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Feb 1, 2012 (Vol. 32, No. 3)

Optimizing Cells and Cultures for Better Productivity

  • Mixtures for the Price of Monoclonals

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    Symphogen is using high-throughput proliferation assays in order to perform lead selection of antibody mixtures.

    At the meeting, Christian Müller, Ph.D., senior scientist at Symphogen, will talk about his company’s work on antibody mixtures. The idea of using multiple antibodies against a specific target is analogous to the antiviral cocktail approach for treating HIV. “The strategy mimics nature as well, where hundreds of antibodies are produced against antigens,” explains Dr. Müller.

    For example Symphogen’s Sym004 Phase II product consists of two antibodies targeting the epidermal growth factor receptor (EGFR) that when combined synergistically inhibit cancer cell growth in vivo and in vitro. In particular, Sym004 leads to extensive internalization/degradation of the receptor. On top of this, Sym004 blocks ligand binding, activation and downstream signaling of the EGF receptor, and activates immune-mediated killing of cancer cells.

    Symphogen’s SympressT technology allows production of two or more antibodies in a single batch. First, cell banks are made of stable CHO cell lines each expressing an antibody of the antibody mixture. The cell lines are then mixed, and polyclonal master- and working-cell banks are made, which is the starting point for manufacturing.

    Why deal with two differently transfected cells and potential problems upstream and downstream? The alternative, to generate the antibodies separately, is almost twice as expensive as through the Sympress process. Cell culture, harvest, and purification in Sympress proceed as they would for a single monoclonal product, and Symphogen has developed an analytical toolkit around characterization and release of the antibody mixture.

    “We use several orthogonal analytic methods drawn from standard antibody work, including ion exchange, reverse phase, and size exclusion analytical chromatography, plus newly developed methods e.g., using mass spectroscopy and quantification of antibody mRNA levels,” says Dr. Müller.

    “We’ve had good feedback from FDA and EMA on both manufacturing and analytics.”

    The difficulty, and the innovation, involve selecting antibodies that potentiate each other’s function. One would not want proteins that target the same epitope on the target cell, for example, or they would compete with instead of augment each other. The other concerns involve determining the optimal antibody ratio, and then matching the productivity of cells for individual proteins with the desired concentration in the final product.

    Since developing medicines like Sym004 requires balancing the effects of two antibodies, it requires some additional preclinical work compared to single antibodies. The potential reward, however, is greater efficacy and much lower cost compared with administration of two separate drugs. So far, according to Dr. Müller, Sym004 “has superior efficacy in preclinical models than cetuximab” (Erbitux), a blockbuster EGFR monoclonal.

  • To License or Not to License?

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    Eden Biodesign follows a fairly typical cell-line optimization strategy: Select the expression system, choose a vector, and pick high-producing clones.

    Eden Biodesign, an integrated biopharmaceutical development organization and part of Watson Pharmaceuticals, follows a fairly typical cell-line optimization strategy: select the expression system (mammalian or microbial), choose a vector, and pick high-producing clones.

    Molecule type and post-translational constraints dictate the expressing organism, which further restrains choices of transfection vector. “If the product is relatively simple, lacking complex glycosylations, we might use a microbial cell line, which grows rapidly and is less expensive than mammalian cell culture,” says Philip Mellors, upstream development manager.

    For CHO cells, the critical decision involves whether to employ public domain or proprietary expression systems. Among the systems requiring a licensing fee Eden has employed are Lonza’s GS (glutamine synthase) System™, Millipore’s UCOE (ubiquitous chromatin opening element), and Selexis’ Genetic Elements™.

    Five products have been approved that use the GS System, including Zenapax® (Roche) and Synagis® (MedImmune). UCOE claims stability over 130 generations, while Selexis’ Genetic Elements improves expression by shielding critical genes from silencing effects, according to the company.

    All commercial systems promise significantly higher titers (greater than 2 g/L) and much shorter development times (as short as two months) compared with standard IP-free expression methods. “Public domain systems take up to 12 months to arrive at a suitable clone,” notes Mellors, “and typically produce between 0.5 and 3 grams per liter of protein.”

    And therein lies the conundrum. Companies short on time or manufacturing capacity might choose a commercial expression system to compress development times and reach the clinic faster, but it will cost more at every stage. Some licensors charge a flat rate plus an additional fee based on the success of the product.

    A royalty-free process, while slower and less productive, is more valuable to a potential partner or purchaser down the road. Thus the decision to license or not involves a complex accounting and statistical exercise.

    “Large companies would tend to go down the proprietary route since they have the money to pay up front and beyond, if necessary,” explains Mellors. For these firms, with potential blockbusters in their pipelines, time to market trumps what is viewed as an incremental cost. “But it’s always a tradeoff,” he says.

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