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Feature Articles : Dec 1, 2012 (Vol. 32, No. 21)

Inventive Approaches Redefine Downstream Ops

  • K. John Morrow Jr., Ph.D.

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.

Single-Use Chromatography

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 Technologies

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.”

E. coli Secretion Technology

Wacker Biosolutions has developed an E. coli-based protein secretion technology, explained Susanne Dilsen, Ph.D., head of production. Referred to as Esetec®, it is a recovery and purification procedure for high-efficiency Fab isolation.

“Our process consists of the Esetec expression system, a fermentation process, a recovery procedure, and a scalable purification process,” Dr. Dilsen said.

Esetec boasts a specially designed E. coli K12 strain, dedicated expression vectors and a toolbox of helper plasmids, supplying chaperones, disulfide isomerases, and foldases. This strain secretes proteins directly into the medium rather than into the periplasm. The accompanying toolbox is an extensive collection of protease deletion strains, signal peptides, chaperones, and secretion components.

An important feature of the process is screening for the most satisfactory clones, working up the ladder in stages. The primary screening stage is a high-throughput, random method, covering 1,000 to 3,000 clones per week, for optimizing titer. The second level of screening focuses on functionality and can handle 30 to 60 clones per week.

Finally, the strain selection for the fermentation component handles only three to nine clones per week and aims at selection for titer, function, and quality.

Nothing is left to happenstance in the configuration process. All inoculation ratios and preculture conditions are standardized and a chemically defined medium free of animal proteins is employed. Temperatures and temperature down-shift before induction have been identified, as have the effects of additives (e.g., reducing agents, stabilizers, antifoaming reagents). These were tested and the cell-harvesting conditions standardized during the initial design phases of the process.

Dr. Dilsen described case studies using the Esetec technology. The first, a collaboration with Bayer HealthCare, focused on the production of a recalcitrant Fab, sporting five disulfide bonds.

“In this case we were aiming for yields in excess of 0.5 g/L in the culture supernatant, favoring large-scale production,” she added. “This required the development of appropriate analytical methods. The first yields were less than satisfactory, so we used the toolbox to improve the heavy chain expression, folding, and secretion.”

The modifications, which increased yields by twofold, included altering the promoter to increase the expression of the heavy chain while at the same time co-expressing helper elements, folding, and secretion of the Fab, which is now correctly assembled and fully functional.

In a second case study, Dr. Dilsen and her colleagues collaborated with Morphosys to construct mono- and bivalent antibody fragment formats with different specificity, framework, and physico-chemical properties. In one series of investigations with the Esetec platform, a 40-fold increase was obtained that generated functional Fab products.

More on Single-Use

The advantages of single-use, disposable technologies are widely acknowledged, as is substantial enthusiasm for therapeutic antibodies. Klaus Kaiser, Ph.D., head of GMP purification, global biological development, Bayer HealthCare, spoke to the issue of the purification of innovative antibodies with new functionalities. Of note are antibody-drug conjugates and modifications of the protein molecule to stabilize it or enhance its activity.

Many companies are targeting biosimilars, given that they are based on approaches that have already been validated. Their biology is well understood. This leads to the potential for shorter time lines, a much desired outcome in an era of approval processes that can run to many years.

With downstream processing a major component of the drug development mix, it is of critical importance to dissect the costs of the various steps in downstream operations. As would be expected, the Protein A purification step is far and away the most costly, at 40% of the total. This means that it would be very difficult to bring down costs significantly without making inroads into this component.

Whereas disposable downstream technologies are highly favored, they are not without their disadvantages and challenges. This includes overly complex construction and mechanical instability. Yet they do not present the high maintenance costs, including decommissioning and dismantling incurred with reusable bioprocessing technologies.

In the long haul the demands of the market will dictate whether existing large-scale reusable plants will be more cost-effective than the small-scale, single-use option.

In designing the facilities plan, Dr. Kaiser discussed the “ballroom” option, in which processes are so tightly closed that there is no need for the traditional spatial segregations between a variety of upstream and downstream tasks. In this configuration one can group upstream and downstream unit operations in a common production ballroom.

Dr. Kaiser finished his analysis with a discussion of another major factor influencing cost containment. That is the emerging discipline of personalized medicine. Given that most drugs are ineffective in a majority of patients, there is a strong push for drugs targeted to those individuals that will actually respond to the agent. This means that accurate biomarkers will be required, a task that has so far frustrated cancer researchers.

Provided that such tests are forthcoming, this will push pharma companies toward smaller volumes and many more individual drugs. This trend will clearly favor the disposable technologies.

“Single-use technologies have many advantages which let them appear ideal for ‘individualized’ production,” he said.

At the end of the conference, Uwe Gottschalk, Ph.D., vp for purification technologies at Sartorius Stedim Biotech, summarized the state of innovation in the downstream processing field.

“Today we are faced with unprecedented challenges that we cannot meet without truly inventive approaches,” he said. “While there is now a general recognition of this reality within the field, unfortunately this has not always been the case.”

Dr. Gottschalk believes that there are drivers coming from several directions that are overwhelming the fear of moving forward that dominated thinking of companies in the past.

“Most significant are cost and regulatory issues. We’re seeing that there is no easy way out of these problems.

“So I think that the take-home message is that the industry is now firmly committed to original ways of thinking that will move us forward.

Novel Filtration Techniques

New technologies in virus filtration, microfiltration, resins, and membrane absorbers are being introduced by Asahi Kasei Bioprocess, according to John Fisher, senior manager for science and technology. “Today many are giving the virus filtration step the respect that it deserves, recognizing that it is not just a simple filter,” he explains.

With increasing titers in the cell culture processing step, there are challenges being forced on traditional downstream unit operations. Asahi, like many other companies, realizes that the virus filtration unit operation is one of the highest cost of goods components in downstream processing.

To lower costs, many companies have adopted longer processing times, thereby reducing the virus filter area requirement. But operating over a longer duration places a greater burden on the virus filter to maintain throughput with minimal flux decay. At the same time, consistent performance during manufacturing and virus validation is essential.

“This strategy places pressure on the validation specialists to create cleaner virus spikes, allowing high capacities and long processing times to be validated,” Fisher continued.

Filter vendors are focusing on filter design and creating robust filters that will tolerate substantial variations in feedstocks.

“Our company is working to characterize the size-exclusion mechanism of virus filtration as a function of the solution and operating conditions using fluorescent labeling and mathematical modeling to provide greater process understanding and to support end-users,” explains Fisher.