Manufacturing recombinant proteins at industrially relevant levels requires technologies that can engineer stable, high-expressing
cell lines rapidly, reproducibly, and with relative ease. Commonly used methods incorporate transfection of mammalian cell
lines with plasmid DNA containing the gene of interest. Identifying stable, high-expressing transfectants is normally laborious
and time consuming. To improve this process, the ACE System has been developed based on pre-engineered artificial chromosomes
with multiple recombination acceptor sites. This system allows targeted integration of single or multiple gene copies and
eliminates the need for random integration into native host chromosomes. To illustrate the usefulness of the ACE System in
generating stable, high-expressing cell lines, we present several case studies covering CHO cell lines expressing monoclonal
Many methods are currently available for producing cell lines that express recombinant proteins. Most of them use plasmid
transfection, or viral transduction procedures, to incorporate DNA sequences containing the gene of interest into mammalian
cell lines. These processes often result in transfectants with highly variable protein expression due to random integration
of the DNA into the host genome. Furthermore, these methods may necessitate time-consuming amplification events, or re-infection,
to boost the cell's productivity. As a result, the process of generating and selecting a high-expressing, stable clonal cell
line suitable for the clinical and commercial manufacture of biopharmaceuticals can be labor intensive and extremely time
To increase the speed and efficiency of generating high-expressing, stable cell lines for the manufacture of recombinant proteins,
Chromos has developed a novel cell-line engineering system, the ACE System, based on the company's proprietary artificial
chromosome technology. The system differs from conventional technologies. It facilitates the targeted and reproducible integration
of multiple copies of genes into specific sites on an artificial chromosome that resides in a production cell line without
amplification. These artificial chromosomes, or ACEs, contain fully functional centromeres and telomeres, and as a result
are as mitotically stable as the host chromosomes. Ultimately, this results in the generation—with minimal screening and reduced
timelines—of high-expressing stable clonal cell lines with high levels of gene expression. ACEs also can be purified to homogeneity
by flow cytometry and readily transferred to a variety of cell types using commercially available transfection agents. This
feature enables the auditioning of alternative cell lines for improved product quality or quantity, thereby providing an option
not typically found in conventional mammalian cell line engineering technologies.
THE ACE SYSTEM
The ACE System was designed so that one or more genes could be reliably and reproducibly loaded, with relative ease, onto
an existing ACE and screened for incorporation and expression. For mammalian cell line engineering, the ACE System consists
of four main components (Figure 1):
1. Platform ACE: A pre-engineered artificial chromosome containing 50–70 recombination acceptor sites, which allows the insertion
of multiple copies of DNA sequences.
2. Platform ACE Cell Line: A manufacturing quality cell line (CHOK1SV, Lonza) containing the Platform ACE that grows to high
cell density under serum-free growth conditions.
3. ACE Targeting Vector (ATV): A plasmid that contains a single recombination donor site for recombination into the acceptor
sites on the Platform ACE, selection marker, and the gene(s) of interest along with all genetic elements required for enhanced
expression in CHO cells.
4. ACE Integrase: A site-specific DNA recombinase that catalyzes the targeted integration of the ATV onto the Platform ACE
residing in the Platform ACE Cell Line.