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公开(公告)号：US20140038234A1
公开(公告)日：2014-02-06
申请号：US14110944
申请日：2012-03-20
Applicant: Yves Durocher
Inventor： Yves Durocher
IPC: C07K16/28 , C07K14/575
CPC classification number: C07K16/2863 , C07K14/56 , C07K14/575 , C12N15/85 , C12N2830/46 , C12N2830/85
Abstract: A short human genomic nucleotide sequence from the SAR3 region of the human interferon α2 gene permits enhances expression stability in the absence of drug selection and permits generation of stable clones or stable pools of cells for producing recombinant proteins. Although stable clones may be generated, the ability to generate stable pools reduces the burden of generating stable clones.
Abstract translation: 来自人干扰素α2基因的SAR3区域的短的人类基因组核苷酸序列允许在没有药物选择的情况下增强表达稳定性，并且允许产生用于产生重组蛋白质的稳定克隆或稳定的细胞池。 尽管可能产生稳定的克隆，但是产生稳定池的能力降低了产生稳定克隆的负担。
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2.发明授权Process, vectors and engineered cell lines for enhanced large-scale transfection 有权过程，载体和工程细胞系，用于增强大规模转染公开(公告)号：US08637315B2
公开(公告)日：2014-01-28
申请号：US12989898
申请日：2009-03-09
Applicant: Yves Durocher , Martin Loignon
Inventor： Yves Durocher , Martin Loignon
CPC classification number: C12N15/85 , C07K14/005 , C07K2319/71 , C12N2710/16222 , C12N2710/16622 , C12N2800/108 , C12N2800/40
Abstract: Processes vectors and engineered cell lines for large-scale transfection and protein production in mammalian cells, especially Chinese Hamster Ovary (CHO) cells are described in which transfection efficiencies are realized through the use of a single vector system, the use of functional oriP sequences in all plasmids, the use of codon-optimized Epstein-Barr virus nuclear antigen-1 (EBNA1) constructs the use of a fusion protein between a truncated Epstein-Barr virus nuclear antigenen-1c (EBNA1c) protein and a herpes simplex virus protein VP16, the use of a 40 kDa fully deacetylated poly(ethylenimine) as a transfection reagent, the use of co-expression of a fibroblast growth factor (FGF) and/or the use of protein kinase B to potentiate heterologous gene expression enhancement by valproic acid (VPA).
Abstract translation: 描述了用于哺乳动物细胞，特别是中国仓鼠卵巢（CHO）细胞中大规模转染和蛋白质生产的载体和工程细胞系，其中转染效率通过使用单一载体系统实现，使用功能性oriP序列 所有质粒，使用密码子优化的爱泼斯坦 - 巴尔病毒核抗原-1（EBNA1）构建了截短的爱泼斯坦 - 巴尔病毒核抗原-1（EBNA1c）蛋白和单纯疱疹病毒蛋白VP16之间的融合蛋白， 使用40kDa完全脱乙酰化的聚（亚乙基亚胺）作为转染试剂，使用共表达成纤维细胞生长因子（FGF）和/或使用蛋白激酶B来加强丙戊酸的异源基因表达增强（ VPA）。
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3.发明申请Method of Inhibiting Angiogenesis, Tumorigenesis and Cathepsin Activity 审中-公开抑制血管发生，肿瘤发生和组织蛋白酶活性的方法公开(公告)号：US20100279937A1
公开(公告)日：2010-11-04
申请号：US12377537
申请日：2007-08-16
Applicant: Maria Moreno , Danica B. Stanimirovic , Marquerite Ball , Yves Durocher
Inventor： Maria Moreno , Danica B. Stanimirovic , Marquerite Ball , Yves Durocher
CPC classification number: A61K38/1754 , A61K38/00 , C07K14/4743
Abstract: Insulin-like growth factor binding proteins (IGFBPs) and variants thereof, including IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, IGFBP-6 and the C-terminal fragments thereof, inhibit angiogenesis, tumorigenesis and cathepsin activity, particularly cathepsin B activity.
Abstract translation: 包括IGFBP-1，IGFBP-2，IGFBP-3，IGFBP-4，IGFBP-5，IGFBP-6及其C末端片段的胰岛素样生长因子结合蛋白（IGFBPs）及其变体，抑制血管发生，肿瘤发生 和组织蛋白酶活性，特别是组织蛋白酶B活性。
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4.发明申请Insulin-Like Growth Factor Binding Protein-4 Compounds and Methods for Inhibiting Angiogenesis and Tumor Growth in Mammalian Cells 审中-公开胰岛素样生长因子结合蛋白-4化合物和抑制哺乳动物细胞血管生成和肿瘤生长的方法公开(公告)号：US20090048158A1
公开(公告)日：2009-02-19
申请号：US11816663
申请日：2006-02-20
Applicant: Maria J. Moreno , Danica B. Stanimirovic , Marguerite Ball , Yves Durocher
Inventor： Maria J. Moreno , Danica B. Stanimirovic , Marguerite Ball , Yves Durocher
IPC: A61K38/00
CPC classification number: A61K38/1709 , C07K14/4743
Abstract: The use of fragments of IGFBP-4 for inhibiting angiogenesis and tumor growth is described.
Abstract translation: 描述了使用IGFBP-4的片段来抑制血管发生和肿瘤生长。
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5.发明申请Expression Vectors Containing a Truncated Epstein Barr Nuclear Antigen 1 Lacking the Gly-Gly-Ala Domain for Enhanced Transient Gene Expression 有权含有截短的爱泼斯坦巴尔核抗原的表达载体1缺乏用于增强的瞬时基因表达的Gly-Gly-Ala结构域公开(公告)号：US20080070232A1
公开(公告)日：2008-03-20
申请号：US11576005
申请日：2005-03-17
Applicant: Yves Durocher
Inventor： Yves Durocher
CPC classification number: C07K14/005 , C12N2710/16222 , C12N2800/108 , C12N2820/60
Abstract: This invention relates to the unexpected discovery that nucleotide coding sequences coding for a truncated Epstein Barr Nuclear Antigen 1 (EBNA1t) protein (lacking the Gly-Gly-Ala domain), when in cells of mammalian origin, are associated with improved growth and increased transient gene expression when compared with cells expressing a complete EBNA1 coding sequence. The expression of EBNA1t also appear to be more stable over time.
Abstract translation: 本发明涉及意想不到的发现，即在哺乳动物来源的细胞中编码截短的爱泼斯坦巴尔核抗原1（EBAS1t）蛋白（缺乏Gly-Gly-Ala结构域）的核苷酸编码序列与改善的生长和增加的瞬时 当与表达完整的EBNA1编码序列的细胞相比时，基因表达。 EBNA1t的表达似乎随着时间的推移更加稳定。
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Patent Agency Ranking
6.发明申请Enhanced production of recombinant proteins by transient transfection of suspension-growing mammalian cells 审中-公开通过瞬时转染悬浮培养的哺乳动物细胞增强重组蛋白的产生公开(公告)号：US20050170450A1
公开(公告)日：2005-08-04
申请号：US10477148
申请日：2002-05-07
Applicant: Yves Durocher , Sylvie Perret , Phuong Pham , Amine Kamen
Inventor： Yves Durocher , Sylvie Perret , Phuong Pham , Amine Kamen
Abstract: Disclosed is a new process for the production of recombinant proteins, by transient transfection of suspension-grown human embryonic kidney cells (293 cell line and its genetic variants) with an expression vector, using polyethylenimine (PEI) as a transfection reagent. In a preferred embodiment, the process uses 293E cells expressing the Epstein-Barr virus (EBV) EBNA 1 protein, in combination with an oriP-based episomal expression vector having an improved cytomegalovirus expression cassette comprising the CMV5 promoter. The process combines in a single step the cell growth, transfection and protein expression, is carried out without changing the culture medium, and allows to achieve high expression levels in a short period of time. The process may be carried out in a serum-free, low-protein culture medium, is easily scalable, compatible with continuous production processes, and fully adapted to high-throughput production of milligram quantities of recombinant proteins.
Abstract translation: 公开了通过使用聚乙烯亚胺（PEI）作为转染试剂用表达载体瞬时转染悬浮培养的人胚胎肾细胞（293细胞系及其遗传变体）来生产重组蛋白质的新方法。 在优选的实施方案中，该方法使用表达爱泼斯坦 - 巴尔病毒（EBV）EBNA 1蛋白的293E细胞与具有包含CMV5启动子的改良的巨细胞病毒表达盒的基于oriP的附加型表达载体的组合。 该过程在一个步骤中结合细胞生长，转染和蛋白质表达，不改变培养基进行，并允许在短时间内达到高表达水平。 该方法可以在无血清的低蛋白培养基中进行，易于扩展，与连续生产过程相容，并且完全适应于高产量的毫克量的重组蛋白质。
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7.发明授权Expression vectors containing a truncated epstein barr nuclear antigen 1 lacking the Gly-Gly-Ala domain for enhanced transient gene expression 有权含有缺失Gly-Gly-Ala结构域的截短的爱泼斯坦巴尔核抗原1的表达载体用于增强瞬时基因表达公开(公告)号：US08551774B2
公开(公告)日：2013-10-08
申请号：US11576005
申请日：2006-03-17
Applicant: Yves Durocher
Inventor： Yves Durocher
CPC classification number: C07K14/005 , C12N2710/16222 , C12N2800/108 , C12N2820/60
Abstract: This invention relates to the unexpected discovery that nucleotide coding sequences coding for a truncated Epstein Barr Nuclear Antigen 1 (EBNA1t) protein (lacking the Gly-Gly-Ala domain), when in cells of mammalian origin, are associated with improved growth and increased transient gene expression when compared with cells expressing a complete EBNA1 coding sequence. The expression of EBNA1t also appear to be more stable over time.
Abstract translation: 本发明涉及意想不到的发现，即在哺乳动物来源的细胞中编码截短的爱泼斯坦巴尔核抗原1（EBAS1t）蛋白（缺乏Gly-Gly-Ala结构域）的核苷酸编码序列与改善的生长和增加的瞬时 当与表达完整的EBNA1编码序列的细胞相比时，基因表达。 EBNA1t的表达似乎随着时间的推移更加稳定。
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8.发明申请Process, Vectors and Engineered Cell Lines for Enhanced Large-Scale Transfection 有权过程，载体和工程细胞系用于增强大规模转染公开(公告)号：US20110039339A1
公开(公告)日：2011-02-17
申请号：US12989898
申请日：2009-03-09
Applicant: Yves Durocher , Martin Loignon
Inventor： Yves Durocher , Martin Loignon
CPC classification number: C12N15/85 , C07K14/005 , C07K2319/71 , C12N2710/16222 , C12N2710/16622 , C12N2800/108 , C12N2800/40
Abstract: Processes vectors and engineered cell lines for large-scale transfection and protein production in mammalian cells, especially Chinese Hamster Ovary (CHO) cells are described in which transfection efficiencies are realized through the use of a single vector system, the use of functional oriP sequences in all plasmids, the use of codon-optimized Epstein-Barr virus nuclear antigen-1 (EBNA1) constructs the use of a fusion protein between a truncated Epstein-Barr virus nuclear antigenen-1c (EBNA1c) protein and a herpes simplex virus protein VP16, the use of a 40 kDa fully deacetylated poly(ethylenimine) as a transfection reagent, the use of co-expression of a fibroblast growth factor (FGF) and/or the use of protein kinase B to potentiate heterologous gene expression enhancement by valproic acid (VPA).
Abstract translation: 描述了用于哺乳动物细胞，特别是中国仓鼠卵巢（CHO）细胞中大规模转染和蛋白质生产的载体和工程细胞系，其中转染效率通过使用单一载体系统实现，使用功能性oriP序列 所有质粒，使用密码子优化的爱泼斯坦 - 巴尔病毒核抗原-1（EBNA1）构建了截短的爱泼斯坦 - 巴尔病毒核抗原-1（EBNA1c）蛋白和单纯疱疹病毒蛋白VP16之间的融合蛋白， 使用40kDa完全脱乙酰化的聚（亚乙基亚胺）作为转染试剂，使用共表达成纤维细胞生长因子（FGF）和/或使用蛋白激酶B来加强丙戊酸的异源基因表达增强（ VPA）。
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公开(公告)号：US20100261275A1
公开(公告)日：2010-10-14
申请号：US12746787
申请日：2008-12-10
Applicant: Yves Durocher , Martin Loignon , Brian Cass
Inventor： Yves Durocher , Martin Loignon , Brian Cass
IPC: C12N5/071 , C07K14/555
CPC classification number: C07K14/56
Abstract: A method of purifying a recombinant interferon protein involves providing an aqueous mixture of the recombinant protein and contaminating proteins; precipitating the contaminating proteins from the aqueous mixture at a pH in a range of from 0.5 to 6; separating the aqueous mixture from the precipitated contaminating proteins; and, eluting the separated aqueous mixture through a cation exchange column using a mobile phase with a salt or pH gradient, the gradient being from lower salt concentration or pH to higher salt concentration or pH, to produce a recombinant interferon protein fraction separated from other components of the aqueous mixture. The method provides for the recovery of recombinant interferon proteins in better yield and purity.
Abstract translation: 纯化重组干扰素蛋白的方法涉及提供重组蛋白质和污染蛋白质的水性混合物; 在0.5至6范围内的pH下从含水混合物中沉淀出污染的蛋白质; 从沉淀的污染蛋白质中分离含水混合物; 并且通过使用具有盐或pH梯度的流动相的分离的水性混合物通过阳离子交换柱洗脱，梯度为较低的盐浓度或pH至较高的盐浓度或pH，以产生与其它组分分离的重组干扰素蛋白级分 的含水混合物。 该方法提供以更好的产率和纯度回收重组干扰素蛋白质。
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10.发明授权公开(公告)号：US10421950B2
公开(公告)日：2019-09-24
申请号：US10477148
申请日：2002-05-07
Applicant: Yves Durocher , Sylvie Perret , Phuong Lan Pham , Amine A. Kamen
Inventor： Yves Durocher , Sylvie Perret , Phuong Lan Pham , Amine A. Kamen
Abstract: Disclosed is a new process for the production of recombinant proteins, by transient transfection of suspension-grown human embryonic kidney cells (293 cell line and its genetic variants) with an expression vector, using polyethylenimine (PEI) as a transfection reagent. In a preferred embodiment, the process uses 293E cells expressing the Epstein-Barr virus (EBV) EBNA 1 protein, in combination with an oriP-based episomal expression vector having an improved cytomegalovirus expression cassette comprising the CMV5 promoter. The process combines in a single step the cell growth, transfection and protein expression, is carried out without changing the culture medium, and allows to achieve high expression levels in a short period of time. The process may be carried out in a serum-free, low-protein culture medium, is easily scalable, compatible with continuous production processes, and fully adapted to high-throughput production of milligram quantities of recombinant proteins.
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