A handful of new approaches that may make bioprocess filtration easier and more effective are emerging. David Delvaille, biotech process sciences project coordinator at Merck Serono, described the application of highly purified diatomaceous earth (DE, Celpure®) in bioprocess filtration.
DE has been used for decades in the food and blood fractionation industries. It is also used in mammalian cell culture downstream processing as a component of depth filters used for cell clarification, in nanofilter prefiltration, and more recently, for removal of contaminants. Celpure has low endotoxin and low impurity levels and is available for GMP manufacturing, according to Delvaille.
“Diatomaceous earth is like the Swiss army knife—the all-purpose tool for downstream processing,” he added. Merck Serono in France manufactures a range of products up to Phase II scale in CHO cells. It is using depth filtration as a low-cost, single-use technology for harvest clarification and precipitation filtration as well as for refiltration for virus removal. Depth filtration is commonly used to remove cell debris from fed-batch culture harvest, where it replaces microfiltration and is typically used after centrifugation and precipitation. Depth filtration is known to remove host cell protein (HCP), DNA, viruses, and endotoxin.
Delvaille noted that depth filtration to remove HCP in purification of an Fc-fusion protein and an antibody led to purification factors of 2.3 and 123, respectively. It is not clear whether the mechanism of contaminant removal in such cases is anion exchange, cation exchange, or by hydrophobic interaction.
For further investigation, the Merck Serono team decided to try out DE to demonstrate if it was capable of reproducing or even improving the performance of depth filtration. The team also wanted to establish the optimum role for DE—whether it is for clarification, polishing, or maybe both. HCP removal was 33–42%, compared to only 8% by depth filter. “Diatomaceous earth can clarify in a single step, and this can be scaled up to 50 liters,” Delvaille noted. The team is now exploring the use of DE for other cell types and scales.
DE has also been evaluated for impurity removal in an antibody and Fc-fusion protein manufacturing process. The experiments tested ionic exchange and hydrophobic interaction mechanisms, three different grades of Celpure, and different capacity and residence times. The first trials on clarified harvests showed the ability of DE to remove up to 50% of impurities, while leaving more than 98% of the antibody/protein in the filtrate.
DE can be used for clarification and impurity removal—and perhaps both at the same time. It allows more efficient use of single-use technologies and could replace centrifugation. DE can also be used in different conditions, either during downstream processing or directly on the cell harvest, by using different interactions. And, there is also the potential for using DE for viral clearance as is done in the blood products industry.
Finally, Sartorius Stedim Biotech scientists presented some of the company’s recent developments in filtration technology. Peter Schmidt, application specialist, spoke about the role depth filters play in recovery before purification. There are three approaches: classic centrifugation followed by depth filtration and membrane filtration, microfiltration and membrane filtration, and two depth filtrations followed by membrane filtration.
The Sartoclear® single-use cellulose-based depth filter can be used for all these approaches. The technology comes in a range of sizes including a drum format and is plug-and-play, easy-to-use, and a low-cost investment, according to Schmidt. Meanwhile, the FlexAct Cell Harvest product, a configurable disposable cell-harvest solution, includes a pressure-controlled filtration process.
For CFF, the Sartocon® Eco increases production capacity, allowing for more filter area in an existing system, thereby reducing consumable and investment costs. “This will enable more economic production and increase production capacities,” Schmidt added.
Another new product is the Sartoflow® Slice, which is automated for R&D and small scale, and the SartoflowAlpha Plus, which is a steel hybrid single-use product appropriate for CMOs or any manufacturer that often changes products.
Amélie Raveneau, a Sartorius Stedim Biotech application scientist, then discussed the importance of orthogonal virus clearance. This multiple approach, increasingly required by the regulatory authorities, involves ultraviolet-C irradiation in combination with membrane chromatagraphy and nanofiltration.
One of the company’s new products in nanofiltration is Virosart® CPV, and Raveneau described some non-GLP virus-spiking studies in orthogonal virus clearance, carried out with WuXi Apptec. She noted that Virosart HC is applicable where Virosart CPV is not, and it will soon be available for blood and plasma viral inactivation.