Demand Strong for Upstream Systems

Process Monitoring and Disposables Are Priorities for Bioreactors and Fermentors

The bioreactor market “has been and continues to be very robust, and demand is certainly up,” says Paul Kubera, vp of engineering at ABEC who, together with Mark Herr, sales manager at Stainless Technology, a division of ABEC, will present workshops on reactor design at the upcoming “ASME” bioprocess technology seminars in October.

For large-volume production of commercial biopharmaceuticals, the demand continues to focus on large, stainless steel, custom-designed, stirred-tank projects in the 20,000-L to 25,000-L range. Customization requirements tend to focus on fitting the reactor system into a defined facility space and design and integrating the system with ancillary equipment obtained from a customer’s preferred suppliers.

Kubera notes that in addition to demands for online sensing to allow for measurement of temperature, pH, pressure, and dissolved oxygen in the tank, some customers request CO2 and cell density monitoring capabilities and analysis of off-gas.

Competition from the disposables market comes into play “at the lower end of the production train”—in the 15-L to 125-L range—“for seeding stirred stainless steel reactors,” says Kubera. For example, Herr describes current projects involving a four-reactor train, in which the two smallest reactors used to produce cell mass to seed the production units are single-use vessels, whereas the larger reactors used to manufacture product are steel tanks.

At commercial volumes, “the economics of disposables starts to melt down,” says Dick Bonyun, vp of sales and service at ABEC.

Biofuel Production

Mike Sattan, marketing director at New Brunswick Scientific (NBS), describes a strong and growing interest in fermentation systems for biofuels production such as ethanol from a variety of agricultural source materials. This work is now largely in pilot scale, with demand for sterizile-in-place (SIP) reactor systems with 150-L to 500-L working volumes.

“A lot of venture capital dollars and government funding are going into bio-fuels development,” says Sattan, with research efforts focusing on identifying novel and more efficient manufacturing processes.”

In the cell culture arena, the vaccine sector remains particularly robust, with a strong worldwide market for stainless steel SIP systems. Interest in NBS’s cell lift impellar technology is especially strong, particularly in Asian markets, notes Sattan.

Kubera also describes strong growth in the vaccine sector. Among the several large projects ABEC has recently embarked on, including vaccine projects, “we are now seeing an aggressive push for time to market. People have talked about it for years, but often the practice hasn’t met the preaching.” However, over the past 18 months or so, timeline demands for equipment validation, process validation, and system operation—based on time to production of product—have become more stringent.

The emphasis on reducing time to market is driving a trend toward shorter lead times. “In general, lead times for delivery of equipment, from approved drawings to delivery to customer, have been reduced by 30% over the last year,” says Kubera.

Optimizing Process Parameters

NBS recently increased the capacity of its large-scale BioFlo® Pro cell culture bioreactors, previously available in a 75-L to 300-L range, up to 650-L total volume. The company also expanded the functionality of the BioFlo Pro bioreactor and fermentor line, incorporating an Allen Bradley process controller manufactured by Rockwell Automation. On the software side, the company upgraded its BioCommand® supervisory control system to include an OPC server that allows for integration of NBS fermentors and bioreactors with a variety of analyzers and other auxillary equipment. “Customers want more information about their cell culture process in real time” to guide decision making in process optimization and scale-up, says Sattan.

Another recent introduction to the NBS product family is the BioFlo 415, a cGMP-compliant, 7–19.5-L benchtop fermentor with a touchscreen controller that allows for control of up to 32 process loops and regulation of one to four gasses. It is capable of automatic SIP without the need for an external steam supply.

“As the industry matures, we are seeing an increasing focus on productivity and cost. People are paying much closer attention to the bottom line,” Kubera says. That translates into a demand for greater efficiency and predictability, using process modeling at small scale, data acquisition and analysis, and Design of Experiment strategies to optimize process parameters, model scale-up, increase reproducibility, and support regulatory findings through clinical trials and the path to market.

Clients are doing more experimental work and are looking to work more closely with equipment manufacturers to model and optimize processes and predict the outcome of scale-up. The emerging objective is “don’t run to recipe, run to results,” says Kubera. This reflects a desire within the bioprocessing sector to understand and define process parameters, modeling processes at small scale, and even microscale, and evaluating growth environments, media options, feed strategies, metabolic characteristics, cell viability, and protein production.

Capitalizing on this growth area, Applikon Biotechnology introduced two new microreactor products. The MicroFlask, available in a 24-well or 96-well disposable format, yields results compatible with standard shake flasks and can be mounted on standard orbital shakers (2-inch stroke provides best results), according to Howard Weber, director of sales and marketing at Applikon.

The company’s Micro-24 bioreactor incorporates a disposable 24-well cassette with built-in sensors and an integrated laptop computer and software package. It allows for individual monitoring and control of pH, dissolved oxygen, and temperature, with agitation (0–800 rpm) used as a single set-point across the plate. The cassette essentially operates as “24 individual 10-mL bioreactors and is ideal for media screening, clone selection, and modeling of processes,” adds Weber.

The newly released ez-Controller, an upgrade to Applikon’s 1010 laboratory scale control unit, offers a color touchscreen interface and capability to monitor pH, temperature, dissolved oxygen, foam/level, and agitation. Built-in expansion capability provides for rotometers, four thermal mass flow controllers, fixed speed pumps, and an AD/DA card that allows users to interface with up to 16 analog devices.

Single-use Technology Matures

“I think we will start to see multimode, or hybrid, single-use bioreactors that use multiple actions, particularly for mammalian cell culture,” says Kevin Auton, Ph.D., chief executive of Cellexus Biosystems. This reflects a change from a one-size-fits-all mentality. Combination modes may incorporate two different mixing strategies or combine perfusion with mixing, for example.

In May, Cellexus launched the CellMaker Lite2, a range of bioreactors that rely on aeration to mix the contents of the company’s disposable Cellexus Bag. “The reactor’s unusual shape allows for mixing with minimum energy input,” says Auton. Mammalian cells, and especially CHO cells, prefer low oxygen levels when growing at low concentrations, Auton explains. The geometry of the CellMaker Lite vessel, compared to a cylindrical reactor, allows for “slow stirring, with aeration purely from gas exchange into the headspace.”

As cell concentrations increase and higher oxygen levels are needed, the company’s CellMaker Hybrid system, introduced in July and having both airlift and stirred tank mixing technology, can switch to a mixed mode, combining airlift and stirred mixing in the HybridBag™. At high cell concentrations, the reactor relies on airlift alone.

“Most of the market is pushing toward high cell concentrations for use in inoculating large stirred-tank bioreactors,” says Auton. Using a passive aeration strategy characteristic of flask-based or rocking culture systems, it is difficult to get enough oxygen into the culture to achieve these high densities, he says.

In September, the company will release the CellMaker PLUS™, initially available in two size ranges, 1–8 liters and 10–50 liters. The system will include disposable sensors for dO2 and pH, dO2 control using both air and nitrogen, automated blending of CO2, and independent gas feeds to the headspace and integrated sparge tube.

Whereas current Cellexus systems all have a maximum working volume of 50 L, by the end of the year the company plans to introduce reactor bags ranging from 100 to 500 L, which, due to the vertical positioning of the reactor bags in the housing, will have a smaller footprint than a similar volume in a rocking system.

Appropriate Technical Resources (ATR) has added the Cellexus CellMaker Lite2 to its U.S. distribution line. New to ATR’s Infors line of traditional bioreactor technology is the X-Controller microprocessor-based control system. It can control up to 16 parameters, linking up to three Multifors units and providing independent control of up to six vessels.

Wave Biotech, a parent of the rocking platform, single-use, bag-based Wave Bioreactor, was acquired in April by GE Healthcare. Prior to the acquisition of Wave, GE’s presence in the cell culture arena focused primarily on its Cytodex microcarrier beads.

Ann O’Hara, general manager of GE Healthcare BioProcess, points to the growing emphasis on Lean Six Sigma in biopharmaceuticals production as a driving force behind GE’s move into the disposable bioreactor space. The company believes that bag-based culture systems offer the potential to minimize the three main forms of waste in manufacturing—time, cost, and defects (out-of-spec product).

Specifically, O’Hara points to the reduced time and cost associated with installation and operation of a facility containing single-use reactors, including elimination of the need to install, run, and validate clean-in-place/SIP systems. The potential to minimize product defects relates to consistency of results and reduced dependence on hardware quality over time, according to O’Hara.

With the acquisition of Wave, GE adds to its product offerings the company’s rocking bioreactor technology and a host of ancillary products for making sterile connections, transporting fluids pre- and post-process, and in-process analytical devices for measuring and monitoring conditions in the bag to facilitate process optimization.

Often, downstream purification equipment sits idle awaiting completion of large batches, says O’Hara. The use of 1,000-L to 2,000-L disposable bioreactors for processing small batches in parallel can help optimize utilization of downstream capacity in a production facility, she contends.

Sartorius most recent entry into the disposable bioreactors space, the Biostat® CultiBag RM, relies on rocking motion mixing technology licensed from Switzerland-based Wave Biotech AG and integrates it with Sartorius BBI controllers. The closed, bag-based, rocking systems include preinstalled, disposable, optical chemical sensors and are available in working volumes ranging from 0.5–1 L, 1–25 L, up to 50–100 L, and up to 300 L. The large bags incorporate scalable mixing and are contained in a stainless steel housing.

“Users will be able to operate the new systems in batch, fed-batch, or perfusion mode,” says Millie Ullah, Sartorius’ product manager in North America for disposable bioreactors. “We have disposable perfusion membrane technology for use with rocking devices, in addition to a number of external perfusion devices,” she adds. Sartorius’ BioWelder™ and BioSealer™ products are designed for sterile fusing and sealing of thermoplastic tubing.

“We are currently not looking to go beyond 300-L working volume with traditional rocking devices,” notes Ullah. “We will focus our larger-scale efforts on disposable stirred-tank bioreactor systems,” and plan to launch a range of systems by the end of 2007. These will initially be intended for cell culture applications and will have a stirring shaft and motor for mixing, with capacities up to 2,000 L.

The systems will incorporate disposable sensors for monitoring pH and dissolved oxygen, heating and cooling capability, over-pressure control (which shuts off the gas when the pressure limit in the bag is reached and then restarts the gas flow when the pressure drops back below the defined upper limit), and will interface with standard controllers. “The 2,000-L mark seems to be the current limit for single-use bag systems,” says Ullah. “The industry wants to drive volumes higher,” but that will require engineering enhancements to improve oxygen transfer, she adds.

Contract manufacturer Xcellerex provides process development and GMP-manufacturing services for biotherapeutics and vaccines using its single-use bioreactor technology. Xcellerex also markets its 1,000-L working volume XDR™ disposable stirred tank bioreactor system, XDM™ stirred tank mixing systems, and PDMax™ high-throughput process development service platform.