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Jun 15, 2012 (Vol. 32, No. 12)

Taming Difficult to Express Proteins

  • Peter Rhode, Ph.D., R&D vp at Altor Bioscience, spoke on maximizing production of interleukin-15. Touted as a possible curative treatment for cancer and viral diseases, IL-15 is poorly expressed in bacterial and animal cells. Production in mammalian cells, explained Dr. Rhode, is controlled at the levels of gene expression, translation, and secretion.

    “Researchers have tried optimizing codons and altering genetic elements, as well as fusing with albumin, but those approaches have not improved expression that much.” The National Cancer Institute has produced GMP-scale quantities of IL-15 in bacterial systems. The protein is excreted in inclusion bodies, which requires re-folding. But issues with protein deamination and amino acid additions have complicated large-scale production.

    Altor scientists found that co-expressing an IL-15 superagonist variant with a soluble IL-15 receptor alpha-IgG1 Fc (IL-15Rα) fusion molecule leads to fully active IL-15:IL-15Rα complex in high yield from CHO cells.

    Compared with the “bare” cytokine, the IL-15:IL-15Rα complex is far more active. “It is the substance that immune cells produce,” noted Dr. Rhode, “and how IL-15 is presented in the body.” The superagonist IL-15 variant alone is as much as 10 times as active as the native cytokine. When combined with IL-15Rα, potency and pharmacokinetics improve over 20-fold.

    The receptor also acts as a molecular chaperone vital to proper expression and secretion of IL-15. “The limitations we’ve observed with IL-15 are likely due to the lack of this ‘partner’ molecule in animal expression cell lines. We recognized early in our development plans that co-expressing IL-15Rα could assist both in expression and efficacy.”

    The IL-15 complex is a potent molecule dosed at microgram/kg compared with multiple mg/kg for monoclonal antibodies. According to Dr. Rhode a “modest-scale GMP run will provide enough material to treat several hundred patients.”

  • Tools of the Trade

    Sabine Geisse, Ph.D., also from Novartis, is director of nuclear localization signal technology. She discussed alternatives to HEK293 and CHO cells for transient protein expression. Derived from human amniocytes, Dr. Geisse’s pet line, CAP-T®, “clearly enhances chances of success” when added to the repertoire of cell lines amenable to transient protein production.

    The line’s developer, Cevec, claims “two weeks from gene to milligram or gram amounts of protein” for early-stage studies. CAP-T has an impressive scientific resume as well, with heavy testing and support from European academics.

    What makes CAP-T cells special is that they are derived from nontumor human amniocytes. Very few nontumor human immortalized cells are able producers when transfected transiently. Dr. Geisse described the cells as “fast, cost-effective, efficient, stable with high-level expression, and highly reproducible.” Her group uses the cells to generate antigens, early antibody candidates, and any type of tool protein and that requires secondary modifications deferred by mammalian cells in the context of research protein production.

    “The benefits, relative to CHO and HEK, are biological characteristics that can lead to higher expression levels for some proteins, or the ability to produce candidate proteins that do not express well in HEK293 and CHO cells.” The drawback, which Dr. Geisse terms “temporary,” is that CAP-T requires “additional fine-tuning and modifications to be user-friendly and applicable on large scale.”

  • Traditional Approach

    Gregory T. Bleck, Ph.D., R&D platform lead at Catalent Pharma Solutions, described an experiment comparing his company’s GPEx® expression and cell-line engineering technology and a more traditional expression in CHO cells.

    Catalent scientists produced GPEx cell lines for one easily expressed protein and three difficult molecules that were either poor producers or exhibited subpar cell-line stability. The “control” process was the in-house platform of a Catalent pharmaceutical partner. The pharmaceutical partner compared cell lines developed by GPEx to cell lines developed using their internal system.

    Investigators did not observe significant differences between the two methods for the easy-to-express antibody. For the other three proteins (two antibodies and a fusion protein), GPEx outperformed their standard system in both productivity and stability.

    Why the difference? “GPEx seems to result in more consistently, high-expressing, stable cell lines than other processes,” said Dr. Bleck. “We have not elucidated the exact mechanism, but we have never encountered a protein that we could not produce at least as well as a more traditional expression system.”

    One possible explanation for GPEx success with difficult proteins is that unlike standard cell-line engineering, which inserts multiple copies of a gene at one location, GPEx introduces many individual gene copies in different locations on the host genome. GPEx also has the reputation for generating extremely genetically stable lines that express proteins reliably and efficiently. But in this particular study, Dr. Bleck admitted, “the reasons for GPEx superiority might be different for each protein.”

  • Click Image To Enlarge +
    Polyplus-Transfection reports that PEI promotes cell attachment to vessels and is an efficient transfection agent, causing DNA to condense into positively charged particles, attach to cell surfaces, and enter through endocytosis.

    Habib Horry, Ph.D., strategic marketing manager at Polyplus-Transfection, discussed the advantages of next-generation polyethyleneimine (PEI) in transient protein expression. A cationic polymer, PEI promotes cell attachment to vessels. It is also an extremely efficient transfection agent, causing DNA to condense into positively charged particles, attach to cell surfaces, and enter through endocytosis. Once inside the cell the DNA finds its way to the nucleus and into the cell’s protein-making machinery.

    During the mid-1990s, PEI became the second significant transfection agent discovered (the first being poly-L-lysine). A spinoff from a Strasbourg, France, research organization, Polyplus has since 2001 held two exclusive patents on the use of PEI for transfection of CHO cells. One patent covers the U.S., the other the rest of the world. Using PEI for transfection requires a license from Polyplus.

    “The advantage of PEI is that it is efficient and low cost,” Dr. Horry said. But not every company employing the reagent for this specific use is aware of Polyplus’ exclusive market position.

    PEI produces transiently transfected CHO cells that churn out between 10 and 100 mg of protein per liter of cell culture—a volumetric productivity that until about a decade ago was considered good for stably transfected cells. Using the reagent, companies can produce a few grams of protein suitable for characterization and preclinical studies. Most developers have settled on a process based on stable transfection before a molecule enters human testing.

    Despite great strides in transient transfection, the technique remains somewhat disconnected from the eventual production-scale process. “Techniques learned at this stage are not directly applicable to stable clones,” Dr. Horry observed. “This still represents a gap in the development of protein therapeutics."


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