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Jun 15, 2008 (Vol. 28, No. 12)

Methods for Maximizing Antibody Yields

New Technologies Could Help Usher in Lower Costs and Increased Availability

  • Among recombinant proteins, antibodies stand out as the strongest candidates for new therapeutic drugs and represent the majority of submissions to the FDA and other regulatory agencies. Total sales of recombinant antibodies reached $15.8 billion in 2006 and are projected to go to $28.6 billion by 2010, according to Rolf Werner, Ph.D., senior vp at Boehringer Ingelheim (www.boehringer-ingelheim.com), who also noted that the biggest selling antibody of 2007 was Remicade, which racked up over $5 billion in sales worldwide.

    The advancement of recombinant protein technology has enabled the humanization of antibody molecules that are well tolerated, safe, and effective. A number of new therapeutic products are in the middle of trials, and although the rate of approvals slowed in 2007, it will probably increase this year.

    Yet even in the midst of abundant rewards, antibody therapeutics face enormous scientific, commercial, and legal challenges. The highly competitive environment, the burdensome pricing, lack of tissue penetration, the large upfront investment costs, and the legal thicket of overlapping patents all conspire against new recombinant antibody products. Nonetheless, a large number of antibodies are moving through clinical evaluation.

    Much of the current excitement over recombinant antibody proteins is because they are the only therapeutics that extend the life of patients with metastatic disease, even though they do not provide a cure. Much discussion at the recent IBC “Antibody Production and Processing Conference” dealt with advances at both the upstream and downstream ends of the production train, which may, in the long run, help to lower costs and increase availability.

  • Engineered Cell Lines

    Even superbly engineered proteins can be hobbled without an optimized synthetic system. Because mammalian cells continue to dominate the market as the vehicle of choice for antibody production, the selection of the optimal cell line becomes more and more critical.

    Blockbuster drugs require huge amounts of material. Cell line manipulation is a long and arduous process, and errors made early in the selection process can result in irreparable deficiencies further down the development road. So a well-researched and carefully reasoned choice of cell line can result in huge cost savings.

    In the early days of recombinant antibody production, murine lymphoid cells and hybridomas were the vehicles of choice. This approach was satisfactory at a time when biotechnologists were content with mg/L of protein yield. Now with yields moving into the multiple grams per liter range, there is pressure to switch to more robust production systems. Unfortunately, the murine lines have subtly different glycosylation patterns and have proven difficult to engineer to higher levels of productivity. For this reason there is great interest in transfected human cell lines and other innovative approaches to cell biosynthesis.

    Currently, the two major contenders are CHO cells and Percivia’s (www percivia.com) PER.C6 line. The CHO line has the virtue of being perhaps the most widely exploited and best understood mammalian cell line in existence but it was never intended to be a protein-production factory. PER.C6, on the other hand, was specifically designed to produce biopharmaceutical products, according to DSM Biologics (www.dsm.com) and Crucell (www.crucell.com), which formed Percivia to promote the PER.C6 platform.

  • CHO Line

    John Birch, Ph.D., CSO at Lonza (www.lonza.com), discussed his group’s efforts to improve protein synthesis capability in the CHO cell line. Manipulation of nutritional conditions, optimization of feeding strategy, and the engineering of the genetic makeup of a particular cell isolate are the critical inputs in maximizing protein production.

    In the last 40 years there have been numerous protein-free and serum-free media developed for the CHO line. The Lonza group has focused its efforts on completely protein-free media and feeds for its fed-batch culture systems. As the technology has improved, there have been increases of orders of magnitude, bringing yields in the range of grams per liters. The drivers of these advances are augmentation in cell density and enhancement in per cell protein production. As the technology matured, Lonza realized a 167-fold boost between 1990 and 2008, according to Dr. Birch.

    In one series of trials, Lonza scientists isolated a variant, the CHOK1SV cell line, that achieved higher cell concentrations and maintained viability longer than another commonly used line (the CHO DG44) when grown under the same conditions. Dr. Birch strived for a simplified screening system that would allow processing of many clones with different genetic backgrounds.

    “We have aimed at a straightforward screening regime,” Dr. Birch stated. “We use the GS expression system, which has a high level of selection stringency, and we have started with the CHOK1SV cell line, which has improved growth characteristics.”

    A particularly favorable property of the cell line is its anchorage-independent growth properties. Ordinarily, CHO cell lines grow attached to the culture vessel, presumably due to the production of adhesive proteins. By selecting for a strain that grows in suspension, the Lonza workers were able to improve performance and save time during the developmental process. Using a relatively simple screening program, the Lonza group is able to routinely generate highly productive cell lines making grams per liter of antibody.

    For Dr. Birch, the take-home message is that the optimal approach to improving protein output is based on ratcheting up a cell’s capacity for protein synthesis. “To do this you need to understand the biological underpinnings of cell performance and perfect your tools for assessing the functional capabilities of the cells,” he posited.


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