A simple, robust and highly efficient transient expression system for producing antibodies

Methods. 2014 Jan 1;65(1):5-10. doi: 10.1016/j.ymeth.2013.07.018. Epub 2013 Jul 17.


Transient expression systems in mammalian cells have become the method of choice for producing research quantities of antibodies. Both the speed and yield of the available transient systems and the natural posttranslational modifications favor these systems above expression in lower eukaryotes, prokaryotes or stable cell lines. We describe an optimized mammalian transient expression system, capable of producing up to 400mg/L of native secreted antibodies in less than a week. The system is composed of commercially available components and is based on expression in the fast growing suspension cell line, FreeStyle™ 293-F (HEK-293F). The method depends on an optimal combination of a gene transfer method, an expression vector and cotransfection with expression enhancing plasmids, encoding the large T antigen of the SV40 virus and the cell cycle inhibitors p21 and p27. Optimization of all components of the expression system, by experimental design techniques, yielded maximal expression levels (including antibody isotypes IgG1, 2, 3, 4 and Fab fragments of various species). Expression volumes were scalable from 0.1 ml up to 1.2L in a simple shaker flask system in animal component free, low protein medium, enabling consistent production of relatively high amounts of a large number of native antibodies.

Keywords: 293Fectin; 293fectin™; Antibody; CMV; Cell cycle; DoE; Experimental design; FreeStyle™ 293 expression medium; FreeStyle™ 293-F; HC; HEK-293; HEK-293F; HEK293; LC; Medium; Optimem; SV40 large T antigen; SVLT; Transient expression; antibody heavy chain; antibody light chain; cyclin-dependent kinase inhibitor 1; cyclin-dependent kinase inhibitor 1B; cytomegalo virus; design of experiments; human embryonic kidney 293 cell line; opti-MEM® I reduced-serum medium; p21; p27; the large T antigen of the SV40 virus.

MeSH terms

  • Cloning, Molecular
  • Gene Expression
  • HEK293 Cells
  • Humans
  • Immunoglobulin G / biosynthesis*
  • Immunoglobulin G / genetics
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / genetics
  • Transfection


  • Immunoglobulin G
  • Recombinant Proteins