Better tools for bioprocessing
By Bruce Flickinger
Although dairy products, biopharmaceuticals, and alternative fuels might be disparate sectors of business, they have one commonality: the need to harvest, manipulate, and transform biological organisms into viable products for market.
Bioengineered goods have become an integral part of our daily lives. Foods, crops, drugs, medical devices, and plant-derived fuels are all made on commercial scales that are subject to the same constraints of efficiency, quality, and profitability that govern any manufacturing process. Biomanufacturing techniques are by and large well characterized and entail myriad steps of extraction, expression, isolation, fermentation, and, not least of all, filtration, separation, and purification—the crucial triad that ultimately yields the final, workable product from a mish-mash of cellular background.
Across the product development continuum, membrane filtration has successfully replaced traditional separation methods as a simple and versatile tool for handling biological materials. One illustration of this is a combined ceramic microfiltration and spiral ultrafiltration process developed by GEA Filtration (Hudson, WI) in collaboration with a manufacturer of industrial enzymes. Prior to the installation of this equipment, the enzymes had gone through the conventional enzyme recovery process of fermentation, centrifugation to harvest whole cells, filtration through a rotary drum vacuum filter (RDVF), and then ultrafiltration to dewater and concentrate the enzymes. The ceramic membrane filtration system replaced both the centrifuge and RDVF steps, greatly simplifying the process and increasing yields by 2 to 4 percent.
“This is largely due to the fact that membrane filtration is a straightforward size exclusion separation,” says Bob Keefe, market manager, biotech/pharma with GEA Filtration. “In this installation, the enzymes are extra-cellular, meaning they are produced outside the cells in the fermentation step, so they can be harvested from the permeate stream while the whole cells are retained in the membrane.”
“Filtration has the advantage of having scalable systems that are easily implemented from bench-scale through to manufacturing,” notes Robert Shaw, program director with Millipore Corp. (Billerica, MA). “Centrifugation is an example of a technique that can be easily used for bench-scale processes but is not as easily implemented at pilot scale because it requires significant capital investment and operational expertise.”
A healthy sector
Equipment costs and operational robustness are the primary metrics used in decision-making for filtration and associated equipment. Upgrade and refurbishment work is a big part of the bioprocess business for suppliers, although completely new lines and facilities also are being built as drug companies gear up to meet clinical and commercial demand. “We’re often called on to complement existing facilities—maybe just add ultrafiltration, for example, or put individual units in,” Keefe says. “It’s not as capital intensive as building new lines or facilities. But for those companies looking to install new production facilities with new recovery equipment, membrane filtration can be very cost effective.”
Several emerging areas of biotechnology research are creating new opportunities for the application of industrial filtration technology. Viral vectors, for example, are a good illustration of how precision laboratory methodologies have been extended into cGMP-compliant processes capable of yielding clinical-grade products. Here, recombinant viral vectors are engineered so that they cannot replicate but can infect cells to introduce foreign genes. The genomes of many different viruses can be used to transfer a gene of interest, and procedures have been devised to produce these vectors in sufficient quantities and of sufficient purity to enable experimentation in animal models and for clinical trials. Commercial entities and universities with vector core facilities are proliferating to serve this burgeoning space.
Similarly, vaccines are entering a phase of increased research and development. Vaccines are produced from viral pathogens responsible for ailments such as polio, influenza, and Lyme disease, or from bacterial pathogens such as pertussis, tetanus, diphtheria, and anthrax. Recombinant fermentation techniques are employed to produce highly purified antigen-specific sub-units that stimulate immunity without the concern about infection. Vaccine production requires several filtration and purification steps because the broths usually have moderate to high solids concentrations that require removal before downstream purification. Final vaccine products also must be highly purified in order to minimize adverse reactions in patients.
“Monoclonal antibodies are templated processes, very similar in nature, while vaccine processes are very dissimilar,” Shaw says. “The containment of infectious agents is a huge issue with vaccines, and while process volumes are lower, the potency of the materials is much higher. You’re working on a much different scale.” As an example, he says influenza vaccines are cultured in eggs, a decidedly low-throughput technique, although there is a trend toward using traditional cell-based influenza cultures.
Production of amino acids for the food and pharmaceutical markets is another recent application for membrane filtration, which can effectively concentrate these products without having to use heat-generating steps such as evaporation. Membranes, specifically nanofiltration, are being used to not only concentrate amino acids but also purify them by passing undesired compounds from the fermentation process, such as sugars and salts, into the permeate.
“Another emerging market we see is the concentration and purification of peptides with membranes either in lieu of more traditional steps like chromatography, or alongside them to reduce load on the columns and speed up the process,” Keefe adds. “The permeate can also be demineralized or dewatered downstream using nanofiltration to increase the purity level.” Membrane filtration in this case is “simpler than ion-exchange chromatography and offers better processing economics.”
Care and feeding of cell lines
Mammalian cell systems are the preferred “cell factories” for the production of complex molecules and antibodies for use as prophylactics, therapeutics, and diagnostics. Cell lines commonly employed include Chinese hamster ovary (CHO), NS0 hybridoma cells, baby hamster kidney (BHK) cells, and PER.C6™ human cells. The CHO and NS0 cell lines, in particular, are relatively easy to genetically engineer: They can be grown at large scale and excrete high titers of recombinant proteins in solution. However, cell viability tends to decline with high protein expression levels, which can contaminate downstream purification steps with cell debris, DNA, host cell protein, and other impurities. Additional media components to support cell growth, such as cholesterols and lipids, also can affect downstream processing.
Filtration and purification play key roles at several points in the handling of cell cultures. Mammalian cells are most commonly grown in bioreactors or fermentation vessels; culture media typically is mixed in bulk, pre-filtered, and then aseptically transferred to the bioreactor. In the bioreactor, cells are lysed and the intracellular material of interest is separated from the cells and other debris in the fermentation broth and clarified.
Growth media used in fermentation must be run through sterilizing-grade filtration to remove bacteria and mycoplasma. pH adjusters and other additives also must be filtered prior to being introduced into the fermenter. Buffer solutions, which are required for a number of purification steps, must be properly filtered to protect chromatography columns and downstream ultrafiltration steps and to ensure the final product is free of endotoxins. Buffer solutions typically are filtered using sterilizing-grade membrane filters; depending upon the salt concentration and buffer properties, pre-filtration might be necessary.
One challenge in bioprocessing is use of high-titered mammalian cell cultures and CHO-based cell lines, work that is beginning to appear in the scientific literature. “The first generation of biologics was produced at very low concentrations, using low numbers of cells,” Millipore’s Shaw says. “Companies now, even large pharmaceutical companies, are publishing research on new generations of cell lines where the number of cells and protein concentrations are extremely high. This means a very challenging separation must occur right at the outset to remove cellular debris and recover protein at much higher yields.” This shift is pushing vendors to provide solutions. For example, “New media are being developed to handle very high cell densities and remove the cells of interest with very low protein binding,” he says.
Ease of use becomes a salient issue in these processes and is embodied in the modular approaches taken in Millipore’s Millistak™ technology and the ÄKTA™ platform from GE Healthcare Bio-Sciences AB (Uppsala, Sweden). Both are designed to allow users to move through successive stages of development using scalable equipment and methods. Millipore offers a family of cartridge, capsule, and disc filters and attendant housings all encompassed in a system of self-contained pods that allow disposable adapters to be quickly connected to and disconnected from process piping.
GE Healthcare’s line similarly includes a range of interchangeable systems that are readily reconfigured for different materials, methods, or volumes. They incorporate hygienic design and are GLP and cGMP compatible. All units can be controlled with GE’s proprietary software, which also offers process modeling capabilities. The software allows users to enter different methods and run them in simulation “so you can scout through different process variations and also build methodologies,” says Vincent Pizzi, filtration group leader in product marketing with GE Healthcare. The program’s ability to acquire and analyze information “lets you develop a fingerprint of the entire process” and that can help mitigate the experimentation and engineering costs involved in scale-up activities.
Across and through
Filters are designed for two overarching uses across the bioprocessing continuum: normal-flow and cross- or tangential-flow filtration. In normal-flow filtration, the fluid stream flows directly toward the filter under the influence of pressure. The term “normal” indicates that the fluid flow is perpendicular (normal) to the filter surface and there is no recirculation of the feed. Smaller particles pass through the membrane and particles larger than the pore size of the filter accumulate at its surface. Common uses for normal-flow filtration include chromatography column and sterile filter protection, clarification, bioburden reduction, virus removal, and liquid sterilization.
“Normal-flow systems are very simple and easy to use because you’re essentially pushing fluid through a filter with a pump,” Pizzi says. “Pressures, pump rates, and other parameters have to be controlled much more carefully in cross-flow applications.”
Normal-flow filtration, however, can result in a build up of “cake” on the filter that needs to be periodically discharged. Cartridge filters are a convenient option because they can be readily discarded and replaced as needed. Additionally, some processes, such as yeast harvesting, are economically prohibitive using normal-flow filtration because of the enormous amount of effluent needed to obtain sufficient quantities of end product.
Cross-flow filtration is based on the pressurized flow of the fluid flowing tangentially over the surface of the filter membrane, with a portion of the feed pushed through the filter and the remainder swept away along the membrane to exit the system without being filtered. Cross-flow filtration can be used for clarification using microfiltration membranes (0.2 or 0.45 μm), or more commonly in purification using ultrafiltration.
“Cross-flow keeps debris and solids in suspension and away from the filter surface. This translates to cross-flow requiring less filter area vs. a normal flow in the same application,” Pizzi says.
Figure 2. A spiral ultrafiltration system from GEA Filtration will run industrial enzymes. Photo courtesy of GEA Filtration.
GEA’s Keefe adds, “The primary advantages of cross-flow separation systems are that the separation steps can be operated continuously to more easily follow downstream steps, and the membranes are essentially being cleaned regularly, allowing them to stay effective for years without having to replace them.”
Contamination is a concern in both configurations, particularly through a phenomenon known as microbiological following. Here, if bacterial growth occurs on membranes, it can “follow” the process flow, contaminating the permeate and subsequent systems. Bioprocessing equipment usually is designed using standard sterile design techniques, where piping and components can be cleaned and sterilized in place and have minimal sites that could harbor microbial growth.
“We also look to reduce holding times, so the process is more or less a continuous system that doesn’t allow bacteria to take hold and grow during the run,” Keefe says. The majority of systems GEA installs are dedicated, but some are used to run different feedstocks; the CIP/SIP function becomes especially important in these circumstances and typically involves aggressive chemicals in addition to hot water. “People need to make sure that their upstream and downstream steps are done with sanitizing capability in mind,” he says.
Another point filtration suppliers emphasize is that expression-system parameters such as percent solids, starting turbidity, and particle size distribution vary widely among microbial strains, so generic filter-sizing specifications are difficult to make.
Protein and yeast broths are particularly problematic media to work with from a filtration and separation standpoint. One challenge is choosing the correct membrane pore size that will retain whole cells but pass proteins into the permeate. “Depending on the specific type of enzymes being produced, they will range in molecular weight from approximately 10,000 to 100,000. The pore size of the membrane is typically 0.1 or 0.2 μm, which allows these enzymes to go through the pores and be recovered in the permeate,” Keefe says.
Even with the correct pore size, perhaps the biggest challenge is operating the membrane filtration system correctly to make sure the gel layer, also called the boundary layer, on the filter membrane stays as thin as possible, Keefe says. The gel layer is a build-up on the active membrane layer of the product being run; if this build-up gets too thick, it can act as the filtration layer and prevent the enzymes from effectively being recovered in the permeate, reducing the yield of the process.
“This control of the gel layer is done by operating the system at the correct TMP [trans-membrane pressure] and recirculation velocity, which helps to promote turbulence within the membrane channels and ???sweep’ the membrane to minimize the thickness of the gel layer,” Keefe says.
Figure 3. Process-scale Millistak™ system from Millipore incorporates self-contained pods that allow disposable filters to be quickly connected to and disconnected from process piping. Photo courtesy of Millipore Corp.
Overall, “we need to work closely with our customers’ technical people to make sure their process is sized appropriately to minimize their costs,” Keefe continues. “Filtration processes can be very sensitive and involve very precise control of operating parameters such as pressure and velocity. This takes a lot of collaboration on the process engineering side.” Initial feasibility/viability testing needs to be conducted to determine appropriately sized membranes that will recover the enzymes the user needs.
Process parameters change proportionally as volumes—and variability—increase. Regardless of how robust a laboratory procedure might be, if it is going to be engineered for larger scale manufacturing it will become more complex and must, among other considerations, be capable of growing vast numbers of producer cells; incorporate closed systems and sampling points that maintain asepsis and do not compromise process integrity; be automated, at least in part; and achieve reproducible yield and purity.
“With any process, there are inherent variabilities that must be considered: those that are random and those that are systematic,” Millipore’s Shaw says. “Random variations occur batch to batch and can be quite large, as there may not be a lot of experience with the process prior to pilot manufacturing, and especially if fermentation is also being scaled up at the same time.” Systematic variabilities include feed-solution handling, filter sources, operating conditions, and scaling effects.
Filtration and handling techniques notwithstanding, feedstock is the most significant source of variability. Even within a company, identical processes can behave differently among different plants and lines. “The user has to be able to share information about their feedstock, because this is what introduces the most variability into a process and affects scale-up most significantly,” Pizzi says. “It is important when scaling up a biological process to make sure that the organism’s protein expression used during the pilot testing, whether it is bacterial, yeast, or fungal, mimics the commercial scale.”
Users need to “make certain the fermentation is conducted as closely as possible on the larger production level as on the pilot scale,” Keefe affirms. When using membrane filtration, he advises conducting lengthy pilot trials over a period of time that will cover any variations in the cell strength or solids level in the production system. “This will help you to better anticipate what changes in performance, if any, the filtration system will see,” he says.
In conducting scale-up planning with its customers, Millipore develops a set of safety factors by unit operation that “allow them to understand which unit operation may face the largest inherent variability and to plan experiments to characterize this variability better,” Shaw says. “As you scale up a process 100 times, for example, you want to know how much safety to build in so that process will still run and won’t exceed its capabilities.” Variability in the process and the feedstock are considered, from which “rules of thumb” are calculated that provide anticipated parameters that bookend the operation: Clarification requires a larger safety factor than buffer filtration, for example, because variability is lower in the latter. Looking at things in this way, Shaw says, provides “useful parameters for people to develop a process they can depend on.”
More than equipment
For many small biotechnology companies without a lot of infrastructure, collaborating with technology suppliers and tapping their expertise are essential to the success of the project. In biopharmaceuticals, the net result of process development and scale-up is being able to provide a formulation at an appropriate dose in an acceptable excipient and in stable final product that can be stored and shipped to clinical sites. The doses involved are small and the costs involved are high. Modern filtration and purification technologies contribute significantly to both the integrity of the final product and the efficiency of the process used to manufacture it.
“It’s important for us to collaborate with our customers in a very detailed manner in order to establish the linkage between unit operations using filtration, purification technology, and chromatography in their processes,” Pizzi says. “Many times, people get gummed up because they are rushing to get to market, so they don’t always have the time and luxury to understand all their process variables.”
“Regulatory and validation support are key, because these companies typically have not had much interaction with the FDA,” Shaw adds. “They want to be able to solve and prevent any problems before taking their processes to that next level.”
Resources and contacts
GE Healthcare Bio-Sciences Corp.