody fragment with an elution buffer.26. The process according to claim 25 , wherein the pH of the washing buffer is identical to the pH of the mixture containing an antibody fragment prior to first chromatography step.27. The process according to claim 25 , wherein the mixture containing an antibody fragment claim 25 , prior to the first chromatography step has a pH of between 4.0 to 5.0.28. The process according to claim 21 , ...

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Номер записи: 32
01-08-2013 дата публикации

Glycosylation of Molecules

Номер: US20130195835A1
Автор: Callewaert Nico Luc Marc, De Pourcq Karen Jacqueline Marcel, Geysens Steven Christian Jozef, Guerfal Mouna, Vervecken Wouter
Принадлежит:

Described herein are methods and genetically engineered cells useful for producing an altered N-glycosylation form of a target molecule. Also described are methods and molecules with altered N-glycosylation useful for treating a variety of disorders such as metabolic disorders. 1. A method of producing an altered N-glycosylation form of a target protein , the method comprising:{'i': Yarrowia lipolytica', 'Arxula adeninivorans, 'providing a or an cell genetically engineered to express a protein, or a biologically active variant thereof, capable of effecting mannosyl phosphorylation of N-glycans; and'}introducing into the cell a nucleic acid encoding a target protein, wherein the cell produces the target protein in an altered N-glycosylation form.2. The method of claim 1 , further comprising isolating the altered N-glycosylation form of the target protein.3. The method of claim 1 , wherein the target protein is an exogenous protein.4. The method of claim 1 , wherein the target protein is an endogenous protein.5. The method of claim 1 , wherein the target protein is a mammalian protein.6. The method of claim 1 , wherein the target protein is a pathogen protein claim 1 , a lysosomal protein claim 1 , a growth factor claim 1 , a cytokine claim 1 , a chemokine claim 1 , or a fusion protein.7. The method of claim 1 , wherein the target protein is a protein associated with a lysosomal storage disorder (LSD).8. The method of claim 7 , wherein the lysosomal storage disorder is Gaucher disease claim 7 , Tay-Sachs disease claim 7 , Pompe disease claim 7 , Niemann-Pick disease claim 7 , or Fabry disease.9. The method of claims 7 , wherein the target protein is glucocerebrosidase claims 7 , alpha galactosidase claims 7 , or galactocerebrosidase.10. The method of claim 7 , wherein the target protein is selected from the group consisting of alpha-L-iduronidase claim 7 , beta-D-galactosidase claim 7 , beta-glucosidase claim 7 , beta-hexosaminidase claim 7 , beta-D-mannosidase claim ...

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Номер записи: 33
01-08-2013 дата публикации

METHOD FOR PRODUCING SURFACTIN AND SALT THEREOF

Номер: US20130197190A1
Автор: Izumida Masashi, Kawasaki Hiroaki, MOROSHIMA Tadashi
Принадлежит: KANEKA CORPORATION

The method for producing a surfactin or a salt thereof according to the present invention comprises the steps of adding an organic solvent containing a branched alkyl alcohol to a culture fluid containing the surfactin or the salt thereof, or to a solution obtained by removing an insoluble component from the culture fluid, and extracting the surfactin or the salt thereof with the organic solvent, 2. The method according to claim 1 , wherein an aqueous layer during the extraction is acidic.3. The method according to claim 2 , wherein a pH value of the aqueous layer during the extraction is not less than 1 and not more than 5.4. The method according to claim 1 , wherein a carbon number of the branched alkyl alcohol is not less than 3 and not more than 10.5. The method according to claim 1 , wherein a ratio of the branched alkyl alcohol in the organic solvent is not less than 30 wt %.6. The method according to claim 1 , wherein the organic solvent further contains an auxiliary solvent claim 1 , and a total ratio of the branched alkyl alcohol and the auxiliary solvent is not less than 0.6 parts by weight and not more than 1.5 parts by weight relative to 1 part by weight of the culture fluid.7. The method according to claim 1 , further comprising the steps of:a) obtaining an aqueous solution containing the surfactin salt by mixing the extract containing the surfactin with a basic aqueous solution and then removing an organic layer; andb) mixing the aqueous solution containing the surfactin salt with an inorganic acid to precipitate the surfactin in a solid form from the obtained mixture.8. The method according to claim 1 , further comprising the step of mixing the extract containing the surfactin with a base to obtain the surfactin salt obtained in a solid form from the obtained mixture.9. A method for producing the surfactin salt claim 1 , comprising the step of obtaining the surfactin salt or a solution thereof by mixing the surfactin obtained by the method according ...

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Номер записи: 34
01-08-2013 дата публикации

METHODS OF REDUCING LEVEL OF ONE OF MORE IMPURITIES IN A SAMPLE DURING PROTEIN PURIFICATION

Номер: US20130197200A1
Автор: Bian Nanying, Chen Jie, Gillespie Christopher, Kozlov Mikhail, Siwak Martin, Stone Matthew T.
Принадлежит: EMD MILLIPORE CORPORATION

The present invention provides novel and improved protein purification processes which incorporate certain types of carbonaceous materials and result in effective and selective removal of certain undesirable impurities without adversely effecting the yield of the desired protein product. 1. A flow-through process for purifying a target molecule from a Protein A eluate comprising the steps of:(i) contacting the eluate recovered from a Protein A chromatography column with activated carbon;(ii) contacting the flow-through sample from step (i) with an anion exchange chromatography media;(iii) contacting the flow-through sample from step (ii) with a cation exchange chromatography media; and(iv) obtaining the flow-through sample from step (iii) comprising the target molecule,wherein the eluate flows continuously through steps (i)-(iii) and wherein level of one or more impurities in the flow-through sample after step (iii) is lower than the level in the eluate in step (i).2. The flow-through process of claim 1 , further comprising subjecting the flow-through sample from step (iii) to virus filtration.3. The flow-through process of claim 1 , further comprising use of an in-line static mixer and/or a surge tank between steps (ii) and (iii) to change pH.4. The flow-through process of claim 1 , wherein the process employs a single skid.5. The flow-through process of claim 1 , wherein the eluate from the Protein A chromatography column is subjected to virus inactivation prior to contacting with activated carbon.6. The process of claim 1 , wherein steps (i)-(iii) may be performed in any order.7. A flow-through purification process for purifying a target molecule from a Protein A eluate comprising contacting the eluate with two or more matrices selected from activated carbon claim 1 , anion exchange media claim 1 , cation exchange media and virus filtration media claim 1 , wherein the flow of the eluate is continuous.8. A method for reducing the burden on one or more ...

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Номер записи: 35
08-08-2013 дата публикации

METHOD FOR PURIFYING ACTIVE POLYPEPTIDES OR IMMUNOCONJUGATES

Номер: US20130202626A1
Автор: Hunter Alan, Linke Thomas, SATHISH Hasige, SHAH Ambarish, Thompson Christopher, WANG William K.
Принадлежит: MEDIMMUNE, LLC

The present invention provides methods for isolating an active polypeptide or immunoconjugate by purification of a solution containing both the active polypeptide or immunoconjugate and an acidic variant thereof, such as a deamidated variant, using anion exchange chromatography. 1. (canceled)2. A method of producing a purified polypeptide from a solution comprising the polypeptide and an acidic variant of the polypeptide , wherein said acidic variant of the polypeptide results in an inhibition of potency of said polypeptide , the method comprising: (a) contacting the polypeptide with an anion exchange (AIEX) chromatography matrix; and (b) eluting the bound polypeptide from the AIEX chromatography matrix with a high salt buffer , thereby separating said polypeptide from the acidic variant and producing a purified polypeptide;{'i': 'Pseudomonas', 'wherein the polypeptide comprises an antibody or antigen binding fragment thereof that binds the cell surface receptor CD22 and a exotoxin, or variant thereof.'}3. (canceled)4. The method of claim 1 , wherein the AIEX matrix contains quaternary amine and tertiary amine ion exchange groups.5. The method of claim 4 , wherein the AIEX matrix contains a quaternary amino (Q) group.6. The method of claim 5 , wherein the AIEX matrix is Q sepharose.7. The method of any of claim 1 , wherein the polypeptide is eluted with a linear or step salt gradient.8. The method of claim 7 , wherein the polypeptide is eluted with a linear salt gradient that is from about 150 mM NaCl in Tris/HCl claim 7 , pH 8.0 to about 300 mM NaCl in Tris/HCl claim 7 , pH 8.0 claim 7 , from about 175 mM NaCl in Tris/HCl claim 7 , pH 8.0 to about 275 mM NaCl in Tris/HCl claim 7 , pH 8.0 claim 7 , or from about 192 mM NaCl in Tris/HCl claim 7 , pH 8.0 to about 245 mM NaCl in Tris/HCl claim 7 , pH 8.0.911-. (canceled)12. The method of claim 1 , wherein between about 75 to about 99% claim 1 , about 80% claim 1 , about 85% claim 1 , about 90% claim 1 , about 95% claim ...

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Номер записи: 36
08-08-2013 дата публикации

Aldehyde Tags, Uses Thereof in Site-Specific Protein Modification

Номер: US20130203111A1
Автор: Bertozzi Carolyn, Carlson Brian L., Carrico Isaac S., Wu Peng
Принадлежит: The Regents of the University of California

The invention features compositions and methods for site-specific modification of proteins by incorporation of an aldehyde tag. Enzymatic modification at a sulfatase motif of the aldehyde tag through action of a formylglycine generating enzyme (FGE) generates a formylglycine (FGly) residue. The aldehyde moiety of FGly residue can be exploited as a chemical handle for site-specific attachment of a moiety of interest to a polypeptide. 2. (canceled)3. The method of claim 1 , wherein the heterologous sulfatase motif is positioned in the non-naturally occurring claim 1 , recombinant polypeptide:at an internal sequence of the polypeptide;at a terminal loop, a C-terminus, or an N-terminus of the polypeptide;on a solvent-accessible region of the polypeptide when folded; and/orat a site of post-translational modification of the polypeptide that is native or non-native to the amino acid sequence of the polypeptide.4. The method of claim 1 , wherein X claim 1 , when present claim 1 , X claim 1 , and Xare each independently an aliphatic amino acid claim 1 , a sulfur-containing amino acid claim 1 , or a polar claim 1 , uncharged amino acid.5. The method of claim 1 , wherein the X claim 1 , when present claim 1 , is L claim 1 , M claim 1 , V claim 1 , S or T.6. The method of claim 1 , wherein Xand Xare each independently S claim 1 , T claim 1 , A claim 1 , V claim 1 , G or C.8. The method of claim 7 , wherein the heterologous sulfatase motif of the polypeptideis positioned at a C-terminus of the non-naturally occurring, recombinant polypeptide;is present at an internal site in a terminal loop of the non-naturally occurring, recombinant polypeptide;is, where the non-naturally occurring, recombinant polypeptide is a transmembrane protein, present at an internal site within an extracellular loop or an intracellular loop of the transmembrane protein;is present at an internal site or at the N-terminus and is solvent-accessible when the non-naturally occurring, recombinant polypeptide ...

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Номер записи: 37
08-08-2013 дата публикации

METHOD FOR PURIFICATION OF COMPLEMENT FACTOR H

Номер: US20130203971A1
Автор: Brandstaetter Hubert, Roemisch Juergen, Schulz Petra
Принадлежит: Octapharma AG

A method for purification of complement Factor H from a complement Factor H containing source such as blood or blood plasma, in particular a caprylate precipitate of a Factor H containing source, which is e.g. obtained by addition of caprylate ions to fractions of blood or plasma, comprising the steps of: 1. A method for purification of complement Factor H from a complement Factor H containing source , comprising the steps of:a. Providing a Factor containing source;b. performing a cation exchange chromatography;c. performing an anion exchange chromatography;d. performing a hydroxyl apatite chromatography;e. followed by ultra/diafiltration to obtain a complement Factor H concentrate.2. The method according to wherein a heparin affinity chromatography is performed.3. The method according to wherein the process comprises at least one of the following methods for pathogen removal and/or inactivation:a. solvent/detergent treatment;b. pasteurization,c. vapor heat treatment;d. dry heat treatment; ore. nanofiltration.4. The method according to wherein the complement Factor H concentrate is lyophilized.5. A complement Factor H obtainable by a method according to .6. The complement Factor H of obtainable by a method comprisinga. reconstitution of caprylate precipitate to provide a complement Factor H containing solution;b. performing virus inactivation by solvent/detergent treatment (S/D treatment).c. performing a cation exchange chromatography chromatographic step under the following conditions:d. binding of complement Factor H to a strong cation exchange resin of the sulphopropyl type, washing with a buffer comprising 20 mM tri-sodium citrate adjusted to pH 6.0 and elution of complement Factor H with an elution buffe r comprising 20 mM tri-sodium citrate and 0.2 M NaCl adjusted to pH 6.0; 'i. applying of the complement Factor H containing solution (conductivity 0.1-0.5 mS/cm) to a strong anion exchange resin of the quaternary ammonium type, washing with a buffer comprising ...

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Номер записи: 38
15-08-2013 дата публикации

WHOLE EGG PROTEIN PEPTIDES, PREPARATION METHOD AND USE THEREOF

Номер: US20130209497A1
Автор: Zou Yuandong
Принадлежит: WUHAN JIUSHENGTANG BIOENGINEERING CO., LTD

Provided are whole egg protein peptides and the preparation method thereof, wherein the whole egg protein peptides are obtained by adopting compound proteases composed of pawpaw protease, fig protease and pineapple protease to enzymatically hydrolyze the whole egg protein powder. The whole egg protein peptides can be used for manufacture of products for enhancing immunity. 1. A method of preparing whole egg protein peptides , comprising:enzymatically degrading whole egg protein powders using a compound plant protease, so as to obtain the whole egg protein peptides;wherein the compound plant protease consists of pawpaw protease, fig protease and pineapple protease.2. The method according to wherein the compound plant proteases have an enzyme activity ratio of pawpaw protease claim 1 , fig protease and pineapple protease of (1-1.2 million)U:(0.3-0.4 million)U:(5.6-7 million)U.3. The method according to wherein the whole egg protein powders are prepared from at least one of following bird eggs as a raw material: chicken claim 1 , duck claim 1 , goose claim 1 , quail claim 1 , sparrow claim 1 , pigeon claim 1 , turtledove and ostrich eggs.4. The method according to wherein claim 1 , in said enzymatically degrading whole egg protein powders claim 1 , the whole egg protein powders are present with water claim 1 , and a ratio of whole egg protein powders to water by mass part is 1:8-10; amounts of enzymes required for enzymatically degrading per gram of the whole egg protein powders are: 50-60 thousand U of pawpaw protease claim 1 , 15-20 thousand U of fig protease claim 1 , and 280-350 thousand U of pineapple protease; and the enzymatic degradation is performed at a temperature of 48-50° C. claim 1 , for a period of 3-4 hours claim 1 , with a pH value of 7.5-8.5.5. The method according to further comprising:sterilizing a hydrolysate obtained from said enzymatic degradation of the whole egg protein powders, and inactivating enzymes in the hydrolysate; andcooling and ...

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Номер записи: 39
15-08-2013 дата публикации

METHOD FOR PURIFYING HUMAN GRANULOCYTE-COLONY STIMULATING FACTOR FROM RECOMBINANT E. COLI

Номер: US20130211054A1
Автор: CHOI Sung Chul, KIM Jin Ki, Lee Jong Soo, OH Young Hak
Принадлежит: HANMI SCIENCE CO., LTD.

The present invention provides a method for purifying a large amount of human granulocyte-colony stimulating factors (hG-CSFs) from a recombinant with high yield and purity. According to the method of the present invention, human granulocyte-colony stimulating factor, identical to the native form expressed in the human body, can be easily purified with high yield and purity without an additional activation process. In particular, according to the purification method of the present invention, hG-CSF variants expressed in are efficiently removed to obtain physiologically active hG-CSFs with high purity. 1E. coli. A method for purifying human granulocyte-colony stimulating factors (hG-CSFs) from a recombinant in a high yield , comprising the steps of:{'i': 'E. coli', '(a) culturing an hG-CSF-expressing recombinant to obtain a cell pellet by centrifugation;'}(b) separating an hG-CSF-containing supernatant from the cell pellet obtained in step (a);(c) treating the supernatant obtained in step (b) with an acid to separate the resulting precipitate by filtration;(d) applying a filtrate obtained in step (c) to cation exchange chromatography;(e) applying an eluate obtained in step (d) to hydrophobic-interaction chromatography; and(f) applying an eluate obtained in step (e) to anion exchange chromatography.2E. coli.. The method according to claim 1 , wherein in step (a) claim 1 , the hG-CSFs are expressed into the periplasm of recombinant3E. coliE. coliE. coliE. coliE. coliE. coliE. coliE. coliE. coliE. coliE. coliE. coli. The method according to claim 2 , wherein the recombinant is one or more selected from the group consisting of BL21(DE3)/pT14SS1SG(HM 10310) claim 2 , BL21(DE3)/pT14SS1S17SEG (HM 10311; Accession No. KCCM-10154) claim 2 , BL21(DE3)/pTO1SG (HM 10409) claim 2 , BL21(DE3)/pTO1S17SG (HM 10410; Accession No. KCCM-10151) claim 2 , BL21(DE3)/pTO17SG (HM 10411; Accession No. KCCM-10152) claim 2 , BL21(DE3)/pTO17TG (HM 10413) claim 2 , BL21(DE3)/pTO17AG(HM 10414) ...

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Номер записи: 40
22-08-2013 дата публикации

SITE-SPECIFIC CHEMICAL MODIFICATION OF PROTEINS AT THEIR N-TERMINI, ENABLING THE FORMATION OF HOMOGENEOUS ADDUCTS

Номер: US20130216479A1
Автор: Krantz Alexander, Yu Peng
Принадлежит:

Site-specific modifications of proteins at their N-termini are provided. In particular, a chemical modification of proteins at their N-termini via a transamination reaction to form homogeneous adducts such as, the corresponding oxime derivatives is provided. Methods of making and using the adducts in radio-labelling, molecular imaging applications, and treatment of disorders such as cancer, Crohn's disease, arthritis, atherothrombosis and plaque rupture are also provided. 3. The compound of wherein Ris hydrogen claim 1 , or methyl.4. The compound of wherein Ris CH.5. The compound of wherein X is O.6. The compound of wherein R is hydrogen claim 1 , or C-Calkyl.7. The compound of wherein R3 is hydrogen or methyl.8. The compound of wherein Z is optionally substituted aryl.9. The compound of wherein n is 3-14.10. The compound of wherein n is 6.1115-. (canceled)16. The compound of further substituted with a reduced oxime linkage claim 1 , wherein at least one oxime linkage is reduced to a corresponding aminoxy group.1922.-. (canceled)23. The compound of further substituted with a therapeutic agent claim 1 , a diagnostic agent claim 1 , a solid support claim 1 , or any combination thereof.24. The compound of claim 23 , wherein the therapeutic agent or diagnostic agent is a radionucleotide claim 23 , small molecule therapeutic agent claim 23 , antibody claim 23 , optical label claim 23 , fluorescent label claim 23 , biosynthetic label claim 23 , or oligonucleotide.25. The compound of claim 23 , wherein the diagnostic agent is a NOTA or DOTA chelate of gallium or technetium claim 23 , Cu-64 claim 23 , Ga-67 claim 23 , Ga-68 claim 23 , Zr-89 claim 23 , Ru-97 claim 23 , Tc-99m claim 23 , Rh-105 claim 23 , Pd-109 claim 23 , In-111 claim 23 , I-123 claim 23 , I-125 claim 23 , I-131 claim 23 , Re-186 claim 23 , Re-188 claim 23 , Au-198 claim 23 , Au-199 claim 23 , Pb-203 claim 23 , At-211 claim 23 , Pb-212 claim 23 , Bi-212 claim 23 , fluorochrome claim 23 , fluorescein claim 23 ...

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Номер записи: 41
22-08-2013 дата публикации

METHODS FOR EXPRESSION AND PURIFICATION OF IMMUNOTOXINS

Номер: US20130217861A1
Автор: JR. David M., Liu Yuan-Yi, Neville, Woo Jung-Hee
Принадлежит: The USA, as represented by the Secretary, Dept. of Health and Human Services

The present invention relates to a method of expressing an immunotoxin in strain mutated to toxin resistance comprising a) growing the in a growth medium comprising an enzymatic digest of protein and yeast extract and maintaining a dissolved oxygen concentration at 40% and above; and b) performing methanol induction with a limited methanol feed of 0.5-0.75 ml/min/IO L of initial volume during induction along with a continuous infusion of yeast extract at a temperature below 17.5° C., antifoaming agent supplied up to 0.07%, agitation reduced to 400 RPM, and the induction phase extended out to 163 h. 126-. (canceled)27. A method of purifying a non-glycosylated immunotoxin comprisinga) loading a solution containing the non-glycosylated immunotoxin onto a hydrophobic interaction column;b) obtaining a first non-glycosylated immunotoxin containing eluant from the hydrophobic interaction column;c) loading the non-glycosylated immunotoxin containing eluant from step (b) onto an anion exchange column;d) obtaining a second non-glycosylated immunotoxin containing eluant from the anion exchange column by eluting the non-glycosylated immunotoxin with a sodium borate solution;e) diluting the concentration of sodium borate in the second non-glycosylated immunotoxin containing eluant from step (d) to about 50 ruM or less;f) concentrating the diluted non-glycosylated immunotoxin containing eluant from step (e) over an anion exchange column; andg) obtaining a purified non-glycosylated immunotoxin from the anion exchange column.28. The method of claim 27 , wherein the non-glycosylated immunotoxin is expressed in yeast.29Pichia pastoris.. The method of claim 28 , wherein the yeast is30. The method of claim 27 , wherein the immunotoxin is a fusion protein.31. The method of claim 27 , wherein the immunotoxin comprises a diphtheria toxin moiety.32. The method of claim 31 , wherein the diphtheria toxin moiety is truncated.33. The method of claim 32 , farther comprising a CD3 antibody ...

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Номер записи: 42
22-08-2013 дата публикации

OPTIMIZED METHOD FOR ANTIBODY CAPTURING BY MIXED MODE CHROMATOGRAPHY

Номер: US20130217866A1
Автор: Falkenstein Roberto, Lemm Thorsten, STRASSER Markus, Yamada Hidenari
Принадлежит: Hoffmann-La Roche Inc.

Herein is reported a method for the purification of an antibody directly captured from clarified cell culture supernatants using Streamline CST and/or Capto MMC, wherein especially product related (aggregates and fragments) and process related impurities (host cell protein, media components) could efficiently be removed, resulting in a preparation with a purity comparable to classical protein A affinity chromatography. 1. A method for producing an anti-IGF-1R antibody comprising the following steps:a) applying a crude mammalian cell culture cultivation supernatant to a multimodal weak cation exchange chromatography material,b) recovering the anti-IGF-1R antibody by applying a buffered solution comprising ethylene glycol and an inorganic salt to the multimodal weak cation exchange chromatography material and thereby producing an anti-IGF-1R antibody.2. The method according to claim 1 , wherein the method comprises the following additional step a-1) prior to step a):a-1) applying a buffered solution comprising an inorganic salt to the multimodal weak cation exchange chromatography material.3. The method according to wherein the method comprises the following additional step a-b) after step a) and prior to step b):a-b) applying a buffered solution to the multimodal weak cation exchange chromatography material, whereby the anti-IGF-1R antibody is not recovered from the multimodal weak cation exchange chromatography material.4. The method according to claim 3 , wherein the step a-b) comprises two steps a-b 1) and a-b2):a-b1) applying a buffered solution comprising an inorganic salt to the multimodal weak cation exchange chromatography material, anda-b2) applying a buffered solution comprising a denaturant to the multimodal weak cation exchange chromatography material,whereby the anti-IGF-1R antibody is not recovered from the multimodal weak cation exchange chromatography material.5. The method according to wherein the denaturant is selected from guanidinium hydrochloride ...

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Номер записи: 43
29-08-2013 дата публикации

RADIOLABELLING METHODS

Номер: US20130224835A1
Автор: ARSTAD ERIK, GLASER MATTHIAS EBERHARD
Принадлежит: HAMMERSMITH IMANET LIMITED

The invention relates to radiodiagnostic and radiotherapeutic agents, including biologically active vectors labelled with radionuclides. It further relates to methods and reagents labelling a vector such as a peptide comprising reaction of a compound of formula (I) with a compound of formula (II): 2. A method according to wherein R* comprises a positron C or F.3. A method according to wherein the vector is a peptide claim 1 , protein claim 1 , hormone claim 1 , cell claim 1 , bacterium claim 1 , virus claim 1 , or small drug-like molecule.4. A method according to wherein the vector is Arg-Gly-Asp peptide.7. A method according to wherein elemental copper is used as source of the Cu(I) catalyst.818-. (canceled)19. The method according to wherein R* is F.20. The method according to wherein a is 1. The present invention relates to radiodiagnostic and radiotherapeutic agents, including biologically active vectors labelled with radionuclides. It further relates to methods and reagents labelling a vector such as a peptide. The resultant labelled conjugates are useful as diagnostic agents, for example, as radiopharmaceuticals more specifically for use in Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT) or for radiotherapy.The application of radiolabelled bioactive peptides for diagnostic imaging is gaining importance in nuclear medicine. Biologically active molecules which selectively interact with specific cell types are useful for the delivery of radioactivity to target tissues. For example, radiolabelled peptides have significant potential for the delivery of radionuclides to tumours, infarcts, and infected tissues for diagnostic imaging and radiotherapy. F, with its half-life of approximately 110 minutes, is the positron-emitting nuclide of choice for many receptor imaging studies. Therefore, F-labelled bioactive peptides have great clinical potential because of their utility in PET to quantitatively detect and characterise a wide ...

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Номер записи: 44
29-08-2013 дата публикации

MICROREACTOR AND METHOD FOR PREPARING A RADIOLABELED COMPLEX OR A BIOMOLECULE CONJUGATE

Номер: US20130225791A1
Автор: Desai Amit V., Kenis Paul J.A., Önal Birce C., Reichert David E., Wheeler Tobias D., Zeng Dexing
Принадлежит: WASHINGTON UNIVERSITY IN ST. LOUIS

A microreactor for preparing a radiolabeled complex or a biomolecule conjugate comprises a microchannel for fluid flow, where the microchannel comprises a mixing portion comprising one or more passive mixing elements, and a reservoir for incubating a mixed fluid. The reservoir is in fluid communication with the microchannel and is disposed downstream of the mixing portion. A method of preparing a radiolabeled complex includes flowing a radiometal solution comprising a metallic radionuclide through a downstream mixing portion of a microchannel, where the downstream mixing portion includes one or more passive mixing elements, and flowing a ligand solution comprising a bifunctional chelator through the downstream mixing portion. The ligand solution and the radiometal solution are passively mixed while in the downstream mixing portion to initiate a chelation reaction between the metallic radionuclide and the bifunctional chelator. The chelation reaction is completed to form a radiolabeled complex. 1. A method of preparing a radiolabeled complex , the method comprising:flowing a radiometal solution comprising a metallic radionuclide through a downstream mixing portion of a microchannel, the downstream mixing portion including one or more passive mixing elements;flowing a ligand solution comprising a bifunctional chelator through the downstream mixing portion;passively mixing the ligand solution and the radiometal solution while in the downstream mixing portion to form a mixed solution and to initiate a chelation reaction between the metallic radionuclide and the bifunctional chelator; andcompleting the chelation reaction to form a radiolabeled complex.2. The method of claim 1 , further comprising claim 1 , prior to flowing the radiometal solution through the downstream mixing portion of the microchannel claim 1 , flowing a precursor radiometal solution comprising the metallic radionuclide through an upstream mixing portion of the microchannel claim 1 , the upstream ...

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Номер записи: 45
05-09-2013 дата публикации

METHOD AND SYSTEM FOR SOLUBILIZING PROTEIN

Номер: US20130231467A1
Автор: Davison Richard, Granda Cesar B., Holtzapple Mark Thomas, Noyes Gary P.
Принадлежит: EE-TERRABON, LLC

A method of solubilizing protein that includes applying an alkali to a protein source to form a slurry; heating the slurry to a temperature sufficient to allow hydrolysis of protein in the protein source to obtain a reaction liquid comprising solubilized proteins, prions, and reactive solids; separating reactive solids from the reaction liquid to produce a separated reaction liquid, wherein the reactive solids comprise unsolubilized proteins; further heating the separated reaction liquid to an elevated temperature and holding for a time period sufficient to destroy prions in the separated reaction liquid, wherein the elevated temperature is between 75° C. and 250° C. and the time period is between 1 second and 5 hours; and neutralizing the reaction liquid with acid or an acid source to produce a neutralized liquid. 1. A method of solubilizing protein comprising:applying an alkali to a protein source to form a slurry;heating the slurry to a temperature sufficient to allow hydrolysis of protein in the protein source to obtain a reaction liquid comprising solubilized proteins, prions, and reactive solids;separating reactive solids from the reaction liquid to produce a separated reaction liquid, wherein the reactive solids comprise unsolubilized proteins;further heating the separated reaction liquid to an elevated temperature and holding for a time period sufficient to destroy prions in the separated reaction liquid, wherein the elevated temperature is between 75° C. and 250° C. and the time period is between 1 second and 5 hours; andneutralizing the reaction liquid with acid or an acid source to produce a neutralized liquid.2. The method of claim 1 , the method further comprising:concentrating the neutralized liquid to produce concentrated liquid and water; andreturning produced water to the slurry before or during the heating the slurry step.3. The method according to claim 2 , wherein the alkali comprises calcium oxide or calcium hydroxide.4. The method according to ...

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Номер записи: 46
12-09-2013 дата публикации

METHOD FOR PRODUCING HIGH-PURITY SOLUBLE THROMBOMODULIN

Номер: US20130237693A1
Автор: OHIGASHI Susumu
Принадлежит:

The aim of the present invention is to obtain soluble thrombomodulin substantially not containing a denatured product of soluble thrombomodulin that is generated under acidic conditions. A method is provided for producing soluble thrombomodulin substantially not containing a denatured product of the soluble thrombomodulin that is generated under acidic conditions, which comprises; subjecting the soluble thrombomodulin-containing material to an anion exchanger or hydroxyapatite; and carrying out linear gradient elution, stepwise gradient elution, or gradient elution in which linear gradient elution is combined with stepwise gradient elution under separation conditions in which the denatured product of the soluble thrombomodulin can be separated, wherein said gradient is a salt concentration gradient, so as to obtain an elution fraction containing soluble thrombomodulin that does not substantially contain the denatured product of the soluble thrombomodulin, either (a) after the position of a fraction has previously been confirmed, or (b) while confirming the elution fraction. 1. A method for producing soluble thrombomodulin substantially not containing a denatured product of the soluble thrombomodulin that may be generated from the soluble thrombomodulin under acidic conditions , which comprises: (0) a step of leaving the soluble thrombomodulin under acidic conditions of pH 5 or less; (1) a step of subjecting a soluble thrombomodulin-containing material that contains or is suspected to contain a denatured product of the soluble thrombomodulin , which is obtained by step (0) , to an anion exchanger or hydroxyapatite; and (2) a step of obtaining a soluble thrombomodulin-containing fraction that does not substantially contain a denatured product of the soluble thrombomodulin under separation conditions in which the soluble thrombomodulin can be separated from a denatured product of the soluble thrombomodulin , wherein the anion exchanger comprises a buffer solution of pH ...

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Номер записи: 47
26-09-2013 дата публикации

HYDROPHOBIZED PROTEIN HYDROLYSATE

Номер: US20130251658A1
Автор: Schilling Martin, Thum Oliver
Принадлежит: EVONIK GOLDSCHMIDT GMBH

The invention relates to enzymatically hydrophobicised protein hydrolysates, their production and use, and to cosmetic preparations comprising these. 2. The alkylated peptide mixture according to claim 1 , wherein said at least one protein is selected from the group consisting of isolated plant storage proteins claim 1 , animal proteins claim 1 , and microbial proteins.3. The alkylated peptide mixture according to claim 1 , wherein the hydrolysis in process step A) is catalysed by at least one acid or at least one enzyme.4. The alkylated peptide mixture according to claim 1 , wherein said protein hydrolysate in process step A) has an average molecular weight of 203 g/mol to 100 claim 1 ,000 g/mol.5. The alkylated peptide mixture according claim 1 , wherein said protein hydrolysate in process step A) is selected from Meripro 810 claim 1 , Meripro 711 claim 1 , Naturalys W claim 1 , Cropeptide W claim 1 , Hydrotriticum 2000 claim 1 , Tritisol claim 1 , Tritisol XM claim 1 , Hydrosoy 2000 claim 1 , Gluadin W20 claim 1 , Gluadin WLM claim 1 , AMCO HCA411 claim 1 , and HLA-198.6Bacillus subtilisStreptoverticillium mombaraensis.. The alkylated peptide mixture according to claim 1 , wherein said transglutaminase in process step B) is isolated from or7. The alkylated peptide mixture according to claim 1 , wherein said radicals Rand Rare independently selected from the group consisting of alkyl and alkenyl radicals.8. The alkylated peptide mixture according to claim 1 , wherein claim 1 , in process step A) claim 1 , the hydrolysis is catalysed by at least one enzyme claim 1 , and process step A) and process step B) are conducted simultaneously.10. A cosmetic claim 1 , dermatological claim 1 , pharmaceutical crop protection claim 1 , care claim 1 , cleaning claim 1 , or surfactant formulation comprising at least one alkylated peptide mixture according to .11. An emulsifier claim 1 , dispersion auxiliary claim 1 , conditioner for skin and hair claim 1 , foam former claim 1 , ...

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Номер записи: 48
26-09-2013 дата публикации

METHOD FOR PREPARING ACTIVE PEPTIDES FROM CORN GERM PROTEINS

Номер: US20130252877A1
Автор: CAI MUYI, Chen Liang, Dong Zhe, GU RUIZENG, JIN ZHENTAO, LIN Feng, LIU WENYING, Lu Jun, Lu Lu, MA YONG, MA YONGQING, PAN XINGCHANG, XU YAGUANG, Yi Weixue
Принадлежит: CHINA NATIONAL RESEARCH INSTITUTE OF FOOD AND FERMENTATION

The present invention discloses a method for producing antihypertensive active peptides with corn germ protein as the material. The method comprises an alkali-heat treatment and continuous enzymolysis of the corn germ protein. The components with molecular weight less than 1000 Da in the active peptides obtained according to the present method account for more than 92%, and alanine-tyrosine (Ala-Tyr, AY) as the characteristic peptide fragments in the antihypertensive peptides accounts for more than 0.6%, so that the active peptides have a good ACE inhibitory activity in vitro as well as stability against temperature, pH and major gastrointestinal digestive enzymes, and have a significant effect of lowering blood pressure on spontaneous hypertension rats in vivo. The active peptides can be applied as a new functional nutrient to development and production of food, health food and pharmaceutical. 1. A method for preparing active peptides from corn germ protein powders , including the following steps:adding the corn germ protein powders to a reaction tank, mixing the corn germ protein powders with water to form a first feed liquid, and the feed liquid being adjusted to be alkalescent, heated to 50˜90° C., and stirred at this temperature for 20˜60 min;centrifugalizing the alkalescent first feed liquid in the reaction tank and collecting slag from the alkalescent first feed liquid;mixing the slag with water to form a second feed liquid, the second feed liquid being heated to 50˜90° C. and centrifugalized, and collecting slag from the second feed liquid; the same processing is repeated at least twice to obtain purified slag;the purified slag being mixed with water at a water-slag ratio of 100:40˜60, stirred, and subjected to a first enzymolysis and a second enzymolysis by alkali protease and compound protease in sequence to obtain an enzymatic hydrolysate, wherein the compound protease is comprised of papain and neutral protease; andheating the enzymatic hydrolysate to ...

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Номер записи: 49
03-10-2013 дата публикации

RADIOCONJUGATION METHOD

Номер: US20130259803A1
Автор: Indrevoll Bard
Принадлежит: GE HEALTHCARE LIMITED

The present invention relates to the field of radiopharmaceuticals for in vivo imaging, in particular to a method of labelling a biological targeting molecule with a radioisotope. The method of the invention is particularly suitable for use with an automated synthesizer apparatus. Also provided are precursors in sterile form, as well as cassettes comprising such precursors useful in the method. 120.-. (canceled)22. The method of claim 21 , where Rand Rare independently Calkyl.23. The method of claim 21 , where the compound of Formula (IA) is used in step (i) claim 21 , so that the aminooxy compound of step (ii) is of Formula (IIA) claim 21 , and the radiolabelled conjugate is of Formula (IVA).24. The method of claim 21 , where Yis H.25. The method of claim 21 , where Q is chosen from F claim 21 , I claim 21 , Tc claim 21 , Ga or Cu.26. The method of claim 25 , where Q is F.27. The method of claim 21 , where the BTM comprises a single amino acid claim 21 , a 3-100 mer peptide claim 21 , an enzyme substrate claim 21 , an enzyme antagonist an enzyme agonist claim 21 , an enzyme inhibitor or a receptor-binding compound.28. The method of claim 27 , where the BTM comprises an Affibody™.30. The method of claim 21 , where steps (ii) and (iii) are carried out simultaneously.31. The method of claim 21 , where the condensation step (iii) is carried out in the presence of aniline.32. The method of claim 21 , which is carried out using an automated synthesizer apparatus.33. The method of claim 32 , wherein said automated synthesizer comprises a single-use claim 32 , disposable cassette which comprises a precursor which comprises the protected compound of Formula (IA) in sterile form.34. A method of preparation of a radiopharmaceutical composition claim 32 , wherein said radiopharmaceutical composition comprises the radiolabelled conjugate of Formula (IVA) or (IVB) as defined in claim 32 , together with a biocompatible carrier in a form suitable for mammalian administration claim ...

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Номер записи: 50
10-10-2013 дата публикации

PLANT PROTEIN HYDROLYSATES

Номер: US20130267683A1
Автор: Berends Pieter, Berger Ralf, Fischer Lutz, Linke Diana, Rabe Swen
Принадлежит: NESTEC S.A.

A membrane reactor for the manufacture of plant protein hydrolysates, the membrane reactor comprising a substrate vessel adapted to provide a plant protein substrate to an enzyme source, a continuously stirred reactor comprising the enzyme source, and an ultrafiltration module comprising a membrane with a molecular cut-off wherein the membrane is adapted to allow passage of the plant protein hydrolysate while retaining the enzyme. 1. A membrane reactor for the manufacture of plant protein hydrolysates , the membrane reactor comprising:a substrate vessel adapted to provide a plant protein substrate to an enzyme source;a continuously stirred reactor comprising the enzyme source; andan ultrafiltration module comprising a membrane with a molecular cut-off wherein the membrane is adapted to allow passage of the plant protein hydrolysate while retaining the enzyme.2. The membrane reactor as claimed in claim 1 , further comprising:a first circulation loop enabling a mixture of the plant protein substrate and enzyme source to be transferred from the continuously stirred reactor to the ultrafiltration module and at least some of the mixture to be returned to the continuously stirred reactor; anda second circulation loop enabling the mixture received from the first circulation loop to be circulated through or over the membrane and at least some of the mixture to be returned to the first circulation loop.3. The membrane reactor as claimed in claim 2 , wherein the first circulation loop operates at or close to atmospheric pressure and the second circulation loop operates at a pressure of 1 to 8 bar.4. The membrane reactor as claimed in claim 1 , comprising a heating device adapted to maintain a temperature of the content of the continuously stirred reactor between 25° C. and 75° C.5. The membrane reactor as claimed in claim 1 , comprising an electro dialysis system.6. The membrane reactor as claimed in claim 1 , comprising a separation device claim 1 , capable of separating ...

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Номер записи: 51
10-10-2013 дата публикации

REMOVAL OF VIRUCIDAL AGENTS FROM BIOMOLECULE PREPARATIONS

Номер: US20130267690A1
Автор: Gagnon Peter S.
Принадлежит: Bio-Rad Laboratories, Inc.

Methods, compositions and kits for chromatography purification of antibodies are provided. In some embodiments, antibodies are purified by hydroxyapatite (HT) or fluorapatite (FT) that is treated with a polycationic agent. In some embodiments, the antibodies are treated with a polycationic agent that is also a virucidal agent prior to purification. 1. A two-stage viral inactivation method , comprising ,incubating a biological sample comprising a target molecule with a positively-charged or neutral virucidal agent under conditions to inactivate viruses in the sample, if present;subsequently contacting the target molecule to an apatite support under conditions resulting in binding of the target biomolecule to the support such that the target biomolecule binds to the apatite and a majority of the virucidal agent flows past the support;washing the support binding the target molecule with a first wash buffer, wherein the first wash buffer comprises at least a second virucidal agent, wherein the second virucidal agent is in sufficient concentration to inactivate viruses, if present, and to dissociate complexes of the positively-charged or neutral virucidal agent and the target molecule, thereby removing at least some residual virucidal agent, if present; andeluting the target biomolecule from the support such that the target biomolecule is substantially free of the positively-charged or neutral virucidal agent.2. The method of claim 1 , wherein the positively-charged or neutral virucidal agent is selected the group consisting of polyethyleneimine claim 1 , ethacridine claim 1 , chlorhexidine claim 1 , benzalkonium chloride claim 1 , tri(n-butyl)phosphate claim 1 , and methylene blue.3. The method of claim 1 , further comprising claim 1 , between the washing and eluting claim 1 , contacting the support with a second wash buffer.4. The method of claim 3 , wherein the second wash buffer has a lower conductivity than the first wash buffer and no chaotropic agents.5. The ...

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Номер записи: 52
17-10-2013 дата публикации

PROTEIN CONCENTRATE FROM STARCH CONTAINING GRAINS: COMPOSITION, METHOD OF MAKING, AND USES THEREOF

Номер: US20130274443A1
Автор: Barrows Frederic T., Bradley Clifford A., Hardy Ronald W., Kearns Robert D., Wasicek Brian D.
Принадлежит:

The present invention relates to methods of producing a protein concentrate from a starch containing grain and uses thereof. In an exemplary embodiment, the protein concentrate produced is used to prepare an aquaculture feed. 1. A process for producing a protein concentrate from a starch containing grain or oil seed , the method comprising:(i) grinding the starch containing grain to produce a ground starch containing grain;(ii) slurrying the ground starch containing grain with water to prepare a slurry comprising starch and glucans;(iii) solublizing starch and glucans comprising the slurry with enzymes to provide a solublized slurry;(iv) adding a fermentation organism to the solublized slurry;(v) fermenting the solublized slurry comprising the fermentation organism until fermentation is complete, thereby producing a fermented slurry;(vi) separating the fermented slurry into solid and liquid fractions;(vii) recovering the solid and liquid fractions;(viii) drying the recovered solid fraction at a temperature below that which would denature or damage proteins;thereby producing a protein concentrate.2. The process of claim 1 , wherein solublizing starch and glucans with enzymes produces glucose.3. The process of claim 1 , wherein the process is a no-cook process.4. The process of claim 1 , wherein the process is a cooking process.5. The method of claim 1 , further comprising:(ix) distilling the recovered liquid fraction to recover a fermentation product.6. The method of claim 5 , wherein the fermentation product is ethanol.7. The method of claim 1 , wherein the starch containing grain is a member selected from the group consisting of barley claim 1 , wheat claim 1 , oats claim 1 , corn claim 1 , rye claim 1 , tritcale claim 1 , sorghum claim 1 , soybeans claim 1 , and soymeal claim 1 , flax claim 1 , camelina claim 1 , or a combination thereof.8. The method of claim 7 , wherein the starch containing grain is barley.9. The method of claim 8 , wherein the barley is from a ...

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Номер записи: 53
17-10-2013 дата публикации

PROCESS FOR PRODUCTION OF FIBRINOGEN

Номер: US20130274444A1
Автор: Gehringer Werner, Pape Rainer, Schulz Petra
Принадлежит: Octapharma AG

The present invention relates to a method or process for the manufacture of a virus and prion save native fibrinogen concentrate of high purity and low amounts of fibrinopeptide A and fibronectin. 1. A process for purifying fibrinogen from a fibrinogen containing source , comprising subjecting a fibrinogen containing solution to chromatography on anion exchange resins , wherein the anion exchange resins are selected from the group consisting of a support material comprising a hydroxylated polymer with grafted tertiary or quaternary amino groups.2. The process of claim 1 , wherein the support material is composed of cellulose claim 1 , agarose claim 1 , silica claim 1 , polymeric or ceramic material.3. The process of claim 1 , wherein the anion-exchange resin is trimethyl-amino groups grafted to a hydroxylated methacrylic polymer via a linking group claim 1 , such as GigaCap Q-650M®.4. The process of claim 1 , wherein the fibrinogen containing source is cryoprecipitate claim 1 , preferably solubilised at about neutral pH .5. The process of claim 4 , wherein the solution is treated with Al(OH)and the resulting gel is removed.6. The process of wherein a virus inactivation is performed by employing a solvent/detergent (S/D) treatment.7. The process of wherein an extraction of S/D reagents with vegetable oil and contacting the water-phase with a TMAE resin at a pH-value of 6.9-7.1 and an osmolality of 570-610 mosmol/l is performed.8. The process of claim 6 , wherein fibrinogen is precipitated by glycine claim 6 , in particular about 1 M glycine claim 6 , and separation of the formed fibrinogen paste.9. The process of wherein the fibrinogen paste is resuspended claim 8 , preferably in about 20 mM TRIS buffer at a pH of about 8.0.10. The process of wherein after filtration the obtained fraction is loaded onto a strong anion exchange resin comprising trimethyl-amino groups grafted to a hydroxylated methacrylic polymer backbone via linking groups and washing off loosely ...

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Номер записи: 54
24-10-2013 дата публикации

METHOD AND KIT FOR PROTEIN LABELING

Номер: US20130280814A1
Автор: Bjellqvist Bengt, Bjerneld Erik, Palmgren Ronnie
Принадлежит: GE HEALTHCARE BIO-SCIENCES AB

The present invention relates to a method for labeling proteins in a sample prior to separation thereof using a protein reactive dye, comprising the following steps a) dissolving the proteins in, or diluting the proteins with, or exchanging an existing protein buffer with, a labeling buffer comprising a dye-reactant (reacting with the protein reactive dye) to form a mixture, b) adding protein reactive dye to said mixture, c) incubating said mixture wherein the labeling of said proteins with said dye can be completed within 5 minutes, and wherein both the proteins and the dye-reactant form measurable reaction products with said dye, and d) separating said reaction products. The invention also relates to a kit for pre-labeling of proteins, comprising a labeling buffer, a dye, a molecular weight marker, and a sample gel loading buffer. 1. A method for labeling proteins in a sample prior to separation thereof using a protein reactive dye , comprising the following steps a) dissolving the proteins in , or diluting the proteins with , or exchanging an existing protein buffer with , a labeling buffer comprising a dye-reactant (reacting with the protein reactive dye) to form a mixture , b) adding protein reactive dye to said mixture , c) incubating said mixture wherein the labeling of said proteins with said dye can be completed within 10 minutes , and wherein both the proteins and the dye-reactant form measurable reaction products with said dye , and d) separating said reaction products.2. The method of claim 1 , wherein the labeling of proteins is completed within 5 minutes.3. The method of claim 1 , wherein the labeling of proteins is completed within 30 seconds.4. The method of claim 1 , wherein the dye-reactant is provided in excess compared to the reactive groups on sample proteins claim 1 , such as amine claim 1 , thiol claim 1 , or carbonyl groups.5. The method of claim 1 , wherein the amount of reaction product from dye and dye-reactant is measured after protein ...

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Номер записи: 55
24-10-2013 дата публикации

PROCESS FOR PRODUCING COELENTERAMIDE OR AN ANALOG THEREOF

Номер: US20130281680A1
Автор: INOUYE Satoshi
Принадлежит:

A process for producing coelenteramide or its analog in a high yield has been desired. The invention provides a process for producing di-O-methylcoelenteramide or its analog of formula (3) 120-. (canceled) The present invention relates to a process for producing coelenteramide or an analogous compound thereof, a process for producing a green fluorescent protein (gFP), and the like.A calcium-binding photoprotein is one of the proteins responsible for bioluminescence. This photoprotein instantaneously emits a flash of light upon specific interaction with Ca. The calcium-binding photoprotein is a complex of a protein having the catalytic function of oxygenation and the peroxide of a luciferin as a luminescence substrate. In the calcium-binding photoprotein, the protein having the catalytic function of oxygenation is called an apoprotein (e.g., apoaequorin). The peroxide of a luciferin is 2-hydroperoxycoelenterazine. Such known calcium-binding photoproteins are those derived from coelenterates, specifically including aequorin, clytin-I, clytin-II, mitrocomin, obelin, etc.Among them, aequorin is a photoprotein isolated from the luminous jellyfish (1: Shimomura, In: , (2006) pp 90-158, World Scientific Pub. Co.; 2: Shimomura et al., (1962) 59, pp 223-240). This aequorin is a non-covalent complex of apoaequorin (21.4 kDa) and a hydroperoxide of coelenterazine (3: Head et al., (2000) 405 372-376). Apoaequorin is a single polypeptide composed of 189 amino acid residues with 3 EF hand motifs characteristic of Ca-binding site (4: Inouye et al., (1985) 82, 3154-3158). In the presence of Ca, aequorin emits blue light (λ=˜460 nm) by an intramolecular reaction and decomposes itself into apoaequorin, coelenteramide and CO(5: Shimomura & Johnson (1972) 11, 1602-1608; 6: Shimomura & Johnson (1973) 2963-2966). The complex of Ca-bound apoaequorin with coelenteramide obtained by this decomposition is known as blue fluorescent protein (BFP) (7: Shimomura & Johnson (1975) 256, 236-238). ...

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Номер записи: 56
31-10-2013 дата публикации

METHODS FOR PURIFICATION OF ALPHA-1-ANTITRYPSIN AND APOLIPOPROTEIN A-1

Номер: US20130289245A1
Автор: Bigler Douglas, BOLLI Reinhard, BRINKMAN Nathan, Foersch Vreni
Принадлежит: CSL Behring GmbH

This invention relates to protein separation and purification methods for both alpha-1-antitrypsin (AAT, also known as alpha-1 proteinase inhibitor, API, and A.sub.1-PI) and Apolipoprotein A-I (ApoA-1) from, for example, a fraction of human blood plasma. In certain embodiments, the invention provides methods for separating AAT from ApoA-1 at the initial stage of purification, so that the same starting material can be used as a source for both proteins. The methods further pertain to providing compositions of AAT and of ApoA-1 suitable for pharmaceutical use and are suitable for large-scale purification. 125.-. (canceled)26. A method for purifying Apolipoprotein A-I (ApoA-I) and Alpha-1-Antitrypsin (AAT) from a single starting human plasma fraction containing both proteins comprising: (a) treating the starting human plasma fraction which is used as the starting material such that ApoA-I and AAT are solubilized;', '(b) adding an ApoA-I binding agent and ensuring an appropriate pH so that ApoA-I binds to the added ApoA-I binding agent, wherein the binding agent is fumed silica;', '(c) separating ApoA-I bound to said fumed silica from the solution containing AAT;', '(d) eluting ApoA-I from said fumed silica; and, '(i) treating a starting human plasma fraction comprising ApoA-I and AAT to separate an ApoA-I containing fraction from an AAT containing fraction, comprising'}(ii) separately purifying ApoA-I and AAT in one or more processing steps.27. The method according to claim 26 , wherein the starting human plasma fraction is selected from one or more of Cohn fractions IV claim 26 , precipitates from Kistler-Nitschmann supernatants A and A+I claim 26 , and ammonium sulfate precipitates.28. The method according to claim 27 , wherein the one or more Cohn fractions IV is Cohn fraction IV.29. The method according to claim 26 , wherein AAT and ApoA-I are not exposed to a pH of 13.69 or above.30. The method according to claim 29 , wherein AAT and ApoA-I are not exposed to a pH ...

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Номер записи: 57
31-10-2013 дата публикации

SINGLE UNIT ION EXCHANGE CHROMATOGRAPHY ANTIBODY PURIFICATION

Номер: US20130289247A1
Автор: Dorst Marijke Yvonne, Kremer Diderik Reinder
Принадлежит: DSM IP ASSETS B.V.

The present invention relates to a method for the purification of antibodies from a protein mixture produced in a bioreactor, at least comprising the steps of intermediate purification and polishing, wherein the intermediate and polishing step comprises in either order in-line anion exchange chromatography (AEX) chromatography and cation exchange chromatography (CEX) chromatography steps in flow-through mode. The present invention further relates to a single operational unit comprising both an anion exchange chromatography part and a cation exchange chromatography part in either order, which are serially connected, wherein the unit comprises an inlet at the upstream end of the first ion exchange chromatography part and an outlet at the downstream end of the second ion exchange chromatography part and wherein the unit also comprises an inlet between the first ion exchange chromatography part and the second ion exchange chromatography part. 111-. (canceled)12. A method for the purification of antibodies from a cell broth produced in a bioreactor , at least comprising the steps of intermediate purification and polishing , wherein the intermediate purification step comprises combined serially connected in-line anion exchange (AEX) and cation exchange (CEX) chromatography steps in either order , both in flow-through and together operating as one single unit operation , wherein the first chromatography step yields as a flow-through fraction a separation mixture , which is directly subjected to the second chromatography step wherein the second step yields as a flow through fraction a purified antibody preparation , and wherein the purified antibody preparation is subjected to at least one further purification step.13. A method according to claim 12 , wherein the AEX chromatography step is carried out first claim 12 , yielding as a flow-through fraction a separation mixture claim 12 , serial in-line followed by a CEX chromatography step yielding as a flow-through fraction a ...

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Номер записи: 58
07-11-2013 дата публикации

METHOD FOR EXTRACTING A PROTEIN FROM MILK

Номер: US20130295646A1
Автор: LEJARS Alain, Nogre Michel, Ollivier Monique
Принадлежит: LFB BIOTECHNOLOGIES

The invention relates to a method for extracting a protein from milk, having at least one hydrophobic pocket and a negative charge to the natural pH of milk, that comprises the following steps: a) skimming and delipidation of the milk; b) passing the delipidated and skimmed fraction containing the protein on a chromatographic substrate on which is grafted a ligand having both a hydrophobic characteristic and an ionic characteristic in pH conditions enabling the protein to be retained on the substrate, the pH being higher than 4.6; c) elution of the protein; d) purification of the eluted fraction by removing the milk proteins from the eluted fraction; and e) recovering the protein. 1. A process for the extraction of a protein present in milk , wherein said protein has at least one hydrophobic patch and a negative charge at the natural pH of milk , comprising:a) skimming and defatting said milk,b) transferring said defatted and skimmed fraction containing said protein to a chromatographic support onto which is grafted a ligand that is simultaneously hydrophobic and ionic, under pH conditions allowing said protein to be captured on said support, said pH being higher than 4.6,c) eluting said protein,d) purifying said eluted fraction by removing milk proteins from said eluted fraction, ande) recovering said protein.2. The process according to claim 1 , wherein after said skimming and defatting step (step a) and prior to step b) claim 1 , a milk clarification step takes place.3. The process according to claim 2 , wherein said clarification step takes place by adding a chelating agent at a concentration such that claim 2 , after mixing with said milk claim 2 , the micellar structure of said milk disappears claim 2 , yielding clarified milk (caseins in solution or lactoserum).4. The process according to claim 1 , wherein after said skimming and defatting step (step a) and prior to step b) claim 1 , casein subunit clusters are precipitated.5. The process according to claim 1 ...

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Номер записи: 59
14-11-2013 дата публикации

COMPOSITIONS AND METHODS OF PRODUCING ENTEROKINASE IN YEAST

Номер: US20130302853A1
Автор: Fotheringham Ian, Sheffield Peter
Принадлежит:

The present specification disclose polynucleotide molecules encoding an enterokinase, yeast expression constructs including a yeast expression vector and a polynucleotide molecules encoding an enterokinase, yeast cells comprising such a yeast expression construct, methods of producing enterokinase using such yeast cells, and method of cleaving or preparing a recombinant polypeptide using an enterokinase produced by such methods. 1. A method of cleaving a recombinant polypeptide , the method comprising the step contacting a polypeptide comprising a cleavage site of SEQ ID NO:1 with an enterokinase , wherein the enterokinase is produced by expressing in a yeast cell comprising a yeast expression construct comprising SEQ ID NO:4 , wherein contacting the recombinant polypeptide with the enterokinase results in specific cleavage of SEQ ID NO:1.2. A method of cleaving a recombinant polypeptide , the method comprising the step contacting a polypeptide comprising a cleavage site of SEQ ID NO:1 with an enterokinase , wherein the enterokinase is produced by expressing in a yeast cell comprising a yeast expression construct comprising SEQ ID NO:6 , wherein contacting the recombinant polypeptide with the enterokinase results in specific cleavage of SEQ ID NO:1. This application is a Continuation of U.S. patent application Ser. No. 13/303,691, filed Nov. 23, 2011, now U.S. Pat. No. 8,497,111, which claims priority pursuant to 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 61/416,622, filed on Nov. 23, 2010, both incorporated by reference.Enterokinase (EK, also known as Enteropeptidase (EP); EC 3.4.21.9) is a heterodimeric glycoprotein produced by cells of the duodenum. Part of the chymotrypsin-clan of serine proteases, it is secreted from intestinal glands (the crypts of Lieberkühn) following the entry of ingested food passing from the stomach and present in the duodenal and jejunal mucosa. Involved in the digestion of dietary proteins, EK catalyzes the cleavage of ...

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Номер записи: 60
14-11-2013 дата публикации

METHOD FOR THE PREPARATION OF SURFACTANT PEPTIDES

Номер: US20130303726A1
Автор: MOZZARELLI Andrea, Pioselli Barbara, Raboni Samanta
Принадлежит:

Surfactant-protein C peptides (SP-C peptides) may be prepared by the heterologous expression of a fusion protein of an SP-C peptide and a maltose binding protein. 1. An expression cassette , comprising:(a) a polynucleotide sequence encoding an SP-C peptide;(b) a polynucleotide sequence encoding an MBP protein; and(c) a polynucleotide sequence encoding a linker peptide,wherein said a polynucleotide sequence encoding a linker peptide encodes a protease cleavage site and is located between said polynucleotide sequence encoding an SP-C peptide and said polynucleotide sequence encoding an MBP protein, andsaid encoding polynucleotide sequence being operatively linked to a promoter sequence suitable for the expression in a prokaryotic cell.2. An expression cassette according to claim 1 , wherein said SP-C peptide is SP-C33(Leu) and has the sequence shown in SEQ ID NO:1.3. An expression cassette according to claim 1 , wherein the MBP protein has the sequence shown in SEQ ID NO:2.4. An expression cassette according to claim 1 , wherein said polynucleotide sequence encoding an SPC peptide has the sequence shown in SEQ ID NO:3.5. An expression cassette according to claim 1 , wherein said polynucleotide sequence encoding an MBP protein has the sequence shown in SEQ ID NO:4.6. An expression cassette according to claim 1 , wherein said linker peptide has the amino acid sequence shown in SEQ ID NO:5.7. An expression cassette according to claim 1 , wherein said polynucleotide sequence encoding a linker peptide has the sequence shown in SEQ ID NO:5.8. An expression cassette according to claim 1 , wherein said polynucleotide sequence encoding an MBP protein is located at the N-terminus.9. An expression cassette according to claim 1 , wherein said polynucleotide sequence encoding an SP-C peptide is located at the C-terminus.10. An expression cassette according to claim 1 , wherein said polynucleotide sequence encoding an MBP protein is located at the N-terminus and said polynucleotide ...

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Номер записи: 61
14-11-2013 дата публикации

Ligand and metal complex having the same

Номер: US20130303728A1
Автор: Chiao-Yun Chen, Gin-Chung Liu, Teng-Wen Li, Yun-Ming Wang
Принадлежит: National Chiao Tung University NCTU

A ligand and a metal complex having the ligand are provided. The ligand and a paramagnetic metal ion form a metal complex with high stability, high relaxivity and high biocompatibility. The metal complex of the present invention is applicable to the preparation of MRI contrast agents for detecting atherosclerosis. The MRI contrast agent includes a peptide sequence specific to a matrix metalloprotease, and can be recognized by a pathological thrombocyte to target a specific site, so as to enhance the imaging contrast.

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Номер записи: 62
21-11-2013 дата публикации

NUCLEOPHILIC CATALYSTS FOR OXIME LINKAGE AND USE OF NMR ANALYSES OF THE SAME

Номер: US20130310546A1
Автор: Haider Stefan, Kosma Paul, Ray G. Joseph, Rottensteiner Hanspeter, Scheinecker Richard, Siekmann Juergen, Szabo Christina, ZHANG Zhenqing
Принадлежит:

The invention relates to materials and methods of conjugating a water soluble polymer to an oxidized carbohydrate moiety of a therapeutic protein comprising contacting the oxidized carbohydrate moiety with an activated water soluble polymer under conditions that allow conjugation and analyzing the conjugation using 2D NMR analysis. More specifically, the present invention relates to the aforementioned materials and methods wherein the water soluble polymer contains an active aminooxy group and wherein an oxime or hydrazone linkage is formed between the oxidized carbohydrate moiety and the active aminooxy group on the water soluble polymer, and wherein the conjugation is carried out in the presence of a nucleophilic catalyst. 1. A method of conjugating a water soluble polymer to an oxidized carbohydrate moiety of a therapeutic protein comprising contacting the oxidized carbohydrate moiety with an activated water soluble polymer under conditions that allow conjugation , wherein the water soluble polymer contains an active aminooxy group and is prepared by a method comprising:a) incubating a solution comprising a water-soluble polymer comprising a reducing end and a non-reducing end with an oxidizing agent to form an oxidized water-soluble polymer comprising a terminal aldehyde group at the non-reducing end of the water-soluble polymer;b) incubating a solution comprising an oxidized water-soluble polymer with an activated aminooxy linker comprising an active aminooxy group under conditions that allow the formation of a stable oxime linkage between the oxidized water-soluble polymer and the activated aminooxy linker, said conditions comprising a time period between about 1 minute and about 24 hours; a temperature between about 2° C. and about 37° C.; in the presence or absence of light, and with or without stirring; thereby forming a water soluble polymer containing an active aminooxy group;c) analyzing the water-soluble polymer containing an active aminooxy group by a ...

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Номер записи: 63
28-11-2013 дата публикации

TEMPERATURE RESPONSIVE ADSORBENT HAVING A STRONG CATION EXCHANGE GROUP AND METHOD FOR PRODUCING THE SAME

Номер: US20130317129A1
Автор: Akiyama Yoshikatsu, Koguma Ichiro, Nagase Kenichi, OKANO Teruo, Okuyama Kazuo, Shigematsu Hiroki
Принадлежит: ASAHI KASEI MEDICAL CO., LTD.

Provided is a temperature responsive adsorbent prepared by immobilizing a copolymer containing at least N-isopropylacrylamide to a base material surface. The copolymer has at least a strong cation exchange group. In addition, the copolymer contains the strong cation exchange group in an amount of 0.01 to 5 mol % relative to N-isopropylacrylamide in terms of monomer. 1. A temperature responsive adsorbent in which a copolymer containing at least N-isopropylacrylamide is immobilized to a base material surface , wherein the copolymer has at least a strong cation exchange group , and contains the strong cation exchange group in an amount of 0.01 to 5 mol % relative to the N-isopropylacrylamide in terms of monomer.2. The temperature responsive adsorbent according to claim 1 , wherein at least a portion of monomer units of the copolymer having the strong cation exchange group is an acrylic acid derivative or a methacrylic acid derivative and has a group represented by the following chemical formula (1) or (2):{'br': None, 'sub': 2', '3, '—CH(—OH)—CH—SOH\u2003\u2003(1)'}{'br': None, 'sub': 3', '2, '—CH(—SOH)—CH—OH\u2003\u2003(2)'}3. The temperature responsive adsorbent according to claim 1 , wherein at least a portion of monomer units of the copolymer having the strong cation exchange group is derived from a vinyl monomer having a sulfonic acid group.4. The temperature responsive adsorbent according to claim 3 , wherein at least a portion of the monomer units of the copolymer having the strong cation exchange group is represented by the following chemical formula (3):{'br': None, 'sub': 1', '2', '3', '3, '—CRR—CR(—SOH)—\u2003\u2003(3)'}{'sub': 1', '2', '3, 'where R, R, Rare each independently H or Me.'}5. A temperature responsive adsorbent in which a copolymer containing at least N-isopropylacrylamide is immobilized to a base material surface claim 3 , wherein the copolymer has at least a strong cation exchange group claim 3 , and wherein the temperature responsive ...

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Номер записи: 64
28-11-2013 дата публикации

METHODS FOR CAPTURING VIRUS LIKE PARTICLES FROM PLANTS USING EXPANDED BED CHROMATOGRPAHY

Номер: US20130317197A1
Автор: Cabrera Rosa, Lihme Allan, Oishi Karen, Vaarst Inga
Принадлежит: PHILIP MORRIS PRODUCTS S.A.

The present invention relates to a method for capturing virus-like particles of interest from a mixture comprising the use of an expanded bed of adsorbent; suitably wherein said method comprises the steps of: (a) providing an expanded bed of adsorbent; (b) contacting the mixture with the adsorbent such that the constituents of the mixture contact the expanded bed of adsorbent; (c) optionally washing the adsorbent; and (d) optionally eluting the particle of interest from the adsorbent. 1. A method for capturing virus like particles of interest from a mixture comprising the use of an expanded bed of adsorbent , wherein said method comprises the steps of: (a) providing an expanded bed of adsorbent; (b) contacting the mixture with the adsorbent such that the virus like particles in the mixture bind the adsorbent; (c) optionally washing the adsorbent; and (d) optionally eluting the virus like particles from the adsorbent.2. The method according to claim 1 , wherein the virus like particles comprises a protein of an influenza virus.316-. (canceled)17. The method of claim 1 , wherein the adsorbent comprises beads that comprise claim 1 , consist or consist essentially of a polysaccharide selected from the group consisting of cellulose claim 1 , agarose and dextran and derivatives thereof or a combination of two or more thereof.1820-. (canceled)21. The method according to claim 1 , wherein the virus like particles comprise a haemagglutinin; suitably a haemagglutinin subtype selected from the group consisting of H1 claim 1 , H2 claim 1 , H3 claim 1 , H4 claim 1 , H5 claim 1 , H6 claim 1 , H7 claim 1 , H8 claim 1 , H9 claim 1 , H10 claim 1 , H11 claim 1 , H12 claim 1 , H13 claim 1 , H14 claim 1 , H15 or H16 or a combination of two or more thereof.22. The method claim 1 , wherein the mixture comprises disrupted cells of a plant; suitably wherein the plant is a tobacco plant infiltrated with nucleic acid molecules that express a protein transiently in the plant claim 1 , said ...

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Номер записи: 65
19-12-2013 дата публикации

PROTEIN PURIFICATION METHODS TO REDUCE ACIDIC SPECIES

Номер: US20130338344A1
Автор: Herigstad Matthew Omon, Ramasubramanyan Natarajan, Yang Hong, YANG Lihua
Принадлежит: AbbVie Inc.

The instant invention relates to the field of protein production and purification, and in particular to compositions and processes for controlling the amount of charge variants, aggregates, and fragments of a protein of interest, as well as host cell proteins, present in purified preparations by applying particular chromatography conditions during such protein purification. 2. The method of claim 1 , wherein the chromatography media is selected from the group consisting of anion exchange claim 1 , cation exchange claim 1 , and mixed mode media.3. The method of claim 1 , wherein the chromatography media is a mixed mode adsorbent material comprising cation exchange and hydrophobic interaction functional groups.4. The method of claim 3 , wherein the mixed mode resin is Capto MMC resin.5. The method of claim 2 , wherein the cation exchange (CEX) adsorbent material is selected from the group consisting of a CEX resin and a CEX membrane adsorber.6. The method of claim 5 , wherein the CEX resin is the Poros XS resin.7. The method of claim 1 , wherein the pH of the loading and wash buffers are lower than the isoelectric point of the protein of interest.8. The method of claim 1 , wherein the chromatography sample contains a reduced level of acidic species.9. The method of claim 1 , wherein the chromatography sample contains a reduced level of protein fragments.10. The method of claim 1 , wherein the chromatographic samples contains a reduced level of host cell proteins.11. The method of claim 1 , wherein the chromatographic sample contains levels of basic variants that differ from the starting load material.12. The method of claim 1 , wherein the protein of interest is an anti-TNFα antibody.13. A pharmaceutical composition comprising a process-related impurity-reduced and/or product-related substance-reduced composition comprising a protein of interest and a pharmaceutically acceptable carrier.14. The pharmaceutical composition of claim 14 , wherein said composition is ...

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Номер записи: 66
16-01-2014 дата публикации

Protein affinity tag and uses thereof

Номер: US20140017797A1
Автор: Adrianus Petrus Josephus Maria De Jong, Geert Petrus Maria Mommen, Gijsbert Zomer, Hugo Derk Meiring, Martinus Richardus Jozef Hamzink
Принадлежит: De Staadt der Nederlanden vert doorde minister van VWS

This invention concerns isotopically coded or non-isotopically coded affinity-tags for analysis of certain target molecules in complex samples, in particular for mass spectrometric analysis of proteomic samples. The affinity-tags have the following general formula X-SPACER-OPO 3 H 2 , wherein X is a functional group or moiety capable of reacting with a functional group of a protein, peptide, DNA, lipid, sugar and/or steroid. These phosphate affinity tags (‘PTAG’) are capable of high but reversible binding to metal-oxides like TiO 2 . Due to this property, tagged sample fractions can be isolated from non-tagged sample fraction by affinity chromatography. The binding of organophosphate to metal-oxides remains intact during multiple washings of preferably acidic solutions to remove non-specifically bound components. PTAG's are also envisaged wherein X is selected such that it is capable of binding proteins, peptides, nucleic acid molecules, lipids, carbohydrates, steroids and the like.

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Номер записи: 67
16-01-2014 дата публикации

METHOD OF INCREASING LIVER PROTEIN SYNTHESIS

Номер: US20140018292A1
Автор: MIURA Susumu
Принадлежит: MEGMILK SNOW BRAND CO., LTD.

A protein synthesis promoter that exhibits a protein synthesis-promoting effect includes a whey protein hydrolyzate having a molecular weight distribution that is within a range of 10 kDa or less and has a main peak of 200 Da to 3 kDa, an average peptide length (APL) of 2 to 8, a free amino acid content of 20% or less, a branched-chain amino acid content of 20% or more, and an antigenicity equal to or less than 1/100,000th of that of β-lactoglobulin. 1. A method of increasing liver protein synthesis in a subject in need of such increase , comprising administering to the subject a protein synthesis promoter comprising a whey protein hydrolyzate , the whey protein hydrolyzate having (1) a molecular weight distribution that is within a range of 10 kDa or less and has a main peak of 200 Da to 3 kDa , (2) an average peptide length (APL) of 2 to 8 , (3) a free amino acid content of 20% or less , (4) a branched-chain amino acid content of 20% or more , and (5) an antigenicity equal to or less than 1/100 ,000th of that of β-lactoglobulin.2. The method according to claim 1 , wherein the whey protein hydrolyzate is obtained by performing a hydrolysis step that hydrolyzes and thermally denatures a whey protein at a pH of 6 to 10 and a temperature of 50 to 70° C. using a heat-resistant protease claim 1 , and an inactivation step that inactivates the protease by heating.3. The method according to claim 1 , wherein the whey protein hydrolyzate is obtained by performing a preliminary hydrolysis step that hydrolyzes a whey protein at a pH of 6 to 10 and a temperature of 20 to 55° C. using a protease claim 1 , a hydrolysis step that hydrolyzes and thermally denatures an unhydrolyzed whey protein at a pH of 6 to 10 and a temperature of 50 to 70° C. using a heat-resistant protease claim 1 , and an inactivation step that inactivates the protease by heating.4. The method according to claim 1 , wherein the whey protein hydrolyzate is obtained by performing a preliminary hydrolysis step ...

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Номер записи: 68
16-01-2014 дата публикации

BUFFER SYSTEM FOR PROTEIN PURIFICATION

Номер: US20140018525A1
Автор: Goklen Kent E., Suda Eric J., Ubiera Antonio Raul
Принадлежит: GlaxoSmithKline LLC

The invention is directed to a method for producing a polypeptide composition comprising: combining a polypeptide with a volatile additive to form a liquid mixture and lyophilizing the liquid mixture to obtain a lyophilized polypeptide composition. 1. A multi component buffer system for the purification of proteins by a series of chromatography steps , where the modes of chromatography are selected from the group consisting of affinity chromatography , anion exchange chromatography , cation exchange chromatography , and mixed-mode chromatography , wherein the modes of chromatography are operated in either bind-elute mode or flow-through mode , where the multi component buffer system comprises an organic acid , an alkaline metal or ammonium salt of the conjugate base of the organic acid , and an organic base and wherein the modes of chromatography are performed using buffers that are made without the addition of NaCl.2. The system of claim 1 , wherein the affinity chromatography is performed using a superantigen immobilized on a solid phase.3. The system of wherein the superantigen is selected from the group consisting of Protein A claim 2 , Protein G claim 2 , and Protein L.4. The system of claim 1 , wherein the organic acid is selected from the group consisting of formic acid claim 1 , acetic acid claim 1 , acid claim 1 , citric acid claim 1 , malic acid claim 1 , maleic acid claim 1 , glycine claim 1 , glycylclycine claim 1 , succinic acid claim 1 , TES (2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid) claim 1 , MOPS (3-(N-morpholino)propanesulfonic acid) claim 1 , PIPES (piperazine-N claim 1 ,N′-bis(2-ethanesulfonic acid)) claim 1 , and MES (2-(N-morpholino)ethanesulfonic acid).5. The system of claim 1 , wherein the organic base is selected from the group consisting of tris base claim 1 , Bis-Tris claim 1 , Bis-Tris-Propane claim 1 , Bicine (N claim 1 ,N-bis(2-hydroxyethyl)glycine) claim 1 , HEPES (4-2-hydroxyethyl-1-piperazineethanesulfonic acid) claim 1 ...

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Номер записи: 69
23-01-2014 дата публикации

INDUSTRIAL PROCESS FOR THE PREPARATION OF N-ALKYL-N-TRIALKYLSILYLAMIDES

Номер: US20140024805A1
Автор: Callens Roland, Collin Andre
Принадлежит: SOLVAY S.A.

The present invention relates to a process for producing N-alkyl-N-trialkylsilylamides from trialkylsilylhalides and N-alkylamides in the presence of a base and in the absence of a solvent. 2. The process according to claim 1 , wherein 0.8 to 2 moles of the organic base [OB] are used per mole of the compound of formula II.3. The process according to claim 1 , wherein R claim 1 , R claim 1 , R claim 1 , Rand Rindependently of each other represent C-Calkyl which can be identical or different.4. The process according to claim 3 , wherein R claim 3 , R claim 3 , R claim 3 , Rand Rindependently of each other represent Cor Calkyl which can be identical or different.5. The process according to claim 1 , wherein R claim 1 , R claim 1 , R claim 1 , Rand Rare the same.6. The process according to claim 5 , wherein R claim 5 , R claim 5 , R claim 5 , Rand Rrepresent methyl.7. The process according to any one of claim 1 , wherein X represents chlorine.8. The process according to claim 1 , wherein the organic base [OB] is a nitrogen containing organic base.9. The process according to claim 1 , wherein the compound of formula I is separated from the salt of the organic base [OB]HXby adding an antisolvent for [OB]HXto the reaction medium so that the salt of the organic base [OB]HXprecipitates and can be removed by filtration.10. The process according to claim 9 , wherein the antisolvent is an alkane having a boiling point in the range from 35 to 80° C.11. The process according to claim 1 , wherein the compound of formula II is reacted with the compound of formula III in a compound of formula II/compound of formula III molar ratio of 1/5 to 5/1.12. The process according to claim 1 , wherein the reaction of the compound of formula II with the compound of formula III is carried out at a temperature in the range from 20 to 50° C.13. A process for the manufacture of a peptide or peptide analogue which comprises{'claim-ref': {'@idref': 'CLM-00001', 'claim 1'}, 'preparing a compound of ...

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Номер записи: 70
13-02-2014 дата публикации

Method for preparing a depleted plasma material consisting of one or more thrombogenic factors

Номер: US20140044742A1
Автор: Monique Ollivier, Philippe Paolantonacci
Принадлежит: LFB SA

The invention concerns a method for preparing a plasma product depleted of one or more thrombogenic factors, comprising the combination of at least two steps chosen from among an ethanol fractionation step, a filtration-adsorption step, a precipitation step with caprylic acid and a chromatography step on ion exchange resin.

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Номер записи: 71
13-02-2014 дата публикации

DRUG SUBSTANCES, PHARMACEUTICAL COMPOSITIONS AND METHODS FOR PREPARING THE SAME

Номер: US20140044759A1
Автор: Liotta Vincenzo, Pridgen Christopher, Zarkadas Dimitrios
Принадлежит:

Drug substances, which comprise a solid amorphous forms of a compound of structural formula I and have a BET specific surface area of up to about 94 m/g, pharmaceutical compositions comprising such drug substances, processes for preparing such drug substances and uses of such drug substances and pharmaceutical compositions are disclosed. 2. The drug substance according to claim 1 , wherein said drug substance has a BET specific surface area of from about 2.9 m/g to about 9.6 m/g.3. The drug substance according to claim 1 , wherein said drug substance has a BET specific surface area of from about 2.9 m/g to about 9.4 m/g.4. A pharmaceutical composition comprising at least one drug substance according to and at least one pharmaceutically acceptable carrier.5. The pharmaceutical composition according to claim 4 , further comprising at least one excipient.7. The process according to claim 6 , wherein said distilling step b) is conducted.8. The process according to claim 7 , wherein said distilling step b) is conducted at a temperature in a range from about −15.0° C. to about 35.0° C.9. The process according to claim 7 , wherein said distilling step b) is conducted at a temperature in a range from about 15.0° C. to about 30.1° C.10. The process according to claim 7 , wherein said distilling step b) is conducted at a temperature in a range from about 15.0° C. to about 24.6° C.11. The process according to claim 10 , wherein said distilling step b) is conducted at a temperature in a range from about 15.1° C. to about 24.6° C. for the first 10 hours of the distilling.12. The process according to claim 7 , wherein said distilling step b) is conducted at a temperature in a range from about −15.0° C. to about 15.0° C.13. The process according to claim 7 , wherein said distilling step b) is conducted over a total of 20 to 30 hours.14. The process according to claim 6 , wherein said filtering step c) is conducted at a temperature in a range from about −15.0° C. to about 15.0° C. ...

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Номер записи: 72
13-02-2014 дата публикации

METHOD OF PURIFYING PROTEIN

Номер: US20140046038A1
Автор: ISHIHARA Takashi
Принадлежит: KYOWA HAKKO KIRIN CO., LTD

The present invention relates to a method for purifying a protein by separating the protein from impurities in a non-adsorption mode using an activated carbon. In particular, the present invention relates to a method for purifying an antibody using the activated carbon instead of protein A affinity chromatography. 1. A method for purifying a protein , wherein the protein is separated from impurities using an activated carbon to obtain the protein with a low content of impurities.2. The purification method according to claim 1 , wherein the protein has a molecular weight of 30000 or more.3. The purification method according to claim 1 , wherein the protein is a glycoprotein.4. The purification method according to claim 3 , wherein the glycoprotein is an antibody.5. The purification method according to claim 1 , wherein the protein is a genetically modified protein.6. The purification method according to claim 1 , wherein the impurities are any one of host cell proteins claim 1 , protein-derived polymers claim 1 , protein-derived degradation products claim 1 , or DNAs.7. The purification method according to claim 1 , wherein the method is carried out in a non-adsorption mode.8. The purification method according to claim 1 , wherein the separation is carried out at pH 3 to 8.9. The purification method according to claim 1 , wherein the activated carbon is an activated carbon from wood.10. The purification method according to claim 1 , wherein the activated carbon has an average micropore diameter of 0.5 to 5 nm.11. A method for preparing a protein claim 1 , comprising the purification method of .12. The preparation method according to claim 11 , wherein protein A chromatography is not used.13. The preparation method according to claim 11 , comprising any one of anion exchange chromatography claim 11 , cation exchange chromatography claim 11 , hydrophobic interaction chromatography claim 11 , and multimodal chromatography.14. The preparation method according to claim 11 ...

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Номер записи: 73
13-02-2014 дата публикации

Method For Selective Derivatization of Oligohistidine Sequence of Recombinant Proteins

Номер: US20140046040A1
Автор: Melman Artem
Принадлежит: CLARKSON UNIVERSITY

Methods and compositions for the selective derivatization of a oligohistidine-tagged recombinant protein. A modifying compound comprised of an imidazole reactive group, a linker, and a ligating group is contacted with the recombinant protein, and a cooperative bond forms between the ligating group and the oligohistidine tag in the presence of a metal cation, and a covalent bond forms between the imidazole reactive group and an imidazole ring of the oligohistidine tag followed by the concomitant separation of the imidazole reactive group from the linker. Addition of a metal chelator results in the dissociation of the ligating group and the oligohistidine tag. 1. A method for the selective derivatization of a recombinant protein , the method comprising: [{'br': None, 'sub': 1', '2', '3, 'XXX,'}, {'br': None, 'wherein'}], 'contacting a recombinant protein comprising a oligohistidine tag with a modifying compound in the presence of a metal cation, wherein the modifying compound comprises the formula'}{'sub': '1', 'Xis an imidazole reactive group;'}{'sub': '2', 'Xis a linking group; and'}{'sub': '3', 'Xis at least one ligating group;'}{'sub': '2', 'wherein said imidazole reactive group comprises a functional group, an electron withdrawing group, and a connector connecting the imidazole reactive group to X;'}{'sub': 3', '1', '1', '2, 'claim-text': {'sub': '3', 'breaking the bond between Xand the oligohistidine tag using a metal chelator.'}, 'and further wherein said contacting is performed under conditions sufficient for the formation of a bond between Xand the oligohistidine tag utilizing said metal cation, and under conditions sufficient for the formation of a covalent bond between Xand an imidazole ring of the oligohistidine tag and concomitant separation of Xand X; and'}3. The method of claim 1 , wherein said electron withdrawing group is selected from the group consisting of an acyl group claim 1 , an ester group claim 1 , an alkylsulfonyl group claim 1 , an ...

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Номер записи: 74
20-02-2014 дата публикации

POLYNUCLEOTIDE AND POLYPEPTIDE SEQUENCE AND METHODS THEREOF

Номер: US20140051842A1
Автор: Baliga Madhuri, Kumar Kirubakaran Naveen, Manoj Mulakkapurath Narayanan, Nair Chandrasekhar Bhaskaran, Subbarao Pillarisetti Venkata, Vasamsetty Venkata Ramachandra Rao
Принадлежит: BIGTEC PRIVATE LIMITED

The present disclosure relates to a field of recombinant DNA therapeutics. It involves the bio-informatics design, synthesis of artificial gene for human insulin precursor including leader peptide coding sequence, cloning in an expression vector and expression in an organism, preferably . The present disclosure also relates to methods of downstream processing for obtaining protein precursor molecules and subsequent conversion of precursor molecules to functional proteins. 1) A polynucleotide sequence as set forth in SEQ ID NO: 2.2) The sequence as claimed in claim 1 , wherein the polynucleotide encodes a fusion polypeptide comprising recombinant Human Insulin Precursor and signal peptide.3) A polypeptide sequence as set forth in SEQ ID NO: 1.4) The sequence as claimed in claim 3 , wherein the polypeptide is a fusion polypeptide comprising recombinant Human Insulin Precursor and signal peptide.5) The sequence as claimed in claim 3 , wherein the polypeptide sequence corresponds to polynucleotide sequence set forth in SEQ ID NO: 2 claim 3 , wherein the polynucleotide is subjected to post-transcriptional modification and codon optimization to obtain corresponding polypeptide of SEQ ID NO: 1.6) A method for obtaining recombinant insulin precursor molecule having polypeptide sequence as set forth in SEQ ID NO: 1 claim 3 , said method comprising steps of:a) synthesizing a polynucleotide sequence set forth in SEQ ID NO: 2 by combining 26 oligonucleotides of SEQ ID NOS: 3 to 28 by assembly PCR, and inserting the synthesized sequence in a vector,b) transforming a host cell with said vector followed by antibiotic screening host selection, andc) fermenting the selected transformed host cell and in-situ capturing of the insulin precursor molecule to obtain said precursor having polypeptide sequence as set forth in SEQ ID NO: 1.7) The method as claimed in claim 6 , wherein the polypeptide is a fusion polypeptide comprising recombinant Human Insulin Precursor and signal peptide.8) ...

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Номер записи: 75
27-02-2014 дата публикации

METHODS FOR REACTING CYSTEINE RESIDUES IN PEPTIDES AND PROTEINS

Номер: US20140054163A1
Автор: Arumugam Selvanathan, BOONS Geert-Jan, Guo Jun, LIN NANNAN, NEKONGO EMMANUEL, POPIK VLADIMIR V.
Принадлежит: University of Georgia Research Foundation, Inc.

Methods for labeling or modifying cysteine residues in proteins and/or enzymes are disclosed. The methods include the reaction of an o-naphthoquinone methide with a thiol group of a cysteine residue of a protein or enzyme, which can be reversible in preferred embodiments. The o-naphthoquinone methide can conveniently be generated by irradiation of a precursor compound, preferably in an aqueous solution, suspension, or dispersion. 1. A method for labeling a protein having a thiol group , the method comprising:generating an o-naphthoquinone methide; andcontacting the o-naphthoquinone methide with the protein under conditions effective to form the labeled protein.2. The method of wherein the protein comprises a cysteine residue having the thiol group.3. The method of wherein the o-naphthoquinone methide is a 2-naphthoquinone-3-methide.5. The method of wherein the labeling method is reversible.6. The method of further comprising:irradiating the labeled protein under conditions effective to remove the label.7. The method of wherein conditions effective to remove the label comprise irradiating the labeled protein in a dilute solution.8. The method of wherein conditions effective to remove the label comprise irradiating the labeled protein in the presence of a polarized olefin.9. The method of wherein the polarized olefin is a vinyl ether.10. The method of wherein generating the o-naphthoquinone methide comprises:providing an o-naphthoquinone methide precursor compound; andirradiating the precursor compound under conditions effective to form the o-naphthoquinone methide.11. The method of wherein the precursor compound comprises a 3-hydroxymethyl-2-naphthol group.13. The method of wherein conditions effective to form the labeled protein comprise irradiating a molar excess of the precursor compound compared to the moles of thiol groups of the protein.14. The method of wherein the precursor compound is irradiated in the presence of the protein.15. A method for inhibiting an ...

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Номер записи: 76
27-02-2014 дата публикации

Polynucleotide and polypeptide sequence and methods thereof

Номер: US20140057318A1
Автор: Chandrasekhar Bhaskaran Nair, Kirubakaran Naveen Kumar, Madhuri Baliga, MULAKKAPURATH NARAYANAN Manoj, Pillarisetti Venkata Subbarao, Venkata Ramachandra Rao Vasamsetty
Принадлежит: Bigtec Pvt Ltd

The present disclosure relates to a field of recombinant DNA therapeutics. It involves the bio-informatics design, synthesis of artificial gene for human insulin precursor including leader peptide coding sequence, cloning in an expression vector and expression in an organism, preferably Pichia pastoris. The present disclosure also relates to methods of downstream processing for obtaining protein precursor molecules and subsequent conversion of precursor molecules to functional proteins.

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Номер записи: 77
06-03-2014 дата публикации

METHODS FOR RECOVERING PEPTIDES/AMINO ACIDS AND OIL/FAT FROM ONE OR MORE ...

Номер: US20140066359A1
Автор: CARLSSON Tomas
Принадлежит: Zymtech Production AS

According to a first aspect, hydrolysis of a protein-containing raw material and separation of amino acids/peptides is carried out, wherein the hydrolysis is effected by using the endogenous enzymes of the protein-containing raw material. The hydrolysate is passed through a membrane filter, wherein peptide/amino acids follow a permeate stream, whilst the active enzymes continuously break down any protein residues that are deposited on the membrane surface. The enzymes are passed together with retentate back to the hydrolysis. Furthermore, an amino acid and peptide product and an oil product are described and the use thereof is disclosed. 1. A method for producing peptides/amino acids with a fat content of less than 0.5% by weight from a protein-containing raw material , wherein the method comprises:(a) grinding the protein-containing raw material;(b) heating the ground raw material to temperatures in the range of 40-62° C.;(c) adding water which has approximately the same, or the same, temperature as the raw material, and wherein the pH of the water is adjusted to 7.0-8.5;(d) hydrolysing the ground raw material with endogenous enzymes in order to prepare a hydrolysate;(e) removing solid particles and non-hydrolysed proteins which can be returned to the hydrolysis from the hydrolysate;(f) periodically or continually separating off fat/oil in order to obtain an oil product;(g) separating off the desired molecular weight fraction of peptides/amino acids by membrane filtration;(h) routing the portions of the hydrolysate that do not penetrate the membrane filter in step (g) back to the hydrolysis in step (d);(i) concentrating and optionally drying the permeate in order to obtain a distillate comprising water, and a concentrate comprising peptides/amino acids and water; and(j) wholly or partly returning the distillate from the concentrating to the permeate side of the membrane filter.2. The method according to claim 1 , wherein the method takes place as a closed process.3 ...

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Номер записи: 78
13-03-2014 дата публикации

METHODS FOR PURIFYING IgY ANTIBODIES

Номер: US20140073766A1
Автор: James Schiltz, Jeremy Vrchota, Subramanian V. Iyer, Thomas A. Henderson
Принадлежит: Avianax LLC

A method for purifying IgY antibodies is provided. The method comprises precipitating IgY antibodies from a sample by contacting the sample with a precipitating agent; and differentially precipitating the IgY antibodies obtained in step (a) using the same or different precipitating agent to separate IgY(Fc) and IgY(ΔFc) antibodies.

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Номер записи: 79
20-03-2014 дата публикации

TUMOUR MARKER PROTEINS AND USES THEREOF

Номер: US20140080736A1
Автор: Graves Catherine Rosamund Louise, Robertson John Forsyth Russell
Принадлежит: Onclmmune Limited

Tumour marker proteins and their preparation from fluids from one or more cancer patients, wherein said fluids are those which collect in a body cavity or space which is naturally occurring or which is the result of cancer or medical intervention for cancer. The present application also relates to preparation of tumour marker proteins from excretions taken from patients with cancer. The tumour marker proteins are useful as immunoassay reagents in the detection of cancer-associated anti-tumour marker autoantibodies. 1. A method of detecting cancer-associated anti-tumor autoantibodies in a sample from an individual , comprising:contacting the sample with an immunoassay reagent; and detecting a presence of complexes formed by specific binding of the immunoassay reagent to any cancer-associated anti-tumor autoantibodies present in the sample,wherein the immunoassay reagent comprises two or more tumor marker proteins, one or more of which having been prepared from a tumor-induced bodily fluid produced in a body cavity or space in the presence of the tumor of one or more cancer patients, wherein the bodily fluid contains more cancer-associated forms of the tumor marker protein than a non-tumor-induced bodily fluid in the same patient, and the bodily fluid is not a fluid from the systemic circulation,wherein the one or more tumor marker proteins prepared from a bodily fluid exhibit selective reactivity with cancer-associated anti-tumor autoantibodies, wherein the tumor marker proteins are over-expressed or altered forms of wild-type proteins, andwherein detection of complexes indicates the presence of cancer-associated anti-tumor autoantibodies in the individual.2. The method of claim 1 , further comprising detecting and/or quantitatively measuring the presence of two or more types of autoantibodies claim 1 , wherein each one of the two or more types of the autoantibodies is immunologically specific to a different tumor marker protein or to different epitopes of the same ...

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Номер записи: 80
20-03-2014 дата публикации

ACID-CLEAVABLE LINKERS EXHIBITING ALTERED RATES OF ACID HYDROLYSIS

Номер: US20140080998A1
Автор: "OKeefe Daniel P.", BEDZYK LAURA A, Fahnestock Stephen R., GRUBER TANJA MARIA, Rouviere Pierre E
Принадлежит: E I DU PONT DE NEMOURS AND COMPANY

An acid-cleavable peptide linker comprising aspartic acid and proline residues is disclosed. The acid-cleavable peptide linker provides an altered sensitivity to acid-hydrolytic release of peptides of interest from fusion peptides of the formula PEP1-L-PEP2. The inventive linker, L, is described in various embodiments, each of which provides substantially more rapid acid-release of peptides of interest than does a single aspartic acid-proline pair. In an additional aspect, a method of increasing the stability of an acid cleavable linkage to acid hydrolysis is also provided. 1. A method of preparing at least one peptide of interest (“POI”) from a fusion peptide comprising at least one POI , comprising:{'claim-ref': {'@idref': 'CLM-00021', 'claim 21'}, 'a) providing a recombinant cell synthesizing the fusion peptide of ;'}b) contacting the fusion peptide with a solution of sufficiently acidic pH so that linker L is cleaved, andc) isolating the at least one POI.2. (canceled)3. The method of wherein the recombinant cell is a recombinant microbial cell.4. The method of wherein the recombinant microbial cell is a recombinant yeast cell.5. The method of wherein the recombinant microbial cell is a recombinant bacterial cell.6. The method of wherein the acid-cleavable linker is cleaved by incubating the fusion peptides at a pH in the range from about pH 1 to about pH 4.7. The method of wherein the acid-cleavable linker is cleaved by incubating the fusion peptides at a pH in the range from about pH 2 to about pH 4.8. The method of wherein the acid-cleavable linker is cleaved by incubating the fusion peptides at a pH in the range from about pH 3 to about pH 4.9. The method of wherein the acid-cleavable linker is cleaved by incubating the fusion peptides at a pH of about 4.10. The method of wherein the acid-cleavable linker is cleaved by incubating the fusion peptides at a temperature of about 40° C. to about 90° C.11. The method of wherein the acid-cleavable linker is cleaved ...

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Номер записи: 81
20-03-2014 дата публикации

Ion Exchange Chromatography with Improved Selectivity for the Separation of Polypeptide Monomers, Aggregates and Fragments by Modulation of the Mobile Phase

Номер: US20140081000A1
Автор: NEUMANN SEBASTIAN
Принадлежит: Hoffmann-La Roche Inc.

Herein is reported a method for producing a polypeptide in monomeric form comprising the following step: recovering the polypeptide in monomeric form from an ion exchange chromatography material by applying a solution comprising a non-ionic polymer and an additive. 1. A method for producing an antibody of the IgG class in monomeric form comprising the following steps:applying a first solution that optionally comprises poly (ethylene glycol) and sorbitol to a cation exchange chromatographic material and thereby equilibrating the material,applying the solution comprising the antibody of the IgG class to the equilibrated chromatography material and thereby loading the chromatography material,producing the antibody of the IgG class in monomeric form by applying a solution to the chromatographic material comprising poly (ethylene glycol) and sorbitol, and thereby desorbing/eluting the antibody of the IgG class in monomeric form from the chromatographic material,whereby the poly (ethylene glycol) polymer has a concentration of about 10% by weight and the sorbitol has a concentration of from 5% to 20% by weight.2. A method for producing an antibody of the IgG class preparation with reduced host cell protein content comprising the following steps:applying a first solution that optionally comprises poly (ethylene glycol) and sorbitol to a cation exchange chromatographic material and thereby equilibrating the materialapplying the solution comprising the antibody of the IgG class to the equilibrated chromatography material and thereby loading the chromatography material,producing the antibody of the IgG class in monomeric form by applying a solution to the chromatographic material comprising poly (ethylene glycol) and sorbitol and thereby desorbing/eluting the antibody of the IgG class in monomeric form from the chromatographic material,whereby the poly (ethylene glycol) has a concentration of about 10% by weight and the sorbitol has a concentration of from 5% to 20% by weight ...

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Номер записи: 82
07-01-2016 дата публикации

CHEMICAL PREPARATION OF UBIQUITIN THIOESTERS AND MODIFICATIONS THEREOF

Номер: US20160002286A1
Автор: BRIK Ashraf, ERLICH Leslie, HAJ-YAHYA Mahmood, KUMAR Ajish, SPASSER Liat
Принадлежит:

The present invention discloses latent thioester functionalities attached to the C-terminus of a first polypeptide, or a first fragment thereof having a Cys residue at its N-terminus, and a process using this functionality for the preparation of polypeptide thioesters, in particular of ubiquitin thioesters, this process comprising preparing a polypeptide or a fragment thereof, being attached to a latent thioester functionality, which can then be ligated with a second polypeptide fragment, followed by selective activation of the latent thioester functionality group, to provide the requested polypeptide thioester. There are also provided the polypeptides obtained by this method, specific unnatural amino acids useful to be incorporated within the polypeptide thioesters, and kits for preparing them. 2. The process of claim 1 , further comprising desulfurization of said ubiquitin thioester to turn said Cys amino acid into an Ala amino acid claim 1 , either before or after step (d).3. The process of claim 1 , wherein said N-S acyl transfer device is a residue of N-methyl cysteine.4. The process of claim 1 , wherein said ubiquitin thioester contains at least one protected δ-mercaptolysine.5. The process of claim 4 , wherein said protected δ-mercaptolysine is a thiazolidine (Thz)-protected mercaptolysine.6. The process of claim 1 , wherein said ubiquitin thioester is prepared of two ubiquitin fragments by native chemical ligation (NCL) claim 1 , wherein said fragment attached to said N-S acyl transfer device is: CGKQLEDGRTLSDYNIQKESTLHLVLRLRGG (Ub46-76) (SEQ ID NO: 8) and said second fragment being in its thioester form is LQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF (Ubl-45) (SEQ ID NO: 4).7. The process of claim 1 , wherein said ubiquitin thioester is prepared of two ubiquitin fragments by native chemical ligation (NCL) claim 1 , wherein said fragment attached to said N-S acyl transfer device is: CKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG (Ub28-76) (SEQ ID NO: 9) ...

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Номер записи: 83
07-01-2016 дата публикации

METHOD OF PURIFYING AN ANTIBODY

Номер: US20160002289A1
Автор: BEIJER DE Thomas Antonius Bernardus, Eppink Michel Hendrikus Maria, KOKKE Bastiaan Pieter Arian, MARZÁ PÉREZ Maria, WIJK-BASTEN VAN Everdina Josephina Wilhelmina
Принадлежит: Synthon Biopharmaceuticals B.V.

A method of purifying an antibody composition comprises application of anion exchange chromatography late in the purification process. An ultrafiltration/diafiltration-purified antibody composition is subjected to anion exchange chromatography (AEX) to form a pharmaceutically-pure antibody composition. 1. A method of purifying an antibody composition , which comprises subjecting a UF/DF-purified antibody composition to anion exchange chromatography (AEX) to form a pharmaceutically pure antibody composition.2. The method of claim 1 , wherein said UF/DF-purified antibody composition has an antibody concentration of at least 1 mg/ml.3. The method of claim 2 , wherein said UF/DF-purified antibody composition has an antibody concentration of at least 10 mg/ml.4. The method of claim 3 , wherein said UF/DF-purified antibody composition has an antibody concentration of not greater than 250 mg/ml.5. The method of claim 4 , wherein said UF/DF-purified antibody composition has an antibody concentration of 20 to 50 mg/ml.6. The method of claim 1 , which further comprises filling said pharmaceutically pure antibody composition into vials.7. The method according to claim 6 , which further comprises aseptically filtering said pharmaceutically pure antibody composition prior to said filling step.8. The method according to claim 7 , which further comprises at least one of the following steps prior to said filling step: (a) adding an excipient to said pharmaceutically-pure antibody composition; (b) concentrating said pharmaceutically-pure antibody composition; (c) diluting said pharmaceutically-pure antibody composition; and/or (d) adjusting the pH of said pharmaceutically-pure antibody composition.9. The method of claim 1 , which further comprises subjecting a partially-purified antibody composition to UF/DF to form said UF/DF-purified antibody composition.10. The method of claim 9 , which further comprises subjecting an antibody cell culture harvest to an antibody capture step and ...

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Номер записи: 84
07-01-2016 дата публикации

MATERIALS AND METHODS FOR REMOVING ENDOTOXINS FROM PROTEIN PREPARATIONS

Номер: US20160002290A1
Автор: GAGNON Peter Stanley, VAGENENDE Vincent
Принадлежит:

A method includes (i) adding allantoin in a supersaturating amount to a protein preparation including a desired protein and at least one endotoxin as a contaminant, (ii) removing solids after the adding step to provide a sample for further purification by void exclusion chromatography on a packed particle bed of electropositive particles in a column, the packed particle bed having an interparticle volume, (iii) applying a sample volume to the packed particle bed, wherein the electropositive particles support void exclusion chromatography, and wherein the sample volume is not greater than the interparticle volume, and (iv) eluting a purified sample including the desired protein and a reduced amount of the endotoxin. The method is optionally carried out with only the allantoin treatment or only the void exclusion chromatography. 116.-. (canceled)17. A method comprising: (i) adding allantoin in a supersaturating amount to a protein preparation comprising a desired protein and an amount of at least one endotoxin as a contaminant; (ii) removing solids from the protein preparation to provide a sample for further purification by void exclusion chromatography using a packed particle bed of electropositive particles in a column , the packed particle bed having an interparticle volume; (iii) applying a sample volume of the sample to the packed particle bed , wherein the electropositive particles support void exclusion chromatography , and wherein the sample volume is not greater than the interparticle volume , and (iv) eluting a purified sample comprising the desired protein and a reduced amount of the at least one endotoxin , wherein the eluted desired protein resides in a buffer to which the column was equilibrated , independently from a buffer content of the sample applied to the column.18. The method of claim 17 , wherein the supersaturating amount of allantoin comprises an amount selected from the group consisting of: (i) about 10% claim 17 , (ii) about 5% claim 17 , (ii ...

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Номер записи: 85
03-01-2019 дата публикации

METHODS FOR THE SITE-SELECTIVE COUPLING OF A FIRST AGENT TO A SECOND AGENT

Номер: US20190002494A1
Автор: Antoatou Eirini, Hoogewijs Kurt, MADDER Annemieke
Принадлежит: Universiteit Gent

The present invention relates to a method for site-selective coupling of a first agent to a second agent, comprising the steps of: contacting a first agent comprising at least one furan moiety with an activation signal and with a second agent comprising at least one hydrazine moiety or at least one hydroxylamine moiety, thereby activating said furan moiety to an activated furan moiety; and reacting said activated furan moiety with the hydrazine moiety or the hydroxylamine moiety, thereby site-selectively coupling said first agent to said second agent. 1. A method for site-selective coupling of a first agent to a second agent , comprising the steps of:contacting a first agent comprising at least one furan moiety with an activation signal and with a second agent comprising at least one hydrazine moiety or at least one hydroxylamine moiety, thereby activating said furan moiety to an activated furan moiety; andreacting said activated furan moiety with the hydrazine moiety or the hydroxylamine moiety, thereby site-selectively coupling said first agent to said second agent.2. The method according to claim 1 , comprising the steps of:a) providing a first agent comprising at least one furan moiety;b) contacting said first agent with an activation signal, thereby activating said furan moiety to an activated furan moiety;c) contacting said activated furan moiety with a second agent comprising at least one hydrazine moiety or at least one hydroxylamine moiety; andd) reacting said activated furan moiety with the hydrazine moiety or the hydroxylamine moiety, thereby site-selectively coupling said first agent to said second agent.3. The method according to claim 1 , comprising the steps of:a) providing a first agent comprising at least one furan moiety;b′) contacting the first agent with a second agent comprising at least one hydrazine moiety or at least one hydroxylamine moiety;c′) contacting the first agent with an activation signal, thereby activating said furan moiety to an ...

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Номер записи: 86
02-01-2020 дата публикации

SYSTEMS AND METHODS FOR PREPARING A POLYPEPTIDE FROM A MIXTURE

Номер: US20200002373A1
Автор: LIVIGINI Isabelle, MATTILA John, McDermott Stefanie, Reilly James
Принадлежит: Regeneron Pharmaceuticals, Inc.

Embodiments of the present disclosure are directed to methods for preparing a target polypeptide from a mixture including the target polypeptide. The method may include contacting the mixture to a hydrophobic interaction chromatography (HIC) apparatus including multiple chromatographic zones. The method may further include passing the target polypeptide through the outlets of at least a first zone and a second zone of the HIC apparatus. A residence time for the mixture including the target polypeptide in a first zone may be approximately the same as a residence time of one or more mobile phases in the second zone. 1. A method for preparing a target polypeptide from a mixture including the target polypeptide , the method comprising:contacting the mixture including the target polypeptide to a first zone of a hydrophobic interaction chromatography (HIC) apparatus, the first zone having one or more chromatographic columns and an outlet;contacting one or more mobile phases to a second zone of the HIC apparatus, the second zone having one or more chromatographic columns and an outlet; andpassing the target polypeptide through the outlets of at least the first and second zones of the HIC apparatus;wherein a residence time for the mixture including the target polypeptide in the first zone is configured to be approximately the same as a residence time of the one or more mobile phases in the second zone.2. The method of claim 1 , wherein the target polypeptide is a monoclonal antibody.3. The method of claim 1 , wherein the one or more mobile phases comprises an equilibration buffer and a wash buffer.4. The method of claim 1 , further comprising passing an effluent including the target polypeptide from the first zone of the HIC apparatus to the second zone of the HIC apparatus.5. The method of claim 1 , wherein contacting the one or more mobile phases to the second zone of the HIC apparatus includes:contacting a wash buffer to the second zone of the HIC apparatus; and ...

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Номер записи: 87
02-01-2020 дата публикации

IMPROVED PROCESS FOR THE PREPARATION OF A DODECAPEPTIDE

Номер: US20200002374A1
Автор: BRIDON Dominique, CABART Frédéric, FAUSTER Christian, Gobron Stephane
Принадлежит:

The invention concerns an improved process for the preparation of the dodecapeptide NX210 of SEQ ID NO: 1 in which the formation of side products is minimized. 115-. (canceled)16. A process for preparing a peptide of SEQ ID NO: 1 , wherein the peptide of SEQ ID NO: 1 is constantly maintained in an aqueous solution comprising an organic solvent throughout the steps of purification and drying , of the peptide of SEQ ID NO: 1.17. A process for preparing a peptide of SEQ ID NO: 1 , wherein the steps of purification and/or drying of the peptide of SEQ ID NO: 1 are carried out in an aqueous solution comprising an organic solvent.18. The process according to claim 16 , wherein the organic solvent is acetonitrile.19. The process according to claim 17 , wherein the organic solvent is acetonitrile.20. The process according to claim 16 , wherein the volume of the organic solvent in the aqueous solution is maintained and/or adjusted in such a way that it represents in proportion from 1 to 90% of the total volume of the aqueous solution during the steps of purification.21. The process according to claim 20 , wherein said proportion is from 1 to 10%.22. The process according to claim 16 , wherein the volume of the organic solvent in the aqueous solution to be dried is maintained and/or adjusted in such a way that it represents in proportion from 1 to 30% of the total volume of the aqueous solution.23. The process according to claim 16 , comprising the following steps:a) grafting of a Fmoc-Gly residue to a resin,b) elongation of the peptide chain with Fmoc-protected amino-acids in which the side chain is protected,c) simultaneous deprotection of the side chains of the amino-acids and cleavage of the polypeptide from the resin, to obtain the polypeptide of SEQ ID NO: 1,d) purification and optional concentration of the peptide of SEQ ID NO: 1,e) drying of the peptide of the peptide of SEQ ID NO: 1 obtained in step e).24. The process according to claim 23 , further comprising a ...

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Номер записи: 88
07-01-2021 дата публикации

METHOD FOR REMOVING N-TERMINAL TRUNCATED AND ABNORMAL VARIANTS IN RHNGF

Номер: US20210002341A1
Автор: Liu Wenchao, Sun Hongliang, Wang Yuesheng, Zhang Yi
Принадлежит:

A method for removing an N-terminal truncated variant and an abnormal variant in recombinant human nerve growth factor (rhNGF) is provided. An rhNGF raw material loaded on a cation-exchange material is washed with a washing liquid to obtain a washed raw material from which an N-terminal truncated variant and an abnormal variant have been removed, where the washing liquid has higher electrical conductivity than the rhNGF raw material. Cation-exchange chromatography (CEC) elution is then performed on the washed raw material with an elution buffer having higher electrical conductivity than the washing liquid. A purified rhNGF product is obtained from the eluate. 1. A method for removing an N-terminal truncated variant and an abnormal variant in recombinant human nerve growth factor (rhNGF) , comprising:1) washing with a washing liquid an rhNGF raw material loaded on a cation-exchange material, thereby obtaining a washed raw material from which an N-terminal truncated variant and an abnormal variant have been removed, wherein said washing liquid is a washing buffer having higher electrical conductivity than the rhNGF raw material; and2) performing cation-exchange chromatography (CEC) elution on the washed raw material of step 1) with an elution buffer having higher electrical conductivity than the washing liquid in step 1), and collecting an eluate from which a purified rhNGF product is obtained.2. The method of claim 1 , wherein the electrical conductivity of said washing liquid in step 1) is 20˜30 mS/cm.3. The method of claim 1 , wherein said washing liquid in step 1) is an NaCl-containing buffer with an NaCl content of 200˜300 mM.4. The method of claim 1 , wherein step 1) comprises: loading the cation-exchange material with said rhNGF raw material claim 1 , washing with the washing liquid claim 1 , and discarding a resulting outflowing liquid.5. The method of claim 1 , wherein said rhNGF raw material in step 1) is a preliminarily purified product obtained by ...

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Номер записи: 89
07-01-2021 дата публикации

TYROSINE-SPECIFIC FUNCTIONALIZED INSULIN AND INSULIN ANALOGS

Номер: US20210002345A1
Автор: Kekec Ahmet, Kevin Nancy Jo, Li Bing, LIN SONGNIAN, Parish Craig A., Tang Weijuan
Принадлежит: Merck Sharp & Dohme Corp.

The present invention relates to tyrosine-specific functionalized insulin analogs and processes of making such tyrosine-specific functionalized insulin analogs using R-3H-1,2,4-triazoline-3,5-(4H)diones (PTAD). 5. The process of claim 2 , wherein the insulin or insulin analog is recombinant human insulin.6. The process of claim 3 , wherein the A14 tyrosine residue on the human insulin is functionalized.7. The process of claim 3 , wherein the A19 tyrosine residue on the human insulin is functionalized.8. The process of claim 3 , wherein the B16 tyrosine residue on the human insulin is functionalized.9. The process of claim 3 , wherein the B26 tyrosine residue on the human insulin is functionalized.10. The process of claim 3 , wherein the A14 and B26 residues tyrosine on the human insulin are functionalized.11. The process of claim 3 , wherein the A14 and B16 residues tyrosine on the human insulin are functionalized.12. The process of claim 3 , wherein the A14 and A19 residues tyrosine on the human insulin are functionalized.13. The process of claim 3 , wherein the solvent system is a mixture of acetonitrile and sodium phosphate buffer.14. The process of claim 11 , wherein the pH is maintained between 7-9. The sequence listing of the present application is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file name “24576WOPCT-SEQTXT-25JAN2019”, creation date of Jan. 25, 2019, and size of 5.05 Kb. This sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.The present invention relates to tyrosine-specific functionalized insulin analogs and processes of making such tyrosine-specific functionalized insulin analogs using R-3H-1,2,4-triazoline-3,5-(4H)diones (RTADs). The present invention further relates to utilization of tyrosine-specific functionalization through R-3H-1,2,4-trizoline-3,5-(4H)diones (RTADs) to modify insulin and insulin analogs for use in imaging or ...

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Номер записи: 90
07-01-2021 дата публикации

METHODS FOR EXTRACTING PROTEINS FROM BLOOD PLASMA

Номер: US20210003552A1
Автор: Curtin Dennis, Zurlo Eugene
Принадлежит:

A method of producing protein products including alpha-1-proteinase inhibitor, gamma globulin, albumin, and other proteins from plasma includes steps of: (1) adding a salt to the blood product to produce a first intermediate, wherein the salt comprises between 11-13 wt % of the first intermediate; (2) separating the first intermediate to produce a first supernatant and a first paste; (3) adding a salt to the first intermediate to produce a second intermediate, wherein the salt comprises between 21-23 wt % of the second intermediate; (4) separating the second intermediate to produce a second supernatant and a second paste; (5) separating a third intermediate from the second supernatant by affinity chromatography; and (6) separating the third intermediate by ion exchange chromatography to produce an eluate containing the protein product. Advantageously, the inventive methods are simple and produce alpha-1-proteinase inhibitor, gamma globulin, albumin, and other proteins in high yields. 1. A method of separating A1P1 from a blood plasma containing product , the method comprising:thawing a frozen blood plasma product followed by stirring at a temperature suitable for dissolving a cryoprecipitate to generate the blood plasma containing product;adding a salt to the blood plasma containing product to produce a first intermediate, wherein the salt comprises between 11-20 wt % of the first intermediate;separating the first intermediate to produce a first supernatant and a first paste;adding a salt to the first supernatant to produce a second intermediate, wherein the salt comprises between 15-30 wt % of the second intermediate;separating the second intermediate to produce a second supernatant and a second paste;separating the second supernatant by affinity chromatography using an A1P1-specific affinity media to generate a flow-through fraction and a first eluate, wherein the first eluate comprises A1P1.2. The method of claim 1 , wherein the step of adding the salt to the ...

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Номер записи: 91
01-01-2015 дата публикации

Norbornene Modified Peptides and Their Labelling With Tetrazine Compounds

Номер: US20150005481A1
Автор: Chin Jason, Deiters Alexander
Принадлежит: NORTH CARLOLINA STATE UNIVERSITY

The invention relates to a polypeptide comprising an amino acid having a norbornene group. Suitably said norbornene group is present as an amino acid residue of a norbornene lysine. The invention also relates to a method of producing a polypeptide comprising a norbornene group, said method comprising genetically incorporating an amino acid comprising a norbornene group into a polypeptide. The polypeptide comprising the norbornene group can be specifically labelled by inverse electron demand Diels-Alder reaction with a tetrazine compound. 1. A polypeptide comprising a single amino acid having a norbornene group , wherein said norbornene group is present as an amino acid residue of a norbornene lysine , wherein said single amino acid is not the N-terminal amino acid.2. (canceled)3. A method of producing a polypeptide comprising a norbornene group , said method comprising genetically incorporating an amino acid comprising a norbornene group into a polypeptide.4. A method according to wherein producing the polypeptide comprises (i) providing a nucleic acid encoding the polypeptide which nucleic acid comprises an orthogonal codon encoding the amino acid having a norbornene group; (ii) translating said nucleic acid in the presence of an orthogonal tRNA synthetase/tRNA pair capable of recognising said orthogonal codon and incorporating said amino acid having a norbornene group into the polypeptide chain.5. A method according to wherein said orthogonal codon comprises an amber codon (TAG) claim 4 , said tRNA comprises MbtRNAand said tRNA synthetase comprises MbPyIRS.6. A method according to wherein said amino acid comprising a norbornene group is a norbornene lysine.7. A polypeptide according to claim 1 , wherein said amino acid is Nε-5-norbomene-2-yloxycarbonyl-L-lysine.8. A polypeptide according to claim 1 , wherein said amino acid having a norbornene group is incorporated at a position corresponding to a lysine residue in the wild type polypeptide.9. A polypeptide ...

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Номер записи: 92
14-01-2016 дата публикации

CHROMATOGRAPHIC PURIFICATION OF ANTIBODIES FROM CHROMATIN-DEFICIENT CELL CULTURE HARVESTS

Номер: US20160009762A1
Автор: GAGNON Peter Stanley, LEE Tze Yang
Принадлежит:

Methods for the improved purification of antibodies and other proteins from protein preparations including the steps of conditioning the protein preparation by contacting it with multivalent organic ions, then applying the conditioned preparation to an adsorptive chromatography medium. 126.-. (canceled)27. A method for the purification of at least one desired protein from a protein preparation comprising the steps of (a) conditioning an impure protein preparation by contacting it with at least one species of soluble or insoluble multivalent organic ion , subsequently removing solids with the result of removing at least 95% of chromatin , thereby providing a conditioned protein preparation; then (b) applying the conditioned protein preparation , optionally after a buffer equilibration step , to an adsorptive chromatography medium for purification of the desired protein.28. The method of claim 27 , step (a) claim 27 , wherein the conditioning of the protein preparation with at least one species of organic multivalent ion comprises contacting the protein preparation with an electropositive organic additive.29. The method of claim 28 , wherein the electropositive organic additive comprises a soluble electropositive organic additive.30. The method of claim 28 , wherein the electropositive organic additive comprises an electropositive organic cation selected from the group consisting of methylene blue claim 28 , ethacridine claim 28 , chlorhexidine claim 28 , benzalkonium chloride claim 28 , and cetyl trimethyl ammonium bromide.31. The method of claim 28 , wherein the electropositive organic additive is present at a concentration of 0.001% to 2% claim 28 , 0.005% to 1% claim 28 , 0.01% to 0.05% or 0.020% to 0.025%.32. The method of claim 27 , step (a) claim 27 , wherein the conditioning of the protein preparation with at least one species of organic multivalent ions comprises contacting the protein preparation with an insoluble electropositive organic additive in the form ...

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Номер записи: 93
09-01-2020 дата публикации

METHOD OF ISOLATING BOTULINUM TOXIN FROM BOTULINUM TOXIN-CONTAINING SOLUTION

Номер: US20200009473A1
Автор: HONG Hyung Pyo, JUNG Hyun Ho, Kim Hack Woo, KONG Joon Chan, Rhee Chang Hoon, RYU Gi Hyuck, SONG Jung Hyun, Yang Gi Hyeok
Принадлежит:

Provided is a method of isolating a toxin type A macro complex from a toxin-containing solution, the method including performing anion exchange chromatography and cation exchange chromatography. 1botulinumbotulinum. A method of isolating a toxin type A macro complex from a toxin-containing solution , the method comprising:{'i': botulinum', 'botulinum, 'contacting the toxin-containing solution with an anion exchange chromatography medium at a pH lower than an isoelectric point (PI) of toxin;'}{'i': 'botulinum', 'contacting the solution, which is not bound to the anion exchange chromatography medium, with a cation exchange chromatography medium at a pH lower than the PI of toxin; and'}{'i': 'botulinum', 'separating the toxin type A macro complex from the cation exchange chromatography medium; or'}{'i': botulinum', 'botulinum, 'contacting the toxin-containing solution with the cation exchange chromatography medium at a pH lower than the PI of toxin;'}{'i': 'botulinum', 'separating toxin from the cation exchange chromatography medium;'}contacting the solution containing the toxin separated from the cation exchange chromatography medium, with the anion exchange chromatography medium at a pH lower than the PI; and{'i': 'botulinum', 'separating the toxin type A macro complex from the solution which is not bound to the anion exchange chromatography medium.'}2botulinum. The method of claim 1 , wherein the pH lower than the PI of toxin is pH 3.5 to 6.0.3botulinumbotulinumbotulinum. The method of claim 1 , further comprising adjusting the pH of the toxin or the toxin-containing solution to a pH lower than the PI of toxin claim 1 , before the contacting of the solution with the anion exchange chromatography medium claim 1 , before the contacting of the solution with the cation exchange chromatography medium claim 1 , or before each of these two processes.4botulinumbotulinumbotulinum. The method of claim 3 , wherein the adjusting comprises mixing the toxin or the toxin- ...

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Номер записи: 94
11-01-2018 дата публикации

MULTISTEP FINAL FILTRATION

Номер: US20180009878A1
Автор: Falkenstein Roberto, Schwendner Klaus
Принадлежит: Hoffmann-La Roche Inc.

Herein is reported a method for the final filtration of concentrated polypeptide solutions comprising the combination of two immediately consecutive filtration steps with a first filter of 3.0 μm and 0.8 μm pore size and a second filter of 0.45 μm and 0.22 μm pore size. 1. A method for producing an immunoglobulin solution comprisinga) providing an immunoglobulin solution with a concentration of at least 100 g/l, andb) applying the immunoglobulin solution to a combination of a first and second filter unit, whereby the first filter unit comprises a pre-filter with a pore size of 3.0 μm and a main-filter with a pore size of 0.8 μm and the second filter unit comprises a pre-filter with a pore size of 0.45 μm and a main-filter with a pore size of 0.22 μm with a pressure of from 0.1 to 4.0 bar, and thereby producing an immunoglobulin solution.2. A method for producing an immunoglobulin comprising the following stepsa) cultivating a cell comprising a nucleic acid encoding an immunoglobulin,b) recovering the immunoglobulin from the cell or the cultivation medium,c) purifying the immunoglobulin with one or more chromatography steps, and providing an immunoglobulin solution,d) optionally adding a sugar, an amino acid and/or a detergent to the solution,e) optionally concentrating the immunoglobulin solution to a concentration of 100 g/l or more with a method selected from diafiltration or tangential-flow filtration, andf) applying the immunoglobulin solution of the previous step to a combination of a first and second filter unit, whereby the first filter unit comprises a pre-filter with a pore size of 3.0 μm and a main-filter with a pore size of 0.8 μm and the second filter unit comprises a pre-filter with a pore size of 0.45 μm and a main-filter with a pore size of 0.22 μm with a pressure of from 0.1 to 4.0 bar, and thereby producing an immunoglobulin.3. The method of or wherein the filter in the first and second filter unit have about the same filter area.4. The method of or ...

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Номер записи: 95
14-01-2021 дата публикации

METHODS OF PURIFYING MONOMERIC MONOCLONAL ANTIBODIES

Номер: US20210009632A1
Автор: Ehamparanathan Vivekh, Lewandowski Angela T., Li Zhengjian, Song Yuanli, Tan Zhijun
Принадлежит:

In certain embodiments, the present invention provides a method of purifying a monomeric monoclonal antibody from a mixture which comprises the monomeric monoclonal antibody and one or more contaminants, comprising: a) subjecting the mixture to cation exchange chromatography (CEX) matrix, wherein the monomeric monoclonal antibody binds to the CEX matrix; b) contacting the CEX matrix with a wash solution at a pH which is between about 7 and about 7.8; c) eluting the monomeric monoclonal antibody from the CEX matrix into an elution solution, thereby purifying the monomeric monoclonal antibody. 1. A method of purifying a monomeric monoclonal antibody from a mixture which comprises the monomeric monoclonal antibody and one or more contaminants , comprising:a) subjecting the mixture to cation exchange chromatography (CEX) matrix, wherein the monomeric monoclonal antibody binds to the CEX matrix;b) contacting the CEX matrix with a wash solution at a pH which is between about 7 and about 7.8; andc) eluting the monomeric monoclonal antibody from the CEX matrix into an elution solution, thereby purifying the monomeric monoclonal antibody.2. The method of claim 1 , wherein the contaminants are selected from aggregates of the monoclonal antibody claim 1 , host cell proteins claim 1 , host cell metabolites claim 1 , host cell constitutive proteins claim 1 , nucleic acids claim 1 , endotoxins claim 1 , viruses claim 1 , product related contaminants claim 1 , lipids claim 1 , media additives and media derivatives.3. The method of claim 1 , wherein aggregates of the monoclonal antibody comprise dimers claim 1 , multimers claim 1 , and an intermediate aggregate species.4. The method of claim 1 , wherein the mixture has been obtained by an affinity chromatography.5. The method of claim 1 , wherein the elution solution is not subjected to a second chromatography step.6. The method of claim 1 , wherein the elution solution is further subjected to a second chromatography step.7. The ...

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Номер записи: 96
14-01-2021 дата публикации

HYBRID LIGAND, HYBRID BIOMIMETIC CHROMEDIA AND PREPARING METHOD AND USE THEREOF

Номер: US20210009635A1
Автор: LIN Dongqiang, LU Huili, YAO Shanjing, ZHANG QILEI, ZOU Xujun
Принадлежит: Zhejiang University

This invention relates to a hybrid ligand, a hybrid biomimetic chromedia and a preparing method and a use thereof, wherein the hybrid biomimetic chromedia takes hydrophilic porous microsphere as a substrate in chromatography, activated with allyl bromide and undergoing bromo-alcoholization with N-bromosuccinimide, then coupled with the hybrid ligands. The sequence of the hybrid ligand is phenylalanine-tyrosine-glutamine-5-aminobenzimidazole. The hybrid biomimetic chromedia has both of the two functional groups of phenylalanine-tyrosine-glutamine tripeptide and aminobenzimidazole, while maintaining the high antibody selectivity of polypeptide ligand, hydrophobic electric charge inductive ligand is introduced to achieve more moderate elution requirement, realizing effective antibody separation. 3. The hybrid biomimetic chromedia according to claim 2 , wherein the substrate in chromatography is sepharose gel or cellulose microsphere.4. A method for preparing the hybrid biomimetic chromedia according to claim 2 , comprising the following steps:1) performing an activating reaction by subjecting the substrate in chromatography with allyl bromide to obtain an activated substrate in chromatography;2) performing a bromo-alcoholization reaction by subjecting the activated substrate in chromatography with N-bromosuccinimide to obtain a bromo-alcoholized substrate;3) performing a coupled reaction by subjecting the bromo-alcoholized substrate and the hybrid ligand to obtain the hybrid biomimetic chromedia.5. The method according to claim 4 , wherein the activating reaction in Step 1) comprises: mixing the substrate in chromatography claim 4 , a dimethyl sulfoxide solution claim 4 , allyl bromide and sodium hydroxide claim 4 , and conducting water bath reaction in a shaker claim 4 , leaching and washing to obtain the activated substrate in chromatography.6. The method according to claim 4 , wherein the bromo-alcoholization reaction in Step 2) comprises: mixing the activated ...

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Номер записи: 97
10-01-2019 дата публикации

METHOD FOR PURIFYING COMPOSITION COMPRISING ANTIBODIES WITH ANIONIC POLYMER

Номер: US20190010188A1
Автор: ITO Seiya, Yanagita Akihiro
Принадлежит:

A composition containing an antibody is prepared in such a state that the composition contains an anionic polymer at pH lower than the pI of the antibody, and impurities insolubilized by the anionic polymer are removed. More preferably, the composition is prepared in such a state that the composition contains an anionic polymer at pH lower than or equal to the pI of the antibody minus one, and impurities insolubilized by the anionic polymer are removed. 1. A method for purifying a composition comprising an antibody , which comprises the steps of:(a) preparing a composition comprising an antibody in such a state that the composition comprises an anionic polymer at pH lower than the pI of the antibody; and(b) removing an impurity insolubilized by the anionic polymer from the composition.2. The method of claim 1 , wherein step (a) is preparing the composition in such a state that the composition comprises an anionic polymer at pH lower than or equal to the pI of the antibody minus one.3. The method of claim 1 , wherein step (a) is preparing the composition in such a state that the composition comprises an anionic polymer at pH of 3.5 to less than the pI of the antibody.4. The method of claim 1 , wherein step (a) is preparing the composition in such a state that the composition comprises an anionic polymer at pH of 3.5 to the pI of the antibody minus one or lower.5. A method for purifying a composition comprising an antibody claim 1 , which comprises the steps of:(a) preparing a composition comprising an antibody in such a state that the composition comprises an anionic polymer at pH of 3.5 to 5.0; and(b) removing an impurity insolubilized by the anionic polymer from the composition.6. The method of claim 5 , wherein step (a) is preparing the composition in such a state that the composition comprises an anionic polymer at pH of 3.8 to 5.0.7. The method of claim 1 , wherein the pI of the antibody is 3.0 to 8.0.8. The method of claim 1 , wherein the pI of the antibody is ...

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Номер записи: 98
14-01-2021 дата публикации

METHOD FOR PREPARING ACTIVE PROTEIN HYDROLYSATES BY HYDROLYZING CEREAL PROTEINS WITH MALT TOGETHER WITH PROTEASES

Номер: US20210010047A1
Автор: Li Junzhong, Liu Xiaolan, Song Mingyang, Zheng Xiqun
Принадлежит:

This disclosure relates to the technological field of nutrient food, particularly relates to a method for preparing active protein hydrolysates by hydrolyzing cereal proteins with malt together with proteases, including the following steps: (1) grinding the malt, obtaining the malt flour; (2) hydrolyzing cereal proteins with the malt flour together with proteases to prepare active protein hydrolysates. The present invention uses the malt flour together with proteases to hydrolyze cereal proteins, which can effectively reduce the consumption of proteases and save the cost, moreover, aminopeptidases and carboxypeptidases in the malt are able to hydrolyze peptide linkages from the ends of proteins, and the bitter hydrophobic amino acids are cut off, thus effectively decreasing the bitterness of the products, on the other hand, which can increase the additional values of the malt, providing its new applications. 1. A method for preparing active protein hydrolysates by hydrolyzing cereal proteins with malt together with proteases , which is characterized in that: including the following steps:(1) grinding the malt, obtaining the malt flour;(2) hydrolyzing cereal proteins with the malt flour together with proteases to prepare active protein hydrolysates.2. The method for preparing active protein hydrolysates by hydrolyzing cereal proteins with malt together with proteases of claim 1 , which is characterized in that: the step (2) was specifically: grinding dry cereal proteins into powders claim 1 , which were prepared into a suspension of cereal proteins claim 1 , hydrolyzed by adding the malt flour first claim 1 , then hydrolyzed by adding proteases claim 1 , upon which they were dried.3. The method for preparing active protein hydrolysates by hydrolyzing cereal proteins with malt together with proteases of claim 2 , which is characterized in that: the substrate concentration of cereal proteins in the step (2) was 5-15% (w/v).4. The method for preparing active protein ...

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Номер записи: 99
09-01-2020 дата публикации

PURIFYING INSULIN USING CATION EXCHANGE AND REVERSE PHASE CHROMATOGRAPHY IN THE PRESENCE OF AN ORGANIC MODIFIER AND ELEVATED TEMPERATURE

Номер: US20200010525A1
Автор: Chow Kartoa, Coleman Michael P., George Shibu T., Ortigosa Allison D., Rauscher Michael A., Sleevi Mark C.
Принадлежит: Merck Sharp & Dohme Corp.

A process is described for purifying insulin and insulin analogs that comprises high-pressure liquid chromatography with an acidic cation exchange medium performed in the presence of a water miscible organic modifier and at an elevated temperature followed by reverse phase chromatography performed in the presence of a water miscible organic modifier and at an elevated temperature. 1. A process for isolating properly folded insulin or insulin analog from an aqueous mixture comprising the insulin analog and related impurities , wherein the process comprises:(a) performing an acid-stable cation exchange chromatography with the aqueous mixture in the presence of a first water miscible organic modifier and at an elevated temperature to yield a first insulin or insulin analog mixture; and(b) performing a reverse phase high performance liquid chromatography on the first insulin or insulin analog mixture in the presence of a second water miscible organic modifier and at an elevated temperature to provide a second mixture comprising the isolated properly folded insulin or insulin analog.2. The process of claim 1 , wherein the insulin analog is an acid-stable insulin analog or pI-shifted insulin analog.3. The process of claim 1 , wherein the acid-stable insulin analog has a pI between 5.8 and 8.0.4. The process of claim 1 , wherein the acid-stable insulin analog is insulin glargine.5. The process of claim 1 , wherein the first water miscible organic modifier is hexylene glycol.6. The process of claim 1 , wherein the first water miscible organic modifier is at a concentration between 20 to 50 percent by volume.7. The process of claim 1 , wherein the second water miscible organic modifier is isopropanol.810-. (canceled)11. The process of claim 1 , wherein the acidic cation exchange chromatography is performed with a differential pressure of less than 1.0 MPa claim 1 , or less than 0.5 MPa claim 1 , or about 0.276 MPa.12. The process of claim 1 , wherein the acid-stable cation ...

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Номер записи: 100