With an increasing number of biologics being introduced into the marketplace, fierce competition has come into play in the bioprocessing industry.
Pressures are building to increase productivity, cut down the cost of goods, bring more products into development, reduce time to the clinic, and implement quality by design—all simultaneously.
A number of bioprocessors are meeting these challenges by changing their platform technologies, including introducing new techniques. Lonza Biologics, for example, has developed a platform process for monoclonal antibody purification, designed to fully optimize the operation.
According to Mardon McFarlane, Ph.D., senior scientist, the goal is to enhance the cost of goods by critically evaluating the polishing steps. Some of the pivotal decisions that were made include elimination of the gel-filtration step and selection of improved cell lines for increasing productivity.
Dr. McFarlane addressed downstream issues at the recent “European Downstream Technology Forum”, sponsored by Sartorius Stedim Biotech in Göttingen, Germany.
Dr. McFarlane stressed that with improvements in fermentation titer, comes the downside of an increased time required for purification and as much as a fourfold increase in demand for raw materials. Buffer needs are especially daunting with such concentrated preparations.
By the mid 2000s the pileup at the downstream end of the process had become acute. To alleviate a downstream bottleneck, the Lonza team adopted less compressible matrices with faster flow rates and higher binding capacities. They now employ the Mab Select SuRe, a high-flow agarose matrix with an alkali-stabilized Protein A-derived ligand. Developed by GE Healthcare, the rigid matrix of this product yielded substantial reductions in buffer demand.
The large-scale requirements for buffer have proven to be the most complex component of the entire process train. In order to meet these needs, Lonza’s prep pilot plant was retrofitted with four 600 kg balances and overhead lighting mixers.
The system employs single-use disposable bags. Their flexibility for different tasks and rapid turnaround time represent important advantages, according to Dr. McFarlane, with the caveat that at times bags will leak, and they must be carefully quality controlled during the purification process.
As a part of the move to single-use technologies, Dr. McFarlane outlined the application of single-use chromatography columns. They come with numerous benefits, including reduction in setup/teardown time, water handling, cleaning and validation requirements, which boils down to huge savings in overall time utilization.
Another advance in purification technology is the move to membrane chromatography and away from traditional column chromatography. In membrane chromatography, the open pores and accessible ligands mean that there is negligible diffusion required for binding and there is no need to pack a column. No post cleaning was required and buffer consumption was greatly reduced.
The larger pores in the membranes insure that flow rates are much higher, resulting in additional time savings in the entire process operation. Loading capacities for Q membranes can be extremely generous, up to 3 grams per mL for some IgGs. This high capacity translates to smaller size for the equipment and cartridges. Over the course of the redesign process it was possible to lower total buffer volumes for a 10 kg purification platform from 50,000 to 14,000 liters.
In summarizing Lonza’s redesign of the platform process, Dr. McFarlane noted that “we have improved its predictability and the changeover to disposables has greatly upgraded our competitive position within the industry.”
Antibody Polishing and Purification
Purification of therapeutically armed antibodies offers a unique bioprocessing challenge, according to Leonardo Giovannoni, Ph.D., head of chemistry, manufacturing, and controls at Philogen. The company is developing antibody products for the treatment of angiogenesis-related disorders, specifically bioactive agents coupled to antibodies for targeted delivery.
For many years, antibody conjugates have not been pursued aggressively, but the technical drawbacks that limited their performance have been largely resolved and many companies have advanced immunoconjugates to clinical trials.
The company has an antibody-TNF fusion protein and an antibody-IL2 fusion protein in development, both now in Phase II trials, which have necessitated the establishment of large-scale antibody production.
Dr. Giovanni discussed in some detail the downstream purification and polishing of the F16-IL-2 conjugate, including affinity chromatography followed by anion and cation exchange, desalting, and a final nanofiltration step.
The Philogen group is partial to the use of Sartobind Q columns for endotoxin, DNA, and Protein A removal. Dr. Giovannoni also compared the use of disposable Sarto S and Sarto Q capsules against conventional ion-exchange chromatography medium packed in reusable columns. The disposable technology was superior, he said, providing shorter total run duration, shorter gradient duration, much less buffer consumption, while avoiding cleaning validation.
Nanofiltration as an alternative bioprocessing platform offers the possibility of cost reduction while achieving effective purification of plasma proteins, reported Merche Faro, Ph.D., of Grifols. Dr. Faro discussed her group’s experiences with nanofiltration-based purification of fibrinogen and intravenous immune globulin, which is used to treat immune disorders and boost the immune response.
According to Dr. Faro, therapeutic products obtained from biological sources pose a particular challenge, as they must be demonstrated to be free of pathogenic agents, both as raw materials and as final products.
Furthermore, regulatory governance bodies require viral safety steps, based on inactivation and removal platforms. Inactivation options include solvents and treatment with caprylate, dry heat, and pasteurization. The removal step detailed by Dr. Faro is achieved by nanofiltration through small (15−50 nm) pore-size membranes.
“This is a robust, reliable, and flexible clearance technology,” stated Dr. Faro, “It permits efficient removal of both enveloped and nonenveloped viruses, and we have achieved high protein recovery with no undesirable effects on the product.”
The company has included a nanotechnology process in its purification train for over 15 years, to a point where it is now a routine step. Proteins filtered through the small virus retentive filter membranes include thrombin, Factor IX, and a number of other candidates.
Dr. Faro explained that each application must be individually tailored for that protein. “One must take into account the features of that individual protein and the manufacturing process to which it will be subjected,” she pointed out.
Not only is the molecular weight of the protein critical, but stability, isoelectric point, and the concentration of the protein solution must all be factored into the protocol. These properties will affect the optimization of the filtration medium and decisions regarding additional polishing steps. In choosing and monitoring membrane performance, membrane structure and filtration conditions will critically impact recovery yield and thereby the cost of goods.
Dr. Faro further discussed her experiences with nanofiltration of the fibrinogen molecule. This 340 KD glycoprotein is a critical component of the coagulation cascade and is widely used as a supplement in surgical procedures. It is structurally complex, containing two sets of three different polypeptide chains.
For this reason it must be handled with care, and Dr. Faro’s team incorporated gentle filtration conditions into their protocol, including the addition of stabilizers to the filtration medium. Addition of a freeze/thaw step greatly improved recovery by removing aggregates that tend to block the filter. This optimized process could be expanded to industrial scale with excellent performance, she said.
The second application of the nanofiltration technology discussed by Dr. Faro was to intravenous immune globulin purification, a major protein found in high concentrations in plasma. It possesses a wide range of critical therapeutic applications, such as treatment of immune deficiencies and autoimmune diseases.
Dr. Faro and her colleagues measured a number of parameters, including different membrane pore sizes and protein concentrations of the filtration solutions. The 20 nm pore size with a 2−3% protein concentration proved to be optimal. Filter size and brand were also important variables that were considered in the optimization process.
“We found that a dedicated optimization protocol is required in the development of a nanofiltration-based platform for each protein,” said Dr. Faro. “Critical aspects that allow optimal nanofiltration performance are unique for each protein, manufacturing process and nanofilter. Once successfully achieved, we observe robust and consistent virus retention capacity under a wide range of process conditions.”