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Higher cell densities are driving innovations in harvesting, including closed systems for intensified processes.
The cell harvest step prepares a clarified, sterile feedstream for downstream purification. The trend toward higher cell densities and the resulting higher biomass coming from the bioreactor create additional challenges for the harvest step, but suppliers aim to meet this and other challenges with various technologies that improve process efficiency
Process robustness and capacity and throughput issues are a challenge, says Vincent Pizzi, BioProcess Upstream strategy leader, GE Healthcare. “Legacy harvest technologies using depth filtration or centrifugation followed by depth filtration have approached this challenge of higher density cell-culture feeds by increasing cycle times or filter area. These approaches have [negatively] impacted the industry process economics and workflow efficiency. Other challenges are the extensive use of water for injection and handling of filter modules with larger volumes,” explains Pizzi.
Madhu Raghunathan, product strategy manager at GE Healthcare, sees increased acceptance and adoption of concentrated fed batch (CFB) and continuous perfusion using tangential flow filtration (TFF) or alternating tangential flow filtration (ATF) for the cell-culture step. “With continuous perfusion, the use of a cell retention device (TFF/ATF) enables direct loading of the product onto the capture chromatography column, bypassing the harvest-clarification steps. However, it is important to choose the correct cell retention device and framework to eliminate and minimize membrane fouling, and to avoid making the perfusion step onerous and labor-intensive,” says Raghunathan.
He says that CFB commonly generates final cell densities greater than 50 million cells/mL, which results in a need for improved harvesting technologies. “At that final density, depth filtration becomes less effective in terms of filtration capacity in liters processed per depth area. Here we see users evaluating the implementation of newer technologies for harvest clarification, such as adding diatomaceous earth to reactors, pH shifts, flocculation, or acoustic separation, among others. These technologies also have challenges, such as the scalability along with system footprint, the need to prove removal of flocculants, and the impact on ion-exchange chromatography steps, for instance.”
Acoustic wave separation enables the continuous removal of cells and cell debris for either batch or continuous bioprocesses. “Pall’s Cadence Acoustic Separator retains recirculating cells from a perfusion bioreactor without the need for a hollow fiber filter device. Having no membrane means no fouling or loss of product, and results have shown simplified integration of the cell retention technology with perfusion bioreactors at cell densities of up to 100 million cells/mL with 100% product transmission under typical process conditions used in the continuous production of biologics,” says Peter Levison, executive director of business development at Pall.
John Bonham-Carter, product line leader for Repligen’s Cell Culture & Clarification Business, says that the company’s XCell Alternating Tangential Flow (XCell ATF) equipment has become an industry standard for cell retention in perfusion and intensified fed batch cultures. “The key advantages are enabling a hollow fiber filter to be used for longer without either cells blocking flow in the lumens or blocking of the filter pores, restricting product harvest. ATF delivers a backflush across the lumen, keeping the pores cleaner, and also flushing cells back to the reactor every 5–10 seconds, [thus] inhibiting blockages,” he explains. “The XCell ATF is used in a N-1 perfusion step in multiple 12–20-kL stainless-steel facilities across the world for several commercial therapies. Additionally, for smaller clinical facilities or for gene therapy manufacturing, the XCell devices are the go-to product for boosting productivity via N-1 perfusion.”
Biopharmaceutical manufacturers are seeking further innovations in cell harvest to improve efficiency. “The variation in cell culture feed experienced in harvesting means titers and yield can be variable, and often more depth filters than might be required are used as a safety factor. As always, people would also like to save time, both on preparation and maintenance of equipment and during the operation of the process step,” says Bonham-Carter.
Repligen has been promoting a relatively new method-high-productivity harvest (HPH) using the company’s XCell ATF equipment-to improve harvest for fed-batch processes. Using ATF eliminates the need for a centrifuge, and it operates as a closed, sterile system. “The HPH method makes a few other changes to reduce impurity build-up and boost protein production. Since the harvest is clarified, no depth filters are required either,” explains Bonham-Carter. He says that Repligen developed the process over several years and is now optimizing and adapting it with interested customers.
Bonham-Carter explains how HPH works: “The sterilized XCell device is attached to the bioreactor in an aseptic manner to keep a closed system. A diafiltration process is started a day or more earlier than at typical harvest, and product starts to be harvested immediately at a slow rate. The media diafiltration has the benefit of keeping cells more viable and so avoiding creation of host cell proteins and other contaminants or byproducts. [Diafiltration also] minimizes degradation of the target protein. On the final day of the fed-batch process, the diafiltration is stopped and the harvest is speeded up, emptying the reactor through the 0.2-micrometer polyethersulfone filter. Depending on initial and final cell concentration, a small diafiltration may be appropriate towards the end of the run, but typically yields are already in excess of 100%, which minimizes the need for complexity or further dilution.”
The yield boost is significant, says Bonham-Carter, and typically 120% to 200% yield is expected. HPH can be used for higher yield (i.e., more protein in the same period of time), or it can be used for faster yield (i.e., harvest earlier to increase throughput). This decision should be balanced with the cost of media, because more media is needed if throughput is higher, notes Bonham-Carter.
HPH is scalable, adds Bonham-Carter. For example, one ATF-10 is running in a GMP facility with a 2000-L bioreactor using fed-batch. A 5000-L fed batch system could use two ATF-10s, he explains.
The XCell ATF is available as either a stainless-steel or single-use system, both of which use a single-use hollow fiber for filtration. “A steel version is often preferred for those people who already have an investment in a large steel facility equipped with steam lines and an autoclave, and those people who expect to run hundreds of batches per year,” says Bonham-Carter. “Single-use devices are preferred by those who don’t have an autoclave, need flexibility and fast start-up/shut down, and run in multi-product facilities.”
Separating microcarrier beads used during adherent cell production is another challenge for cell harvest. “Current techniques require significant capital, routine maintenance, an open system and long cycle times, and could potentially yield low recovery rates,” says Jarv Campbell, senior product manager, Single Use Technologies, Thermo Fisher Scientific. The company’s single-use Harvestainer Microcarrier Separation System was designed to separate microcarrier beads from the cell debris and virus found in the cell culture supernatant in a single-step, closed system that reduces cross-contamination concerns while maintaining high yields, says Campbell.
The system is available in multiple sizes. The 3-L and 12-L Harvestainer systems consist of a preassembled 2D bioprocess container (BPC) and tray that acts as the secondary containment device. The 25-L and 50-L systems consist of a 200-L 3D BPC with a drum as the secondary containment device.
Vol. 32, No. 8
When referring to this article, please cite it as J. Markarian, "Enhancing Cell Harvest," BioPharm International 32 (8) 2019.