Filtration is a key step in many stages of downstream processing, from product recovery to removal of viral contamination. The last few years have seen many developments in filtration science, including the increasing adoption of single-use filters. Sartorius Stedim Biotech recently hosted its sixth “European Downstream Technology Forum” at its headquarters in Goettingen, Germany, where experts discussed the latest developments and challenges in downstream processing.
Alexander Caliebe, Ph.D., head of production at Richter-Helm-Biologics, described how filtration techniques, such as crossflow filtration (CFF), play a role in improving purification. CFF is used in harvesting crude supernatant, protein dissolution, buffer exchange after refolding or chromatography, and removal of aggregates. There are big differences in these product streams in both physicochemical properties and purity of the product.
In one case involving purification of a chimeric protein made from a Fab fragment with an effector molecule, Richter-Helm-Biologics took on the production process after Phase I, intending to scale up to Phase II. It faced problems with high viscosity because of high cell density and needed to add antifoam, which led to low flux and fouling of membrane filters. The challenge of scaling up and improving this purification, without changing the techniques used, was achieved by preclarification with a depth filter.
“We then went on to make more changes,” Dr. Caliebe explained. “Viscosity and high load were still the main challenges so we went for in-line dilution. We were able to eliminate two CFF steps by introducing expanded bed absorption chromatography with no loss of product or quality. We also reduced the process time by 50 percent. But this was an unusual case. Cross flow filtration is still a powerful technique.”
Another study reviewed at the meeting involved a recombinant human cytokine that is produced as an inclusion body in E.coli. The original process involved depth filtration and 20-fold concentration with CFF, buffer exchange with CFF, and then dead-end filtration. Unfortunately, high salt concentration led to low-flux and high-buffer consumption. Rinsing the filter and better temperature control led to lower viscosity and better control of the process.
As a result, the purification became robust and efficient with 50% reduction in buffer consumption, reduction of membrane area by 20%, and no loss of product. “Here a small adjustment had a big effect.”