August 1, 2014 (Vol. 34, No. 14)

As GEN readers are well aware, single-use bioreactors (SUBs)—already accepted at research scale for screening and optimization, as well as at pilot scale and seeding—are now ready for GMP-compliant manufacturing.

“Improved automation and monitoring, with better noninvasive sensors and quality-by-design approaches, has led from an unmonitored small-scale process to a controlled process,” notes Roman Rodriguez, global market development manager for single-use at EMD Millipore.

The need for personalized medicines and the emergence of biosimilars promise a great future for SUBs. “The advent of 2,000 L SUBs has created incentives related to process optimization and overcoming challenges in mass transfer, heat exchange, sensing technologies, and plastic film technologies,” Rodriguez says. “I anticipate new services in process optimization, SUB validation, and accessories such as computational studies, media optimization, scalability, and shear stress.”

A technician configures the EMD Millipore Mobius® SensorReady external loop, which is used to control and monitor the company’s CellReady bioreactor.

Related is the trend toward continuous processing through perfusion bioreactors. Most current processes remain fed-batch, but many manufacturers are examining combinations of SUBs with a single-use perfusion culture. This creates a need for more automation and new inline sensing methodologies.

The quest for high cell densities in smaller volumes in shorter time periods have made SUBs the technology of choice in many instances, but new challenges have emerged.

Scalability, operation at high cell density and power input, oxygen transfer efficiency, price, supplier dependability, and overall robustness of SUBs are still issues. “There have been recent advances at small scale to address these, and I have no doubt that vendors will soon overcome those challenges at all scales,” Rodriguez states.

Issues of scalability have been as critical for single-use equipment as they were for glass and stainless-steel bioreactors, according to Ken Clapp, senior product manager at Xcellerex-GE Healthcare Life Sciences. These issues, he avers, are inescapable when small-scale or fractional single-use bioreactors are used for quality-by-design studies and process optimization.

“The never-ending quest for higher product titers, greater cell densities (approaching and even exceeding 100 million cells per milliliter), and higher throughput will create new demands,” asserts Clapp. “Moreover, these demands, which will run the gamut from materials to supply chain capacity, will bring about a new level of integration within the manufacturing infrastructure of biologics companies.”

Bioreactors with sufficiently high turn-down ratios should support the elimination of intermediate vessels and help manage consumables costs. Higher densities and increased throughput will likely require improved process equipment, such as perfusion devices, during the production phase.

“Downstream, as on the production side,  there will there be a need for tighter integration,” adds Clapp. “Regulatory compliance will require tight coordination of the unit operations and the consolidation of process data under existing automation infrastructure.”

Scaledown and DOE

Bioprocess development strategies frequently include some form of small-scale modeling, or scale-down modeling, with the aid of design of experiment (DOE). Several products on the market distinguish themselves through various features.

For example, the ambr™ from TAP Biosystems has working volumes of 10–15 mL and uses 24 or 48 disposable reactor wells. Dasgip’s parallel bioreactor products are larger—up to 100 mL—and are controllable through mobile devices. Pall’s Micro-24 Microreactor uses much smaller volumes (3–7 mL) in a single-use cassette format.

The micro-Matrix microbioreactor from Applikon Biotechnology makes a good case for keeping reactor volumes smaller, not larger. micro-Matrix provides 24 independent microreactors with working volumes of 2–4 mL, in a microplate footprint.

When Applikon designed the micro-Matrix, the prime concern was that results flawlessly scale to the gold-standard 3 L benchtop bioreactor and beyond. “We produce systems as large as several thousand liters,” explains Erik Kakes, the company’s director of international sales and marketing. “Whatever we do at smaller scales must be directly transferrable to larger scales.”

As in many micro- and minireactors, each reactor well has individual control of temperature and pH. In addition, each of the 24 wells has PID (proportional-integral-derivative) control similar to benchtop reactors. “They’re real bioreactors,” Kakes says. To preserve volumetric fidelity during pH, media, and feed adjustments, Applikon also developed a microvale liquid dispenser that provides nanoliter-scale additions.

Applikon has run kLa (a measure of bioreactor performance) and scalability studies comparing its microbioreactors, minireactors, and lab-scale reactors, and it has “achieved exactly the same results,” according to Kakes.

“In the near future, the majority of all new cell culture processes will run in single-use format,” says Davy De Wilde, director of marketing, fermentation technologies at Sartorius Stedim Biotech.

Adoption will occur based on the usual benefits of lower costs and improved flexibility, and is further supported by the availability of single-use bioreactors founded on conventional stirred-tank design. These vessels will apply established design principles, accounting for vessel geometry in the design of impellers and spargers. This, De Wilde says, will shorten scaleup time and ultimately reduce time to market.


A schematic of an Applikon micro-Matrix bioreactor. Each micro-Matrix cassette has 24 of these bioreactors on the standard SBS format plate. The total volume per bioreactor is 10 mL with working volumes from 1–7 mL.

The Sartorius Stedim Biotech single-use portfolio stretches from 250 mL to 2,000 L, enabling a fully disposable process from development through commercial manufacturing. While process development was typically conducted at 2–10 L scale, development-stage reactors have trended downward in volume. The ambr bioreactor family from TAP Biosystems, a Sartorius Stedim Biotech company, provides fully automated workstations operating up to 24 bioreactors in parallel at 250 mL.

“Its conventional design ensures that the data generated are representative of pilot-scale bioreactors,” De Wilde says. “Good comparability to production scale is expected as well.”

The minibioreactor, provided experimental tools are designed for smart experimental planning, allows process development at reduced material cost, resource need, and development time. Multivariate data analysis provides real-time feedback on batch performance in comparison to a model batch. For media or clone selection, working volumes as small as 15 mL enable rapid screening while producing consistent data. “This ultimately will help to reduce variability, deviations, and lost batches,” De Wilde observes.

Fermentation and Single Use

Xcellerex-GE’s Ken Clapp calls microbial fermentation the new frontier in single-use process equipment. By contrast, single-use stirred-tank reactor technology is widely adopted in cell culture. “Biologics manufacturing, at scale, has never relied so heavily on the technology as it does today.”

Single-use bioreactors have not caught on with fermentation to the degree they have with cell culture. “Achieving heat removal capacity and rates of mass transfer and oxygen transfer adequate for high-density fermentations has been difficult,” notes Richard Mirro, executive director, portfolio management for bioprocess at Eppendorf. “Bioprocessors have had to rely on exhaust gas cooling to avoid filter fouling or high evaporation.”

Mammalian cell culture is more forgiving. The relatively gentle conditions required are achievable in either rigid-walled or flexible containers, whereas the extreme agitation, baffled mixing, and gas-flow requirements demanded by high density microbial cultures is much better suited to a rigid-walled container.

Eppendorf’s BioBLU f vessels combine the advantages of disposability with the reliability of conventional glass or stainless-steel bioreactors for fermentation. “They are the first single-use bioreactors designed to mimic traditional stirred-tank fermentation vessels,” Mirro adds.

BioBLU f features Rushton-type impellers and high-performance magnetic agitation for efficient mass transfer and improved mixing; cooling baffles for heat removal; and innovative liquid-free Peltier exhaust condensers to enable cultivation of bacteria, yeast, and fungi, even at high densities. The rigid-walled stirred-tank design, sensor technology, and scalable geometries can allow for smooth process transfer and transition between reusable and single-use systems.

Thermo Fisher Scientific launched its first single-use bioreactor for mammalian cell culture in 2006. “Since then, we have seen a demand from the market where customers have been asking for single-use fermentation products as well,” says Millie Ullah, the company’s product management leader for single-use products.

Fermentation differs from mammalian cell culture in several respects. Bacteria and yeast cultivations are typically much denser and more oxygen-demanding processes that can withstand high shear forces, especially Escherichia coli and Pichia processes. Mammalian cells are slow growing and shear sensitive, so agitation and gassing must be gentle.

“We have customers who use our single-use bioreactors today modified for fermentation applications,” Ullah notes.

At this stage, the company has a cell culture product that operates at a gas flow of 0.1 VVM (vessel volumes per minute) in a standard product, which can be increased through customization to meet the demand for fermentation, but limited when compared with stainless-steel fermenters. Agitation can also be modified to be higher than conventional bioreactors.

Thermo Fisher Scientific achieved this by designing spargers that introduce more gas into the system, increasing the size or number of exhaust filters, adding condensers to the exhaust line, and increasing the motor size and agitation rate. Condensers cool exhaust flow to reduce condensation and avoid filter blocking. Materials of construction are the same as for single-use bioreactors.

A number of customers use these bioreactors today for moderate-density microbial applications. Scientists from Allergan and the Keck Graduate Institute presented a case study on a variant of this system at the Flexible Facilities conference earlier this year.

“We’ve already achieved very high cell densities, but we’re looking to improve performance even more,” Ullah tells GEN.

A Cleaning Alternative

Rapid, efficient process development is vital for every company, and even at universities and research institutes. “Hence, low-maintenance bioreactors that are easy to operate and always ready-to-use are an emerging requirement for many users,” says Dirk Hebel, Ph.D., product manager for bioreactors at Infors.

The factor most limiting bioreactor throughput is the time spent for cleaning and sterilizing the vessels. While clean-in-place and steam-in-place are standard features of larger stainless-steel bioreactors, this functionality has traditionally been inaccessible for compact, lab-scale vessels.

Infors has recently bridged this capabilities gap with its LabCIP system, which is now available as an option for its Labfors 5 glass bioreactor with a working volume of 0.5–10 L. LabCIP began as a special project for Novartis, an Infors customer, and is now fully commercialized.

LabCIP automatically conducts CIP and SIP at the push of a button. The process is configurable and offers options for cleaning the vessel with base or acid. In contrast to steel bioreactors, sterilization occurs chemically at 60°C. A network of valves ensures contact between cleaning solution and every product contact surface within the bioreactor.

“The Labfors 5 with LabCIP allowed us to double the throughput from 8 to 16 protein expressions per week,” says Alvar Gossert, Ph.D., a research investigator and lab head at Novartis.

Labfors 5 bioreactors belong to the Infors incubation shaker and bioreactor portfolio, with volumes from 1 mL to 1,000 L. Within this range, the Labfors 5 is suitable for process optimization after screening in smaller, parallel bioreactors.

BoB Project

The Bolt-on Bioreactor (BoB) project is an independent initiative founded in 2013 and aimed at providing the future standard system for the culture of adherent cells for biopharmaceutical applications, according to Marcos Simon, founder and inventor of the technology behind the project.

“The successful solution should provide a huge surface area optimum for cell attachment, contained in a reduced volume, supplied as a sterile and ready-to-use device that provides automated and continuous culture of adherent cells even in nonclassified laboratories,” explained Simon, who’s also the CEO running the project.

Having secured the intellectual property and following an on-going implementation of design features provided by the market through an open design initiative, Simon said the BoB team expects to conclude a funding round and will strive to bring the product to market by the end of second quarter 2015. The BoB project is currently established in northern Spain and plans to open offices in Singapore, Scandinavia, and the United States.

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