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.
Backliwal
Gaurav
G
Ecole Polytechnique Fédérale de Lausanne, Laboratory of Cellular Biotechnology, Institute of Bioengineering, Faculty of Life Sciences, Lausanne, Switzerland.
Hildinger
Markus
M
Hasija
Vivek
V
Wurm
Florian M
FM
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