Charles Meadows (product manager at Sartorius Stedim Biotech) joined BPI for an Ask-the-Expert webinar on 13 June 2019 featuring the MYCAP CCX cell culture expansion system. He showed how a closed aseptic cell expansion process can decrease the rate of batch failure from contamination, in turn reducing waste and overhead costs.

Meadows’s Presentation
Contamination is an inherent risk of open cell culture expansion systems. Each passage from one flask to the next potentially exposes cellular material. A recent BioPlan Associates survey noted that batch failures from contamination occur once every 9.4 months, costing US$1–2 million. Contamination is especially troubling for facilities that make <1,000-L batches, with 2.3% of them lost to culture corruption. But expensive countermeasures such as biosafety cabinets cannot eliminate the risk of contamination.

With those challenges in mind, Sartorius Stedim Biotech developed a “lean” expansion process to reduce the need for biosafety cabinets while limiting contamination risk. The MYCAP CCX system combines two simple technologies: vented caps and integral tubing. Individually, they do not support aseptic cell expansion. Large membranes in vented caps enable adequate gas exchange but prevent access to material without opening flasks. Integral tubing makes aseptic fluid transfer possible but restricts air flow at the hose barb, preventing adequate gas exchange for cell cultures.

The MYCAP CCX system enables both adequate gas exchange and aseptic liquid transfer. It features a gas exchange cartridge with a surface area of 361.9 mm2, which is significantly larger than that of traditional disc filters and vented caps. That maximizes the filter’s membrane and footprint, permitting gas exchange and tube embedding while reducing contamination factors.

Evaluation results are promising. Parallel expansion studies using Chinese hamster ovary (CHO) cells on 500-mL to 3-L flasks have showed no significant differences in culture doubling times between traditional flasks and the MYCAP CCX system, although results vary by cell line. Sartorius Stedim models also indicate a 9% reduction in operating costs through decreased labor based on market prices for material and testing services. Facilities using this system can expect to pay more for consumables, but cost–benefit models show decreases in other spending.

The MYCAP CCX system offers substantial benefits with modest changes. The system demonstrates that it is possible to expand cell growth with closed, aseptic fluid transfer while eliminating the need to work under the hood of a biosafety cabinet.

Questions and Answers
What is the void volume in this sampling line? Current designs specify small-diameter tubing for the sampling line, so the hold-up volume is quite low. On 250-mL and 1-L flasks, that void volume is in the range of 0.5–0.6 mL. With 2-L and 3-L flasks, it gets to about 0.9 mL.

It is important that this volume be low, especially for smaller flasks, because cultures are left in the line after the first sampling. The void volume needs to be purged before the final day’s sample is collected. A small volume in the tubing will limit total losses and purge volumes needed before evacuating a line.

Do flasks need to be stabilized in an incubator to prevent tipping? Yes. The MYCAP CCX system uses standard Corning Erlenmeyer flasks, so it will fit into whatever stabilization method is used. The system is top-heavier than traditional flasks because of tubing in the fittings. But Sartorius Stedim experiments and feedback from customers suggest that something as simple as double-sided sticky tape will be enough to stabilize a flask while it is shaken in the incubator.

Do dip tubes cause any problems? They need to be resting in the culture because liquid must be drawn out of the flask. The tubes are made from silicone, which is commonly used in production processes, so there is little concern about it negatively affecting cell growth. Because the tubes sit in a culture while it is shaken, we wanted to see whether that might cause any physical damage. We studied cell growth rates in the MYCAP CCX system and in traditional flasks with different shaking speeds to create more and less turbulence around those dip tubes. We found no difference in the growth rates.

How should inoculum be measured inside the flasks? We recommend placing a receiving flask on a balance and making transfers by weight. Sartorius Stedim made an application specific for this expansion process, so the balance can calculate the volume of media and culture to be added to each flask. The balance is connected directly to a pump and will control the starting and stopping of that pump once it has achieved the targeted volume. Effectively, this application automates cell passaging to remove operator error risk and create good documentation of transfer volumes throughout the passages.

More Online 
The full presentation of this webcast can be found on the BioProcess International website at the link below.

Watch the full webcast now.