Recombinant Protein Production Yields from Mammalian Cells: Past, Present, and Future - The biopharmaceutical industry has been making rapid progress in yield improvement. - BioPharm International


Recombinant Protein Production Yields from Mammalian Cells: Past, Present, and Future
The biopharmaceutical industry has been making rapid progress in yield improvement.

BioPharm International Supplements

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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