Increasingly users of traditional bioreactor and fermentor systems are looking to integrate disposable reactors and system components, or even make the switch to completely disposable systems in order to minimize the need for complex cleaning, sterilization, and validation procedures.
Disposable reactor bags, mixing and storage units, and the necessary connectors and valves needed to link these components are becoming standard fare in the industry.
Wave Biotech (www.wavebiotech.com) introduced its Wave Bioreactor, an inflated plastic bag that relies on wave-induced agitation for fluid mixing and oxygenation, in 1998 (see Technology Review on p. 74). Seven years later, disposable reactors and related accessories grace the catalogs, websites, and new-product announcements of suppliers better known for their steel tanks, spinner flasks, and glass benchtop reactors.
Though serving a limited (but rapidly growing) sector of the bioprocess market, disposables can no longer be considered niche products suitable only for small-scale projects.
Vijay Singh, Ph.D., founder of Wave Biotech, envisions the day when pipeless manufacturing facilities will be the norm. When a start-up company that needs to produce 10 g of protein for a clinical trial will simply convert a warehouse into a clean environment, roll in disposable culture equipment, and, in 1012 weeks, be able to operate a pilot-scale facility and produce GMP material.
At the other end of the spectrum, making tailored cell-based therapeutics for individual patients, versus large-scale manufacturing of one-size-fits-all therapeutics, Dr. Singh anticipates a sizeable potential market for disposable technology.
He talks of disposable reactors as medical devices in a "one person/one bioreactor" scenario in which cells are removed from a patient, expanded, and in some way treated or modified, and then returned to the patient.
Producing enough material to yield a therapeutic effect will require high density cell culture systems capable of producing hundreds, and perhaps even thousands or millions of cells, Dr. Singh predicts. He believes that stem cell-based therapies will also require greater numbers of cells than are currently thought to be needed.
Toward the end of this year, Wave Biotech plans to introduce a 2,000-L bag system with a working volume of 1,000 L. This larger reactor will operate on the company's existing mechanical rocking unit.
The latest product development at Wave is its FlexMixer 1,000-L, single-use mixing bag. It contains a perforated disk that moves into and out of the headspace to promote mixing. The company has also introduced reuseable fiber optic dissolved oxygen probes for online monitoring.
The DOOPT-PROBE can be inserted into the Oxywell2 port of Wave's Cellbags. Optical sensing as a means of noninvasively monitoring control parameters in disposable reactor bags is the wave of the future, and Dr. Singh sees it as a key growth business in coming years.
In their short history, disposable reactors have primarily targeted human and animal cell culture for GMP protein production.
But Dr. Singh is seeing a trend emerging in which disposables are spilling over into the fermentation arena for growing bacteria, yeast, and other fungi. Accompanying this shift, Dr. Singh sees growing interest in disposable reactor technology outside the biotechnology sector and, in particular, in the food and beverage industry.
A Renaissance in Yeast Fermentation
To take advantage of the beneficial growth characteristics of yeast, its protein expression potential, and the ease of growing yeast in large-scale cultures, much interest and research have been focused on this organism in recent years, and this effort is beginning to bear fruit.
Chris Julien, director of sales and marketing at Sartorius BBI Systems (www.sartorius-bbi-systems.com), points to the clinical trials currently under way involving human-like proteins produced in Pichia pastoris as evidence of the "tremendous progress that has been made."
The advantages of producing recombinant proteins in yeast include faster growth than mammalian cells, ability to grow on basal media supplemented with trace materials, and elimination of the need for costly serum and animal-derived growth factors, thereby reducing the extent of downstream purification needed and alleviating some regulatory issues.
Furthermore, with current technology, yeast can express proteins at concentrations of up to 15 g/L. This emerging potential for high density cultures is having a "significant impact on the fundamental design factors of reactors," says Julien.
Even as contract manufacturers were investing in new, larger-scale production facilities over the past few years to address a projected capacity crunch, the realization was emerging that at some point it would be more efficient and cost-effective to invest in research aimed at making more from lessproducing more product with the existing capacity.
This sparked a renewed focus on increasing fermentation and cell culture yields by improving recombinant strains, optimizing expression systems, and maximizing process output.
As a result, whereas expression levels of 1 g/L were the norm for monoclonal antibody production a few years ago, 5 g/L is now the expectation, and, according to Julien, "some believe expression levels of 10 g/L will be achievable in the next decade."
The emphasis in bioreactor/fermentor design has taken a 180 shift away from scale-up to large 10,00020,000 L systems, and is now focused on "scale-down," according to Julien. He describes scale-down as the desire to mimic eventual manufacturing-scale processes on benchtop units, doing process design and validation and optimizing process parameters at a smaller, 210 L scale, saving time, resources, and labor.
Customers have demonstrated that a well-defined small-scale system can make scale-up sufficiently predictable that, for example, it is possible to optimize a 12 L process and scale it directly to 600 L, without devoting the time and effort to take the process through a series of intermediate scale-up volumes.
"The segregation of duties has been challenged," says Julien, explaining that the lines between R&D, process development, and production are blurring. The goal is to modify processes in their infancy so they can run at manufacturing scale without significant changes and redesign.
The staff responsible for production is now reaching down into the process-development venue to share their expertise and collaborate on the selection of first-tier bioreactors and fermentors and on early-stage process design.
One result of this trend is increased demand for online sensing and more sophisticated monitoring and control strategies, requiring instrumentation and methods that have typically been limited to larger systems.
Julien cites frequent requests by customers for greater functionality on benchtop units, and, in particular, more advanced approaches to controlling dissolved oxygen.
Sartorius has responded to these market demands by integrating its controllers and bench-top systems with an increasing number of auxiliary sensors that enable online monitoring and control, to support the Process Analytical Technology (PAT) initiative, and generate the data needed to validate a system at small scale, simplifying validation and regulatory compliance at manufacturing scale.
Increasing adoption of PAT and demand by customers for greater integration of system components led Sartorius to introduce an OPC driver that enables its systems to communicate with any OPC-based instrument.
The needs of the research community are driving the trend toward downsizing, concurs Stephen Mitchell of ATR (www.atrbiotech.com). With few real differences in the design of the various bioreactor and fermentor units on the market today, innovation centers on the control components. Mitchell identifies one of the key challenges in bioprocess scale-down as the need to develop smaller-scale sensing devices, with the emphasis being on fiber optic sensing technology.
In late summer, ATR will launch the Twinfors benchtop reactor, a modified version of the company's Sixfors product. Whereas the Sixfors has six individual bioreactors in a single chassis, with each reactor vessel available in either a 300 mL or 500 mL maximum working volume, Twinfors offers the same small working volumes in a dual-vessel configuration.
Users can link up to three Twinfors using a single electronics package. ATR has also added a larger vessel option with a maximum working volume of 1 liter, widening the working volume range per reactor to 80 mL up to 1 L.
Depicting the current market as a battleground between traditional stainless steel tanks and disposables, with the latter being considered a replacement for the former, is a misrepresentation of the true market landscape, in the view of Julien. "Neither technology has all the answers," he states. "The true power is in the integration of the two."
Sartorius recently signed a worldwide distribution agreement with TC Tech (www.tc-tech.com), giving it access to the company's disposable mixing technology.
About six months ago Sartorius introduced its SuperSpinner incubator-based cell culture device with disposable membrane aeration.
The FibraStage cell culture device is New Brunswick Scientific's (NBS; www.nbsc.com) newest entry into the disposables market. This small-scale system can accommodate four 500-mL bottles, each containing 10 g of FibraCel support matrix. At the base of each bottle are collapsible bellows that "push media up through the packed bed of the FibraCel disk," as it is compressed, explains Mike Sattan, director of marketing at NBS.
The inoculate becomes entrapped in the support matrix as essential nutrients are forced through the culture and waste products are pushed out. When the bellows expand, air is pulled through the cell bed, facilitating oxygen transfer and gas exchange.
A controller allows for variable speed settings to control the up-and-down movement of the bellows and can be programmed to incorporate "hold times" for exposure of the cells to the media or gases.
A single, disposable FibraStage reactor bottle can produce the equivalent amount of product as 2040 roller bottles, according to Sattan. The cells remain in the disk bed, media replacement and product recovery can be achieved by decanting the media from the bottle. On the horizon, Sattan sees a need for perfusion-type disposable devices.
NBS has expanded its line of modular BioFlo Pro sterilizable-in-place systems to include fermentation vessels up to 3,000 L and has increased the maximum volume of its bioreactor vessels from 150 to 500 L.
Commenting on the increasing penetration of disposables into the bioprocess market, Mitchell points to ATR's ongoing pilot program in which the company is evaluating various forms of disposable reactors. "Bags are huge," he says. "They have working volume limitations, but are perfect for the research/small-scale side." The use of disposables can eliminate about "50% of the up-front and post-product clean-up," he adds.
Optical detection to monitor parameters such as dissolved oxygen, pH, and biomass will rely on fiber optics, predicts Mitchell, "because it is so inexpensive." Technological advances in sensing will enable optimization of processes in smaller-scale vessels.
Applikon Biotechnology (www. applikonbio.com) has linked up with Stedim (www.stedim.com), producer of the Flexboy disposable container systems, and will be launching a line of bioprocessing bags under the Applikon name toward the end of September.
First on the market will be a 20-L bag, followed by a 100-L container, together with a rocking platform that will accommodate either size reactor, according to Howard Weber, director of sales and marketing.
The AppliFlex system "will have a higher oxygen transfer rate and more sophisticated controllers than other disposable bag systems," says Weber. It will link to Applikon's ADI 1010 BioController.
Standard equipment developed for monitoring and control of bioprocess systems is becoming more sophisticated, in Weber's view, reducing the need for customization.
Applikon's BioConsole XL incorporates standardized bioreactor control software and is adaptable for use with a selection of off-the-shelf Programmable Logic Controllers (PLCs), including Allen Bradley and Siemens PLCs and Delta V's DCSs, as well as Applikon's proprietary PLC.
The BioConsole can control two bioreactors independently, working off a single touch screen. It requires no additional configuration for use with standard PLCs and enables scalable control parameters and validation capabilities for scale-up from R&D to manufacturing.
In late summer, Millipore (www.millipore.com) launched the Lynx sterile-to-sterile (S2S) connector, a single-use device for linking presterilized disposable fluid paths in bioprocess systems.
Roberta Landon, group product manager for disposable manufacturing at Millipore, describes this product and the company's broader Mobius line of disposables, including disposable assemblies, Opticap capsule filters, the Acerta dispensing system, and the Lynx ST connector, as not only a physical link between fluid paths, but also as representative of the company's role in providing solutions to bridge the gap between traditional stainless steel or glass reactors with fully integrated disposable bioprocess systems.
Landon envisions continued growth in the demand for disposable components that reduce cleaning requirements in existing bioprocessing facilities.
"Companies don't want to throw out their investment in existing equipment," she notes. They are gradually integrating disposables in areas such as media preparation, fermentation, reactor vessels, process monitoring, separations, clarification, and fill and finish, and are looking for disposable solutions to link these operations, with the ultimate goal being fully disposable plug-and-play units.
Staking a claim to the market for disposable spinner flasks, Corning Life Sciences' (www.corning.com/ lifesciences) 125-mL and 500-mL single-use flasks contain an ISO 10993 polystyrene vessel, shaft, and paddle with an integrated magnet. These units are intended for growth of suspension cell lines and attachment-dependent cultures using microcarrier beads.
The unit design minimizes heat build-up in the vessel through the use of a specially designed flange that raises the vessel off the surface of standard laboratory stir-plates.