Recombinant products find a wide application in pharmaceutical, veterinary food industries, as well as in the production of pesticides and detergents. These substances represent approx. 30% of the pharmaceutical products or more than 90% from all commercially available recombinant products. Even though bacterial and yeast cell can be considered as an attractive option for expressing certain biopharmaceutical proteins, many biopharmaceutical molecules are too large and complex and need to be expressed in mammalian cells.
Which cell lines can used for production of biopharmaceuticals?
The most reported cell lines used for industrial production of biopharmaceuticals are HEK293, CHO, NS0 and Sp2/O-Ag14 cell lines. HEK293 became one of the preferred cell lines for transient or stable protein expression because of their application because of its high transfection efficiency. Genetic manipulation also includes new techniques for directing the integration of the foreign gene of interest to highly expressed chromosomal sites by RMCE (recombinase-mediated cassette exchange).
How the HEK293 cell lines can be used for production of recombinant proteins for pharmacological applications?
A team of scientists compared three strategies for recombinant protein production in HEK293 cells.
The first strategy is based on the rAdV plasmid production expressing the protein of interest. Viral DNA has been isolated from field and the gene of the capsid (CapPCV2) was cloned into Adh5 genome using AdEasyXL kit. The production of viral particles and protein of interest was performed by infecting HEK293-F6 suspension cells.
The second strategy is observed with table cell line establishment by random gene transfection. Following this strategy, the scientists have used Cap protein from the capsid of porcine circovirus serotype 2 (CAP-PCV2) as a protein of interest. This virus is involved in infection that causes post weaning multisystemic wasting syndrome (PMWS). CapPCV2 gene sequence has been codon optimized for mammalian cell expression. The obtained sequence was cloned into pIRESpuro3 bicistronic vector. HEK293-F6 suspension cells have been transfected via Polyethylenimine (PEI)- DNA method. The positive cells were collected by the addition of puromycin to the culture media.
The third strategy lies on the site-specific integration using RMCE technology. Three targeting vectors that encode for different promoters were transfected by electroporation to three different HEK293 master cell lines. The selection of the positive clones has been done by the addition neomycin and ganciclovir addition to the culture media. Clones were isolated by single colony pick up. RMCE was applied to 293T cell line upon the selection of the best promoter. Positive clones have selected by puromycin addition to media and single colony isolation have been carried out. The selected clone has been transferred and cultured as a suspension in new media.
Which strategy leads to highest cell productivity?
According the results the best strategy is the application of site directed integration using RMCE technology. This bioprocess based on site-integrated stable suspension cell line showed several advantages as highest specific production and easy construction of each step of the process in contrast to adenovirus infection. The production rates are reproducible for each process and the protein of interest can be easily substituted.