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Oct 15, 2009 (Vol. 29, No. 18)

Enhancement of Cell Culture Techniques

Emerging Insights on Metabolomics, Media, and 3-D Structures Could Lead to Improvements

  • Perfusion Process

    Click Image To Enlarge +
    Percivia’s XD™ process takes advantage of the PER.C6® cell’s ability to withstand high shear and grow to high concentrations.

    Kathryn Golden, associate scientist III at Percivia, presented data on a high-titer perfusion process for PER.C6 cells. The process is based on the high-titer, high-density XD™ perfusion process, which was developed by DSM and is being commercialized by Percivia, a joint venture between DSM and Crucell.

    XD reportedly achieves cell densities of more than 200 million cells/mL—about ten times the density of conventional cultures—and antibody production titers of 40 g/L of cell-free supernatant over a 17-day culture. Because the cell volumes are substantial, the volumetric productivity are only 27 g/L of harvest when the correction is made, reported John Chon, Ph.D., director of process development.

    “Moreover, the cells are healthy throughout the culture time,” he noted. “Cells in standard fed-batch cultures tend to be quite healthy in the beginning, but die off as a result of not removing metabolites.” In the XD process, metabolites are constantly removed and the cells are recirculated. Unlike standard perfusion cultures, product is retained within the medium, which provides substantial benefits during purification by keeping volumes low, Dr. Chon added.

    XD has been demonstrated at the 50 L scale which, due to the high cell density and volumetric productivity, performs about as well as a reactor ten times as large, Dr. Chon said. Percivia is working to bring the culture volume up to 250 L.

    XD is based on the ATF™ cell culture system from Refine Technology. ATF consists of a pump and hollow fiber membrane that removes media from the culture but returns cells and products. The ATF system connects to any bioreactor (stainless steel or disposable) and can handle cultures of up to 2,000 L working volume. “The great benefit is that you don’t need to retool your manufacturing plant to use XD technology,” noted Dr. Chon.

  • Minimal Media

    Ferruccio Messi, Ph.D., CEO of Cell Culture Technologies, another speaker at the Cambridge event, is a huge advocate of what he calls minimal cell culture media. Minimal media contain only water and small molecule chemicals with Chemical Abstract Services or European Inventory of Existing Commercial Chemical Substances numbers—no animal-derived sera, extracts, peptones, hydrolysates, or other undefined substances. “We only use compounds with a defined chemical identity,” said Dr. Messi.

    According to Dr. Messi, 15 companies have performed “remarkable work” on the cultivation of CHO cells in minimal media. He described one line, the CHO-easyC (derived from CHO-K1), as “one of the most promising host cell lines for development of next-generation bioprocesses.

    “Many people talk about chemically defined media, but when you try to go beyond that definition you realize that chemically defined means everything and therefore nothing. People are using chemically defined lipid mixtures, and the user doesn’t know what’s in there. In my presentation, I showed the complete composition of a minimal medium, and everybody knew what I was talking about.”

    Even more than media that take half-measures to achieve chemical definition, minimal media replace all vestiges of black boxes and “fairy dust” from cell culture with hard science—only what cells actually need to thrive and produce.” The most obvious benefit is batch-to-batch consistency and the opportunity for science-based optimization—goals that can only be achieved when processors know precisely what media consist of. Minimal media are also less likely to require esoteric purification techniques and more readily satisfy regulators.

    Processors add extracts, hydrolysates, and other ingredients, explained Dr. Messi, because their basal media are not optimized. “They didn’t fulfill the cell’s basic nutritional requirements, so they compensated by adding boosters. But you can easily grow any kind of cell in minimal media if you knew the identity of every molecule required by cells, and their proper balance.”

    Cell Culture Technologies provides its expertise through long-standing collaborations with client companies. The average such relationship, which involves scientists from Dr. Messi’s company working at clients’ facilities, lasts 2.7 years. “It’s a lot more complex than filling a bottle with media and selling it,” Dr. Messi explained. 

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