By Bruce Flickinger

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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.

Figure 1. A small-volume ceramic membrane filtration system with two ceramic modules. Ceramic membrane filtration can replace centrifuge and RDVF steps in processes such as industrial enzyme recovery. Photo courtesy of GEA Filtration.Click here to enlarge image

“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.Click here to enlarge image

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.

Size matters

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

By David C. Eagleson, The Baker Company, Inc.

Class II biological safety cabinets (BSCs) are vital pieces of laboratory equipment in many life science applications requiring contamination control. In addition to providing a workstation with aseptic conditions for product protection, a BSC also helps protect laboratory personnel from exposure to aerosols of hazardous substances and prevents the release of such hazards into the environment. Field certification is the method by which proper BSC operation is verified over time, to be sure that product, personnel, and environmental protection are maintained.

Requirements for Class II biological safety cabinets in the United States are established by NSF International and published in NSF/ANSI Standard 49.¹ Class I and Class III cabinets also exist and are generally used only for special applications. Class II cabinets are by far the most prevalent, and NSF 49 is specific to Class II BSCs. Therefore, this article will focus on Class II BSCs, and all subsequent uses of “BSC” are in respect to Class II BSCs.

NSF 49 includes definitions of the types and function, acceptable materials, design and construction requirements, and performance requirements for Class II BSCs. NSF International manages a program of type testing and product certification for Class II BSCs. Annex F of the standard includes specific requirements for field certification. To ensure that individuals performing certification are properly qualified, NSF International also administers a program for Biosafety Cabinet Field Certifier Accreditation.²

This article will review the function and designs of Class II BSCs and describe the field tests that should be performed during certification. The relationship between certification and commissioning will then be discussed, and the process and requirements for accreditation of field certifiers will be reviewed.

Types of Class II biological safety cabinets

All Class II biological safety cabinets offer product, personnel, and environmental protection from biological and other aerosolized contaminants. Product protection is offered by unidirectional (commonly called “laminar”) downflow air in the work chamber, generated by the cabinet blower pushing air through the supply HEPA filter. Personnel protection comes from the intake air pulled into the front access opening of the cabinet. Environmental protection is provided by HEPA filters in the exhaust air stream of the cabinet. The airflow pattern in a generic Class II BSC is shown in Fig. 1.

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Different types of Class II BSCs are utilized when protection from chemical vapor hazards is also a concern, as HEPA filters do not capture gases or vapors.^3,4 Type B1 and B2 cabinets must be directly connected to the building’s exhaust system for venting to the outdoors via a hard connection (see Fig. 2). Type A1 and A2 cabinets usually return their filtered exhaust air to the room but may optionally be connected to the building exhaust system with a canopy, formerly called a “thimble,” connection (see Fig. 3). Note that many existing installations of exhausted Type A cabinets may utilize a direct connection to the exhaust system. However, more recent revisions of the NSF 49 standard recommend use of the canopy exhaust, and in the future hard connections for Type A cabinets will most likely not be allowed at all. See Table 1 for a summary of the definitions of different cabinet types.

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Field tests for biological safety cabinets

BSC field tests should be performed by the certifier upon installation and relocation of cabinets, after major maintenance is conducted, changing of HEPA filters, and at regular intervals thereafter. NSF 49 recommends a maximum interval of 12 months between certification, though many organizations re-certify more frequently. For example, pharmacies certify their BSCs every 6 months as required by USP Chapter <797> on sterile compounding.

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In order to verify proper operation of a biological safety cabinet, the following tests are performed.¹ These tests are related to the containment and product protection provided by the cabinet, and results must correlate to the values obtained by NSF for type testing of that particular make, model, and size of cabinet.

• Downflow velocity profile test
• Inflow velocity test
• Airflow smoke pattern tests
• HEPA filter leak test
• Cabinet integrity test (for Type A1 cabinets with positive-pressure contaminated plenums only)
• Alarm function verification
• Blower interlock (for Type B1 and B2 cabinets)
• Exhaust system performance (for any cabinet connected to the building exhaust)

While not part of Class II BSC field certification to NSF Standard 49, for certain installations additional validation tests may be required. For example, most cGMP compliant facilities will verify air cleanliness with a particle counter to the required ISO class per IEST protocols. Any such validation tests should be performed in conjunction with
field certification.

The following tests, related to worker comfort and safety, may optionally be performed. These tests verify functions of the cabinet not directly related to containment or product protection.

• Lighting intensity test
• Vibration test
• Noise level test
• Electrical tests (leakage, ground circuit resistance, and polarity)

Certification and commissioning

The goal of commissioning is to verify and document that the facility and its systems meet defined objectives and criteria for performance.⁵ Commissioning is performed prior to occupying new laboratory facilities to ensure that systems are operating as specified. Many organizations also repeat commissioning, or at least some portion thereof, at regular intervals to ensure continued proper operation of building systems. This “re-commissioning” is a good practice to guarantee that all building systems continue to function as required.

One of the most important systems considered in the commissioning process for laboratory facilities is the HVAC (heating, ventilation, and air conditioning) system. This is where commissioning and certification have a vital overlap and interdependency. Proper HVAC design and operation is crucial to the proper operation of BSCs. While the majority of BSC installations simply return cabinet exhaust to the room, they may still impact, or be impacted by, the function of the HVAC system.

From a heating and cooling standpoint, a typical 4-ft Class II, Type A2 cabinet may generate 2,000 to 3,000 BTUs/hr. Air conditioning and heating systems should be verified to maintain desired environmental conditions when BSCs are in operation, as well as when cabinets are turned off.

More crucial to the safety of laboratory workers and the integrity of aseptic work processes is the possible impact of air currents in the room on the performance of the BSC itself. Location of room air supplies and returns is critical, as cross-drafts may negatively affect the performance of BSCs.⁶ This dependency should be taken into account when designing room air ventilation and should be verified during the commissioning process.

Another level of complexity is introduced when BSCs are vented to the outdoors via the building exhaust system. With such an installation, the BSC itself becomes the first piece of the system ductwork and needs to be designed for and tested as such. In addition to the possible impact of cross-drafts, the commissioning process for BSCs should also verify that the required exhaust and room supply air to operate the BSC are available and that HVAC controls are interfaced with the BSC controls.

1. Supply air

Supply air requirements for BSCs are often overlooked. However, they can be just as critical as exhaust air requirements to proper cabinet function. Whatever volume of air is exhausted by the BSC must also be supplied to the room in order to avoid “starving” the cabinet of air. The supply air available to the BSC should be verified as well as the supply air to maintain desired room pressurization and air exchange rates. A BSC cannot be certified if a lack of supply air causes a low or inconsistent inflow or downflow velocity.

2. Exhaust airflow and static pressure

Biological safety cabinets are constant volumetric airflow devices, and the fan energy to exhaust the cabinet must be provided by the building exhaust system. In addition to the exhaust airflow, the static pressure requirements for hard connected Type B cabinets are relatively high (up to

By Fran McAteer, Microbiology Research Associates, Inc., and Ryan Burke, Acusphere, Inc.

Environmental monitoring (E/M) is a surveillance system for microbiological control of cleanrooms and other controlled environments. The process provides monitoring, testing, and feedback regarding the microbiological quality levels in aseptic environments. Environmental surveillance monitors the effectiveness of various controls for microbiological contamination, sources of which can come from air, personnel, equipment, cleaning agents, containers, water and compressed gases, among other things.

Sound monitoring requires understanding the stringent regulatory specifications put into effect by organizations such as the U.S. Food and Drug Administration (FDA), International Standards Organization (ISO), Parenteral Drug Associates (PDA), European Union (EU), and United States Pharmacopeia (USP). Tables 1

Purdue University researchers have reported a new technology for potential biosafety and food safety applications that can simultaneously screen thousands of samples of food or water for several dangerous food-borne pathogens within a couple of hours.

The technique also can estimate the amount of microbes present and whether they pose an active health risk. This could help neutralize potential threats and improve food processing techniques, says Arun Bhunia, a professor of food science at Purdue University.

“For food safety and biosecurity purposes, you need a quick test—a first line of defense—to be able to tell if there is something pathogenic in the food or water,” Bhunia says.

The detection method employs live mammalian cells that release a measurable amount of a signaling chemical when harmed. According to Bhunia, optical equipment and computer software can then be used to analyze the chemical to estimate the amount of harmful microbes present, which is important because there is an effective dose or threshold that many toxins or pathogens need to pass before setting off a red flag for addressing the problem.

The technology can recognize very small amounts of Listeria monocytogenes, a bacterium that kills one in five people infected and is the leading cause of food-borne illness. It also recognizes several species of Bacillus, a non-fatal but common cause of food poisoning, says Pratik Banerjee, a Purdue researcher and first author of a study published in the February issue of the journal Laboratory Investigation detailing the technology. The study was funded by the U.S. Department of Agriculture and Purdue’s Center for Food Safety Engineering.

Purdue researcher Pratik Banerjee, at left, measures fluid as he and professor of food science Arun Bhunia work in the lab. Their technology uses common lab materials to quickly screen food and water samples for several food-borne pathogens and toxins. Photo by Tom Campbell/courtesy of Purdue Agricultural Communication.Click here to enlarge image

The cells are suspended in collagen gel and put into small wells within multi-well plates, which may allow them to be prepared in a central location and shipped to a processing location for on-site testing. This suspension of live mammalian cells within a collagen gel is unique, according to the researchers.

The technology tests for bacteria and toxins that attack cell membranes. For this reason, researchers used cells with high amounts of alkaline phosphatase, the signaling chemical released upon damage to the cell membrane. Researchers could conceivably employ other types of cells within this framework to detect additional types of pathogens.

Samples of food and water are added to biosensor wells before being incubated for 1 to 2 hours. To each well, a chemical is added that reacts with the biosensor’s alkaline phosphatase, yielding a yellow product quantified by a special camera and a computer. However, a precise calculation may be unnecessary sometimes.

“When a large amount of pathogen is present, you can literally see the color change taking place before your eyes,” Banerjee says.

According to the researchers, this technology can actively identify harmful pathogens but ignore those that are inactive, or harmless. Some comparable tests lack this ability and have a tendency to produce false alarms; the incubation period to grow out any living microbes is also rather long with such tests. The new technology also could help optimize processes to kill harmful microbes or deactivate toxins.

The cells currently only live between 4 to 6 days within the gel; Bhunia says this time span could be expanded to 2 weeks, the shelf life he deems necessary for commercial application.