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Dec 1, 2012 (Vol. 32, No. 21)

Inventive Approaches Redefine Downstream Ops

  • E. coli Secretion Technology

    Click Image To Enlarge +
    A Wacker Biotech technician works on expanding Fab-producing clones that were developed using the Esetec expression system.

    Wacker Biosolutions has developed an E. coli-based protein secretion technology, explained Susanne Dilsen, Ph.D., head of production. Referred to as Esetec®, it is a recovery and purification procedure for high-efficiency Fab isolation.

    “Our process consists of the Esetec expression system, a fermentation process, a recovery procedure, and a scalable purification process,” Dr. Dilsen said.

    Esetec boasts a specially designed E. coli K12 strain, dedicated expression vectors and a toolbox of helper plasmids, supplying chaperones, disulfide isomerases, and foldases. This strain secretes proteins directly into the medium rather than into the periplasm. The accompanying toolbox is an extensive collection of protease deletion strains, signal peptides, chaperones, and secretion components.

    An important feature of the process is screening for the most satisfactory clones, working up the ladder in stages. The primary screening stage is a high-throughput, random method, covering 1,000 to 3,000 clones per week, for optimizing titer. The second level of screening focuses on functionality and can handle 30 to 60 clones per week.

    Finally, the strain selection for the fermentation component handles only three to nine clones per week and aims at selection for titer, function, and quality.

    Nothing is left to happenstance in the configuration process. All inoculation ratios and preculture conditions are standardized and a chemically defined medium free of animal proteins is employed. Temperatures and temperature down-shift before induction have been identified, as have the effects of additives (e.g., reducing agents, stabilizers, antifoaming reagents). These were tested and the cell-harvesting conditions standardized during the initial design phases of the process.

    Dr. Dilsen described case studies using the Esetec technology. The first, a collaboration with Bayer HealthCare, focused on the production of a recalcitrant Fab, sporting five disulfide bonds.

    “In this case we were aiming for yields in excess of 0.5 g/L in the culture supernatant, favoring large-scale production,” she added. “This required the development of appropriate analytical methods. The first yields were less than satisfactory, so we used the toolbox to improve the heavy chain expression, folding, and secretion.”

    The modifications, which increased yields by twofold, included altering the promoter to increase the expression of the heavy chain while at the same time co-expressing helper elements, folding, and secretion of the Fab, which is now correctly assembled and fully functional.

    In a second case study, Dr. Dilsen and her colleagues collaborated with Morphosys to construct mono- and bivalent antibody fragment formats with different specificity, framework, and physico-chemical properties. In one series of investigations with the Esetec platform, a 40-fold increase was obtained that generated functional Fab products.

  • More on Single-Use

    The advantages of single-use, disposable technologies are widely acknowledged, as is substantial enthusiasm for therapeutic antibodies. Klaus Kaiser, Ph.D., head of GMP purification, global biological development, Bayer HealthCare, spoke to the issue of the purification of innovative antibodies with new functionalities. Of note are antibody-drug conjugates and modifications of the protein molecule to stabilize it or enhance its activity.

    Many companies are targeting biosimilars, given that they are based on approaches that have already been validated. Their biology is well understood. This leads to the potential for shorter time lines, a much desired outcome in an era of approval processes that can run to many years.

    With downstream processing a major component of the drug development mix, it is of critical importance to dissect the costs of the various steps in downstream operations. As would be expected, the Protein A purification step is far and away the most costly, at 40% of the total. This means that it would be very difficult to bring down costs significantly without making inroads into this component.

    Whereas disposable downstream technologies are highly favored, they are not without their disadvantages and challenges. This includes overly complex construction and mechanical instability. Yet they do not present the high maintenance costs, including decommissioning and dismantling incurred with reusable bioprocessing technologies.

    In the long haul the demands of the market will dictate whether existing large-scale reusable plants will be more cost-effective than the small-scale, single-use option.

    In designing the facilities plan, Dr. Kaiser discussed the “ballroom” option, in which processes are so tightly closed that there is no need for the traditional spatial segregations between a variety of upstream and downstream tasks. In this configuration one can group upstream and downstream unit operations in a common production ballroom.

    Dr. Kaiser finished his analysis with a discussion of another major factor influencing cost containment. That is the emerging discipline of personalized medicine. Given that most drugs are ineffective in a majority of patients, there is a strong push for drugs targeted to those individuals that will actually respond to the agent. This means that accurate biomarkers will be required, a task that has so far frustrated cancer researchers.

    Provided that such tests are forthcoming, this will push pharma companies toward smaller volumes and many more individual drugs. This trend will clearly favor the disposable technologies.

    “Single-use technologies have many advantages which let them appear ideal for ‘individualized’ production,” he said.

    At the end of the conference, Uwe Gottschalk, Ph.D., vp for purification technologies at Sartorius Stedim Biotech, summarized the state of innovation in the downstream processing field.

    “Today we are faced with unprecedented challenges that we cannot meet without truly inventive approaches,” he said. “While there is now a general recognition of this reality within the field, unfortunately this has not always been the case.”

    Dr. Gottschalk believes that there are drivers coming from several directions that are overwhelming the fear of moving forward that dominated thinking of companies in the past.

    “Most significant are cost and regulatory issues. We’re seeing that there is no easy way out of these problems.

    “So I think that the take-home message is that the industry is now firmly committed to original ways of thinking that will move us forward.

  • Novel Filtration Techniques

    New technologies in virus filtration, microfiltration, resins, and membrane absorbers are being introduced by Asahi Kasei Bioprocess, according to John Fisher, senior manager for science and technology. “Today many are giving the virus filtration step the respect that it deserves, recognizing that it is not just a simple filter,” he explains.

    With increasing titers in the cell culture processing step, there are challenges being forced on traditional downstream unit operations. Asahi, like many other companies, realizes that the virus filtration unit operation is one of the highest cost of goods components in downstream processing.

    To lower costs, many companies have adopted longer processing times, thereby reducing the virus filter area requirement. But operating over a longer duration places a greater burden on the virus filter to maintain throughput with minimal flux decay. At the same time, consistent performance during manufacturing and virus validation is essential.

    “This strategy places pressure on the validation specialists to create cleaner virus spikes, allowing high capacities and long processing times to be validated,” Fisher continued.

    Filter vendors are focusing on filter design and creating robust filters that will tolerate substantial variations in feedstocks.

    “Our company is working to characterize the size-exclusion mechanism of virus filtration as a function of the solution and operating conditions using fluorescent labeling and mathematical modeling to provide greater process understanding and to support end-users,” explains Fisher.

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