August 1, 2011 (Vol. 31, No. 14)
K. John John Morrow Jr. Ph.D. President Newport Biotech
Researchers Incorporate Innovative Technologies into Processes to Address Purification Challenges
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.”
Scientists at ImClone Systems, a wholly owned subsidiary of Eli Lilly & Co., are using Sartorius Stedim Biotech’s antibody polishing platform, Sartobind Salt Tolerant Interaction Chromatography (STIC) to improve the platform process, according to Kenneth Kang, Ph.D., a senior scientist and the head of the purification team in the bioprocess sciences department.
In order to process higher titer cell culture and meet higher production requirements, Dr. Kang and his team evaluated higher capacity protein A resin as well as alternatives. They also worked to integrate a higher capacity AEX polishing step while incorporating single-use devices, reducing the process steps and developing a buffer that was both “process- and product-friendly.”
Sartobind STIC is based on the use of a polyallylamine ligand covalently coupled to a double-porous membrane. This feature allows a higher flow rate and a shorter processing time, speeding the purification process and possibly reducing costs. In order to achieve the optimum conditions, Dr. Kang and his team set up a Sartobind 96-well plate configuration with a vacuum manifold. They tested four different human antibodies under a wide range of pH and ionic strength conditions in order to arrive at the most effective purification configuration.
The beauty of 96-well plates is that each well constitutes a micro-experiment, allowing a multidimensional plot to be assembled resulting in a fine-tuning of conditions that would not be possible through conventional macroscale evaluation. Considering each mAb has a unique physical-chemical profile, the approach followed by ImClone scientists facilitates the development of optimal purification conditions for each individual antibody.
The results of these screening and optimization protocols allowed Dr. Kang and his co-workers to design an alternative mAb polishing step, while reaching an acceptable process performance. A critical modification of the process was the elimination of the post protein A tangential flow filtration step.
“These newly improved modifications, including the developed polishing step, can be easily integrated into current mAb purification platforms if needed, especially for problematic candidates that do not behave well in the current production process,” Dr. Kang added.
John Milne, Ph.D., technical manager at Biouetikon, discussed his company’s experiences with single-use technologies in the contract manufacturing of biologics. “In principle we would anticipate lower initial capital expenditures, shorter facility development time, lower validation costs and expenditure of time resources, and fewer constraints on the existing facilities layout,” Dr. Milne indicated.
Every step in downstream processing has separate challenges for single-use technologies. For buffer preparation, magnetically coupled stirrers allow the system to remain closed and sterile. Single-use bioreactors are now available in sizes up to 2,000 L. These are highly complex systems, as many parameters must be monitored in the course of a run.
“Many case studies have now shown that single-use stirred tank bioreactors can mimic reusable bioreactors in geometry, design, cell growth, viability, and specific product expression. The technology has advanced to the point that a completely disposable process manufacturing train is now possible.”
In membrane chromatography, single-use technologies can offer some dramatic advantages, such as 95% less buffer consumption, simple setup protocols, and higher throughput for trace impurity removal than traditional packed columns. There is no packing, no regeneration, and no re-use validation required.
However, concerns have been voiced regarding a number of aspects of disposable implementation, such as high cost and a lack of continued innovation within the industry.
There is also the issue of extractables and leachables, compounds that could migrate from the disposable unit into the biologic material being processed. A thorough evaluation of all the materials that the product comes in contact with is difficult in earlystage development, given time requirements and the rapid turnover of different artificial materials. It is an ongoing issue that will require analytical expertise and the use of a stringent risk-assessment framework.