文献类型： 会议论文

第一作者： Mart Ustav Jr.

作者： Mart Ustav Jr. ¹ ; Kadri Kangro; Lauri Peil; Meelis Kadaja; Margit Ool; Radi Tegova; Karl Mumm; Anne Kalling; Kerttu Murumets; Gaily Kivi; Kristiina Karro; Tiiu M?nnik; Andres Tover; Andres M?nnik; Urve Toots; Mart Ustav;

作者机构： 1.Icosagen Cell Factory Oü, Eerika tee 1, Kambja vald, Tartumaa, 61713 Estonia

会议名称： Protein Engineering Summit.

主办单位：

页码： 171-171

摘要： Fast growth of therapeutics field over the last few years has created an urgent need for efficient and scalable methods that would be suitable for the production of antibodies, including afucosylated antibodies, and other recombinant proteins for the pre-clinical studies. We have developed the QMCF technology - a stable episomal production system in CHO cells, which can either be used transiently or for producing from stable cell pools and from cell banks. Two viral factors, Mouse Polyomavirus (PyV) Large T antigen (LT) and Epstein-Barr Virus (EBV) EBNA1 protein are expressed from the CHO cellular genome to maintain and replicate the expression vectors through their respective DNA elements - PyV origin of replication and EBV FR elements for plasmid segration. LT initiates plasmid replication from the PyV core origin during S-phase of the cell cycle, whereas EBNA1 protein tethers the plasmid to the mitotic chromatin through EBNA-1 multimeric binding sites in Family of Repeats (FR)-and thereby assures the equal segregation of expression vectors to daughter cells. Due to this system, Icosagen QMCF plasmids are maintained at the level of few hundred copies per cell under antibiotic pressure, unlike conventional plasmids that get lost in dividing CHO cells. Latest addition to our QMCF technology is the ability to produce afucosylated antibodies, with afucosylation yields reaching 98% or better. To conclude, we have developed CHOEBNALT85 cell line that replicates and maintains pQMCF expression vectors in proliferating cells. Plasmid maintenance leads to higher copy number, which translates into greater productivity of antibodies, reaching 1 g/L or higher levels without expensive media formulations. Stable episomal cell pools can be generated as fast as 10 days while scaling up the cell culture from 10 ml to 400 ml, these pools can then be used to generate production cell banks.

分类号： q81