High-density transfection with HEK-293 cells allows doubling of transient titers and removes need for a priori DNA complex formation with PEI.
Recombinant proteins are of great commercial and scientific interest. Yet, most production methods in mammalian cells involve the time- and labor-consuming step of creating stable cell lines. Production methods based on transient gene expression are advantageous in terms of speed and versatility; yet, depending on the transfection protocol, transient transfection faces some bottlenecks such as a priori complex formation, limitations in terms of transfection and production media used and the need for medium exchange prior to and/or after transfection. Published protocols for transfection of suspension-adapted HEK-293 cells with polyethyleneimine have shown great promise in overcoming some of these bottlenecks, but still require a priori complex formation for optimal yields and limit the choice of transfection and production media. Here, we report successful in situ transfection of suspension-adapted HEK-293 cells with 25-kDa linear polyethyleneimine at densities up to 20 x 10(6) cells/mL in complex media followed by production at lower cell densities (1 x 10(6) cells/mL). After concentrating cells to such high densities, transfection of HEK-293 cells becomes possible in most commonly used media and is not restricted to a specific medium. Furthermore, there is no need to make transfection complexes a priori, a step that prevents inline sterile filtration of the DNA bulk for transfection, an important consideration when scaling processes up to 100 or 1,000 L. Finally, transfecting HEK-293 cells at high density in complex media is superior to existing transfection protocols and doubles yields of recombinant protein obtainable by transient gene expression.
Ecole Polytechnique Fédérale de Lausanne, Laboratory of Cellular Biotechnology, Institute of Bioengineering, Faculty of Life Sciences, Lausanne, Switzerland.
Gaurav
G
Backliwal
Markus
M
Hildinger
Vivek
V
Hasija
Florian M
FM
Wurm
D002460:Cell Line; D004247:DNA / Q000737:chemistry* / Q000235:genetics*; D004337:Drug Carriers / Q000737:chemistry*; D006801:Humans; D007668:Kidney / Q000502:physiology*; D011094:Polyethyleneimine / Q000737:chemistry*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
D004337:Drug Carriers; D011994:Recombinant Proteins; D011094:Polyethyleneimine; D004247:DNA