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    METHODS FOR PRODUCING MODIFIED GLYCOPROTEINS
    5.
    发明申请
    METHODS FOR PRODUCING MODIFIED GLYCOPROTEINS 审中-公开
    生产改性糖蛋白的方法

    公开(公告)号：US20130295604A1

    公开(公告)日：2013-11-07

    申请号：US13934551

    申请日：2013-07-03

    申请人： Tillman U. Gerngross

    发明人： Tillman U. Gerngross

    IPC分类号： C12P21/00

    CPC分类号： C12P21/005 ,  C07K2319/04 ,  C07K2319/05 ,  C12N1/14 ,  C12N9/1048 ,  C12N9/2488 ,  C12N15/79 ,  C12N15/80 ,  C12N15/81 ,  C12Y302/01 ,  C12Y302/01113

    摘要： Cell lines having genetically modified glycosylation pathways that allow them to carry out a sequence of enzymatic reactions, which mimic the processing of glycoproteins in humans, have been developed. Recombinant proteins expressed in these engineered hosts yield glycoproteins more similar, if not substantially identical, to their human counterparts. The lower eukaryotes, which ordinarily produce high-mannose containing N-glycans, including unicellular and multicellular fungi are modified to produce N-glycans such as Man5GlcNAc2 or other structures along human glycosylation pathways. This is achieved using a combination of engineering and/or selection of strains which: do not express certain enzymes which create the undesirable complex structures characteristic of the fungal glycoproteins, which express exogenous enzymes selected either to have optimal activity under the conditions present in the fungi where activity is desired, or which are targeted to an organelle where optimal activity is achieved, and combinations thereof wherein the genetically engineered eukaryote expresses multiple exogenous enzymes required to produce “human-like” glycoproteins.

    摘要翻译： 已经开发了具有遗传修饰的糖基化途径的细胞系，其允许它们进行模拟人类中糖蛋白的加工的酶反应序列。 在这些工程化宿主中表达的重组蛋白可以产生与其对应物更相似的糖蛋白（如果基本上不相同）。 通常产生含有高甘露糖的N-聚糖（包括单细胞和多细胞真菌）的低等真核生物被修饰以产生N-聚糖如Man5GlcNAc2或沿人糖基化途径的其它结构。 这是使用以下工程和/或选择的组合实现的：不表达产生真菌糖蛋白特征的不合需要的复合结构的某些酶，其表达选择在真菌中存在的条件下具有最佳活性的外源酶 其中需要活性或靶向实现最佳活性的细胞器，以及其组合，其中遗传工程真核生物表达产生“人样”糖蛋白所需的多种外源酶。

    Methods for producing modified glycoproteins
    6.
    发明授权
    Methods for producing modified glycoproteins 有权
    生产改性糖蛋白的方法

    公开(公告)号：US07981660B2

    公开(公告)日：2011-07-19

    申请号：US12549062

    申请日：2009-08-27

    申请人： Tillman U. Gerngross

    发明人： Tillman U. Gerngross

    IPC分类号： C12N1/15 ,  C07K14/00

    CPC分类号： C12P21/005 ,  C07K2319/04 ,  C07K2319/05 ,  C12N1/14 ,  C12N9/1048 ,  C12N9/2488 ,  C12N15/79 ,  C12N15/80 ,  C12N15/81 ,  C12Y302/01 ,  C12Y302/01113

    摘要： Cell lines having genetically modified glycosylation pathways that allow them to carry out a sequence of enzymatic reactions, which mimic the processing of glycoproteins in humans, have been developed. Recombinant proteins expressed in these engineered hosts yield glycoproteins more similar, if not substantially identical, to their human counterparts. The lower eukaryotes, which ordinarily produce high-mannose containing N-glycans, including unicellular and multicellular fungi are modified to produce N-glycans such as Man5GlcNAc2 or other structures along human glycosylation pathways. This is achieved using a combination of engineering and/or selection of strains which: do not express certain enzymes which create the undesirable complex structures characteristic of the fungal glycoproteins, which express exogenous enzymes selected either to have optimal activity under the conditions present in the fungi where activity is desired, or which are targeted to an organelle where optimal activity is achieved, and combinations thereof wherein the genetically engineered eukaryote expresses multiple exogenous enzymes required to produce “human-like” glycoproteins.

    摘要翻译： 已经开发了具有遗传修饰的糖基化途径的细胞系，其允许它们进行模拟人类中糖蛋白的加工的酶反应序列。 在这些工程化宿主中表达的重组蛋白可以产生与其对应物更相似的糖蛋白（如果基本上不相同）。 通常产生含有高甘露糖的N-聚糖（包括单细胞和多细胞真菌）的低等真核生物被修饰以产生N-聚糖如Man5GlcNAc2或沿着人糖基化途径的其它结构。 这是使用以下工程和/或选择的组合实现的：不表达产生真菌糖蛋白特征的不合需要的复合结构的某些酶，其表达选择在真菌中存在的条件下具有最佳活性的外源酶 其中需要活性或靶向实现最佳活性的细胞器，以及其组合，其中遗传工程真核生物表达产生“人样”糖蛋白所需的多种外源酶。

    N-Acetylglucosaminyltransferase III Expression in Lower Eukaryotes
    8.
    发明申请
    N-Acetylglucosaminyltransferase III Expression in Lower Eukaryotes 审中-公开
    N-乙酰葡糖胺基转移酶III在低等真核生物中的表达

    公开(公告)号：US20100016555A1

    公开(公告)日：2010-01-21

    申请号：US12540915

    申请日：2009-08-13

    申请人： Piotr Bobrowicz ,  Stephen R. Hamilton ,  Tillman U. Gerngross ,  Stefan Wildt ,  Byung-Kwon Choi ,  Juergen H. Nett ,  Robert C. Davidson

    发明人： Piotr Bobrowicz ,  Stephen R. Hamilton ,  Tillman U. Gerngross ,  Stefan Wildt ,  Byung-Kwon Choi ,  Juergen H. Nett ,  Robert C. Davidson

    IPC分类号： C07K16/00 ,  C07K14/435

    CPC分类号： C07K14/39 ,  A61K38/00 ,  C07K2319/05 ,  C12N9/1051 ,  C12P21/005 ,  Y02A50/396

    摘要： The present invention relates to eukaryotic host cells having modified oligosaccharides which may be modified further by heterologous expression of a set of glycosyltransferases, sugar transporters and mannosidases to become host-strains for the production of mammalian, e.g., human therapeutic glycoproteins. The process provides an engineered host cell which can be used to express and target any desirable gene(s) involved in glycosylation. Host cells with modified lipid-linked oligosaccharides are created or selected. N-glycans made in the engineered host cells exhibit GnTIII activity, which produce bisected N-glycan structures and may be modified further by heterologous expression of one or more enzymes, e.g., glycosyltransferases, sugar transporters and mannosidases, to yield human-like glycoproteins. For the production of therapeutic proteins, this method may be adapted to engineer cell lines in which any desired glycosylation structure may be obtained.

    摘要翻译： 本发明涉及具有修饰的寡糖的真核宿主细胞，其可以通过异源表达一组糖基转移酶，糖转运体和甘露糖苷酶进一步修饰，以成为用于产生哺乳动物例如人治疗性糖蛋白的宿主菌株。 该方法提供了可用于表达和靶向参与糖基化的任何所需基因的工程化宿主细胞。 制备或选择具有修饰的脂质连接寡糖的宿主细胞。 在工程化的宿主细胞中制备的N-聚糖表现出GnTIII活性，其产生二等分的N-聚糖结构，并且可以通过异源表达一种或多种酶（例如糖基转移酶，糖转运蛋白和甘露糖苷酶）进一步修饰，以产生人样糖蛋白。 为了生产治疗性蛋白质，该方法可适用于工程化可能获得任何所需糖基化结构的细胞系。

    Production of modified glycoproteins having multiple antennary structures
    9.
    发明申请
    Production of modified glycoproteins having multiple antennary structures 审中-公开
    具有多个触角结构的修饰的糖蛋白的生产

    公开(公告)号：US20090226959A1

    公开(公告)日：2009-09-10

    申请号：US12313636

    申请日：2008-11-21

    申请人： Piotr Bobrowicz ,  Stephen R. Hamilton ,  Tillman U. Gerngross ,  Stefan Wildt ,  Byung-Kwon Choi ,  Juergen Hermann Nett ,  Robert C. Davidson

    发明人： Piotr Bobrowicz ,  Stephen R. Hamilton ,  Tillman U. Gerngross ,  Stefan Wildt ,  Byung-Kwon Choi ,  Juergen Hermann Nett ,  Robert C. Davidson

    IPC分类号： C12P21/00 ,  C12N1/19 ,  C12N1/15 ,  C12N15/63 ,  C07K2/00

    CPC分类号： C12P21/005 ,  C12N9/1051

    摘要： The present invention relates to eukaryotic host cells, especially lower eukaryotic host cells, having modified oligosaccharides which may be modified further by heterologous expression of a set of glycosyltransferases, sugar and sugar nucleotide transporters to become host-strains for the production of mammalian, e.g., human therapeutic glycoproteins. The process provides an engineered host cell which can be used to express and target any desirable gene(s) involved in glycosylation. Host cells with modified lipid-linked oligosaccharides are created or selected. N-glycans made in the engineered host cells exhibit GnTIII, GnTIV, GnTV, GnT VI or GnTIX activity, which produce bisected and/or multiantennary N-glycan structures and may be modified further by heterologous expression of one or more enzymes, e.g., glycosyltransferases, sugar, sugar nucleotide transporters, to yield human-like glycoproteins. For the production of therapeutic proteins, this method may be adapted to engineer cell lines in which any desired glycosylation structure may be obtained.

    摘要翻译： 本发明涉及具有修饰的寡糖的真核宿主细胞，特别是较低的真核宿主细胞，其可以通过异源表达一组糖基转移酶，糖和糖核苷酸转运蛋白进一步修饰，以成为用于产生哺乳动物的宿主菌株， 人类治疗糖蛋白。 该方法提供了可用于表达和靶向参与糖基化的任何所需基因的工程化宿主细胞。 制备或选择具有修饰的脂质连接寡糖的宿主细胞。 在工程化宿主细胞中制备的N-聚糖表现出GnTIII，GnTIV，GnTV，GnT VI或GnTIX活性，其产生二等分和/或多元N-聚糖结构，并且可以通过异源表达一种或多种酶，例如糖基转移酶 ，糖，糖核苷酸转运蛋白，以产生人样糖蛋白。 为了生产治疗性蛋白质，该方法可适用于工程化可能获得任何所需糖基化结构的细胞系。

    Methods and apparatus for the production of amorphous polymer suspensions
    10.
    发明授权
    Methods and apparatus for the production of amorphous polymer suspensions 有权

    公开(公告)号：US06228934B1

    公开(公告)日：2001-05-08

    申请号：US09328783

    申请日：1999-06-09

    申请人： Daniel Horowitz ,  Tillman U. Gerngross

    发明人： Daniel Horowitz ,  Tillman U. Gerngross

    IPC分类号： C08F216

    CPC分类号： C08J3/03 ,  C08G63/06 ,  C08G63/88 ,  C08J2367/04

    摘要： Methods and apparati have been developed for producing a suspension of predominately amorphous polymer particles, wherein the method includes thermally treating a suspension that includes crystalline or semi-crystalline polymer particles. The thermal treatment includes (a) heating a suspension of polymer particles of an appropriate size to a temperature effective to cause the polymer to become amorphous, and then (b) cooling the suspension of amorphous polymer particles below the melting point of the polymer at a rate effective to prevent substantial coalescence of the polymer particles. The method and apparati are effective for use with a variety of polymers having suitable crystallization parameters, although polyhydroxyalkanoate (PHA) polymers are preferred, particularly in an aqueous suspension medium. For PHA polymers, the polymer particles subjected to treatment preferably are of a size of less than 5 &mgr;m, or more preferably less than 1.5 &mgr;m in diameter.