December 1, 2010 (Vol. 30, No. 21)
Susan Aldridge, Ph.D.
Tools that Make Process Easier and More Effective Have Positive Impact on Purification
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.”
Jodee Lewis, technology engineer in Pfizer’s biomanufacturing services group, presented work on screening tangential flow filtration (TFF) filters. Ultrafiltration/diafiltration (UF/DF) is ubiquitous to purification of polysaccharide-protein conjugate vaccine intermediates such as Prev(e)nar® (a pneumococcal saccharide conjugate vaccine against Streptococcus pneumoniae). This product is made by conjugating pure polysaccharide and pure carrier and purifying the vaccine. Not surprisingly, it involves a complex manufacturing process with many purification processes.
The UF/DF operation is seen as a source of risk because it is so sensitive to change, therefore, experimental evaluation is required to understand and maybe mitigate, the impact of changes in ultrafilter characteristics as well as to qualify alternate or replacement ultrafilters prior to validation. The application of quality-by-design highlights the impact of small-scale work on regulatory communications throughout a product’s life cycle, Lewis noted.
Lewis discussed an ultrafilter that had to be replaced because the supplier was planning to discontinue the product. “Our strategy was to mitigate risk by looking at a small-scale evaluation and then scaling up.” Lewis’ group devised a small-scale model and then screened a narrow pool of 13 potential filters, thereby generating a large matrix of data.
One or two filters emerged from this exercise for further evaluation. The team then carried out design of experiments with risk assessment, which brought the choice down to one filter. The next stage was to see how this performed in scale up.
Lewis noted some roadblocks and recommendations. For example, analytical testing should be completed during evaluation so that there are no surprises in full scale. Water runs should be performed, although that is not always representative because of the viscosity of product streams. “Tech transfer of the process and knowledge sets a clear path for filter evaluation for submission.”
Transgenic plants are a novel route for production of protein-based APIs, including antibodies. Martin Lobedann, Ph.D., now a process expert at Bayer Technology Services. described work done for his doctoral thesis at the Fraunhofer Institute on the purification of a recombinant anti-HIV antibody, 2G12, which is manufactured in transgenic tobacco plants.
These plant-made pharmaceuticals (PMPs) may offer an economic route to biopharmaceuticals in the future. Partnered with the University of Aachen, this work is part of the EU-funded Pharma-Planta project that aims to advance PMPs.
Dr. Lobedann described the various downstream processing steps he developed for the purification of 2G12. First there was a dispersion and extraction step, followed by a four-step filtration. The first filtration stage removed fiber from the antibody-containing crude extract, followed by clarification with disposable filters. Antibody capture by protein A affinity chromatography, followed by ion-exchange chromatography in combination with virus filtration, was used for 2G12 isolation.
Finally, Dr. Lobedann carried out an UF/DF step for buffer exchange and concentration adjustment. Overall yield was 60%, which is 3.6 g of antibody from 216 kg of tobacco leaves; and 250 kg of leaf material can be processed in one working day. 2G12 is expected to enter Phase I soon.
The Pharma-Planta project, which has 39 partners, is not trying to compete with mammalian systems but rather aims to show that plants can produce antibodies under GMP conditions to initiate clinical trials. Possibilities for the future include using plants as vehicles for making vaccines as commodities or for manufacturing them simply and cheaply in emerging countries.
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.
Susan Aldridge, Ph.D. (firstname.lastname@example.org), is a freelance science and medical writer specializing in biotechnology, pharmaceuticals, chemistry, medicine, and health.