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

Abstract

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.

Affinity of human IgG subclasses to mouse Fc gamma receptors

“…All IgGs were produced by transient transfection of HEKfreestyle cells (Thermo Fisher Scientific), as previously described by Vink et al 47 and Dekkers et al 48 After 5 days, IgG-containing cell supernatant from these cells was harvested by spinning twice at maximum speed (> 4000 g) and subsequent filtration with 0.45 nm puradisc syringe filter (Whatmann, GE Healthcare, 10462100).…”

Glycosylation pattern of anti‐platelet IgG is stable during pregnancy and predicts clinical outcome in alloimmune thrombocytopenia

“…A single-gene vector encoding IgG1 was subsequently generated by ligation of the BamHI-NotI fragment from pEE6.4 [including a cytomegalovirus (CMV) promoter, IgG1 heavy chain, and poly (A)] into the light-chain-encoding pEE14.4 vector. The plasmids were then transfected in the HEK FreeStyle 293 expression system (Life Technologies) with co-transfection of vectors encoding p21, p27 and pSVLT genes, as described by (Vink et al, 2014). Antibodies were purified on a protein A (WT IgG1) HiTrap HP column (GE Life Sciences, Westborough, MA, USA) and dialysed against phosphate-buffered saline overnight.…”

An Antibody–Drug Conjugate That Targets Tissue Factor Exhibits Potent Therapeutic Activity against a Broad Range of Solid Tumors

“…NP001984), cloned into the mammalian expression vector pEE13.4 (Lonza Biologics), and transfected into Freestyle 293-F cells (HEK-293F, Invitrogen) or NSO cells as described (15). To generate recombinant His-tagged soluble tissue factor, PCR was used to amplify the part encoding the extracellular domain (aa 1-251) of tissue factor from the construct, adding a C-terminal His tag containing six His residues (TF-ECDHis).…”