Generating Cell lines with High Specific Productivity
Obtaining high-producing recombinant cell lines remains among the top priorities for protein manufacturers. Cell lines are
generated following delivery of the gene of interest and the selection gene—on a single plasmid or on separate plasmids—into
host cells by transfection. The most widely used selection markers are dihydrofolate reductase (DHFR), an enzyme that produces
a cofactor for thymidylate synthetase, and glutamine synthetase (GS).3 These two markers are mainly used for selection in CHO and NS0 cells, respectively. In both cases, selection occurs in the
absence of the appropriate metabolite(s): glycine, hypoxanthine, and thymidine for DHFR and glutamine for GS. Cells surviving
selection are characterized by the integration of one or more copies of the transfected plasmid(s) at a single site in the
cell's genome. The DHFR and GS selection markers have the advantage of supporting amplification of the copy number of the
integrated DNA by exposure of the selected cells to increasing amounts of methotrexate (MTX) or methionine sulphoximine (MSX),
respectively.2 Usually, no effort is made to integrate the transfected DNA to a specific site of the host cell's genome. Instead, this
process is allowed to proceed randomly, and the selected cell lines are then screened for protein productivity and growth
rate. The superior cell lines are also eventually analyzed for the stability of protein production over time. This strategy
for cell line generation depends on the screening of a large number (100s to 1,000s) of recombinant cell lines for the desired
characteristics. Strategies to increase the percentage of high-producing cell lines in the population of transfected cells
include increasing the stringency of selection or amplification.2
Integration of Plasmid DNA
The structure of the chromatin at the site of integration is also a critical regulatory factor with regard to expression of
the integrated plasmid DNA. Integration of the plasmid DNA into actively transcribed regions of the genome (euchromatin) favors
high and stable expression of the recombinant gene(s), whereas integration into condensed, and therefore, transcriptionally
inactive regions of the genome (heterochromatin) results in the repression of gene expression. Including cis-regulatory elements
such as insulators, scaffold and matrix attachment regions (S/MARs), ubiquitous chromatin opening elements (UCOEs), and antirepressor
elements near the promoter or enhancer of the recombinant gene has proven to be an effective strategy for limiting heterochromatin
formation at the site of plasmid DNA integration.4 Methods to target integration of transfected plasmid DNA to transcriptionally active sites of the genome have been developed
and may have been used to generate high-producing cell lines by some manufacturers.5 However, convincing evidence showing that targeted integration consistently yields cell lines with higher specific productivities
and with increased long-term expression stability than those generated by random integration has not yet been published. Indeed,
the recent exploitation of chromatin regulatory elements, as described above, appears to have reduced the need for targeted
integration.
Screening Tools
The recovery of cell lines with high specific productivity also has been improved through the development of high-throughput
screening tools.6 By increasing the number of candidate cell lines screened, the probability of finding a few highly productive ones is increased.
The new methods are mainly based on the recovery of individual cells by fluorescence-activated cell sorting (FACS). As an
example, genes for the protein of interest and a reporter protein such as green fluorescent protein (GFP) can be co-transfected
into cells. If the two genes are expressed from a bicistronic mRNA having a internal ribosome entry site (IRES) for translation
of the reporter gene at the 3' end of the mRNA, then cells selected by FACS for a high level of GFP are expected to be high-producers
of the recombinant protein of interest. One drawback of this approach is that the intracellular accumulation of GFP may be
limited by several factors and may have negative effects on cell growth and cell survival. To avoid these secondary problems,
it is preferable to use high-throughput methods that directly measure the level of the recombinant protein of interest without
using a reporter protein.
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