Transient Expression

In transient expression systems or ''transient transfections,'' DNA is introduced into the cell and maintained/replicated as an extrachromasomal unit. Besides the strength of the promoter driving expression of the product gene, transfection efficiency (percentage of cells taking-up and expressing DNA) is the most important factor in determining the overall expression level, and several methods have been recently developed which yield reproducibly high transfection efficiencies (~50%, see following text). The average production life of the transient expression system is usually limited by toxicity of the rapidly multiplying DNA, or the loss of DNA from the cell population during division. While not suitable for long-term production or commercial manufacture, expression levels in transient systems may range from >1 to 100mg/L, making these systems extremely useful for rapid production

Cell Line Development Flow Chart

□ Clone product gene cDNA

□ Subclone into suitable mammalian expression vector containing sequences encoding a selectable marker

□ Transfect host cells with plasmid

□ 24-48 hr later, expose to selection reagent

Confirm sequence & verify correct linkages

□ Pick 10s to 1000s clones/colonies of surviving cells

□ Screen for productivity in multi-well tissue culture plates

□ Identify < 10% of clones for further analysis

Optional iterative process

Expose high producers to increased concentrations of amplification reagent

□ Scale and introduce cells to suspension growth in serum-free or protein-free media, if required.

□ Final screening of productivity, growth characteristics, product quality

Full characterization for freedom from adventitious agents

Figure 1 Cell line development flow chart.

of small quantities of research materials for drug candidate identification and in vivo evaluation. Moreover, methods for transient transfection of larger batch cultures, that will potentially enable scalability to at least the 100 L scale, are presently in development (1).

Stable Expression Systems

DNA containing the product gene must be integrated into the host cell genome for stable product production. Besides promoter strength, the frequency of DNA integration into the chromosome (copy number) as well as the position of integration, rather than the transfection efficiency are the more important factors in determining overall expression levels. There are no differences in transfection methods per se between transient and stable transfection. What does differ is the need to identify and select cells that have integrated the plasmid into the genome. This is most often accomplished by transfecting a sequence encoding a marker gene into the host cell along with the expression vector containing the product gene(s). Transfected cells are then cultured under defined conditions that allow for direct selection of cells that have incorporated the marker sequence into the genome and are expressing its gene product (2,3). Over the past decade, many vector design strategies have been developed which help ensure cointegration of the selection marker and the product gene. Thus, it is expected that most of the selected cells will also express the product gene(s).

Depending on the cell type, the selection system used, and the desired levels of product expression, the specific productivity of the initially selected stable cells may not be optimal. In this case there may be the possibility to further enhance protein expression by subjecting the cells to repetitive rounds of exposure to a selective agent that often results in gene amplification. Individual clones are generally screened for productivity at the 96-well scale, and lead candidates further evaluated for performance in conditions which model high cell density cell culture process conditions. In most instances it is also desirable to make some early evaluation of the biochemical quality of the product being produced. Based on data collected, a single cell line is chosen for continued process development. The cell line is banked and extensively characterized in accordance with specific Regulatory guidelines for freedom from adventitious agents to ensure suitability for cGMP manufacturing.

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