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Oct 1, 2011 (Vol. 31, No. 17)

Protein Expression Technology Review

Improvements in Microbial and Mammalian Systems Design Drive Advances in the Field

  • Click Image To Enlarge +
    John R. Birch, Ph.D.

    We now take it for granted that we can use recombinant DNA technology to express a wide range of proteins for use in many research and commercial applications, particularly in the field of human healthcare.

    Before the recombinant revolution, there were a few proteins such as insulin and human growth hormone that could be manufactured by extraction from natural tissues, but many proteins of potential therapeutic interest could not be produced in sufficient quantity. A good example of this is interferon.1

    Discovered in the 1950s, this family of proteins generated a huge amount of interest in the 1970s as potential antiviral and anticancer agents. There was a global effort to produce sufficient quantities in cell culture and from natural tissues but the quantities that could be made were small. In addition, it was a real challenge to produce enough material for clinical use.

    Researchers at the Frederick Cancer Research Center, for example, reported that it required several thousand liters of cell culture to produce tens of milligrams of interferon. The picture changed dramatically in the 1980s when the genes for the different interferon species were cloned and expressed in microbial systems and the first recombinant interferon product made it to the market in 1986.

  • Plethora of Products

    Click Image To Enlarge +
    Scanning electron micrograph of chinese hamster ovary cells: The CHO cell line is one of the most commonly used in the biotechnological production of substances such as monoclonal antibodies. [Eye of Science/Photo Researchers]

    There are now over 200 biopharmaceuticals on the market in the U.S. and EU2 and many hundreds more in development. The first recombinant product, insulin3 (Eli Lilly), produced in E. coli, was approved in the U.S. in 1982. This was followed by other products expressed in that bacterium (human growth hormone-1985, interferon-a-1986) and in the yeast Saccharomyces cerevisiae (hepatitis B vaccine-1986).

    While both of these organisms proved valuable and have been used extensively in the ensuing years, it became apparent quite early on that they would not be appropriate for the expression of all proteins. The first generation of products were typically small proteins, lacking post-translational modifications such as glycosylation that are commonly found on many mammalian proteins of therapeutic interest.

    For large complex molecules, and particularly for proteins requiring mammalian-type glycosylation to be effective as therapeutics, microbial expression was not a viable option. For these proteins attention turned to mammalian cell culture and the first recombinant product (tissue plasminogen activator from Genentech) was approved in 1987.

    As it turned out, mammalian cell culture would be used to produce a wide range of enzymes, blood factors, monoclonal antibodies and other proteins. In fact, cell culture is now used to produce more than half of the licensed proteins.

    Monoclonal antibodies have had a particularly big impact on the development of mammalian cell expression technology. There was great interest in the therapeutic use of antibodies following the development of the monoclonal technology in the 1970s and a nonrecombinant murine antibody (OKT®3), produced in mouse hybridoma cells was licensed in 1986.

    However, the more general use of antibodies was limited by their immunogenicity, a problem that was resolved using novel engineering approaches to produce chimeric, humanized, and eventually, fully human recombinant antibodies. There are now around 25 antibody therapeutics on the market, and as a class they represent approximately two-thirds of the biopharmaceutical proteins in development4.

    In addition to numbers of products, antibodies have also had an impact in terms of quantities required. Because of the large doses needed for some products, manufacturing requirements can be high—tonnage quantities in a few cases. On the one hand this has led to a requirement for extremely large-scale culture systems (up to 20,000 liters in scale).5 On the other hand, it has been a potent driver for improving the efficiency of cell culture technology.

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