Process Sciences and Production, Novartis Biologics, Basel, Switzerland.
Appl Microbiol Biotechnol. 2010 Jul;87(4):1517-24. doi: 10.1007/s00253-010-2625-0. Epub 2010 May 12.
The recently described 2A/furin technology combines both chains of the antibody in a single open reading frame. Upon translation and secretion, the peptide is processed by the cell to generate native fully functional IgG antibodies. Here, we describe the results of an evaluation study of this technology for an industrial CHO cell line development process. The 2A/furin expression cassette setup was combined with a Novartis vector system. A transfection, selection, and cloning procedure in chemically defined media was established at Novartis and applied for a monoclonal test antibody. The productivity of 2A/furin-vector-derived clones in non-optimized generic shake flask fed-batch models was in a comparable range with clones derived from the reference control vector. Higher clonal production stability was seen for the majority of clones generated with the 2A/furin technology compared to the clones generated with the reference control vector. Product quality was analyzed by SDS-PAGE and no significant difference was detected between the two systems. Thus, it was shown that the 2A/furin technology can be successfully combined with a Novartis CHO expression system and platform. Due to the single ORF setup, the 2A/furin technology may therefore offer a suitable approach to reduce vector size and complexity.
最近描述的 2A/furin 技术将抗体的两条链结合在单个开放阅读框中。在翻译和分泌后，该肽被细胞加工以产生天然的全功能 IgG 抗体。在这里，我们描述了该技术在工业 CHO 细胞系开发过程中的评估研究结果。2A/furin 表达盒的设置与诺华载体系统相结合。在诺华建立了在化学成分确定的培养基中的转染、选择和克隆程序，并应用于单克隆测试抗体。在非优化的通用摇瓶补料分批模型中，2A/furin 载体衍生克隆的生产力与参考对照载体衍生克隆相当。与参考对照载体生成的克隆相比，大多数用 2A/furin 技术生成的克隆的克隆生产稳定性更高。通过 SDS-PAGE 分析产品质量，在这两个系统之间未检测到显著差异。因此，证明 2A/furin 技术可以成功地与诺华 CHO 表达系统和平台结合。由于单个 ORF 设置，因此 2A/furin 技术可能提供了一种降低载体大小和复杂性的合适方法。
