Upstream Processing:Vendor Notes: Generating Stable, High-Expressing Cell Lines for Recombinant Protein Manufacture - - BioPharm International


Upstream Processing:Vendor Notes: Generating Stable, High-Expressing Cell Lines for Recombinant Protein Manufacture

BioPharm International
Volume 20, Issue 3


Figure 2. The ACE System process overview. Single-load and double-load maximum batch titers with candidate cell-line generation times from transfection of the Platform ACE Cell Line.
Transfection, or loading of the Platform ACE, is accomplished by cotransfecting the Platform ACE Cell Line with both the ATV and ACE Integrase plasmid, using standard transfection methods. The ACE Integrase is only transiently expressed and not incorporated into the host chromosome. To generate high-expressing, stable, clonal cell lines, a "single-load" or "double-load" process can be used, depending on the required expression level and generation time (Figure 2). ATV construction can take between one and two months, depending on the target gene DNA source. Single-load candidate cell lines can be generated in three to four months from ATV transfection into Platform ACE Cell Line, using standard transfection reagents and methods and employing simple screening methodologies to determine expression and protein yield. Briefly, loading of ATVs onto the Platform ACE in the Platform ACE Cell Line is accomplished by a lipid-mediated cotransfection of ATV and expression plasmid containing the coding region for ACE Integrase under adherent conditions. After transfection of the Platform ACE Cell Line, drug-resistant colonies are switched to a basic serum-free media, selected, and expanded from 96-well and 24-well cultures to shake flask, and serially screened for growth characteristics and productivity by ELISA or HPLC Protein A chromatography. This process takes between one-and-a-half and two months and results in the generation of individual pools of cells (primary transfectants) that can be used to produce material for research programs (Figure 2). Selected primary transfectants are subsequently single-cell subcloned by limiting dilution, expanded to shake flask, and subjected to performance testing in terminal shake-flask cultures. This requires an additional one-and-a-half to two months to complete. Candidate clonal cell lines are selected, based on growth, yields, and stability of expression and take between three and four months to generate from ATV transfection.

Because not all recombination acceptor sites on the Platform ACE are targeted in a single-load process, a second transfection, or double load, can be performed if the user desires higher levels of expression. In the double-load process, single-load primary transfectants are loaded with a second ATV containing the gene(s) of interest and an alternate drug resistant marker. As with the single-load process, individualized pools of drug-resistant transfectants are generated, screened, and single-cell subcloned to identify clonal lines for performance testing in terminal shake-flask cultures. Although the double-load process requires an extra one-and-a-half to two months to generate clonal candidate cell lines, compared with the single load process, it has routinely resulted in titers that are >50 percent higher than those obtained with a single load.

The ACE System has been used to generate a number of CHO cell lines expressing a variety of different recombinant proteins. For monoclonal antibodies, the single-load process routinely produces clonal cell lines with yields of 300 to 700 mg/L in non-fed (unsupplemented CD-CHO medium from Invitrogen), batch terminal, shake-flask cultures, and 500 to 1,000 mg/L for the double-load process. Several cell lines have been subject to growth optimization and scale-up, in which 2–5 fold gains in performance have been noted.

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