Abstract
BackgroundHuman 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.ResultsPrimary 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.ConclusionWe 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.
Highlights
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
Primary amniocytes can be efficiently transformed by adenoviral E1-functions In order to test if primary amniocytes can be used to establish permanent, therapeutic protein expressing cell lines, amniocytes were cotransfected with the E1-expressing plasmid pGS119 containing E1A, E1B- and pIX-functions plus the plasmid pGS116 expressing the human alpha-1 antitrypsin glycoprotein
About 20–30 days after transfection of approximately 1 × 106 cells, numerous transformed cell colonies appeared on all dishes, and cells on the dishes were expanded as 10 pools (Z171-1A, B – Z171-5A, B)
Summary
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. Even though some modifications can occur in yeast and bacterial expression systems, mammalian and preferably human cells are the host of choice for proteins that require authentic glycosylation or other post-translational modifications. For screening purposes and for maintenance of protein expression potent selection markers need to be used and producer cells have to be constantly cultivated in medium containing the respective selective agent. By transfection and subsequent constant selection in the appropriate selection medium producer cell lines are selected which express high levels of the protein of interest. Genes that confer resistance to cytotoxic drugs can be used like kanamycin, neomycin, geneticin and blasticidin [10]
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