October 1, 2012 (Vol. 32, No. 17)

Vicki Glaser Writer GEN

Single-use bioprocessing product lines made their debut mainly in the form of disposable bags for mixing and buffer supply. They gradually moved into the heart of the bioprocess stream with the emergence of single-use bioreactor systems.

Single use has more recently made inroads in downstream process flows for separation, purification, and fill and finish applications. This evolution in the industry is clearly illustrated at bioprocessing conferences, in which presentations highlight the range of product areas and biopharmaceutical R&D and manufacturing applications targeted by single-use technologies.

Examples include IBC’s recent “Single-Use Applications for Biopharmaceutical Manufacturing” conference in San Francisco, and Visongain’s upcoming “Single Use Bioreactor” conference in London. Compared to early single-use bioreactors, which were essentially disposable bags mounted on platforms to achieve mixing, the emerging, more complex generation of single-use systems more closely mimic conventional stirred-tank glass and stainless steel bioreactors. They incorporate impellers, sparge lines, and sensors for better control of process parameters and have aspect ratios that mirror those of traditional stirred tank reactors.

With the launch of its new ambr 250™ mL automated, scalable, single-use bioreactor system in August, TAP Biosystems aimed to increase the complexity and functionality of single-use bioreactors for use in process development while maintaining their ease of use in terms of set-up, operation, and user interface.

Andrew Tait, product development scientist at TAP Biosystems, will describe the company’s new technology and how it achieves these dual goals in his presentation at the Visongain conference.

Meeting its initial goal meant providing core stirred tank bioreactor technology for both microbial and mammalian applications, with an impeller and width-to-height ratios of conventional steel bioreactors. The system’s single-use bioreactor incorporates gas and liquid supply lines with connectivity to the bioreactor control unit.

The system is configurable for automated, independent, parallel control of 12 or 24 bioreactors, each with a maximum working volume of 250 mL. Each bioreactor can be individually controlled for temperature, pH, and dissolved oxygen, has four liquid feed lines, and off-gas analyzers for microbial applications. Up to 24 bioreactors are integrated into a dedicated biosafety cabinet.

Facilitating Flexibility

The main advantages of single-use systems are flexibility and speed to a functional, multiproduct facility, reduced footprint, elimination of clean-in-place, sterilize-in-place protocols, reduced water use, and increased overall throughput and productivity, according to Joe Makowiecki, senior manager, purification process development at Xcellerex.

The primary limitation at present is scale. “We are at the 2,000 liter single-use bioreactor scale already, and then from harvest all the way down to bulk drug substance it is single-use, except for the chromatography resins,” he said.

Single-use pumps are gradually replacing peristaltic pumps, offering low shear and flow rates >150 L/min, with tubing sets available up to 1 inch in diameter. Flow capacity for sensors is lagging a bit behind—ranging from about 20–80 L/min—but is slowly increasing.

Makowiecki pointed to several key trends driving technology development in single-use bioprocessing, including high-titer expression systems resulting in increasing amounts of biomass for downstream processing, and higher flow rates leading to, for example, the development of membranes with higher binding capacity.

Emerging on the market are new types of sensors, multicolumn chromatography, and “smart” mixers for automated adjustment of pH and conductivity. “Smart mixers will be ubiquitous in downstream processing,” predicted Makowiecki.

The FlexFactory flexible platform enables biomanufacturers to accelerate deployment of new manufacturing capacity while lowering risk, decreasing time to market, and reducing capital costs, according to Xcellerex, which reports that the approach enables the deployment of new production facilities in 9 to 18 months at a total cost less than 50% that of conventional plants.

“Flexibility is one of the hallmarks of single-use, and that bleeds into economics,” said William Whitford, senior manager at Thermo Fisher Scientific. “Single-use lends itself to varying production schedules,” easing not only normal operations, but making it easier and faster to mothball equipment or establish surge capacity.”

Minimizing up-front expenses reduces risk if a product fails, noted Whitford. In a new or expanded facility, single-use systems can reduce initial plumbing needs, the cost of having to validate complex cleaning systems, and the personnel needed to operate and maintain those systems.

“As a CMO, we have to be as flexible as possible,” to be able to operate a multiproduct facility at different scales, said Kai Lipinski, Ph.D., head of cell culture and virus production at Vibalogics. “We do not want to invest in equipment for one client that we might not use again.”

There are a “lot of restrictions currently for scalable adherent cell culture for virus and vaccine production,” continued Dr. Lipinski.

Several commercially important anchorage-dependent cell lines are not adaptable for suspension culture, noted Dr. Lipinski, including WI-38, A549, MRC-5, VERO, and CEF cells. Existing planar systems that support adherent cell culture such as roller bottles, T-flasks, and cell factories are labor intensive.

Vibalogics has performed studies using VERO and A549 as model cell lines to assess virus production when the cells are grown in the ATMI iCELLis™ semi-single use cGMP nano bioreactor system. Instead of containing a donut-shaped basket as in ATMI’s fully single-use, commercial-scale iCELLis 500, the nano system has a cylindrical fixed bed composed of the same polyethylene terephtalate microfibers. Linear scalability between the systems is given as the bed height is the same, explained Dr. Lipinski.

Thermo Scientific HyClone Single-Use Bioreactors shown in a process- development lab illustrating their modularity and scale-out potential.

Tracking Trends

Higher volumetric productivity is contributing to smaller scale manufacturing. In addition, noted Whitford, volume demand is decreasing, with emerging products such as personalized medicines with companion diagnostics targeted to screened populations, and cancer vaccines generally required in substantially smaller amounts.

Another trend described by Whitford is “scaling out” instead of scaling up, also known as distributed manufacturing. Instead of running one 10,000 L batch, a company might opt to run five 2,000 L reactors in parallel.

Among values inherent in this flexibility, this minimizes the potential loss if one batch were to become contaminated or unusable for some other reason. Single-use systems also simplify geographic distribution of manufacturing capacity. To duplicate a facility in another location would require only purchase of the same devices and transfer of the process, whether to an adjacent building or another country.

Manufacturers of single-use devices are pursuing R&D to create new, cleaner materials for single-use products and new ways of producing these materials. For example, Paul Killian, Ph.D., senior scientist at EMD Millipore, described research under way on new methods for sterilizing single-use materials that would reduce the amount of leachables created. At present, most single-use systems are gamma-irradiated, a process that generates small oxygenated compounds that contribute to the leachables load.

Raising the ceiling for “large-scale” processing in single-use bioreactors from 1,000 L vessels to the new generation of 2,000 L vessels has expanded the utility of single-use systems for commercial-scale batch production. Sartorius Stedim Biotech plans to introduce a 2,000 L scale single-use bioreactor in 2013. Davy De Wilde, director of marketing for fermentation technologies for the company does not anticipate any volume increase beyond that.

“Improved cell strains, media, and process conditions have led over the past years to a significant increase in product yields per volume,” he says. “This enables the industry today to reach their required product volumes already at 1,000 L or 2,000 L scale, while previously required bioreactor volumes were up to five times higher.”

This is helping to drive uptake of single-use systems overall, according to Alison Rees-Manley, fermentation application specialist at Sartorius Stedim Biotech.

Other factors contributing to the increase in adoption of single-use bioreactors, in De Wilde’s view, are reduced cost of ownership and increased flexibility, thus allowing users to switch more easily between processes and to increase capacity rapidly due to reduced lead times and utility requirements.

User needs are a critical driver of technology and product development, and in response Sartorius Stedim Biotech plans to introduce a point-of-use integrity test for single-use bioreactor bags, beginning with a test for its bioreactors up to the 200 L system by the end of 2012 and shortly followed by tests for bags up to 1,000 L.

Need for Standardization

The issues and uncertainty surrounding extractables and leachables from single-use systems remain an ongoing topic of discussion. “The risks change depending on where the material is used,” said Dr. Killian.

An overall lack of standardization and regulatory guidance continues to present an obstacle to more rapid adoption of single-use technology.

“The regulatory agencies still put the onus on drug companies to demonstrate that there is no or low risk to the patients,” continued Dr. Killian. With increased use, confidence in the materials is growing among biopharmaceuticals producers.

As the industry has matured, companies have used their experience to modify their approach to performing extractables and leachables studies and evaluating the data.

For example, whereas before companies might have carried out leachable studies across all single-use devices—an expensive and challenging task—now they might take an extractables-to-leachables approach in which they would perform leachable studies only on devices identified as high-risk, explained Dr. Killian.

Similarly, they may be able to limit the scope of the studies required by comparing the results of some initial tests to established standards and use these quick evaluations to define what areas require more extensive data collection and analysis.

Downstream Adoption Is Looking Up

Unlike for the single-use bag systems developed for upstream buffer preparation, mixing, and storage, and designed to replace conventional glass and stainless steel bioreactors and fermentors, when it comes to downstream processing systems, “single-use” may not mean intended for disposal after a single run.

“We like to call it ‘single-use, batch-dedicated,’ or ‘single-batch,’” explained Makowiecki.

“Prepacked does not mean single-use,” emphasized Paul Lynch, production manager for Life Technologies’ prepacked Poros® chromatography resins. The main advantage of prepacked columns is time savings, as they are ready to be dropped into a process without the need for validation.

“They save about 80% of the set-up time,” Lynch said, as well as the associated overhead of personnel needed to pack and validate the column.

At the June conference in San Francisco, Richard Garretson, business development manager at Life Technologies, led a workshop in which he described the advantages of the company’s GoPure™ columns prepacked with POROS chromatography resins.

Garretson compared prepacked columns to disposable membrane absorbers, which can be used in place of anion exchange chromatography, for flow-through polishing of monoclonal antibody preparations to remove DNA, viruses, and host-cell proteins. The high mass transfer capability of the POROS prepacked resin allows for high flow rates, short bed lengths, and small column sizes.

“The resin has the same sort of mass transfer profile as a membrane absorber,” said Garretson. Once users have selected the ideal column bed length for the separation they want to achieve, they can then increase or decrease the diameter of the column as desired for scale-up or scale-down, Garretson explained.

In contrast, scale-down—for process modeling, process characterization, and viral clearance studies, for example—is “problematic with filters,” he added, due to more limited availability of membrane sizes.

In terms of single-use capability, the prepacked chromatography resin, like a traditional self-packed column, can be re-used multiple times or replaced after a single use. It would typically be used for a single production campaign. In contrast, a functionalized filter is a consummable product intended to be disposed of after one use.

Producing relatively small drug batches or material for toxicology studies or clinical trials can mean switching product streams, and thus chromatography columns, more frequently. In this scenario, the use of prepacked columns can save time and money, contended Michael Killeen, business development manager, GE Healthcare.

Killeen’s presentation in San Francisco was entitled “‘Out of the Box’ Thinking for Process Chromatography.” The company’s ReadyToProcess™ prepacked columns can be used for multiple runs; however, the resins cannot be removed and repacked and are intended for use in a single campaign.

When combined with GE Healthcare’s ÄKTA™ ready disposable chromatography flow path, there is no need to clean or validate the system before use, pointed out Killeen. With flow rates up to 510 L/h, the systems are appropriate for pilot to small batch production.

A side-by-side analysis that compares the cost of processing one batch with a prepacked versus traditional column at pilot scale “can show significant savings,” added Killeen. “If you can get ten molecules produced instead of eight,” that is a good value proposition for the customer.”

ReadyToProcess™ columns are high-performance bioprocessing columns that come prepacked, prequalified, and presanitized, according to GE Healthcare. The ÄKTA™ ready is a liquid chromatography system that incorporates ready-to-use, disposable flow paths for process scale-up and production for early clinical phases.

Editor’s Note: As GEN went to press Eppendorf North America reported the release of the first single-use vessel to incorporate New Brunswick’s proprietary packed-bed impeller system. The New Brunswick CelliGen® BLU 5L comes pre-loaded with 150 g of Fibra-Cel® disks.

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