May 1, 2011 (Vol. 31, No. 9)

Transitioning from Bench to Production Floor via Gas-Permeable Technology

In the development of a new biological product, it is important to remember that a successful R&D development project needs to be converted from a bench-scale concept to a bioproduction platform. This process-development stage requires identifying ways to increase cell numbers in order to meet annual dosage demands, as well as to minimize direct contact with the cell product by developing a closed system for all culture manipulations. Identifying a bench-scale platform that can scale to cGMP production scale eases the scaling process.

Corning Life Sciences first introduced the use of gas-permeable film surfaces for cell culture with the HYPERFlask® Cell Culture Vessel and recently expanded to include the newly developed HYPERStack Vessels. These cell culture vessels simplify the transition from bench to production floor. The HYPERStack™ Vessels have been designed for closed system use to meet the needs of large-scale adherent cell culture.

The gas-permeable technology and product design differs from traditional cell culture products as the cells attach to an ultrathin, gas-permeable polystyrene film, instead of a relatively gas-impermeable, injection-molded polystyrene surface. The gas-permeable film allows for the elimination of the air gap within the flask resulting in an overall reduced vessel height. For optimal cell attachment and growth, the gas-permeable film is Corning CellBIND® Surface treated to create a more hydrophilic, oxygen-rich surface.

In order to optimize cell growth in an aerobic environment, gas exchange between the culture medium and cells needs to be optimized. The gas-permeable film is used to build growth chambers, as depicted in Figure 1, where the gas exchange between the cells and the atmosphere occurs in the tracheal spaces located directly beneath the cell layer. This differs from the traditional culture methods wherein the gas exchange occurs between the media surface and a large air gap above the cultures, such as in a traditional T-flask. The ultrathin gas-permeable film technology ensures not only efficient gas exchange between the medium and the cells but also enables even gas exchange across the entire surface area.

The design also allows for the stacking and manifolding of multiple growth chambers in a compact design. For comparison purposes, a HYPERStack-36 Vessel, which provides 18,000 cm2 growth surface area, requires a similar amount of incubator space as the Corning CellSTACK® 10 layer Culture Chamber, which consists of 6,360 cm2 cell growth surface area. This results in the HYPERStack Vessels having high efficiency with regard to cell growth surface area per unit volume occupied. Additionally, because the dimensions of the HYPERStack Vessels maintain the perimeter footprint of the traditional Corning CellSTACK Culture Chambers, these vessels are compatible with existing automation equipment.

Figure 1. Corning HYPERStack Cell Culture Vessel principle of operation: The cell growth surface area for each gas-permeable layer is 500 cm2. The layers are joined through manifolds to form equivalent cell growth chambers designed to contain approximately 100 mL of fluid (0.217 mL/cm2).

In order to scale up production without significantly increasing cost, ideally there is no requirement to significantly add equipment or space to the production area (i.e., incubators, cleanroom space). The data in Figure 2 demonstrates that the product output from HYPERStack Vessels is approximately three times higher when compared to vessels that utilize traditional multilayered flask technology while occupying the same space in the incubator or warm room.

MDBK cell growth in the traditional two-layer multilayered vessel was compared to growth in the HYPERStack-12 Vessel (Figure 2A). CHO-K1 and Mesenchymal Stem Cells (MSC) were cultured in either the HYPERStack-36 Vessel or traditional 10-layer vessels and total cell yield compared (Figure 2B and 2C, respectively). This is significant for large-scale manufacturing, as the end result is an increase in the product throughput per manufacturing area, thereby controlling costs.

Another key feature incorporated into this vessel design making the scaling to production level easier, is the use of BSE/TSE-compliant C-Flex tubing for liquid manipulations. All liquid transfer is conducted through the liquid-handling tubing connected to one manifold while air escape and entry occurs through the other manifold. (Figure 1). For closed systems, addition and removal of fluids occurs between the HYPERStack Vessel tubing set and various auxiliary vessels, such as media bags, tube welding, or the use of aseptic connectors.

Working with an optimized set of flexible bioprocess containers, such as TheraPEAK™ bioprocess containers by Lonza, a product expansion project can be conducted from vessel seeding through culture expansion and harvest in a completely closed system. Utilizing a closed system not only greatly reduces the risk of culture loss due to contamination, but makes the process more suitable for cGMP manufacturing.

The Lonza Walkersville cell therapy development team has developed closed systems allowing up to 70 vessels to be handled in a single harvest, yielding 10 billion cells or greater harvests for many cell types.

The HYPERStack cell culture vessel family has been developed as a modular system, with the HYPERStack-12 as the single module. These modules can be stacked, allowing the process to be scaled as required for the manufacturing facility and the annual dosage requirements of the product. Currently, the HYPERStack-12 and HYPERStack-36 Vessels are available, and the HYPERStack-120 Vessels are expected to be available by the end of 2011.

Figure 2. Total cell yield comparison between Corning HYPERStack cell culture vessel and traditional multilayered vessels: (A) MDBK cells were seeded at 3,000 cells/cm2 in either a traditional two-layer vessel or the HYPERStack-12 vessel and cultured for 96 hours prior to harvest and counting. (B) CHO-K1 cells were seeded at 3,000 cells/cm2 in either a traditional 10-layer vessel or the HYPERStack-36 vessel and cultured for 96 hours prior to harvest and counting. (C) MSCs were seeded at 5,000 cells/cm2 in either a traditional 10-layer vessel or the HYPERStack-36 vessel and cultured for seven days prior to harvest and counting.

The modularity and stacking ability of the HYPERStack Vessel allows process-development departments the option of increasing the lot size by using either multiple smaller HYPERStack Vessels or a single larger HYPERStack Vessel. The Table depicts a potential scaling strategy based on the amount of product required.


The Corning gas-permeable family of products, which began with the HYPERFlask Cell Culture Vessel and has recently expanded with the addition of the HYPERStack Vessel, are useful for easing the scaling process from benchtop to cGMP production.

The gas-permeable film construction of the HYPERStack Vessels ensures equal gas exchange for all cell culture compartments in the vessels. The “tracheal space” eliminates the need for an internal air gap, making the vessels more compact, saving laboratory and production space.

Depending on the optimal lot size for production requirements, either the HYPERStack-12 with 6,000 cm2 of cell growth surface area or the HYPERStack-36 with 18,000 cm2 of surface area can be selected. For manufacturing-scale production, the 60,000 cm2 HYPERStack-120 layer Vessel may be the optimal choice.

Elizabeth Misleh is a research associate II at Lonza, Jacob Pattasseril is engineering manager, CT R&D at Lonza, Cindy Hunt is alliance manager at Lonza, Pilar Pardo is cell biologist at Corning Life Sciences, Renee Gallagher is cell scientist at Corning Life Sciences, Vitaly Klimovich ([email protected]) is assistant product line manager at Corning Life Sciences, and Greg Martin is senior product development engineer at Corning Life Sciences.

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