August 1, 2015 (Vol. 35, No. 14)

Equipped with Unobtrusive Biosensors and Sophisticated Control Software, Bioreactors Are More Efficient

Although single-use bioreactors are increasingly common in small-scale cell culture, many users still rely on glass bioreactors for screening, quality by design (QbD), and design-of-experiment (DOE) studies.

“There’s quite a bit of growth for bioreactors in the 250 mL and smaller range, of up to 3 or 4 L,” says Parrish Galliher, chief technology officer for upstream products at GE Healthcare. “Growth is driven primarily by QbD studies and scale-down efforts aimed at optimizing and increasing productivity of much larger processes.”

Such studies are in line with regulatory trends. For example, the Food and Drug Administration and the European Medicines Agency are encouraging bioprocessors to specify process design space—the combined parameters that allow a process to run reliably and produce consistent product at acceptable yield. More and more commercial registrations include QbD studies that define process design space and other critical parameters.

“Vendors of single-use equipment are aware of these trends [toward lower volumes] and are trying to make inroads into that market. There are several systems available from a variety of vendors,” Galliher adds. Small-scale reactors consume fewer resources than larger vessels, and risk is lower should something go awry. “You can also do a large number of experiments with small systems, so you get more data points.”

A disadvantage, however, is that at lower scale, mixing and mass transfer rates become less representative of production reactors. One factor contributing to the mismatch is the presence of sensors and probes that, due to their physical dimensions relative to vessel volume, contribute disproportionately to mixing patterns in small vessels but have negligible effect at large scale.

“As you get down to less than 10 L, all those probes and sensors suspended in the tank create their own mixing patterns,” Galliher explains. “That means processors must be careful with scale-down modeling and process optimizations in smaller systems whenever mixing and mass transfer rates are important. In those instances, smaller vessels do not provide the same condition as larger scale systems.”

Galliher notes a similar effect in chromatography where wall effects tend to dominate as columns get smaller.

One way to avoid suspended sensor effects is to embed probes within the bioreactor walls, but these devices tend to foul over time by cells adhering to them. Such fouling can be prevented by high turbulence around free-standing probes.

Microbials Lagging

Fermentation is characterized by large volumes, high titers, and low production costs. Developers of single-use fermentation products face many of the challenges encountered by mammalian cell culture specialists during the early 2000s, specifically high regulatory risk and the lack of reliable platform processes. As fermentation economics evolve, particularly with respect to nontraditional products, prospects for single-use processing in that arena will improve, but this will require the reengineering of single-use bioprocessing specifically for microbial cultures.

Single-use cell culture bioreactors are characterized by low mixing and power input, limited oxygen transfer, restricted exhaust capacity, and poor foam management.

“Conventional plastic bioreactors are suited to perhaps the least challenging 5% of fermentation processes, which excludes most current commercial processes,” explains Surendra Balekai, senior product manager for single-use systems at Thermo Fisher Scientific. “And since existing single-use microbial bioreactors are limited to a maximum OD600 of between 50 and 100, far lower than for fixed-tank fermenters, simply redesigning or retrofitting a bioreactor for fermentation cannot work.”

Single-use microbial fermenters should duplicate the physical characteristics of stainless steel systems with an aspect ratio of 3:1 and a high turndown ratio. A turndown ratio of 5:1, for example, allows operation at 20% nominal volume with efficient mixing.

Water-jacketing provides the most efficient removal of high heat generated during fermentation, and disposable fermenters need to exhaust two vessel volumes per minute, which is standard in steam-in-place systems. And because single-use in the age of process analytical technology (PAT) and QbD demands close monitoring and control, features such as sensors for foaming, dissolved oxygen, pH, and temperature must be standard.

Several vendors now sell disposable reactors that meet the high demands of fermentation. Among them are GE Healthcare, Sartorius Stedim Biotech, and Thermo Scientific, whose HyPerforma™ single-use fermentor was designed from the ground up to duplicate the performance of fixed-tank fermentation vessels.

The Thermo Scientific HyPerforma Single-Use Bioreactor (S.U.B.) TK, 50 L, is a turnkey bioprocessing solution for large-scale cell culture growth and bioproduction processes. It combines the functionality of a single-use stirred-tank bioreactor with a state-of-the-art controller system.

Next-Level Sensing

Bioreactor development, says Dirk Hebel, Ph.D., product manager, bioreactors at Infors HT, is striving to achieve three key goals: faster bioprocess development, shorter time-to-market, and greater bioprocess understanding. “One way to achieve [these goals] is to add more sensors to the bioreactors, and not just hardware,” observes Dr. Hebel. “Researchers are looking into reliable soft-sensors that provide additional insight into what is happening inside bioreactors.”

Using data from classical hardware sensors (for example, the off-gas analyzer for oxygen and carbon dioxide), soft sensors calculate in real time additional parameters such as respiratory quotient, biomass concentration, and substrate uptake rates. These parameters are subsequently employed for control and automation in the supervisory control and data acquisition (SCADA) software. A well-understood bioprocess can be scaled up much more easily while still meeting all relevant quality criteria.

“I see increasing demand for open access systems, that is, bioreactors that are shared between many different workgroups,” continues Dr. Hebel. “While this is most common in universities, more and more open access bioreactors are now found in commercial institutions. The goal is obvious: minimize capital expenses and maximize equipment utilization.”

For this strategy to succeed, bioreactors need to be designed for maximum usability and minimum training requirements. “Minimal manual work for preparation of the bioreactor is a big plus and, consequently, systems for the automatic cleaning and sterilization,” notes Dr. Hebel. Similarly, systems that can automate cleaning and sterilization are advantageous.

The same focus on usability is also required for designing a bioreactor’s human-machine interface (HMI). The HMI will incorporate expert settings for fine-tuning the controller for specific bioprocesses, but for guest operators the interface should be as simple as possible.

“Usability and the software accompanying the bioreactor are becoming more and more important. While a one-button bioreactor might seem a bit far-fetched, the bioreactor should present only the information and options that are currently required,” asserts Dr. Hebel.

“This again requires a more intelligent software that provides online help and adapts to current tasks such as calibrating probes before the process begins. Additionally, the HMI can mimic the look and feel of the mobile and web applications with which today’s users are already acquainted.”

The Labfors 5, a benchtop bioreactor from Infors HT, has clean-in-place (CIP) and sterilize-in-place (SIP) functionality. When the Labfors 5 is teamed with the LabCIP accessory, CIP/SIP routines can be instigated at the push of a button and timed to run overnight, for example. The next morning, the bioreactor can begin another microbial bioprocess.

Real-Time Monitoring, Control

Single-use technologies, continuous processing, and QbD/PAT initiatives are driving much of the innovation in bioreactor design according to Mark Givens, senior solutions consultant, Rockwell Automation.

“Real-time monitoring and ongoing control of cell culture are what matter today, instead of a manufacturer completing a batch and analyzing it afterward for two or three days,” Givens explains. “The combination of continuous monitoring and analytics enables a product to move from stage to stage in the manufacturing process more efficiently.” Integration of bioreactors with instrumentation, analytics, and informatics therefore becomes critical, particularly for continuous cultures with no well-defined endpoint.

With expertise in automation and process control, Rockwell Automation has worked closely with leading bioreactor companies, as well as large and small biomanufacturers. Original equipment manufacturers and end users seek to leverage scalable process automation technologies that are “information enabled” while avoiding the burdens of traditional distributed control systems. For example, such systems may come with  onerous licensing terms. There is immense value in implementing control strategies from early-phase design to commercial-scale manufacturing when the product is ready.

One goal of these innovations is to provide basic control of standards such as pH, dissolved oxygen, and temperature. But at another level these innovations should help with decision-making across the enterprise. “It is of great importance that frontline operators be able to use these analytics to determine, based on appropriate culture factors, whether a cell culture process is beginning to fall out of specification, and what might be done to bring it back,” Givens insists.

Again, the availability of real-time analytics matters a great deal, and concurrent multivariate closed-loop control is critical to making sound, timely decisions. As Givens emphasizes, a more mature information and solution set is needed to advance biomanufacturing to the next level.

Continuous Upstream Processing

Continuous manufacturing, parallel mini-bioreactors, and sensor technology are the most significant trends in single-use bioreactors, says Christel Fenge, Ph.D., vp fermentation technologies at Sartorius Stedim Biotech (SSB). “Many companies are looking into continuous upstream processing not only in the U.S. and Europe but also in China and other regions of the world,” she says. “The main drivers are reducing facility footprint by increasing its output.”

Among the pioneers in continuous processing are Bayer, Sanofi/Genzyme, and Patheon. By achieving process intensification and high cell densities, continuous cell culture now allows the production of Phase III and commercial material in 2,000-L single- use bioreactors that are operated in continuous mode.

Some challenges remain, however, e.g., dealing with mass and oxygen transfer, foaming, and venting carbon dioxide especially in such high cell density cultures. SSB’s single-use Biostat® STR bioreactor system addresses these via a special sparger design, excellent mixing, and a large headspace, from 50-L to 2000-L, according to Dr. Fenge.

Continuous processing in single-use systems changes the game for sensing and monitoring. SSB recently introduced sensors for online monitoring of biomass, which is useful in controlling cell density, e.g. via a bleed pump. Another analytical target in continuous cell culture is glucose, whose concentrations are useful in controlling feed rate.

As companies increasingly rely on highly automated parallel mini-reactors for process development and as scale-down model systems, Dr. Fenge sees the need for providing such products in continuous-culture formats. “Customers are asking if the ambr®250 can be used with a cell retention system that will mimic larger-scale perfusion culture,” she notes.

Achieving this will require miniaturization in cell retention technology and integration into fully automated multiparallel bioreactor systems. Dr. Fenge mentions different approaches, including alternating tangential flow and classical tangential flow technology as options. She also observes that industry has begun focusing seriously on downstream continuous processing as well.

Installation of a single-use bag into a Sartorius Biostat STR bioreactor (500 L).

Bioreactor Trends

Karl Rix, vice president for bioprocess sales and support, Europe, Eppendorf, notes the following trends in bioreactors:

•  More built-in sensors and controls. Improvements in sensor technology, such as the digital options and the advanced features of Mettler’s ISM and Hamilton’s Arc (“smart”) sensors, are enhancing current bioreactor technology. The possible addition of new in situ sensor types (such as glucose/lactate) that withstand autoclaving and gamma irradiation will help promote process analytic technology and allow for more hands-off operation.

•  Easier sampling methods for in-process QC or analytics. Improvements in sampling through automation or improved in situ analysis can help to standardize processes and reduce human interaction—and the variability that comes with it.

•  Emergence of better control software. Software, not only for control but also data collection and design of experiments, continues to assume more importance. Flexibility to connect to and use input/data from third-party devices continues to be important. Rix sees rising demand for software capable of providing common interfaces to devices that have slightly different target groups, devices such as freezers and bioreactors.

•  More construction materials made of plastic. At smaller scales, below 10 L, glass has always been more common than steel with only a few custom systems produced using steel vessels. Rix, however, can definitely see an increase in single-use material.

•  Impact of single-use on glass/stainless vessels. The demand for glass and steel vessels remains strong even after the past decade of single-use growth. Perhaps a more significant change will be observed as single-use fermentation options become more readily available. Rix perceives that customers are interested in systems that can accommodate both glass and single-use technology without significant retrofitting.

•  Single-use encroaching more on development-stage bioreactors. More customers are evaluating single-use technology and selecting it during process development. It is still not right for every process or facility, Rix notes. He adds, however, that having systems flexible enough to accept single-use or re-usable components of various sizes has been important to many Eppendorf customers.

•  Progress in single-use systems for microbial applications. Single-use systems are being developed to meet the special demands of microbial processes. For example, BioBLU rigid-walled vessels from Eppendorf can handle high agitation and high flow rates. Eppendorf anticipates that its line of BioBLU single-use vessels will continue to expand, positioning the company as a leader for small-scale single-use fermentation.

Eppendorf’s BioFlo® 320 bench-scale bioprocess control station features autoclavable and single-use vessel flexibility, intelligent sensors, and IP network communication for multi-unit control.

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