For many therapeutic proteins post-translational modifications,
proteolytic processing and oligomerization of multiple chains are
important for protein functionality, stability and efficient secretion.
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 production of biopharmaceuticals,
there is a permanent demand for improved methods for cell line
development featuring shorter time lines, higher productivity, improved
consistency and genetic stability.
Rapid production of small quantities of protein can be achieved by
transient transfection of the appropriate mammalian cell line. In
contrast, large-scale protein production depends on a stable cell line
with the protein expressing genetic construct integrated in the host
genome. The development of a permanent production cell line and the
manufacturing process for a recombinant protein usually follows a
well-established scheme. 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. The two expression cassettes – one
expressing the protein of interest, the second containing the selection
marker – can either be located on different plasmids, or can be
incorporated in one plasmid, preferably expressed from the same
promoter by taking advantage of an internal ribosomal entry site (IRES)
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. Classical selection markers like
glutamine synthetase (GS) [2,3], dihydrofolate reductase (DHFR) [4,5], hypoxanthine guanine phosphoribosyl transferase (HPRT) [6,7] or herpes simplex virus TK [8,9]
genes can only be used in cells deficient for the respective gene.
Alternatively, genes that confer resistance to cytotoxic drugs can be
used like kanamycin, neomycin, geneticin and blasticidin .
Usually, the development and selection of an optimized permanent
producer cell line is a very time-consuming procedure which can last
for months and includes selection for cell clones with highest
expression levels, limited dilution to obtain genetically identical
cell clones and testing for stability of expression during multiple
passages. Expression levels of the protein of interest depend on
numerous factors including the promoter, cellular levels of relevant
transcription factors, presence of factors transactivating the
promoter, the number of gene copies within the cell and the chromatin
structure at the integration site .
The site of integration has a major effect on the transcription of
the gene of interest. Therefore, integrations into transcriptional
active chromatin sites are preferred. However, very frequently the gene
of interest is rapidly inactivated and thus silenced [12,13].
Several strategies to overcome this position effect have been developed
and include the use of regulatory elements like matrix attachment
regions and insulators flanking the gene of interest .
In addition, specific targeting of the gene of interest into
transcriptionally active sites of the genome using yeast or phage
recombinases seems to be a possible option [15,16].
The production of authentic human proteins is best addressed by the
use of human cell lines, because they are not expected to add
potentially immunogenic glycan structures to the protein of interest.
However, there are only a few human cell lines described and restricted
access or deficient documentation limit their use in biopharmaceutical
production. Among the cell lines used are HEK293 (E1-transformed human
embryonal kidney/neuronal cells) , HKB11 (HEK293 cells fused with a Burkitt's lymphoma cell) , PerC6 (E1-transformed human embryonal retina cells) , and E1-transformed human amniocyte cells [20,21].
We describe here a novel method for rapid generation of human
production cell lines capable of secreting high levels of proteins
without any need for antibiotic selection. Our approach was to
co-transfect primary human amniocytes with two plasmids, one expressing
adenoviral E1-gene products and a second expressing a therapeutic
protein. Transformed cell clones were obtained which show high and
stable expression of the glycosylated therapeutic protein. Development
of these cell lines did neither require genetic knock-out of internal
marker genes, nor cotransfection of a selection marker, nor selection
of clones in medium containing antibiotics.