Aglycosylated antibodies and antibody fragments produced in a scalable in vitro transcription-translation system

作者：Yin Gang; Garces Eudean D; Yang Junhao; Zhang Juan; Cuong Tran; Steiner Alexander R; Roos Christine; Bajad Sunil; Hudak Susan; Penta Kalyani; Zawada James; Pollitt Sonia; Murray Christopher J^*

来源：mAbs, 2012, 4(2): 217-225.

DOI：10.4161/mabs.4.2.19202

• cell-free protein synthesis
• Fab antibody
• aglycosylated antibodies
• HER2
• trastuzumab

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-13 alpha 1R, respectively. In addition we demonstrate production of aglycosylated immunoglobulin G (IgG1) 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.

• 出版日期2012-4