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Feature Articles : Aug 1, 2009 (Vol. 29, No. 14)

Bioreactor and Fermentor Market Trends

Vaccine and Biopharmaceutical Product Developments Boost Technological Innovation
  • Vicki Glaser

Despite the struggling economy, “the bioreactor/cell culture market remains relatively strong,” claims Mike Sattan, vp of marketing at New Brunswick Scientific (NBS), an Eppendorf company.

The substantial uptick in interest in mammalian cell culture technology is largely being attributed to renewed interest in vaccine development and production, dating back to the events of 9/11. Additional market drivers include the emergence of infectious agents such as West Nile virus and other pathogens affecting humans, pets, and livestock, and most recently growing concerns surrounding pandemic influenza. The vaccine industry is rapidly trying to move away from traditional egg-based production to more rapid, flexible, and cost-effective cell-based techniques.

Sattan notes a rigorous trend toward single-use systems, and NBS will enter that increasingly competitive market in the fourth quarter of this year with a small-scale, 1–10 L working volume bioreactor designed for mammalian cell culture. It will feature a rigid plastic stirred tank vessel intended to mimic a traditional stirred tank reactor (STR).

The product launch will include 5 L and 14 L single-use vessels with a preinstalled fluorescent disposable pH sensor and traditional dissolved oxygen probe that utilize a membrane technology for online sensing without the need for aseptic connections. The full feedback control system can sparge in four gases or activate pumps for acid/base addition to control pH.

There is also a demand for the company’s packed-bed FibraCel disks, according to Sattan, particularly among generic drug manufacturers looking for higher yields than can be achieved with traditional roller bottle technology. The disks can be used in the company’s CelliGen® 310, CelliGen 510, and BioFlo® Pro bioreactors and the FibraStage disposable cell culture system.

NBS recently redesigned its small-scale, glass autoclavable bioreactor and introduced the BioFlo/CelliGen 115 benchtop system, which features a 1.3–10 L working volume, a touchscreen controller, enhanced control functions, and interchangeable vessels. During the third quarter, NBS will launch the BioFlo 610, a mobile, compact, pilot-scale fermentation system with a total volume of 65–125 L.

Expanded Portfolio

At “ACHEMA,” Sartorius Stedim Biotech announced an expansion of its single-use bioreactor portfolio, which currently features the BIOSTAT® CultiBag STR 200 stirred tank disposable system and the BIOSTAT CultiBag RM product line, which is based on a rocking motion for mixing. The company’s two new systems will include a rotationally oscillating bioreactor and an orbital shake bioreactor.

The oscillating bioreactor is being developed in collaboration with Bayer Technology Services and will initially be designed for mammalian cell culture. ExcellGene is the partner for development of the orbital shake bioreactor, which will range in volume from 200–1,000 L and provides a more gentle mixing method appropriate for cells sensitive to the shear forces that can be generated by stirred tank technologies.

Sartorius Stedim is also expanding the CultiBag STR family with the addition of  50 L and a 1,000 L bag volumes by the end of 2009, according to Maik Jornitz, group vp marketing. The STR stirrer is based on the centered agitator design found in tradiational reusable systems. In addition, the company introduced its membrane technology into CultiBag RM systems for fermentation in perfusion mode.

Additional automation, more elegant sensing and control strategies, and a renaissance of perfusion approaches that rely on single-use technologies are the main trends reported by Daniella Kranjac, vp of sales and marketing at GE Life Sciences. Accordingly, GE is incorporating its ReadyMate genderless, aseptic connectors into the disposable Cellbags™ used in the company’s rocking motion Wave Bioreactor™ systems. The sterile connectors are also being used upstream in media preparation.

Mixing It Up

“Bioprocess is all about mixing and mass transfer,” says Hans van den Berg, director of business development at CELLution Biotech. The first-generation single-use bioreactors relied on a rocking motion to mix the contents of a pillow-shaped bag. They represented a major advance and opened the door to the development of a disposable bioprocess workflow.

However, claims van den Berg, “with wave-type bioreactors the mixing is poor and the mass transfer limited. With stirred (agitated) single-use bioreactors, in theory, it is possible to improve this dramatically. However the properties of plastics are such that a good, reliable, and robust agitator still cannot be introduced.”
Available stirred single-use bioreactors still face some problems, in van den Berg’s view: “sterility (dynamic seals) and limited mass transfer (due to a plastic agitator sealing).”

CELLution patented the CELL-tainer® disposable bioreactor, which combines horizontal and vertical movement to yield a 70-fold increase in mass transfer, according to van den Berg. Compared to a mass transfer coefficient (kLa) of <15/h for a wave-type reactor, and about 30/h for a typical stirred tank reactor, the CELL-tainer can achieve a kLa <200/h and with oxygen up to 700/h in a pillow-shaped bag at a working volume of 10 L, van den Berg says.

With a higher kLa the reactor “can support higher cell densities; the higher mass transfer rate also reduces CO2 buildup,” which increases the cell density potential and productivity of the process, he adds. In a recent poster, CELLution demonstrated growth of PER.C6® cells up to densities as high as 20–25 x 106 cells/mL in fed-batch mode with minimal process control, making it possible to produce 60–80 g of IgG per batch in a 10 L single-use bioreactor.

The CELL-tainer can also support microbial fermentation, and the company reports rising interest in the system for process development and inoculants preparation for fermentation applications.

To allow for low-volume operations, the company mounted the disposable sensors for pH and dissolved oxygen in the bottom of the bag. The bioreactor bag can be clamped, dividing it into three compartments to allow for starting volumes (inoculants) as low as 150 mL. When sufficient cell density is achieved, the clamps can be removed, facilitating scale-up in the same bag from 150 mL to 15 L. “The microenvironment remains the same,” says van den Berg.

CELLution is developing new online sensors and plans to introduce a glucose and lactate sensor during the fourth quarter of 2009. “New technologies are needed to measure viable biomass,” and van den Berg reports that the company is exploring the use of near-infrared sensing devices under single-use conditions.

Microbial Culture Market

Until recently, Xcellerex focused its capabilities on single-use mammalian cell culture technology, but the company has expanded into the microbial culture market with the launch of a 30 L fermentor; a 200 L fermentor is in the prototype stage of development.

In addition to strong interest in the new small-scale, disposable fermentor, the company reports growing demand for its mammalian cell systems, according to Geoff Hodge, vp of process technology and development. Xcellerex recently introduced 2,000 L and 50 L units, rounding out the range of its bioreactor product line.

Xcellerex developed its biomolecule manufacturing platform based on the modular FlexFactor™ systems and XDR single-use bioreactors. The company recently sold a FlexFactory production line to Crucell for use in its manufacturing facility in Leiden, The Netherlands, for vaccine and monoclonal antibody production.

According to Andrew Bulpin, Ph.D., vp for upstream processing at Millipore, “single-use stirred tank bioreactors have been gaining favor for pilot- up to commercial-scale production in volumes ranging from 50–2,000 L. Disposable technologies eliminate sterilize-in-place/clean-in-place steps, offer multipurpose and process flexibility, and enable streamlined validation and reductions in overall turnaround times. “Single-use STRs can be used within a fully disposable or hybrid disposable-reusable production scheme and are ideally suited for mid-scale (1,000–2,000 L) cGMP manufacturing,” adds Dr. Bulpin.

Millipore developed the Mobius® CellReady 3L stirred-tank bioreactor, which was launched at “ESACT,” in partnership with Applikon Biotechnology. Designed for cell culture applications, the unit operates with existing Applikon motors and controllers.

All parts of the rigid 1/4-inch thick reactor vessels, including the plastic head plate, are disposable. Larger size vessels will follow, according to Dr. Bulpin. The units are delivered preassembled and gamma irradiated and contain multiple fluid addition ports above and below the liquid level and an integrated harvest line at the bottom of the vessel to ensure sterile transfer and full removal of contents.

Applikon’s bioprocessing product line, which includes the AppliFlex line of single-use bioreactors, is available with 10 L, 20 L, or 50 L bags, and monitors pH, temperature, and dissolved oxygen using a choice of disposable or reuseable sensors. In the first quarter of 2010, the company will introduce a new form of shaker that uses acoustic energy to achieve high oxygen transfer rates and enhanced expression levels. For the biofuels market, Applikon recently introduced a six-pack, 500 mL fermentation system designed for feedstock selection applications.

When Smaller Is Better

To accelerate product development, drug companies are scaling down early-stage bioprocesses to analyze and better define a range of parameters and to optimize media preparations and process conditions. Miniaturization represents a strong trend in the bioprocess industry today as companies look for innovative ways to save time, labor, and resources.

In the area of small-scale systems, Applikon offers the MicroFlask which, company officials say, can replace orbital shake flasks for the rapid growth of microbial strains, clonal libraries, and cell lines in 24- or 96-well plates. The firm’s Micro 24 bioreactor system can perform high-throughput fermentation or cell culture in 7 mL volumes with automated, independent monitoring and control of 24 individual microreactors on one microtiter plate, explains Howard Weber, director of sales and marketing.

Frank Kensy, managing director at m2p-labs reports strong demand from industry for miniaturized fermentation systems for clone screening and media development. The company’s BioLector system can perform 48 parallel fermentation runs at a scale of 300 to 1,500 µL, with online optical monitoring of biomass, pH, and dissolved oxygen, and the ability to detect fluorescent proteins. Being able to measure labeled biomolecules allows users to “study protein expression, characterize promoters, and study regulation by culturing different knock-outs on different substrates,” says Kensy. The benchtop system can be integrated with automated liquid-handling platforms or operated manually.

Kensy also reports interest from the biofuels sector, which is using the BioLector to study the effects of different gases in the bioreactor chamber, to develop anaerobic processes, to screen clones for desired metabolic pathways, and to study various carbon sources to identify less costly media preparations.

Last year, m2p released the Flowerplate microbioreactor platform for microbial cell culture, which features flower-shaped well geometry to improve mixing and gas/liquid mass transfer. The flower-shaped wells function akin to baffles in shaker flasks to disrupt the liquid stream and increase gas exchange during orbital shaking. The company reports achieving an oxygen transfer rate of 0.2 mol/L/h. The Flowerplate has 48 wells to enable parallel batch fermentation, with filling volumes of 0.3–0.5 mL.

Dasgip introduced a new edition of control software for the Dasgip Parallel Bioreactor System, the Dasgip Control 4.0 OPC Edition, which supports the openness, productivity, and collaboration (OPC) standards for network-wide exchange of real-time data across third-party hardware platforms.

The Dasgip control system was designed for use with glass bioreactors from 50 mL to 5 L and supporting the Thermo Scientific Hyclone S.U.B. for scale-up features mass flow controlled gas mixing, up to six liquid feeds per reactor, overpressure detection and gas flow cutoff, and integration with Hyclone temperature and agitation control.

Falk Schneider, Ph.D., director of software engineering at Dasgip, identifies two key trends that his company’s technology is addressing: miniaturization and parallelization, to allow for scaled-down applications such as cell line screening, media optimization, and stem cell applications. The goal is to minimize process volumes, maximize the number of runs in the shortest timeframe, while still generating ample process information.

The trend toward OPC control of the bioreactor system is driven by the FDA’s PAT initiative, and control technology, including online and at-line autosamplers, sensors, analytical devices, and integrated control software, is rapidly moving into smaller scale bioreactor systems.

A critical gap “in many installations exists between the bioreactor data and the sampling data,” says Dr. Schneider. “We need to integrate analyzer values into process controllers in real time.” Automated process monitoring is not enough; it is important “to close the feedback loop for nutrients as an example,” he adds.

HexaScreen Culture Technologies, a spin-off from Universitat Autònoma de Barcelona, Universitat Politècnica de Catalunya and Fundació ASCAMM, plans to introduce its first commercial bioreactor for mammalian cell culture in January. The company’s patented technology mimics traditional T-flask cultures in single-use plastic plates, each housing six individual minibioreactors, combined with noninvasive temperature, pH, and optical density control.

“The 10 to 15 mL working volumes are small enough to save a lot of preparation time and money, but are still sufficient to collect samples for quality assurance measurements, as well as obtaining reliable data for bioprocess optimization,” says Francesc Gòdia, a founder of the company and professor of chemical engineering at the University. He describes the minibioreactors as homogeneous systems in which the internal liquid gradients and microenvironments that can form in microwell plates are avoided.

Gòdia believes customers will find the parallel bioreactor system especially useful for replicate experiments, screening of cell lines, scale-down studies, media development, and toxicity testing. HexaScreen anticipates introducing a second model capable of performing automated fed-batch fermentation and pH control in early 2011.

Flu Vaccine Drives Demand

Customer feedback to Thermo Fisher Scientific points to a “migration to modular production systems” that can be up and running quickly, moved from site to site, and give facilities multipurpose manufacturing capabilities, according to Brandon Pence, associate director of market management for Thermo Scientific cell culture and bioprocessing. Thermo’s single-use customer base is focusing on the 500–2,000 L scale, with 1,000 L being “the sweetspot right now,” says Pence.

For pandemic flu vaccine production, for example, “we are working with companies responding to H1N1, and they are asking for process development and optimization support in single-use bioreactors” for seed train development. They want speed and ease of use “without compromising protocols. Instead of purchasing one 10,000 L system and having centralized production at one site, they want ten 1,000 L reactors that they can move to regional sites where there is need.”

Ideally, companies would like a complete single-use process workflow, including upstream mixing of liquids, operation of the bioreactor, and harvesting of product, asserts Pence. At the core of Thermo’s disposable bioprocess product line is the stirred tank S.U.B., available in 25 L to 1,000 L working volumes, with a 2,000 vessel in development.

The process development market, at scales of 50–250 L also remains strong, according to Pence. The company’s application center works with customers to develop and optimize processes and to customize media for a client cell lines.

ATMI LifeSciences plans to double its global production capacity of disposable bioprocess vessels for life science applications by upgrading and modifying its manufacturing facility in Bloomington, MN, and continuing production at its plant in Brussels. ATMI’s expertise is focused on single-use mixing, storage, and bioreactor technology.

Last year the company acquired LevTech, with its Newmix product line of scalable, single-use mixing systems for benchtop to industrial-scale applications. Subsequently, ATMI granted exclusive distribution rights to the LevTech mixing technology to Sartorius Stedim Biotech for use in its disposable bag mixing systems.

Last December, ATMI purchased assets related to the Nucleo™ single-use bioreactor and Jet-Drive™ disposable mixing system from Artelis. Artelis and ATMI, in partnership with Pierre Guerin Technologies, jointly developed the Nucleo™ line of disposable bioreactors.

“Artelis is focused on process intensification,” says Nicolas Havelange, business development director. He defines process intensification as scaled-down manufacturing with increased cell density and fewer unit operations. The company is developing a perfusion-based bioreactor called the iCELLis™, in which either adherent or suspension cells can be cultivated on a matrix that allows them to grow to densities of 108 up to 2x108 cells/mL.

Havelange anticipates particular interest from the viral vaccine and cell therapy markets and reports that Artelis has a contract with GlaxoSmithKline Biologicals for vaccine production. iCELLis single-use vessels come presterilized and prepacked with matrix ready-to-use. “We have also developed our own process controller for the system, says Havelange.

The company’s mixing technology relies on a centrifugal pump driven by a magnetic motor. The falling film allows efficient gas transfer for oxygenation. A 25 L fixed-bed iCELLis bioreactor “is equivalent to a 20–30-fold increased volume in a traditional stirred tank reactor, or a 500–800 L working volume,” says Havelange.

Artelis has a co-development agreement with Cardio3 BioSciences, a company developing regenerative therapies for treating heart failure, to build a fully enclosed GMP system for producing autologous mesenchymal stem cells.

“We believe there is a strong need for process technologies and bioprocess intensification in stem cell production,” adds Havelange.

Pierre Guerin Technologies’ single-use Nucleo bioreactors and fermentors can accommodate disposable bags ranging in scale from 25 L to 500 L working volume, with a 1,000 L capacity bioreactor due on the market during the third quarter of this year and a 1,000 L scale fermentor expected toward the end of 2009 or in early 2010. The systems are compatible with industry-standard PLC controllers and supervisory platforms, according to Doro Felezeu, director of marketing and business development at Pierre Guerin.

Felezeu anticipates growing interest in the production of high density cell cultures, or “cell concentrates,” for use in feeding bioreactors and for cell therapy applications. “Imagine the advantages if you could get the same results with a 100 L bioreactor versus a 1,000 L bioreactor,” says Felezeu. Very high density cell growth would require optimal process conditions and new types of sensors to allow for accurate control of a broader range of parameters.

Scale to Meet Demand

Felezeu views newer disposable and traditional CIP/SIP bioprocess systems as being complementary technologies, with disposables meeting market needs up to 1,000 L scale and traditional systems taking over from there.

The company has longstanding expertise in the development of stainless steel, modular, customized industrial-scale bioprocess systems that are assembled on-site. It also offers a line of laboratory-scale bioreactor systems with autoclavable glass vessels. At its manufacturing facilities in France and the U.S., Pierre Guerin offers customized skid design services and pressure vessels ranging from 5–25,000 L for bioreactor systems and up to 30,000 L for microbial applications.

Bioengineering’s product offerings range from lab- to pilot- to production- scale fermentors and bioreactors and include autoclavable models and traditional CIP/SIP stainless steel tank systems.

“All Bioengineering bioreactors—from laboratory to production scale—are of modular design; every newly developed component is designed as a module and can be seamlessly installed in established bioreactors,” explains Karin Koller, Ph.D., a scientific associate at the company.

Noting that single-use systems have limitations in terms of process control, the possibility of leaching of extractables, mass and heat transfer on scale-up, and the production of waste materials, Dr. Koller contends that, “more expenditure in capital costs, validation and automation for steel fermentors signifies more flexibility for process development and more safety in the production process.

The Medicel Explorer is a parallel cell culture reactor system that features 15 bioreactors, automated sample withdrawal, and control software. The system contains a cultivation unit that houses 15 independently controlled 200 mL or 500 mL bioreactors, with monitoring and individual control of temperature, pH, and dissolved oxygen, as well as agitation, aeration, and feeding.

Optional features include gas mixing for three additional gases, a liquid pulse module for rapid feeding and perturbation, gas analysis capabilities, in-line HPLC, and an in-line microscope to perform cell count and viability analyses.

Infors HT’s Multifors line of benchtop reactors features a modular design to allow for bacterial fermentation at working volumes of 500 mL or 1,000 mL and for cell culture at working volumes of 500 mL and 700 mL.

The company’s Minifors system combines a shaker flask culture system with probes for process monitoring, and automatically performs data-logging, graphing, and report generation. The Labfors 4 system contains four pumps for independent, computer-controlled operation of multiple bioreactors.