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1. 发明申请

US20130011875A1 METHODS FOR THE PRODUCTION OF RECOMBINANT PROTEINS WITH IMPROVED SECRETION EFFICIENCIES 审中-公开
标题翻译：用改良的分泌效率生产重组蛋白的方法
公开(公告)号：US20130011875A1
公开(公告)日：2013-01-10
申请号：US13503707
申请日：2010-10-25
申请人： Michael Meehl , Heping Lin , Byung-Kwon Choi
发明人： Michael Meehl , Heping Lin , Byung-Kwon Choi
IPC分类号： C12N1/19 , C12P21/00
CPC分类号： C07K14/39 , C12N15/67 , C12N15/81 , C12P21/005 , C12P21/02
摘要： The present invention is related to methods and for producing higher titers of recombinant protein in a modified yeast host cell, for example Pichia pastoris, wherein the modified yeast cell lacks vacuolar sorting activity or has decreased vacuolar sorting activity relative to an unmodified yeast host cell of the same species. In particular embodiments vacuolar sorting activity is reduced or eliminated by deletion or disruption of a gene encoding Vps10 or a Vps10 homolog. The invention is also related to the modified yeast cells which are modified in accordance with the methods disclosed herein.
摘要翻译：本发明涉及在修饰的酵母宿主细胞例如巴斯德毕赤酵母中产生较高滴度的重组蛋白的方法，其中所述修饰的酵母细胞相对于未修饰的酵母宿主细胞缺乏液泡分选活性或具有降低的液泡分选活性相同的物种在具体实施方案中，通过缺失或破坏编码Vps10或Vps10同源物的基因来减少或消除液泡分选活性。本发明还涉及根据本文公开的方法修饰的修饰的酵母细胞。

2. 发明申请

US20120213728A1 GRANULOCYTE-COLONY STIMULATING FACTOR PRODUCED IN GLYCOENGINEERED PICHIA PASTORIS 审中-公开
标题翻译：在GLYCOENGINEER PICHIA PASTORIS生产的GRANULOCYTE-COLONY刺激因子
公开(公告)号：US20120213728A1
公开(公告)日：2012-08-23
申请号：US13504528
申请日：2010-10-25
申请人： Michael Meehl , Sandra Rios , Sujatha Gomathinayagam , Huijuan Li , Piotr Bobrowicz
发明人： Michael Meehl , Sandra Rios , Sujatha Gomathinayagam , Huijuan Li , Piotr Bobrowicz
IPC分类号： A61K38/19 , C12P21/02 , C12N15/62 , C12N1/19
CPC分类号： C12N15/81 , C07K14/535 , C12P21/005 , C12P21/06
摘要： Compositions comprising granulocyte-colony stimulating factor (GCSF) produced in a strain of Pichia pastoris glycoengineered to produce a GCSF wherein greater than 18% of the molecules comprise an 0-glycan with one mannose per (0-glycan is described. In particular aspects, the GCSF is PEGylated at the JV-terminus.
摘要翻译：在巴斯德毕赤氏酵母菌株中产生的产生GCSF的粒细胞集落刺激因子（GCSF）的组合物，其生产GCSF，其中大于18％的分子包含每个（O-聚糖）含有一个甘露糖的O-聚糖， GCSF在JV端聚乙二醇化。

3. 发明授权

US08715963B2 Method for increasing N-glycosylation site occupancy on therapeutic glycoproteins produced in Pichia pastoris 有权
标题翻译：增加毕赤酵母中产生的治疗性糖蛋白的N-糖基化位点占有率的方法
公开(公告)号：US08715963B2
公开(公告)日：2014-05-06
申请号：US13579972
申请日：2011-02-23
申请人： Natarajan Sethuraman , Byung-Kwon Choi , Bianka Prinz , Michael Meehl , Terrance Stadheim
发明人： Natarajan Sethuraman , Byung-Kwon Choi , Bianka Prinz , Michael Meehl , Terrance Stadheim
IPC分类号： C12P21/06 , C12P21/04 , C12P1/02 , C12N15/74 , C12N5/07 , C12N1/00
CPC分类号： C07K16/2863 , C07K14/44 , C07K16/1027 , C07K16/32 , C07K2317/14 , C07K2317/21 , C07K2317/41 , C12N15/81 , C12P21/005
摘要： Described is a method for increasing the N-glycosylation site occupancy of a therapeutic glycoprotein produced in recombinant host cells modified as described herein and genetically engineered to express the glycoprotein compared to the N-glycosylation site occupancy of the therapeutic glycoprotein produced in a recombinant host cell not modified as described herein. In particular, the method provides recombinant host cells that overexpress a heterologous single-subunit oligosaccharyltransferase, which in particular embodiments is capable of functionally suppressing the lethal phenotype of a mutation of at least one essential protein of the yeast oligosaccharyltransferase (OTase) complex, for example, the Leishmania major STT3D protein, in the presence of expression of the host cell genes encoding the endogenous OTase complex. The method is useful for both producing therapeutic glycoproteins with increased N-glycosylation site occupancy in lower eukaryote cells such as yeast and filamentous fungi and in higher eukaryote cells such as plant and insect cells and mammalian cells.
摘要翻译：描述了增加如本文所述修饰的重组宿主细胞中产生的治疗性糖蛋白的N-糖基化位点占有率的方法，并且与在重组宿主细胞中产生的治疗性糖蛋白的N-糖基化位点占据相比，进行遗传工程改造以表达糖蛋白未经本文所述修饰。特别地，该方法提供过表达异源单亚基寡糖基转移酶的重组宿主细胞，其在特定实施方案中能够功能性地抑制酵母寡糖转移酶（OTase）复合物的至少一种必需蛋白质的突变的致死表型，例如，利什曼原虫主要STT3D蛋白，在表达编码内源性OTase复合物的宿主细胞基因的存在下。该方法对于在低等真核生物细胞如酵母和丝状真菌以及高等真核细胞如植物和昆虫细胞和哺乳动物细胞中产生具有增加的N-糖基化位点占有的治疗性糖蛋白是有用的。

4. 发明申请

US20140235537A1 N-GLYCOSYLATED INSULIN ANALOGUES 审中-公开
标题翻译： N-糖苷类胰蛋白酶类似物
公开(公告)号：US20140235537A1
公开(公告)日：2014-08-21
申请号：US14237369
申请日：2012-08-03
申请人： Michael Meehl , Natarajan Sethuraman , Sandra Rios
发明人： Michael Meehl , Natarajan Sethuraman , Sandra Rios
IPC分类号： C07K14/62
CPC分类号： C07K14/62 , A61K38/00 , A61K38/28
摘要： Compositions and formulations comprising N-glycosylated insulin analogues are described. In particular embodiments, the glycosylated insulin analogues are produced in vivo and comprise one or more the N-linked N-glycans selected from high mannose or fucosylated or non-fucosylated hybrid, paucimannose, or complex N-glycans. In other embodiments, the N-glycan comprising the high mannose or fucosylated or non-fucosylated hybrid, paucimannose, or complex N-glycan is attached to the insulin analogue in vitro. Examples of N-glycans include but are not limited to a molecule having a structure selected from N-glycans in the group consisting of Man(1—9)GlcNAc2; or selected from N-glycans in the group consisting of GlcNAc(1—4)Man3GlcNAc2; or selected from N-glycans in the group consisting of Gal(j. 4)GlcNAc(1—4)Man3GlcNAe2; or selected from N-glycans in the group consisting of NANA({umlaut over (ι)}_4)Gal(1—4)GlcN Ac(1—4)Man3 GlcN Ac2—
摘要翻译：描述了包含N-糖基化胰岛素类似物的组合物和制剂。在具体实施方案中，糖基化胰岛素类似物在体内产生，并且包含一种或多种选自高甘露糖或岩藻糖基化或非岩藻糖基化杂合物，低聚西南糖或复合N-聚糖的N-连接的N-聚糖。在其它实施方案中，包含高甘露糖或岩藻糖基化或非岩藻糖基化的杂交体，低聚甘露糖或复合N-聚糖的N-聚糖在体外与胰岛素类似物连接。 N-聚糖的实例包括但不限于具有选自Man（1-9）GlcNAc2组中的N-聚糖的结构的分子; 或选自由GlcNAc（1-4）Man 3 GlcNAc 2组成的组中的N-聚糖; 或选自由Gal（j.4）GlcNAc（1-4）Man3GlcNAe2组成的组中的N-聚糖; 或选自NANA（{umlaut over（ι）} _4）Gal（1-4）GlcN Ac（1-4）Man3 GlcN Ac2-

5. 发明申请

US20120328626A1 METHOD FOR INCREASING N-GLYCOSYLATION SITE OCCUPANCY ON THERAPEUTIC GLYCOPROTEINS PRODUCED IN PICHIA PASTORIS 有权
标题翻译：在PICHIA PASTORIS生产的治疗性GLYCOPROTEIN上增加N-糖苷化位点的方法
公开(公告)号：US20120328626A1
公开(公告)日：2012-12-27
申请号：US13579972
申请日：2011-02-23
申请人： Natarajan Sethuraman , Byung-Kwon Choi , Bianka Prinz , Michael Meehl , Terrance Stadheim
发明人： Natarajan Sethuraman , Byung-Kwon Choi , Bianka Prinz , Michael Meehl , Terrance Stadheim
IPC分类号： C12P21/02 , A61K39/395 , A61P31/14 , C12N1/15 , C12N5/10 , A61K39/42 , C12N1/19
CPC分类号： C07K16/2863 , C07K14/44 , C07K16/1027 , C07K16/32 , C07K2317/14 , C07K2317/21 , C07K2317/41 , C12N15/81 , C12P21/005
摘要： Described is a method for increasing the N-glycosylation site occupancy of a therapeutic glycoprotein produced in recombinant host cells modified as described herein and genetically engineered to express the glycoprotein compared to the N-glycosylation site occupancy of the therapeutic glycoprotein produced in a recombinant host cell not modified as described herein. In particular, the method provides recombinant host cells that overexpress a heterologous single-subunit oligosaccharyltransferase, which in particular embodiments is capable of functionally suppressing the lethal phenotype of a mutation of at least one essential protein of the yeast oligosaccharyltransferase (OTase) complex, for example, the Leishmania major STT3D protein, in the presence of expression of the host cell genes encoding the endogenous OTase complex. The method is useful for both producing therapeutic glycoproteins with increased N-glycosylation site occupancy in lower eukaryote cells such as yeast and filamentous fungi and in higher eukaryote cells such as plant and insect cells and mammalian cells.
摘要翻译：描述了增加如本文所述修饰的重组宿主细胞中产生的治疗性糖蛋白的N-糖基化位点占有率的方法，并且与在重组宿主细胞中产生的治疗性糖蛋白的N-糖基化位点占据相比，进行遗传工程改造以表达糖蛋白未经本文所述修饰。特别地，该方法提供过表达异源单亚基寡糖基转移酶的重组宿主细胞，其在特定实施方案中能够功能性地抑制酵母寡糖转移酶（OTase）复合物的至少一种必需蛋白质的突变的致死表型，例如，利什曼原虫主要STT3D蛋白，在表达编码内源性OTase复合物的宿主细胞基因的存在下。该方法对于在低等真核生物细胞如酵母和丝状真菌以及高等真核细胞如植物和昆虫细胞和哺乳动物细胞中产生具有增加的N-糖基化位点占有的治疗性糖蛋白是有用的。

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