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Nov 1, 2011 (Vol. 31, No. 19)

Getting the Most from Process Operations

A Strong, Predictive Expression System Is Essential to Maximize Productivity

  • Alternatives to Cell Culture

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    According to Alder Biopharmaceuticals, shorter cycle times with P. pastoris fermentations more than make up for lower volumetric productivity.

    Of major alternatives to expression in mammalian cells, yeast, particularly P. pastoris, are attractive for their long history of large-scale processing for therapeutic proteins and other products at scales of up to hundreds of thousands of liters.

    Alder Biopharmaceuticals makes a good case for P. pastoris as a production host. When the company began operations in 2004, the founders avoided production systems that lacked an FDA track record.

    “We didn’t want to face novel regulatory issues,” notes John Latham, Ph.D., CSO. Instead they set out to “change the timing, cost structure, and scale of manufacturing intact, glycosylated, fully humanized antibodies for chronic indications.”

    Dr. Latham would not directly address questions about the kinds of titers Alder achieves in P. pastoris fermentations, saying only that they were “somewhat lower” than the 3–6 gram/L levels achieved with many mAb processes in CHO cells today. Shorter cycle times make up for lower volumetric productivity, however. “If you think in terms of grams of productivity per 10,000 liters per month, our first-generation process is about as good for mAbs as cell culture.”

    Companies specializing in CHO culture rarely introduce the time factor when discussing titers since cell cultures take several weeks. When comparing CHO to yeast, perhaps the term “productivity cycle” makes more sense than “volumetric productivity.”

    Yeast cultures have one drawback: very large harvest volumes. But for antibodies the disadvantage is minimized since initial capture occurs in flow-through mode, and recently developed capture resins allow very high flow velocities. Once the product is captured, the process resembles cell culture purification. “Except we don’t have to deal with viral inactivation or removal validation, which eats into yield due to aggregate formation. So downstream processing is actually simplified,” Dr. Latham says.

    Short cycle times have the advantage of improving plant utilization. Upstream, where manufacturers plan a 10–20% failure rate, recovery and readiness to process the next batch is more rapid with yeast, and since fermentations are of short duration, downstream equipment never sits idle for very long.

    Dr. Latham described Alder’s development-stage molecules as being small molecule-like in their activity. “They are designed to block pharmacologic activity of a ligand or receptor, and do not work through cytotoxicity or complement activation.” The molecules also lack N-glycosylation in the Fc region. “We engineered that out.”

    Lack of glycosylation in critical regions eliminates safety issues and reduces heterogeneity. Mannose, which is abundant in yeast glycosylation, is viewed by the human system as foreign. “Eliminating mannose reduces the complexity of what lies ahead, particularly with regulators.”

    By contrast GTC Biotherapeutics lets its expression system—rabbits and goats—carry out their native glycosylation. GTC was a pioneer in transgenics during the 1990s. Like most such companies it eventually ran out of money and was purchased by the French plasma products manufacturer LFB Group. The company has one marketed product, Atryn (recombinant human antithrombin), which is used to prevent clotting during surgery and as a result of several conditions. Previously, all antithrombin was isolated from plasma.

    Processing throughput for transgenics has generally been good relative to conventional cell culture and fermentation, so cost of goods was not a factor in the demise of transgenics firms. The big hurdle was regulations.

    “One of the concerns with recombinant proteins manufactured by transgenics was potential differences in glycosylation,” says Harry Meade, Ph.D., senior vp of research. “But analysis shows the sugars to be the same, and they are not immunogenic.”

    That’s not to say that “volumetric productivity” never mattered. Expressing therapeutic proteins in field-grown corn is inexpensive; bringing plants indoors to prevent cross-pollination with nontransgenic plants raises serious cost issues.

    Milk-expressed proteins provide a level of safety and economics. Over the years GTC has reportedly produced many of the top antibody products currently marketed.

    Goats can routinely produce proteins, including blockbuster mAbs, at about one-tenth of a large biotech firm’s upstream cost. The two methods are on par for purification costs. “This means that we can compete with very large cell-culture processes no matter what the production scale,” Dr. Meade adds.

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