Efficient and reproducible generation of high-expressing, stable human cell lines without need for antibiotic selection.
BACKGROUND
Human cell lines are the most innovative choice of host cell for production of biopharmaceuticals since they allow for authentic posttranslational modification of therapeutic proteins. We present a new method for generating high and stable protein expressing cell lines based on human amniocytes without the requirement of antibiotic selection.


RESULTS
Primary amniocytes from routine amniocentesis samples can be efficiently transformed with adenoviral functions resulting in stable human cell lines. Cotransfection of the primary human amniocytes with a plasmid expressing adenoviral E1 functions plus a second plasmid containing a gene of interest resulted in permanent cell lines expressing up to 30 pg/cell/day of a fully glycosylated and sialylated protein. Expression of the gene of interest is very stable for more than 90 passages and, importantly, was achieved in the absence of any antibiotic selection.


CONCLUSIONS
We describe an improved method for developing high protein expressing stable human cell lines. These cell lines are of non-tumor origin, they are immortalized by a function not oncogenic in human and they are from an ethically accepted and easily accessible cell source. Since the cell can be easily adapted to growth in serum-free and chemically defined medium they fulfill the requirements of biopharmaceutical production processes.
Schiedner
Gudrun
G
CEVEC Pharmaceuticals GmbH, Gottfried-Hagen-Str, 62, 51105 Cologne, Germany. schiedner@cevec-pharmaceuticals.com
Hertel
Sabine
S
Bialek
Corinna
C
Kewes
Helmut
H
Waschütza
Gero
G
Volpers
Christoph
C
D000653:Amniotic Fluid / Q000166:cytology*; D000900:Anti-Bacterial Agents; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line / Q000166:cytology* / Q000502:physiology*; D049109:Cell Proliferation; D024861:Genetic Enhancement / Q000379:methods*; D006801:Humans; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism*; D014162:Transfection / Q000379:methods
D000900:Anti-Bacterial Agents; D011994:Recombinant Proteins