A high-quality product begins with efficient upstream process equipment. Ten years ago, manufacturers were still warming up to single-use bioreactors, which were mainly rocking-bag–based solutions. The benefits relating to cleaning and validation were clear, but their use as bioreactor vessels was still new, and stainless steel systems up to 20,000 L in scale were still needed. Today’s facilities are a hybrid of sophisticated single-use components and stainless steel equipment, the mechanisms of both having undergone improvement during the past decade.

Thanks to increasing cell culture titers, vessels can be smaller, and the large tanks of the past are more of an exception. Add that factor to a healthcare industry that is leaning toward paying for only treatments that “work” (fueling efforts toward “personalized” medicine). Biotech facilities of the future appear poised to have smaller footprints with flexibility and “just-in-time” manufacturing in mind. Advanced Stainless Steel Designs

Don’t expect stainless steel vessels to disappear completely. Experts have argued that steel units are preferable to single-use systems for dedicated processes and retrofitting facilities (1). During the past decade, BPI authors have described continuing work to improve the efficiency of stainless steel bioreactors and large-scale fermentation vessels to facilitate scale-up (2, 3).

Most stainless steel bioreactors are stirred-tank vessels for fluid mixing. The configuration of a vessel, especially the design of its impeller, can affect quality and yield, which makes choosing the right design a critical task (4). To facilitate that decision and optimize processing, manufacturers are turning to computer simulation and other powerful process modeling tools (5,6,7). Those approaches can help optimize parameters such as mixing, gas hold-up and mass-transfer coefficients, and gas distribution (to prevent foaming) within a vessel as well as the effects of hydrodynamic and mixing during scale-up.

Modes of bioreactor system operation are batch, fed-batch, continuous, and perfusion processing. Although perfusion processing is still relatively limited in application, during the past decade, interest has grown because of perfusion’s ability to produce a high number of cells in a bioreactor that is small compared with those for batch or fed-batch systems (7,8,9,10). One challenge, however, is that such cultures require cell-retention devices. Modeling systems for perfusion bioreactors can provide data for optimizing a particular process (10). Current perfusion systems are available from FiberCell, ZellWerk (Glen Mills in the United States), Biovest, ATMI, Refine Technologies, AmProtein, Xcellerex (a GE Healthcare Company), Applikon, and Wave Biotech, LLC (11). Single Use Platforms

Without a doubt single-use technologies have profoundly affected the way manufacturers design their upstream processing. Among the benefits of single-use systems are reduced capital expenditure, low risk of contamination, quick changeover, and elimination of cleaning validation (12, 13).

Disposable mixing systems for media and buffer preparation offer modularity, operation in unclassified gray spaces, and strong agitation for thorough hydration of powdered media (14,15,16,17,18). Current models use a number of mixing technologies, including levitated magnetic stirrers, magnetically driven stir bars, motor-driven “wands” or impellers, motion rockers, and bellows systems that force perforated plates through solution.

Single-use bioreactors entered the mainstream bioprocessing industry early as orbitally shaken flasks (e.g. CultiFlask 50 model, Sartorius), bag-in-vessel systems, and stirred-tank motor-driven models. Wave Biotech (now part of GE Healthcare) introduced a revolutionary wave-motion system in 1996 that eliminates problems with shear force, followed by other wave-motion systems (e.g., Tsunami, CatchMabs BV; Appliflex, Applikon Biotechnology; and Biostat CultiBag RM, Sartorius-Stedim) (19,20,21,22). A 2006 BPI article exemplified the speed, efficiency, and other advantages of single-use bioreactors during an “eight-week scale-up challenge” comparing stainless steel and single-use stirred-tank systems (23).

During the past decade, industry experts have worked toward improving single-use bioreactor performance and developing novel systems. Configurations include a frustoconical–bottom shaker vessel based on a new oxygen-transfer method (24), a single-use system for microcarrier cultures (Nucleo, ATMI LifeSciences, Pierre Guerin-Biolafitte, and Artelis) (25), bioreactors for both cell and microbial cultures (CELLution, Biotech BV; and Cell-tainer), high-cell density models (iCellis-ATMI), bioreactors that can be integrated as part of larger platforms (XDR for FlexFactory, Xcellerex), cube-shaped vessels (ECube, Corning), and “microscale” systems (e.g., ambr, TAP Biosystems). Single-use perfusion bioreactors have entered industrial application as well (26). Such configurations have facilitated process characterization and scale-up (27, 28).

Researchers have also worked on improving connection technologies for use with single-use bioreactors. Advances in the use of thermoplastic resins help bring about several types of heat-weldable Class VI tubing brands (e.g., C-Flex, Consolidated Polymer Technologies; Sanipure, Saint-Gobain Performance Plastics). Such tubings can be fused and/or sealed when heated to provide customized options (29). Solutions for connecting stainless steel and single-use systems have also been developed (30). Process Monitoring and Control

Bioreactor parameters to monitor include mixing time, pH, oxygen mass-transfer coefficient (k_La), volumetric power input, and heat-transfer rate. Accurate understanding and routine measurement of such parameters can improve performance (31). Industrial near-infrared spectroscopy methods can be used for rapid and real-time monitoring of pH and osmolality (32,33,34), and radio-frequency impedence methods have been developed for controlling and monitoring microbial fermentation and biomass (35, 36).

Disposable alternatives to traditional process monitoring components include tubing pinch valves along with single-use dissolved oxygen (DO) sensors, pressure sensors, and flow meters (37, 38). Sensor technology has evolved from probes to single-use sensors as s
mall as 1 mm in size. On-line pH and DO monitoring with optical sensors for shake-flask systems also can provide data for comparison with bioreactors (39). Cell Therapy Systems

One challenge associated with cell therapy culture vessels is scale-up (larger batch sizes) and scale-out (increasing same-size batches) (40). However, scaling out autologous therapies (processed with relatively small-scale equipment) greatly increases production costs because it requires increased handling of laboratory-scale equipment, automation to handle a large-volume batches, and high levels of testing for each patient’s cells (41).

Multitray configurations have been used for large-scale cell culture for more than 30 years. During the past decade, their use has expanded to the growth of therapeutic cells. Multilayer systems for scaling up a dendritic cell process first appeared in BPI in a 2006 article describing the Cell Factory system from Nalge Nunc International (42). Other systems include Cell Stacks multilayer vessels (Corning), planar flasks, and packed-bed bioreactors (PBRs), in which adherent or nonadherent cells are immobilized to a substrate that is enclosed in a packed bed within a bioreactor system. PBRs offer several advantages, including beneficial biological attributes of cell cultures in three-dimensional scaffolds (43).

Such systems, along with increasing titers and expanded use of single-use systems and components are likely to continue to play significant roles in the trend toward smaller batch sizes overall. Improved efficiencies in stainless steel reactors should ensure their place in future process chains and hybrid configurations.

Author Details
Maribel Rios is managing editor of BioProcess International; mrios@bioprocessintl.com.

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