- Efficient and reproducible generation of high-expressing, stable human cell lines without need for antibiotic selection

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) [1]. 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 [10].

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 [11].

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 [14]. 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) [17], HKB11 (HEK293 cells fused with a Burkitt's lymphoma cell) [18], PerC6 (E1-transformed human embryonal retina cells) [19], 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.

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