New drug approvals in the U.S. and EU continue in the range of 20 to 25 per year, with vaccines and monoclonal antibodies accounting for the lion's share. While this rate is lower than what was anticipated, the ongoing development and introduction of new antibody-based pharmaceuticals is putting pressure on the industry to improve bioproduction processes.
Tool companies, including Sartorius Stedim Biotech, are continually introducing new products to surmount antibody purification challenges. The Sartorius Downstream Forum, held recently in Goettigen, Germany, presented some of the company's new technological offerings as well as case studies detailing their application.
Frank Meyeroltmanns, head of product management crossflow, and Anika Meyer, product manager for viral clearance, discussed the relative advantages and disadvantages of single-and double-layer filtration devices in processing proteinaceous solutions. Efficient methods of cell and debris removal are critical for downstream processing. Whereas double-layer filters have greater dirt-holding capacity per square meter, the build-up of material can cause problems and slow the filtration process.
Meyeroltmanns focused on crossflow filtration, in which the influent stream (feed) is divided into two effluent streams, defined as the retentate and the permeate. The concentrate or nonfiltered portion “crossflows” over the membrane at high linear velocities. This flow creates a continuous self-cleaning sweeping action.
In an optimization procedure the crossflow efficiency is maximized, thus allowing the use of higher transmembrane pressures, which result in increased flux, translating to higher permeate rates, he said. The goal is to reach an optimal flux with a minimal decline during filtration.
Downstream single-use technologies have been slow to mature, saddled by low capacity, high cost, and poor scalability. However, according to Meyer, Michael Kuczewski, scientist I in downstream process development at Percivia, and his team found that single-use Sartobind® filter units meet the challenges posed by the large amount of antibody generated upstream by a suspension culture of PER.C6 cells.
Meyer also discussed integrated and orthogonal viral-clearance technologies. There are a number of options available to meet various purification protocols, she said. Inactivation with the UV-C system is highly effective for small, nonenveloped viruses, while the FlexAct VI system targets large enveloped viruses.
Ablynx' signature product is the Nanobody®, which is based on the heavy chain antibodies produced by members of the camel family. It is the smallest antibody unit that has full functional binding ability, according to Willem van de Velde, Ph.D., a scientist team leader, and for this reason is of great interest in the area of antibody engineering.
One of the notable achievements of the Ablynx program has been the fast tracking of Nanobodies directed against cancer target CXCR4, Dr. van de Velde said. This cell surface molecule plays a role in immune signaling and its overexpression has been correlated with poor response in various types of cancer.
Several years ago Ablynx developed a number of formatted, functional Nanobodies, including potent antagonists of CXCR4. One of these, ALX-0651, has moved through clinical testing in non-Hodgkin lymphoma and multiple myeloma, and an IND filing with the FDA is expected during the course of 2011.
Dr. van de Velde reported that Nanobodies have many advantages over conventional monoclonal antibodies, including the fact that the molecules are more physically stable and characterized by a lower propensity for aggregation.
Using Aggresolve™ software designed by Lonza, Dr. van de Velde and his colleagues were able to select the amino acid sequences for the Nanobodies that showed the least chance of misfolding and forming aggregates. The program can suggest sequence changes to reduce aggregation and stabilize engineered proteins. It can be used to either engineer sequences de novo or correct an already generated framework.
Nanobodies have other auspicious properties including resistance to pH extremes, high temperatures, and proteases. This is not surprising given the fact that they are much smaller than traditional antibody molecules. Because of their small size, manufacturing costs are also reportedly less, typically one-third the cost of conventional antibody production.
When Dr. van de Velde and his co-workers tested a number of antibodies, they found a range of production over six orders of magnitude. Pichia is clearly preferable to E. coli as a host, in that a typical Pichia production run yields greater than 95% pure Nanobody.
Considering the overall process, Dr. van de Velde argued that Nanobodies offer a considerable range of advantages over other antibody options. “Host creation is less time consuming, growth of cell lines uses no animal products, Nanobodies are secreted and there is no viral clearance required, and, finally, Nanobodies show long-term stability and are homogeneous and simple molecules.”