Multiple Gene Expression—Potential for Metabolic Engineering
Another unique feature of the ACE System is its ability to integrate several copies of different genes onto the same ACE (essentially
a double load with an ATV containing a different gene or genes). To highlight this novel feature,4 a study was conducted in which the DG44 CHO Platform ACE Cell Line was loaded with both green fluorescent protein (GFP)
and human erythropoietin (EPO). The Platform ACE was initially loaded with an ATV encoding GFP, resulting in a cell line expressing
GFP at levels detectable by fluorescence microscopy and flow cytometry. This GFP-expressing cell line was then loaded with
a second ATV encoding EPO. The resulting cell lines maintained parental GFP expression levels and also expressed EPO at levels
greater than 400 IU/106 cells/day as measured by ELISA. The ability of the ACE System to incorporate and stably express different genes by sequential
loading provides the basis for potential metabolic engineering applications. For instance, genes encoding growth factors,
anti-apoptotic factors, or factors affecting post-translational modifications or protein secretion could be sequentially loaded
onto an ACE expressing a product gene, thereby enhancing the growth characteristics of that cell line for clinical and commercial
manufacture in a bioreactor. Alternately, a Platform ACE could be loaded with multiple copies of metabolic factors, sorted
and purified, and transferred into a pre-existing production cell line. Cell lines containing multiple ACEs have been generated
and preliminary data have demonstrated that both ACEs are quite stable. Metabolic engineering studies like those discussed
above are currently ongoing.
The ACE System is a potent biological engineering system that is being applied to the engineering of mammalian cells for the
clinical and commercial manufacture of biopharmaceuticals. Using the ACE System, stable, high-expressing clonal cell lines
can be generated in three to six months with minimal screening. Depending on the process selected, candidate cell lines are
generated with antibody titers in terminal, non-fed, shake-flask cultures of 300 to 1,000 mg/L and specific productivities
in the range 20 to 40 pg/cell/day. These candidate cell lines had a stable gene expression for over 90 generations and were
amenable to media and growth optimization and scale-up, to which 2–5 fold gains in yields have been noted. These results show
that ACE System candidate cell lines can be generated faster and perform as well, if not better, than those generated by existing
technologies. The ACE System allows the downstream genetic engineering of cell lines and the auditioning of alternative cell
lines to be performed more readily and routinely with minimal effort. It is clearly an alternative to conventional methods
of cell line generation
Malcolm L. Kennard is director of cell line engineering at Chromos, Burnaby, British Columbia, Canada, 604.415.7100, email@example.com
Danika L. Goosney is an associate for research planning and reporting at the Canadian Institutes of Health Research; and Harry C. Ledebur, Jr., is vice president of scientific affairs at Chromos.
1. De Jong G, Telenius AH, Telenius H, Perez C, Drayer J, and Hadlaczky G. Mammalian artificial chromosome pilot production
facility: large-scale isolation of functional satellite DNA-based artificial chromosomes. Cytometry 1999;35:129-33.
2. Lindenbaum M, Perkins E, Csonka E, Fleming E, Garcia L, Greene A. A mammalian artificial chromosome engineering system
(ACE System) applicable to biopharmaceutical protein production, transgenesis and gene-based cell therapy. Nucleic Acids Res.
3. Perez CF, Vanderbyl SL, Mills KA, and Ledebur HC. The ACE System: A versatile chromosome engineering technology with applications
for gene-based cell therapy. Bioprocessing J. 2004 July/August;61-8.
4. Vanderbyl S, Sullenbarger B, White N, Perez CF, MacDonald GN, Stodola T. Transgene expression after stable transfer of
a mammalian artificial chromosome into human hematopoietic cells. Experimental Hematology 2005;2005;33:1470–6.