June 15, 2006 (Vol. 26, No. 12)

New Components and Systems Improve Process Reliability and Reduce Cost

With the rise of biotherapeutic molecules, especially antibodies, the demand for innovations in bioreactor technology has become widespread. Early efforts to produce proteins expressed by bacteria, yeast, and mammalian cells in commercial quantities employed costly, re-useable hardware, requiring extensive cleaning and re-sterilization. This longstanding approach places heavy demands on standard operating procedures and inevitably entails high costs and extensive personnel training.

Disposable components and systems are increasingly favored, both for improved process reliability and for the economic advantages they offer. For this reason many biotech producers of protein molecules are moving to disposable modules, presterilized and qualified according to regulatory requirements. Disposable components are accessible at a moment&#8217s notice, and their ready availability makes it easy to change cell lines and target proteins in a production process quickly and inexpensively.

These and other advantages have been seized upon by the industry. According to Bettina Berendsen, business development manager at Sartorius(www.sartorius.com), “Benefits of flexible bag containers include faster facility set-up, reduction of down time, simplified validation, and more efficient use of plant floor space. Disposable bags greatly reduce the risk of cross contamination.”

Integrated Bioreactor Systems

The bioreactor has progressed immeasurably, as the early, noncomputerized models have evolved into smart descendants. Today&#8217s sophisticated technology allows microorganisms to be cultivated under controlled conditions so that their cells or metabolic products may be harvested for the manufacture of pharmaceuticals. Sartorius has developed bioreactors as disposable systems to meet the regulatory requirements of the biopharmaceutical industry. The company&#8217s goal is a vertical expansion of its product line, outfitting bioreactors with computer software, disposable components, and all the accoutrements necessary for a total, integrated system.

Sartorius signed a licensing deal with Fluorometrix (www.fluorometrix.com), a spin-off of the University of Maryland, Baltimore, for disposable bioreactor and mixing vessel sensors, another step in its quest for a fully integrated disposable process line.

Govind Rao, Ph.D., professor of chemical and biochemical engineering at the University of Maryland and co-founder of Fluorometrix, has developed oxygen, pH, and carbon dioxide sensors. The devices use patches immobilized with pH-, CO2- and O2-responsive dyes, and the signals are optically and noninvasively measured with commercially available LEDs and photo-detectors.

The control system consists of the patch, a coaster, an interface, and a computer. The patch, a hydrophobic silicone membrane containing dyes, utilizes a hydrogel. The patches are monitored by the coaster, which is a miniature fluorimeter, able to measure either excitation ratio for CO2 and pH or fluorescence lifetime for O2. This device, which measures the excitation ratio of the patch, is positioned outside the bioreactor and does not need to be sterilized. The patch assembly is sterilizable and ready to use.

The sensors are inexpensive thus they can be incorporated into disposable components of the bioreactors. They are precalibrated and do not require individual calibration. Low-cost sensors are essential for the standardization process when new products are in development, since many runs may be required. Such sensor technology will be useful for the FDA&#8217s Process Analytical Technologies initiative as well.

Reinhard Vogt, senior vp of sales and marketing for the Sartorius Biotechnology Division, calls the new licensing contract an important milestone on the path ultimately leading to disposable solutions for the entire production process. “Disposable sensor technology is the backbone of any disposable process. Without disposable sensor technology, the existing status quo remains, which means either cleaning in-place, discarding expensive re-usable sensors, or avoiding sensors, which makes the process uncontrollable,” says Vogt.

Florian Wurm, Ph.D., of the Swiss Federal Institute of Technology in Lausanne, working in collaboration with TPP (www.tpp.ch), developed specially designed, 50-mL tubes to be used as minibioreactors. “The TubeSpin reactor system is in our hands the closest scale-down system to the real bioreactor,” he states. “Using tubes with semi-permeable caps for CO2 exchange we were able to achieve high throughput and mimic both batch and fed-batch processes.”

Both TPP and Sartorius sell the TubeSpin bioreactors. The Tube Spin technology is based on orbital shaking, which will be the main principle for new larger volume bioreactors (1-100 L) under development with Sartorius.

Advanced Mixing Technology

LevTech (www.levtech.net) is already a Sartorius partner with a disposable mixing option. Its DB-200 series Disposable Mixing System, which uses superconductive magnetic technology to levitate and drive a disposable impeller inside a single-use bag, has a broad installed base in the biopharmaceutical industry. Unlike other mixing technologies, there are no shafts, seals, or bearings, and there is no friction, particle shedding, or stress on the flexible bags. The advantages to the end-user are scalability and reduced contamination, operating costs, and downtime associated with cleaning and validation of conventional mixing systems.

According to Jeffery Craig, Ph.D., chairman and CBO, the LevTech drive unit utilizes superconducting material that when placed in a permanent magnetic field and cooled below 77K will couple with that field and retain the permanent magnets&#8217 position. This allows the drive unit to levitate the impeller off the bottom of the bag and transmit scalable, rotational torque.

The LevTech drive unit delivers sufficient torque for efficient mixing and includes a variable speed controller allowing the impeller to be run at the desired speed. LevTech impellers are available in multiple configurations. A single drive unit can service multiple tanks at variable sizes and with an infinite number of bag designs. The splash-proof drive unit can accommodate vessel change in under seven minutes.

“The adoption of levitated mixing applications was driven by our customers,” reports Dr. Craig. “We determined early that our mixture performance is efficient on the pilot scale. Our subsequent development has focused on simple designs, already proven in established stainless steel mixing vessels.”

Disposable Bag Technology

A number of companies, including Applikon (www.applikonbio.com), obtain their bag systems from Stedim (www.stedim.com), which specializes in single-use plastic bags and does not produce integrated systems. “Our strength is in plastics, not electronics, so we function primarily as a supplier,” explains Bob Smith McCollum, director of marketing.

However, Stedim&#8217s newest product is a single-use, impeller-based mixing system, based on the Flexel 3-D bag system, which includes, for the first time in the company&#8217s history, an electro-mechanical component. “The shaft and impeller are reusable and can be cleaned and sanitized using standard chemicals and procedures,” McCollum adds. “With various size options, up to 500 L can be accommodated.”

Another player in the disposables market is SAFC Biosciences (www.safcbiosciences.com), which manufactures Bioeaze disposable bags and disposable bioprocessing systems. According to Denise DeTommaso, marketing manager biodisposables.

“We provide standard and application-specific systems, including bag assemblies, rigging sets, bioreactor kits, containers, and other components, DeTommaso states. The bags, riggings, and other components are available separately, or assembled as total systems. “We can engineer custom systems according to the client&#8217s specific design.”

HyClone (www.hyclone.com) also markets a line of single-use BPC products, including single-use bioreactors and single-use mixers. Its single-use bioreactor is available in 50-L and 250-L formats, and a 1,000-L unit will come on line later this year. All hardware and control panels are available, and other components are disposable, including the sparger systems, filters, impellers, and bearing assemblies.

Specially designed probe-insertion kits allow pH and dissolved oxygen probes to be reused at the client&#8217s discretion. “The single-use bioreactor has been designed and developed utilizing traditional bioreactor components in a single-use format. This facilitates customer conversion from traditional stirred tank bioreactors, while maintaining ease-of-use conveniences and product drivers employed by today&#8217s single-use technologies,” explains Justin Hutchinson, BPC market development manager for HyClone.

HyClone also markets multiple mixing technologies, including the Mixtainer. HyClone will be introducing additional single-use mixing products later this summer, based on the same stirred tank mixing technologies as the single-use bioreactor. These mixing products will also be scalable to 1,000-L volumes and beyond.

Cell Culturing Device

Metabios (www.metabios.com) developed a disposable cell culturing device, Optima-mini, for growing mammalian, insect, fungal, and plant cells. According to general manager Hong Zhu, Ph.D., “We target our products at the academic market. Our concept is a simple, economical, and flexible product that will handle up to 10 L of volume.”

With a 500-mL working volume, the standard Optima-mini model is designed to fit most laboratory shakers and rocking platforms for suspension, perfusion, and microcarrier culture. Disposable bags are convenient and usually require no specialized equipment. The gentle wave motion induced by agitation provides mixing and oxygenation for cell growth.

The Optima-mini product line also offers greater flexibility in gas transfer between the bag headspace and the environment and it is capable of both gas diffusion and continuous gas flush. Gas diffusion through the built-in, microporous membrane on the screw-cap provides sufficient gas exchange for most cell culture needs. If required, pressurized air or gas can be added through one of the Luer ports and vented out through the membrane cap.

The company also offers a line of single-use bioreactors known as BelloCell, developed and manufactured by Cesco Bioengineering (www.cescobio.com). This cell culture device features an artificial lung design, capable of supporting high cell density growth in a low shear stress, high aeration rate, and foam-free culture environment. The device grows immobilized cells in porous matrices, which are packed in the culture chamber. Culture media are located in the lower chamber with compressible bellows, which raise and lower the medium, alternatively, to submerge and expose the matrices.

During the exposing phase, cells immobilized in the porous matrices are uncovered to the gaseous environment through a thin liquid film as shown in the diagrams. During the submerging phase, cells are bathed in the new liquid surface for uptake of nutrients and removal of metabolic wastes from the cells.

&#37The matrices are particularly useful, since they greatly increase the effective surface area for cell growth,&#37 says Dr. Hong. &#37In fact, one gram of matrix material has the equivalent surface area for cell growth of 4,500 cm2.&#37

Changing Industry Mindset

Vijay Singh, Ph.D., president of Wave Biotech LLC (www.wavebiotech.com), developed disposable bioreactor and mixing components in the late 90s. Dr. Singh describes his company as a research-based operation, focused on moving the industry mindset from stainless steel process to single-use disposable systems. Its best-known products are a series of disposable bioreactor bags, going from a few mLs to 1,000 L.

The System 200 is primarily suited to pilot- and production-scale applications, while System 20/50 units are employed in research, development, and small-scale production.

The Wave Bioreactor System 1000 is used for commercial-scale operations in cGMP environments for up to 500-L culture volume. Batch, fed batch, and perfusion operations of all cell types may be performed in a presterilized disposable Wavebag.

Wave Biotech LLC has moved forward, developing disposable mixing and aseptic fluid transfer devices, such as the Wave Mixer, Sterile Tube Fuser, and Hot Lips Tube Sealer devices. According to Dr. Singh,”It is our view that we have taken disposable bioreactors from a laboratory curiosity to mainstream GMP manufacturing of biologics.”

Wave Biotech AG(

href=”http://www.wavebiotech.net”>www.wavebiotech.net), which was originally part of Wave Biotech LLC and was formed to service Switzerland and several other European countries, became an independent company in 2004, although Dr. Singh still owns 20% of Wave Biotech AG. The firm has also dealt with the problem of sterile fluid transfer from bag to bag or from the bag to conventional reactors and media tanks in a closed system without the use of laminar flow. The company&#8217s ReeWelder 3 and Reesealer can be used for sterile welding of thermoplastic tubing. Interchangeable tube holders make it possible to change the tube diameters to be welded or sealed.

Recently, Wave Biotech AG and Sartorius inked a partnership. Marcel Roell, CEO of Wave Biotech AG, says, “We will work jointly with Sartorius to develop disposable bioreactors incorporating disposable sensors, sterile connectors, and mixing and control technologies.”

Last month, ATMI Life Sciences (www.atmi-lifesciences) and Pierre Guerin-SAS (www.pierreguerin.com) formed an alliance to design, manufacture, and market disposable fermentors and bioreactors. ATMI Life Sciences brings its ultraclean film manufacturing expertise and single-use mixing technology to the table, while Pierre Guerin will contribute years of experience in the design and manufacture of fermentors and bioreactors, related control equipment, and software to the ATMI deal.

“Our initial focus has been to establish our scalable single-use mixing technology,&#37 explains Mario Philips, ATMI&#8217s product line managing director. “Now, in our alliance with Pierre Guerin, we are extending our mixing technology platform to fermentation and cell culture applications.”

“Biotechnology companies are searching for comprehensive validation documentation and strong aftermarket service,” adds Doru Felezeu, sales and marketing director of Pierre Guerin-Biolafitte. “We&#8217ll be able to provide customers customized, as well as off-the-shelf solutions.”

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