Optimizing Process Development
What was an emerging trend five to ten years ago is now an accepted fact: by doing better quality experiments upstream of your bioreactor you can develop higher-titer and better quality products and processes,” says David Laidlaw, small-scale technologies manager at Applikon. Users want to run more experiments in the same bench space to gain a better understanding of their processes, and to perform cell-line screening and selection at higher throughputs and smaller scales, explains Laidlaw. By “upstream” he means before moving into a 3 L stirred tank vessel, where much process-development work has traditionally been done.
Applikon recently introduced the first version of its new miniBio small-scale stirred tank reactors, with a 250 mL vessel capable of supporting 50 mL to 200 mL working volumes. Subsequent vessels will be available in 500 mL, 1,000 mL, and 3 L volumes. Introduced along with the miniBio is Applikon's new my-Control ™ controller that offers the same capabilities as the Applikon EZControl unit in a smaller footprint. “Five complete systems—vessels and controllers—can now fit in one meter of bench space,” notes Laidlaw.
Also new from Applikon for early-stage cell culture work including cell-line creation and screening is a 24-well deep-well plate. Made of ultrapure polystyrene, this new cassette is gamma-irradiated, features a pyramid-shaped bottom, and can be used with the uFlask well plate closures in place of shake flasks.
Companies continue to embrace the principles of process analytical technology (PAT) and incorporate them into their processoptimization strategies. The latest version of Sartorius Stedim Biotech's multifermentor control software, for example, offers design of experiments (DoE) and multivariant analytical tools to support PAT.
Ferraro of GE reports that the company's new WavePOD II control module is the result of two years of collaboration with clients to understand the relative sensitivity of various process parameters and “to make PAT more realistic for the end-user” and more of a plug-and-play operation. The controller integrates all of the readouts from the Wave Bioreactor 20/50 system, including DO, optical pH, and CO2/O2 gas mixing controls and automatically varies the rocker speed or oxygen concentration supplied to the Cellbag to maintain critical process parameters within the desired ranges.
The ability of a control system to monitor process parameters in real-time and to make changes in response to those measurements is particularly critical for perfusion processes, a growth area in the bioprocess market, according to Ferraro. With the continuous addition of media and extraction of product over periods as long as weeks, cell density can reach a high level and the system may exchange thousands of liters of media. Perfusion systems are very sensitive to small changes and “can become very unstable.”
Single-use sensors are another evolving area. Ferraro describes all of GE's sensors as single-use: they either have direct product contact and are disposable, or they are inserted into a cell bag through a sleeve or sheath, do not directly contact the product, and can be re-used without cleaning. The company is moving toward adding more embedded sensing capability into its bags to meet the demand for monitoring processes such as cell density, glucose, and other types of metabolic parameters.
The main challenge, Ferraro explains, is fabricating single-use sensors capable of making these measurements that can withstand the gamma irradiation used to sterilize the bags.
Focusing on the single-use cell bag, GE plans a redesign of its Wave bag by the end of this year. The new line of standard cell bags will incorporate in-demand features of its customized versions, including more connections and greater use of aseptic connectors. One goal of the new bag design will be to accommodate more intensive cell culture processes such as perfusion cultures and to enable increased monitoring of acid/base additions and feeding and harvesting of the cultures, for example.
Additionally, companies such as GE are taking a close look at the film/plastic used to manufacture the disposable cell bags, as the FDA and their customers are becoming increasingly concerned about the effects of extractables and leachables.
Innovating Process Streams
Market demand for parallel unit operations for process design and optimization studies was the underlying principle for the design of HexaScreen's automated multiple minibioreactors system, sold under the brand name Telstar HexaScreen.
The system supports animal cell culture on a scale of 10 mL to 15 mL. With the HexaBatch model, users can perform six experiments in parallel in single-use interchangeable plates, with online monitoring of temperature, DO, and pH.
Earlier this year Infors adapted its Labfors 4 Cell bioreactor system to be compatible with Millipore's single-use Mobius™ CellReady 3L Bioreactor vessel, and a single XDDC control unit can operate four parallel bioreactors.
A collaboration between Infors Benelux and Wageningen University led to the design for a flat panel bioreactor for growing phototrophic microalgae and cyanobacteria, which is now in use at the Algae Production and Research Centre in Wageningen. Commercial versions of the photo-bioreactor are being produced by Infors in Switzerland and Appropriate Technical Resources (ATR) in the U.S.
The 2 L (total volume) autoclavable vessel connects to a standard Labfors console. The flat panel design of the photo-bioreactor supports high biomass densities. The system can monitor and control temperature, pH, DO, antifoam, and feed parameters, and is also capable of off-gas measurements.
ATR will also produce and market the CELLutions Biotech CELL-tainer® single-use bioreactor in North America. The two-dimensional mixing movement of the CELL-tainer supports high cell growth rates and allows the system to support both cell culture and microbiall fermentation processes, according to Steve Mitchell, CEO of ATR. It can accommodate 500 mL to 15 L working volumes and includes embedded pH and DO probes.
Felezeu of Pierre Guerin described the three largest markets for his company's bioreactors and fermentors over the past year as vaccine manufacturing (from seed culture at 5 to 10 L volumes up to 1,000 L production scale), enzymes (largely for use in biofuels production), and a center in Ireland called the National Institute for Bioprocessing Research & Training, where people receive hands-on training to work in cGMP manufacturing facilities.
Felezeu also reports on the company's ongoing development of software modules to enable more complete automation of bioprocesses. With mergers and acquisitions continuing in the pharmaceutical sector, creating extra bioprocessing capacity, Felezeu describes a healthy market for refurbishing, updating, and optimizing existing facilities, equipment, systems designs, and software to meet the needs of new users and new applications and to be in accordance with the changing regulatory landscape.
ATMI reports evidence of a deliberate focus on process intensification driven by the need for faster, less expensive, and more efficient cell culture technologies. With the acquisition of Artelis, ATMI has expanded its integrity bioreactor platform to include the iCellis perfusion-based reactor, which uses an inert substrate to immobilize attachment-dependent and suspension cells.
The Xpansion reactor from Artelis utilizes stacked disks to create a high density perfusion environment for growing stem cells and other cell therapy products. The PadReactor Mini is a benchtop and scalable version of ATMI's PadReactor, which combines a square reactor vessel and the gentle PadMixer that incorporates sparging technology. Earlier this year, ATMI entered an agreement with Finesse Solutions to provide additional plug-and-play controller options that readily integrate with the Integrity PadReactor.