Changing industry dynamics, advances in technology and the benefits of continuous processing are driving renewed interest in perfusion.
Perfusion cell culture is not a new concept. It was first introduced in the early 1980’s as a means for improving the productivity of cell-culture processes, which at the time generally suffered from low titers and inefficiencies. Advances in fed-batch processes, including improved cell lines and expression systems, enhanced media and feeds and optimized operating conditions resulted in dramatic improvements in process performance, eliminating this driver of interest in perfusion. As a result, it has over the past 20 years largely been used for unstable/labile proteins that degrade under batch and fed-batch process conditions.
All of that is changing, however. Biopharmaceutical manufacturers face significant pressure to reduce development times and production costs, leading to the need for new approaches to processing. Continuous manufacturing has been demonstrated in many industries to provide more consistent and better product quality in smaller production footprints with reduced setup times, waste and often capital and operating expenses. With encouragement from regulatory agencies and access to advanced technologies, notably single-use systems, the biopharmaceutical industry is actively exploring the potential of continuous manufacturing, with most activity to date focused on upstream perfusion cell-culture processes.
Perfusion Offers Many Potential Advantages
In batch and fed-batch cell culture processes, the cells remain in the bioreactor for the length of the run, which can be up to several weeks. In fed-batch processes, additional nutrients and media are provided until the culture conditions are no longer optimal for protein expression. In perfusion processes, fresh media is continually added to the bioreactor while spent media and product are removed, leaving the bulk of viable cells for further expression.
Under perfusion conditions, cell culture can proceed for many weeks to months. Because the product is regularly removed from the bioreactor, it is not exposed to increasing concentrations of toxic byproducts generated during cell culture process, avoiding concerns about degradation, particularly for sensitive/labile biologics. The replacement of spent media with fresh material, in fact, prevents buildup of toxic byproducts and allows for operation of cell culture under optimized, steady state conditions, leading to more consistent processes and thus product quality. The milder cell culture conditions that exist in perfusion processes, including reduced shear due to easier mixing, also result in more consistent protein expression in terms of product forms, glycosylation patterns and reduced production of undesired variants.1 Higher and more consistent product quality means downstream purification processes are often less complex.2
Perfusion processes also typically operate with much higher productivities, expressed as grams/L of bioreactor working volume per day, than can be achieved in batch or fed-batch processes.3 Higher productivities allow the use of smaller bioreactors to produce similar product quantities, leading to reduced capital expenses, according to Chris Hwang, Senior Director of Late-Stage Process Development at Genzyme.3 Scale-up is also simplified; perfusion cell-culture processes can be scaled-out (rather than scaled up) using the same equipment setup and process conditions identified during development, reducing the considerable risks associated with technology transfer.3
Single-use bioreactors, which are ideal for flexible, multiproduct facilities can often be employed. Perfusion processes themselves are well-suited for multiproduct manufacturing because the quantity of product produced can be easily adjusted to meet demand by shortening or lengthening the cell culture run.4
Important Advances in Harvesting Technology
One of the keys to implementing successful perfusion cell-culture processes is access to effective technologies for the separation of product and spent media from viable cells, which either remain or are returned to the bioreactor. Advances in continuous harvesting technologies have been essential to the wider adoption of perfusion cell culture in the biopharmaceutical industry.
A number of technologies are possible. Langer and Rader identify gravity settling, pumping through internal filters, external loop flow-through filters, and centrifugation as the most widely used methods. In some cases, adherent cells are attached to microcarriers, such as magnetic microbeads, or various other surfaces, such as hollow capillaries, flat plates, sponge-like materials, or different types of fibers and membranes as fixed beds. Filtration removes the media and product and leaves the cells in the bioreactor.1
The introduction of XCell™ alternating tangential flow (ATF) filtration technology (developed by Refine Technology, now Repligen) for continuous removal of spent media was influential in increasing the practical application of perfusion cell culture. Unlike centrifugation, which requires further filtration, ATF allows for complete clarification.2
Researchers from Eppendorf recently compared perfusion processes using an ATF filtration device and a packed-bed bioreactor with cells immobilized on a solid support matrix.5 The ATF run provided the highest yields of both viable cells and monoclonal antibody (mAb), but required more space, daily sampling and monitoring and consumption of a large quantity of medium. The packed-bed setup was much simpler and involved simple sampling, and produced peak cell densities only slightly lower than that obtained using the ATF.
ATF has other limitations as well. According to Timothy D. Hill, Director of Upstream Process Development for FujiFilm Diosynth Biotechnologies USA, setting up an ATF is not easy, nor is sterilization and operation of stainless-steel systems.6 Fouling of the hollow fibers is also often a problem at large scale. As importantly, the separation process is slow and can be a bottleneck. Efficiency improvements are also needed, particularly for intensified perfusion processes with very high cell densities.4
Acoustic wave separation (AWS) technology has recently been introduced as an alternative to centrifugation and filtration methods. At least two companies – Applikon Biotechnology and Pall Life Sciences (through an exclusive license to the AWS technology developed by FloDesign Sonics) – offer solutions for biopharmaceutical manufacturing. With AWS, cells are separated using acoustic waves rather than via a physical method. The waves cause the cells to clump together and eventually settle due to gravity.
Applikon’s BioSep acoustic perfusion system7 is scalable from the lab to the process development and production scales and can be operated in continuous mode for months. Pall launched its Cadence™ Acoustic Separator at Interphex. “We launched our continuous processing vision at the end of 2015, introducing the concept of acoustic wave separation technology as an enabling tool for customers to bridge the critical gap between the bioreactor and the downstream processing train,” said Vice President and General Manager of Biopharmaceuticals, Michael Egholm. “The CAS technology is truly revolutionary, eliminating reliance on centrifugation for cell culture clarification, reducing buffer requirements by around 75%, and creating a continuous feed stream for direct integration with our full portfolio.”8
Single-Use Systems Facilitate Continuous Cell Culture
As mentioned above, single-use systems are ideal for multiproduct facilities that need flexibility in production. They have also made it possible for perfusion processes to be more easily evaluated due to the reduced cleaning and validation requirements.2 Scientists from Sartorius Stedim Biotech believe that with the high titers possible with perfusion, “even blockbuster drugs could be manufactured using 2,000-L single-use bioreactors, and no additional scale-up step after process validation in Phase III would be needed.”9
Sartorius has developed Biostat STR single-use bioreactors that provide the level of aeration needed for perfusion cell culture. To demonstrate its applicability, the production of two mAbs – one that requires typical culture conditions and one that involves much higher cell densities and thus more oxygen. The Biostat STR 2000 system provides equivalent performance to that of an existing single-use bioreactor for the conventional process, but with reduced need for gassing and thus less risk of exhaust-filter blockage due to foaming and pressure build-up. For the more challenging process, cell-culture performance was equivalent to that obtained in a stainless-steel bioreactor.9 The authors also noted that incorporation of sensors such as Sartorius BioPAT ViaMass sensors into single-use bioreactors allows for automated, real-time monitoring and control while reducing the need for manual sampling.
AcuSyst single-use perfusion bioreactors from Cell Culture Company (C3), meanwhile, have been designed to specifically overcome issues faced during the production of difficult-to-express proteins, such as bi- and tri-specific antibodies and other unnaturally occurring glycoproteins.10 Cellulose acetate fibers in the bioreactors create extracapillary (EC) and intracapillary (IC) compartments that allow protected cell growth and high rate media flow, respectively. Pores in the fibers allow exchange of nutrients and removal of waste, and product is continually harvested from the EC space, which contains a small amount of media.
In a recent paper, AcuSyst perfusion cell culture of a TNF-β fusion protein, an anti-HIV-1 replication recombinant protein (Mut-F), an HC3 recombinant protein and the human–murine chimeric granulocyte-macrophage colony-stimulating factor (GM-CSF) fusion protein was reported to provide higher yields than in traditional culture in all cases.10 It was also estimated that cost savings for these low-producing cell lines would be significant, largely due to a reduction in seed train materials and labor. The perfusion bioreactors required significantly less inoculum per cartridge compared to stirred tank bioreactors.
It is also worth noting that in October 2016, Repligen launched a single-use version of its XCell™ ATF system for continuous harvesting.11 The single-use format aligns with continuous processing workflows, offering ease of use and faster implementation (over 80% reduction in time) with equivalent performance to the stainless steel line, according to Tony J. Hunt, Repligen President and CEO.
Questions Still Need to be Addressed
Despite the numerous advantages of continuous bioprocessing, the biopharmaceutical industry has been slow to widely adopt this manufacturing approach. That should not be surprising given the risks to patient safety associated with any significant change in manufacturing technologies. It is also unlikely that established processes will be switched from batch/fed-batch to perfusion cell culture given the extensive work that must be completed to demonstrate equivalency.
There are other issues as well. In its 2013 10th annual industry survey, BioPlan Associates found that respondents identified most issues commonly encountered during bioprocessing as being more serious with perfusion than with batch processing, and that the perception of problems with perfusion increased since the previous survey conducted in 2010.1
In addition, while the evidence does seem to be mounting, the question remains as to whether or not perfusion cell culture does indeed provide cost savings and reduced development times compared to batch/fed-batch processes.2 Advances are still needed in process analytical technology to ensure the level of real-time monitoring and control necessary to achieve truly optimum perfusion processes, such as for the direct determination of protein folding, aggregation, glycosylation, oxidation, and contamination, according to Parrish Galliher, CTO of Xcellerex, a part of GE Healthcare’s Life Sciences business.3
But the Prospects for Perfusion Seem Bright
These issues are coming into focus, however. FDA actively encourages the adoption of continuous processing and is working closely with the biopharma industry to develop analytical, control and other solutions.12 Notable advances are being made by equipment vendors. Pumps, feedback controls and online analytical tools are continuously improving, allowing for improved processes.4 Equipment vendors such as Pall Life Sciences, Applikon Biotechnology, Sartorius, AcuSyst, GE Healthcare - and many others - are developing effective, single-use bioreactors, sensors and other systems to simplify setup and operation of perfusion cell culture.
Researchers at Patheon Biologics proposed a four-step approach to reducing the complexity of perfusion processes: confirm that perfusion is the best processes; determine the optimum media composition to provide good culture performance at the lowest cost; optimize the seed, ramp and perfusion processes (ATF, AWS, etc.); and link the perfusion and purification processes.13 Using a quality-by-design with a design of experiments approach facilitates the development process and reduces development timelines.
In fact, the complexity of perfusion processes can be one of perspective, according to Weichang Zhou, Senior Vice President of Biologics Development and Manufacturing at WuXi Biologics. In an interview in December 2016 with Nick Hutchinson, Technical Content Marketing Manager at Sartorius Stedim Biotech, Zhou indicated that the relative degree of complexity of fed-batch and perfusion cell culture depends on which manufacturing approach an operator is used to.14 In addition, development can be faster with a lower initial investment because perfusion processes are smaller scale, and once a perfusion platform is developed, it is possible to construct additional modules in different locations to meet global supply, rather than build one large facility. Wuxi Biologics installed two 1000L single-use bioreactors at its facility in Wuxi city for increased flexibility to produce both stable and unstable proteins. Zhou noted that biopharma manufacturers in emerging markets that are constructing new facilities are not tied to legacy infrastructure and have the opportunity to leverage the benefits of perfusion technology.
Media designed specifically for perfusion cell culture that enables very high cell densities and specific productivities at low perfusion rates with improved waste management and reduced cost of goods4 are becoming available. MilliporeSigma, for instance, launched its EX-CELL® Advanced HD Perfusion Medium in May 2017.15 The products, according to the company, is the first off-the-shelf medium designed to facilitate high productivity at low perfusion rates, increasing production yields and reducing costs. "This launch is a major milestone on the road to truly enabling next generation processing," said Udit Batra, CEO, MilliporeSigma.
- E.S. Langer and R.A. Rader, “Continuous Bioprocessing and Perfusion: Wider Adoption Coming as Bioprocessing Matures, ” BioProcessing Journal, Spring 2014. http://www.bioinfo.com/Continuous_Bioprocessing.pdf
- C.A. Challener, “A Look at Perfusion: The Upstream Continuous Process,” Bioprocess International Continuous Processing Supplement June 2016. http://www.bioprocessintl.com/upstream-processing/perfusion-cell-culture/a-look-at-perfusion-the-upstream-continuous-process/.
- C.A. Challener, “The Potential of Perfusion,” Pharmaceutical Technology, 39 (8), pp 36-38 (2015).
- B. Lehr and D. Lyons, "Perfusion in the 21st Century," BioPharm International 29 (8), pp 24-26 (2016).
- M. Dorceus et al., “ Comparing Culture Methods in Monoclonal Antibody Production: Batch, Fed-Batch, and Perfusion,” BioProcess International, March 20, 2017. http://www.bioprocessintl.com/analytical/upstream-development/comparing-culture-methods-monoclonal-antibody-production-batch-fed-batch-perfusion/
- C.A. Challener, “High Titers and Perfusion Processes Challenge Cell Harvesting Systems,” Biopharm International, 28 (9), pp 28-31,39 (2015).
- Applikon Biotechnology, BioSep Acoustic Perfusion System, http://www.applikon-bio.com/en/news2/itemlist/category/52-biosep
- European Pharmaceuticlal Review, “Pall Life Sciences Launches Commercial Cadence Acoustic Separator for Intensified Bioprocess Clarification and Purification Applications,” News, May 3, 2016. https://www.europeanpharmaceuticalreview.com/news/40821/pall-life-sciences-acoustic-separator/.
- C. Fenge et al., “Continuous Cell Culture Operation at 2,000-L Scale,” BioProcess International, November 17, 2016. http://www.bioprocessintl.com/2016/continuous-cell-culture-operation-2000-l-scale/
- S. Waninger et al., “Difficult-to-Express Proteins: Resolving Bioprocessing Challenges with a Scalable Perfusion Bioreactor,” BioProcess International, May 19, 2017. http://www.bioprocessintl.com/manufacturing/continuous-bioprocessing/acusyst-scalable-perfusion-bioreactor-difficult-express-proteins/.
- Repligen, “Repligen Launches Single-Use XCell™ ATF System for Bioprocess Intensification,” Press Release, Ovtober 5, 2016.
- Lawrence Yu, FDA Voice, April 12, 2016 http://blogs.fda.gov/fdavoice/index.php/2016/04/continuous-manufacturing-has-a-strong-impact-on-drug-quality.
- P. Jorjorian and D. Kenyon, “How to Set Up a Perfusion Process for Higher Productivity and Quality,” BioProcess International, April 17, 2017. http://www.bioprocessintl.com/upstream-processing/perfusion-cell-culture/set-perfusion-process-higher-productivity-quality/
- N. Hutchinson, “Perfusion Cell Culture Processes: WuXi’s experience from China: WuXi Biologics sees an opportunity to start with a clean slate,” BioPharma Asia, December 19, 2016. https://biopharma-asia.com/featured-article/perfusion-cell-culture-processes-wuxis-experience-from-china/
- MilliporeSigma, “MilliporeSigma Launches Industry's First Off-the-Shelf Cell Culture Media for Perfusion Processes ,” May 15, 2017. http://www.biospace.com/News/milliporesigma-launches-industrys-first-off-the/456567.