May 15, 2014 (Vol. 34, No. 10)

Choosing the Appropriate Technological Approach to Achieve High Cell Densities

As the GEN article “Prospects for Downstream Production” notes, perfusion cell culture is most often associated with  upstream continuous processing operations.

The two main perfusion techniques are microcarrier-based processes (e.g., iCELLis™ from Pall/ATMI) and those in which cells flow across the membrane through which product is harvested (e.g., GE Healthcare’s hollow fiber microfiltration). A third method, based on centrifugation, was under investigation during the 1990s and early 2000s, but has fallen out of favor.

Benefits of perfusion over fed-batch cultures include higher productivity and, at least theoretically, lower cost of goods.

According to Pall, iCELLis combines the “advantages of single-use technologies with the benefits of a fixed-bed system.” This design, which relies on microcarriers, provides a 500m2 growth area (approximately equal to 3,000 roller bottles) within a 25L volume.

Because cell multiplication occurs in a fixed bed, iCELLis bioreactors are inoculated at low density, with reduced manual operation. iCELLis is available as a fully-disposable product, which scales from bench to production.

“Perfusion cell culture provides higher productivity from a smaller set of assets,” says Nick Hutchinson, market development manager at Parker Hannifin Manufacturing. “Quality may also improve because cells are in a steady state throughout the culture.”

Recent improvements in cell density and product titers should have lessened the appeal for continuous cell culture, but this has not been the case. Hutchinson says “there’s more noise than ever” over perfusion methods.

Perfusion’s major challenge compared with fed-batch is maintaining a controlled, sterile, cell-friendly environment for weeks or months.

“Fed batch is much simpler, and doesn’t require moving volumes of cell culture and product-containing media around,” adds Hutchinson. Movement of process fluids increases the risk of contamination, leakage, and equipment failure.

Perfusion cultures raise additional concerns of handling contamination, specifically the cutoff for retaining or discarding product once contamination is detected. It also requires more intense process development than fed-batch culture.

Development time becomes a serious issue when processors face the need to test numerous culture conditions, which could result in delayed time-to-clinic or time-to-market.

There is also some disagreement on productivity calculations. “OK, you’ve reduced your fixed asset footprint, and all the utility and other requirements that entails, but perfusion cultures use a great deal more cell culture media, which is not cheap,” says Hutchinson. “If you view product titer for overall media usage, perfusion doesn’t always look that great.”

Systems such as GE Healthcare Life Sciences’ ReadyToProcess WAVE 25 can support perfusion cell culture for a range of applications, including seed train.

High Cell Densities

About four years ago Eva Lindskog, Ph.D., upstream marketing leader at GE Healthcare, began a collaboration with Sweden’s Royal Institute of Technology to test the capabilities of hollow fiber cell culture within a WAVE Bioreactor. “The result exceeded all our expectations,” says Dr. Lindskog.

Her team achieved what was arguably, at the time, the highest cell density observed in a single use bioreactor: 214 million cells/mL. “It was not a process I’d recommend for production. But by pushing the limits we came to understand the system’s capabilities, and for which processes it was suitable, she adds.”

Dr. Lindskog recognizes that even under ideal conditions, perfusion is not right for every company or every process. Bioprocessors comfortable with fed-batch processes, who may lack the proper equipment and mindset to adopt perfusion, usually stay away.

Firms experienced with perfusion for certain product categories, for example enzymes and blood factors, might consider perfusion even for monoclonal antibodies that may otherwise be produced through low-risk platform processes.

Seed Train Applications

The seed train is one area where perfusion could help companies simplify cell culture-based manufacturing, particularly if their comfort zone includes single-use bioprocessing. Disposable bioreactor systems are available with high mixing rates and oxygen transfer capacity, and they can be combined with a simple, disposable retention system. The result is a convenient perfusion reactor capable of supporting very high-density cultures.

“A very high-cell density perfusion seed culture in small-scale can be used to inoculate a production bioreactor directly, with a split ratio of up to five hundred,” notes Dr. Lindskog, while skipping intermediate bioreactor scales in the seed-train. “An intensified seed culture strategy also makes it possible to begin the production bioreactor at higher cell densities, and thereby shorten process times in manufacturing scale.”

The total time to scaleup for a biomanufacturing process depends on the culture population doubling time. However, lags can occur when cells enter a new environment, such as when scaling up to a new bioreactor stage. By skipping intermediate links in the seed chain, perfusion methods can avoid such cell growth interruptions.

Cells remain in an optimized, stable environment until they are needed. Cultures are no longer interrupted when moving from bioreactor to bioreactor, thereby reducing complexity in the seed train.”

Dr. Lindskog dismisses the potential riskiness of switching from a traditional seed train based on increasingly voluminous reactors to high-density perfusion reactors.

“Traditionally, the regulatory challenges have been how to define a batch, how to handle contamination, and validating the manufacturing process,” she explains. “The simplified seed train approach doesn’t change any of that. It only affects the seed train, not the production process per se.

Even regulators are on board. In 2011, FDA’s Janet Woodcock, M.D., noted at the AAPS annual meeting that bioprocessing had barely changed in the previous half-century. “…[bio] manufacturing will change in the next twenty-five years as current manufacturing practices are abandoned in favor of cleaner, flexible, more efficient continuous manufacturing.”

The nature of regulatory oversight in biotech is such that the only chance for continuous processing, including perfusion cell culture, is in new processes. In this regard Genzyme has become something of a leader, particularly in perfusion culture.

While interest remains keen in perfusion cell culture as a tool to increase efficiency in different parts of the biopharmaceutical process, it is clear that established fed-batch platforms still offers a convenient route for manufacturing at scale.

“Fed-batch will remain the workhorse for the majority of processes, at least in the near term,” maintains Dr. Lindskog.

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