Yin Gang, Garces Eudean D, Yang Junhao, Zhang Juan, Tran Cuong, Steiner Alexander R, Roos Christine, Bajad Sunil, Hudak Susan, Penta Kalyani, Zawada James, Pollitt Sonia, Murray Christopher J
Sutro Biopharma, Inc.; South San Francisco, CA USA.
MAbs. 2012 Mar-Apr;4(2):217-25. doi: 10.4161/mabs.4.2.19202. Epub 2012 Mar 1.
We describe protein synthesis, folding and assembly of antibody fragments and full-length aglycosylated antibodies using an Escherichia coli-based open cell-free synthesis (OCFS) system. We use DNA template design and high throughput screening at microliter scale to rapidly optimize production of single-chain Fv (scFv) and Fab antibody fragments that bind to human IL-23 and IL-13α1R, respectively. In addition we demonstrate production of aglycosylated immunoglobulin G (IgG 1) trastuzumab. These antibodies are produced rapidly over several hours in batch mode in standard bioreactors with linear scalable yields of hundreds of milligrams/L over a 1 million-fold change in scales up to pilot scale production. We demonstrate protein expression optimization of translation initiation region (TIR) libraries from gene synthesized linear DNA templates, optimization of the temporal assembly of a Fab from independent heavy chain and light chain plasmids and optimized expression of fully assembled trastuzumab that is equivalent to mammalian expressed material in biophysical and affinity based assays. These results illustrate how the open nature of the cell-free system can be used as a seamless antibody engineering platform from discovery to preclinical development of aglycosylated monoclonal antibodies and antibody fragments as potential therapeutics.
我们描述了使用基于大肠杆菌的开放无细胞合成（OCFS）系统进行抗体片段和全长无糖基化抗体的蛋白质合成、折叠和组装。我们利用DNA模板设计和微升规模的高通量筛选，快速优化分别与人白细胞介素-23（IL-23）和白细胞介素-13α1受体（IL-13α1R）结合的单链Fv（scFv）和Fab抗体片段的生产。此外，我们还展示了无糖基化免疫球蛋白G（IgG 1）曲妥珠单抗的生产。这些抗体在标准生物反应器中以分批模式在数小时内快速产生，在扩大到中试规模生产时，产量呈线性可扩展，在高达100万倍的规模变化中，产量可达数百毫克/升。我们展示了从基因合成的线性DNA模板对翻译起始区域（TIR）文库进行蛋白质表达优化，从独立的重链和轻链质粒对Fab进行时间组装优化，以及完全组装的曲妥珠单抗的优化表达，其在基于生物物理和亲和力的测定中与哺乳动物表达的材料相当。这些结果说明了无细胞系统的开放性如何能够用作一个无缝的抗体工程平台，用于从无糖基化单克隆抗体和抗体片段的发现到临床前开发，作为潜在的治疗药物。
