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Dec 1, 2010 (Vol. 30, No. 21)

Exploring Future of Cell-Culture Processes

Scaleup, Process Performance, and Product Quality Serve as Key Points for Discussion

  • Improving Transfection

    The production of recombinant proteins always involves either transient or stable transfection. The former is rapid but is not inheritable, while the latter results in low transfection. Integrating vector DNA into the genome of the host cell tends to be random in frequency and efficiency, and resulting productivity unpredictable due to the need for selection and screening for the optimized clone.

    Hence the great interest in meganucleases, zinc finger nucleases, tag-and-exchange, and other techniques. One such alternative, scaffold/matrix attachment region (S/MAR) minicircle DNA, was the subject of a presentation by Bernd Rehberger, a scientist at Rentschler.

    Minicircles are circular, superhelical DNA vectors resulting from homologous recombination of “normal” parental plasmids. Characterized by the absence of unwanted bacterial sequences and other potentially interfering elements originating from the parental plasmid, minicircle DNA is viewed as a way to achieve high stable transfection rates, particularly when combined with S/MAR elements.

    According to Rehberger, whose company sells S/MAR minicircle reagents, minicircles result in transfection rates as high as 90%, with 1% of plasmids entering the genome.

    The potential for generating stably transduced cell lines with more predictable productivity and growth behavior within a short time make the S/MA minicircle vectors an interesting alternative to conventional transfection since they eliminate the need for time-consuming selection and screening. Incorporation of the desired gene can be monitored using cell sorting based on green fluorescent protein.

    Minicircles are also under investigation as gene-therapy agents and DNA vaccines, although Rehberger said “it will still take some years to achieve the levels of quality and reliability required for human therapeutics.”

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