Maik W. Jornitz

Total cost of ownership, including operating costs, must be considered when deciding between flexible and traditional cleanroom systems.

Articles

Cleanroom and Facility Planning: Getting Beyond Cost Per Square Foot

Biopharmaceuticals commonly cannot be terminally sterilised, as such aseptic processing using sterilising grade filtration is essential. Heat sterilisation, or any energetic input into biotech drug products, causes undesired product degradation so the only available option is sterilised filtration of the biopharmaceutical fluid. Sterilising membrane filtration has been used for decades, and there is a very high experience and expertise level within the industry. As such, the process is reliable and well understood when properly validated. Filtration is also often required to reduce bioburden levels within process streams to avoid potential biofilm formation; for example within purification steps and/or elevated endotoxin levels.  

Process validation of sterilising grade filters is a fundamental task to ensure and verify the performance of the filter used within the process conditions and fluid stream. Process validation means that the specified membrane filter is subjected to a bacteria challenge test in accordance with ASTM 838-05, utilising the process parameters of the filtration process and, if possible, the actual fluid product. If the fluid has properties that are detrimental to the challenge organisms, a placebo as close as possible to the actual drug product, is required. The effect of the fluid stream on the organisms needs to be tested using viability tests. 			

An additional validation test is chemical compatibility to check whether or not the filter membrane and construction is suited for both the product to be filtered and the process conditions. Leachable and particulate matter tests may also be necessary to determine any foreign subject matter release into the drug product. Often forgotten, but essential for biopharmaceutical filtration, are unspecific adsorption studies, which not only determine the fouling rates of the membrane polymer, but also the yield losses onto the filter membrane. These tests can be performed when filters are tested and scaled in filterability trial work. When filters are in line steam sterilised, the sterilisation process requires qualification to prove that it does not damage the filter. Other qualification data, such as gamma sterilisation robustness and efficiency, endotoxin level, integrity test limits and oxidisable substances can be obtained from the filter manufacturer.

Over-sterilisation is sometimes cited as a concern. Beside other key attributes, sterility is key to an injectable drug product; therefore I would hesitate to utilise the word "over-sterilisation". Safety and sterilisation robustness are essential, and one has to ensure that sterility is achieved by appropriate measurements, qualification and validation. If one wants to classify something as "over sterilised", it could be through the use of either redundant filtration or filter ratings, which are inappropriate for the application.  

More important than adding filters or tighter pore sizes are process validation procedures, which verify that the filter does what it is supposed to do: retain the organism level and species found in the upstream fluid and create a sterile effluent. When a sterilising grade filter is properly validated and evidence has been obtained that it performs to required specifications, why would one need a second one or move to a tighter pore size?  

The only reason would be insurance, which creates an immediate insecurity notion. Another "over-sterilisation" aspect could be the European restriction of a maximum 10 cfu/100 mL in front of a sterilising grade filter. Why are end-users of sterilising grade filter bound to such a restriction, when the filters are commonly challenged with 10

? If one utilises the example of a 1000 L volume with a bioburden of 10 cfu/100 mL being filtered through a 10 in. filter element of 0.6 m

, the challenge would be 16 organisms per cm

, far below the validation challenge test level of 10

The main concerns of regulators when it comes to sterile filtration depend on which regulator is consulted. Critical regulators very much stress the benefits of heat sterilisation 

 sterilised filtration, a known fact acknowledged by everyone who has any scientific sense. Having said that, it would be an advantage if regulators, in contrast, acknowledged the fact that there are drug products that cannot be heat sterilised; in these instances, filtration is the best option.  

acknowledge the thousands of process validations performed by the industry with sterilising grade filters, the billions of litres filtered and the decades of experience? If similar scepticism were attached to flying, planes probably would only be used for short distance flights. The regulatory promotion of redundant filtration, pre-use integrity testing or the use of a tighter pore size should be left to the end user, as the decision to use either or all of the aforementioned is an economic decision after the filtration process has been properly validated.  

The requested process validation (by all regulators) of sterilising grade filters is necessary and welcomed by filter manufacturers and end users alike. The stated tasks required during process validation are of help to any validation exercise and create a robust performance analysis of a sterilising grade filter. However, it would be good if regulators relied on their stated requirements when the industry performs the requested tasks and documents the outcome and reliability. 

Optimising and improving current processes

The sterilising filtration of biopharmaceuticals generally means that a high value, low volume fluid is filtered. As such, oversized filtration systems, redundant filtration systems and, in single-use instances, tubing length, all contribute to excessive hold-up volume, i.e., valuable product losses. Therefore, the size and design of filter systems should not be neglected because these can impact the production process.  

Again, unspecific adsorption, which is often neglected, should be tested very early in the drug development process to understand which filter membrane polymer is optimal for a particular fluid or application. Yields are very much influenced by unspecific adsorption and can vary greatly between membrane polymers. Total throughput — the volume that can be filtered through a specified filter device — also has an influence on yield. In some application, yield can drop dramatically when the filter starts blocking. This drop-off has to be determined in small-scale studies utilising flat disc filter composites — so-called indicator trials. When a filter needs to be scaled to production requirements, however, indicator trials are not sufficient and verification trials with small scale pleated devices should be used. The data of these trials are commonly reliable to scale the filter appropriately; again, the goal is not only to find a filter system that is able to process the entire volume, but also not to over-size. High flow rate is the desired parameter in some applications, but flow rate cannot be tested with 47 mm disc filters because the results would be meaningless. The flow dynamics and the filtration area within a 10 in. element requires testing in high flow applications. The interaction between the filter adapter and housing recess, as well as pipe work, the filter pleat behaviour under pressure and possible compression, can only be tested when the actual filter element is used.  

In essence, a filter system can be compared to a well-defined recipe; too much as well as not enough can spoil it. Similarly, too much filtration area is as undesirable as too little. 

 is Senior Vice President, Marketing, at Sartorius-Stedim North America, Inc.  

Biopharmaceuticals commonly cannot be terminally sterilised, as such aseptic processing using sterilising grade filtration is essential.

The role of sterile filtration in the biopharm industry

For the past decade, the development and approval of biological drugs has increased rapidly. This increase highlights the significance of aseptic processing because biologics commonly cannot be heat sterilized. Because aseptic processing receives greater regulatory scrutiny than heat sterilization does, the industry developed, in addition to risk-management and risk-analysis activities, new processing equipment and technologies to overcome potential weaknesses (1). These technologies' main purpose was the avoidance of human intervention, which is one of the most substantial risks in aseptic processing. The technologies included environmental-containment solutions such as isolators and restricted-access barrier systems, and new filling strategies such as blow–fill–seal. Disposable solutions have changed, and their applications have broadened, during the past several years. 

The most far-reaching changes in the technology's early days affected processing equipment, which gradually was converted into single-use equipment (2). The benefits of single-use equipment not only enable a potentially higher sterility-assurance level within process steps than was possible before, but also enable production-utilization enhancements, cost savings, and end-user protection. Single-use equipment does not require setup times of 8–10 h or copious amounts of water for cleaning purposes (3). Furthermore, it protects end users from contact with potentially hazardous fluids. This article will focus on recent advances in single-use technologies. 

The first disposable process parts to be used were tubing sets. They were followed by filtration devices called capsule filters. Capsule filters were only available in small effective-filtration-area units, commonly between 0.5 and 2 ft

. The introduction of single-use components for mixing and fluid containment required larger filter capsules, and end users requested small-scale pleated devices for scaling studies. These two factors led the filter-capsule range to be expanded to an area of 0.1 to 30 ft

 (4–6). Current filter capsules are gamma-sterilizable and commonly connected to tubing sets or bag assemblies (see Figure 1).  

Figure 1 (IMAGES ARE COURTESY OF THE AUTHORS) 

Single-use technology for mixing and holding tanks also emerged (e.g., single-use bag devices). Single-use bags are available in volumes of 20 to 3000 L and can replace glass vessels, carboys, and stainless-steel tanks. Before single-use bags could be introduced into the production process, they needed to demonstrate mechanical stability, chemical resistance, a barrier to oxygen transfer, and low leachable levels, even when the bags were gamma sterilized at 50 kGy. New polymeric films laminated onto each other made these characteristics possible and have become common (see Figure 2).  

Single-use bags are used most often in an assembly format, either with or without a filter, as part of a multibag system. The entire assembly is gamma irradiated in the packaging and is ready to use. 

Single-use systems, though, are not restricted to bags and filters. Various equipment components are available that can be combined into a single-use system or process step. Such equipment components include the following:  

Mixing systems of various designs and volumes

Sensors (e.g., for dissolved oxygen, pH, and conductivity)

Sterile fluid-transfer connectors and tubing

Bioreactors of different designs, volumes, and agitation technologies) 

Aseptic-transfer systems for fluids and solids 

Membrane-chromatography (e.g., ion exchange) columns (7, 8)

Viral-clearance and ultraviolet-inactivation filters.

At first, single-use components were designed to be connected to a traditional unit operation. The newest advance in technology combines single-use components into a single-use unit operation. One day, these unit operations likely will be connected together to form a single-use process (see Figure 3).  

Single-use unit operations currently are available for buffer and media preparations, ultra- and diafiltration, virus filtration, and bioreactors. Other process steps will follow soon, and low-volume streams inevitably will be processed within single-use designs in the near future. Large volumes probably will still need to be processed in hybrid solutions, which connect reusable and single-use process steps.  

New disposable sensor technologies will ensure that process analytical technology needs are not neglected. Radio-frequency identification tags will enable the end user to connect systems safely and track systems during inventory-management or transfer activities. 

Limitations such as mechanical instability at large volumes and cost factors within affinity chromatography will restrict the development of single-use processes (see Figure 4). Despite these limitations, the advantages of reduced setup times, the elimination of cleaning activities, and end-user protection justify converting even a small number of process steps to single-use technologies. Eliminating end-user intervention with presterilized, interconnected unit operations potentially can enhance safety within aseptic processing. Single-use technology inevitably will be an essential component within biopharmaceutical processes, even if the majority of components in a process is not single-use.  

The validation requirements for a single-use aseptic process are the same as those for a traditional stainless-steel process. Because single-use systems are preassembled and presterilized by the vendor, the validation work requires a high level of vendor involvement. The end user works closely with the vendor to determine the specific validation and qualification parameters and activities needed to ensure the expected product performance for a given application. The vendor, therefore, must have an in-depth understanding of current regulatory requirements and good manufacturing practices. Moreover, the vendor must be able to adjust to the end user's needs. Every application and process stage has different specifications and critical aspects. These differences must be taken into consideration to avoid working too much or too little to satisfy validation requirements. 

Most single-use systems are gamma-irradiated at 25–50 kGy. The irradiation sterilization makes short- and long-term stability studies necessary to evaluate potential polymeric degradation, which could raise leachable levels or lower mechanical resistance. Irradiation commonly reduces the shelf life of polymeric devices, and personnel must determine the radiation limit that a given device can withstand. Qualification tests, defined by the vendor, are performed before any single-use unit or system is distributed to the industry. These qualification tests are manifold and are designed to meet regulatory requirements (see Table I). The end user can rely on the results of the vendor's qualification tests, but should consider process-specific validation requirements.  

Table I: Common qualification tests performed by single-use equipment vendors. 

Process-validation studies would, for example, use a model solvent and simulate the process parameters within the end user's specifications. Leachable testing cannot always be performed with the product because the product would mask potential peaks. For this reason, model solvents that are similar to the solvent used within the product stream often are chosen. The possible influence of the environmental conditions within the end user's processes should be tested, however, to ensure that the disposable device performs to specifications. Process-validation steps vary according to disposable devices' purposes. Sterilizing-grade filters must undergo a product bacteria-challenge test under end users' process conditions. The influence of the process conditions and fluid on the challenge organisms or separation mechanisms should be determined. Product hold bags or mixing bags do not have to undergo bacteria-challenge tests, but possibly bacteria-ingress tests. Both filter and bag systems must be tested for leachables and extractables. As mentioned previously, the end user should request the vendor's assistance. 

Many single-use devices already are available, most commonly as individual entities. More and more filter–bag and bioreactor–filter systems have been introduced, and single-use unit operations recently have become available. Components' rapidly developing connectivity will enhance the development of integrated systems and, potentially, totally disposable processes. Some limitations, especially in the filling area still must be overcome (e.g., fill precision). The last step in aseptic processing has not reached the level of disposability that one finds in the intermediate steps.  

Single-use components have many benefits within aseptic processes. For example, cleaning deficiencies are a common regulatory observation that would be eliminated by single-use equipment. The equipment also greatly reduces the risk of cross contamination. Moreover, disposable, aseptic connectivity will reduce end-user manipulation within the process and therefore improve safety. Some in the industry have wondered whether the benefits of single-use systems are outweighed by their environmental impact. Further studies and thorough evaluations, however, might show that the environmental impact of cleaning and sterilizing stainless-steel equipment is greater than that of single-use systems. This topic will likely be addressed in the near future. 

 is vice-president of marketing FT/FRT, and 

 is global director of marketing IS, both at Sartorius Stedim North America, 5 Orville Dr., Bohemia, NY 11716, tel. 631.254.4249, 

. Jornitz also is chair of the Parenteral Drug Association.  

*To whom all correspondence should be addressed. 

Concerning Medicinal Products for Human and Veterinary Use,

 Vol. 4, Annex 1, p. 12 (European Union, Brussels, 2008). 

 2. P.M. Priebe, PDA SciTech Conference (Orlando, FL, 2004). 

 5. PDA, "Liquid Sterilizing Filtration," Technical Report 26 (Parenteral Drug Association, Bethesda, MD, 2008). 

The authors describe the origins of single-use components and explain their application to aseptic processes. They also show how disposable devices have changed over time and offer a glimpse of the future.

The Evolution of Single-Use Technologies in Aseptic Processing

 Patent expirations, stiff competition, plant utilization, product safety, and supply assurance are some of the challenges facing the biopharmaceutical industry. The manufacturing landscape has changed dramatically during the past several years—and it's still evolving.    

 Figure 1: Single-use cell culture system. (ALL FIGURES ARE COURTESY OF THE AUTHORS) 

Novel approaches for treating specific diseases such as cancer are very promising. However, the days of biotechnology innovations being measured purely on gaining blockuster status (i.e., reaching more than $1 billion in revenues) are largely gone. The capacity constraints predicted in the late 1990s, combined with insufficient development pipelines and tougher regulations, caused a major market consolidation, evidenced by acquisitions, alliances, and outsourcing.  

Similar to what the semiconductor industry already experienced, drug manufacturers face a changing environment that requires innovation and efficiency. They must manufacture products economically and efficiently while also improving quality and safety. Understanding cost drivers within the manufacturing and development processes is key to assessing the impact of new business models and technologies.  

The costs associated with constructing and operating manufacturing capacities as applied to clinical phases and large-scale manufacturing call for challenging current paradigms. Single-use technologies represent one option to achieve operational excellence without compromising quality.  

Single-use technologies are being used more in biopharmaceutical manufacturing and that use is expected to increase substantially in the coming years. With the further penetration of disposables into larger volumes and more critical areas of the biomanufacturing process, however, companies involved in biopharmaceutical development and the production of new molecules must closely examine the advantages, limitations, and facility implications of single-use manufacturing scenarios.  

The challenge is to design and implement application-specific, integrated-process solutions that follow the same principles as conventional process designs regarding quality, engineering, automation, and control. Single-use technologies are not an "one-size-fits-all" solution. They must be intelligently integrated into process designs.  

Process analytical technologies (PAT) and quality by design (QbD) are major initiatives in the industry that will influence the future technology penetration significantly. It's important to examine additional improvement opportunities offered by the increased implementation of single-use solutions to exploit advantages. Production facilities, traditionally, were built to house stainless steel-based production scenarios. Does this mean that new facilities are required to be built the same way? What about cleanroom classifications to operate individual process units? Single-use solutions are presterilized, closed systems that can reduce, if not eliminate the need for extensive cleaning and steaming utilities. Aseptic-processing requirements that benefit from single-use solutions through the reduction of cross-contamination successfully support the shift to multipurpose manufacturing designs and concepts. One might think that disposing such solutions adversely affects carbon-footprint evaluation. However, there is no standardized approach to such calculations and studies published in the past two years demonstrate that the opposite is true. The authors contend that understanding the advantages provided by single-use technologies, mitigating the limitations, and exploring new opportunities is the most-appropriate approach for operational excellence.  

The "single-use" concept came loaded with huge expectations of finding new solutions to address the needs of the changing biopharmaceutical industry. Suppliers of single-use equipment have had difficulty living up to these expectations thus far, partly because the expectations were not properly defined and partly due to intellectual property (IP) and supply-chain issues. 

In a hard-piped manufacturing process, the bioprocessing flow path is only qualified once. With single-use technology, a new flow path might be supplied various times, especially in multipurpose scenarios, and the qualification of the new flow's components is often in the hands of the supplier. The supplier is urged to provide disposable flow paths using the same components and properties for years to come.  

Figure 3: Water-consumption percentage differences: Stainless steel versus single-use equipment (Data from Ref. 9).  

To meet this request, however, the supplier must own all of the critical pieces of a particular assembly. For this reason, many suppliers are aligning with, or acquiring, component suppliers and IP owners. Unfortunately, a lack of standards and competition in this arena has promoted a vast array of equipment items, assemblies, and validation-procedure differences. These varieties, marketing-driven product and validation claims cause more insecurities and questions, than solid standardized solutions. 

Promising new development, in the form of single-use unit operations, is moving away from isolated technical solutions and toward new, highly-integrated concepts. End-users of disposable bags for media initiated this trend in an effort to move from a fixed process to a single-use process that includes mixing, filtration, temperature, recipe, and level control.  

The integration of disposable sensors helped develop fully-disposable, cell-culture systems and opened the door for single-use applications for most of the unit operations within a manufacturing process. New approaches are supplying single-use technology platforms along the typical manufacturing process. 

The application know-how determines the design of those platforms, but standardization of components such as tubing and connectors, ensures interconnectivity at the same time. These concepts allow a clean and streamlined process with a unified automation, control, and validation concept and, finally, a high level of operational excellence and security.  

The single-use approach demands the highest level of technology integration possible. However, the economic and operational advantages of the disposable option apply mostly to Green Field Facilities (i.e., facilities planned from scratch) with high-value, low-volume products and might not be an option for existing manufacturing processes. This restriction to new facility design is a purely economical one as most of the capital investments within existing processes are made and the equipment is written off. Nevertheless, the flexibility of single-use manufacturing philosophy is attractive for expansion projects within existing facilities. In addition, so-called "hybrid" systems merge the best out of both worlds by integrating single-use unit operations such as buffer and media-hold steps, into existing production facilities.  

Single-use equipment and its support of QbD

QbD has found success within pharmaceutical processes and gained momentum in the production of biologics (2). Knowing the quality attributes and properties of a product creates the opportunity to design quality into the process with process analytical technology (PAT) tools. The very well-known saying, "to design quality into a process instead of testing it in" and Janet Woodcock's statement, "a maximally efficient, agile, flexible pharmaceutical-manufacturing sector that reliably produces high-quality drug products without extensive regulatory oversight" (10) are very much supported by single-use technologies, especially interconnected unit operations.   

Once the critical quality attributes are known, process-development activities can include small-scale, single-use process steps to evaluate the reliability of the equipment and its potential effect on the product. Furthermore, single-use sensor technologies can be deployed to allow for process controls, creating a dependable and robust output. Such small-scale systems, however, must be scalable to avoid surprises in process scale.  

It is, therefore, important for single-use vendors to supply a scaling-basis system supported by various single-use sensor technologies and appropriate qualification documentation. The benefits of single-use unit operations include less human intervention and fewer possible set-up failures. The process step is assembled and ready-to-use coming as soon as it comes out of the package. The assembly is fitted into the supporting hardware systems, which is simpler than disassembling a process step, cleaning the components, assemblying the components, and sterilizing the system before moving to point-of-use (POU).  

Single-use process steps are unfolded at POU and interconnected aseptically by validated-connection systems. Any designs within a hardware system might be complex to disassemble, clean, and then reassemble. An interconnected single-use operation is less complex because the end-user does not have to assemble multiple components. Such design benefits and ever-advancing, single-use sensor technologies create opportunities to support QbD initiatives with PAT.  

A PAT approach must take into account any out-of-specification situation within a process step. Sensor technologies in single-use format include pressure, pH, conductivity, dissolved oxygen, and temperature sensors (7). However, more needs to be done within the sensor-technology field. The qualification documentation, which commonly goes together with any single-use components or assemblies, can be used to determine any obstacles to critical quality attributes and potential influences on product quality. Vendors of single-use technologies have a thorough understanding of their raw material, finished goods, and assemblies. Furthermore, they support validation requirements with their service organization. End-user process-development resources should exploit service support opportunities by vendors to establish appropriate qualification and validation activities. 

Biopharmaceutical manufacturing typically involves numerous process steps. The sequence, number, or size os steps differs by application and individual bioprocesses (8). To reduce the time and engineering effort required during the design and construction of production facilities, process platforms are increasingly used to define cost-effective solutions.  

Process platforms are well-defined sequences of processes or process steps, which enable progress in biopharmaceutical development and production as they expedite time-to-market. Significant benefits associated with process platforms include efficient-engineering workflows and more precise cost determinations and cost allocations, particularly in the early design phases. Furthermore, projects can be implemented faster and investment decisions can be postponed until the drug candidate shows positive results.The use of process platforms also offers potential for process optimization, implementation support of QbD initiatives, accelerated production start-ups, and improved supply security. 

The prerequisite is closed, preconfigured, single-use unit operations. These units serve as process platforms while supporting  bioprocess and user safety. By maintaining flexibility and allowing different configurations, process complexity is significantly reduced, which is often considered a key success factor by the end user.  

Several suppliers have already launched their first configurable disposable solutions or systems (CDS). These solutions address the different volume needs, within the development cycle to production-capacity needs for buffer or media preparation, from 50 to 1000 L. The integration of monitoring and control features for pH, pump-speed, and fluid-level control is an additional milestone in the development and implementation of process-relevant, single-use equipment. The integrated controls allow end users to perform other tasks during, for example, buffer-preparation operations.  

Whether a production facility is a retrofit or green field, the process and workflows must always be defined to assist in developing the facility layout. Logistics for material handling, segregation of critical steps, space requirements for work-in-progress and storage, plus provision of the utilities required for process and cleaning needs at every critical process step, must be fully specified. 

Finally, support spaces required and their interactions with the process path must be clearly defined (5). Employing single-use equipment generally requires more space to accommodate movement and handling. For this reason, horizontally-positioned production operations located on one floor, with sufficient space for material and personnel transfer, are the best layout for disposable-production facilities. Although this may require more floor space than production facilities which are installed vertically, this arrangement is recommended for bag-based transport and storage of raw materials, semi-finished, and finished products. 

Vertically arranged equipment (common in many current biopharmaceutical-production facilities) tends to be more cost-effective, due to the compact-building design possible and the resulting low ground-area footprint. However, in the conventional arrangement, where both medium and buffer preparation take place on the floor above the process suites, a vertically-designed, disposable production facility is at a disadvantage because transporting medium and buffer bags must be done over one floor or the fluid transport must be accomplished between different floors and, thus, involve piping. In the latter case, the potential advantages associated of storage and transporting media and buffers in disposable bags would be negated. 

Environmental impact of single-use technologies

More often, biopharmaceutical companies are confronted with the need of utility reduction, recycling, and effective waste treatment (3). Several studies have evaluated the impact of single-use technologies applied in biomanufacturing scenarios. Single-use based facilities have the ability to reduce the overall environmental impact despite the creation of solid plastic waste, as demonstrated in a case study that compared stainless steel-based facilities to single-use-based facilities (1). A cost-of-goods model presented an even higher economic efficiency for single-use-based facilities.   

Water, water-for-injection, and pharmaceutical-grade water are some of the largest cost and environmental contributors in stainless-steel plants. Clean-in-place (CIP) and steam-in-place (SIP) are major operating procedures in stainless-steel scenarios combined with a high consumption of energy, water, and chemicals. Implementing single-use technologies in these situations can reduce water usage between 50–80%, and energy consumption up to 30%, depending on the process and application (9). 

 Because single-use systems are presterilized, mobile, and considered closed systems, they can reduce space requirements and the need for large cleanroom floor space, which is a high-energy consumer. Aseptic-transfer ports separating higher-classified production areas from lower or nonclassified utility areas can easily be applied. Corresponding heating, ventilation, and air conditioning  costs could be decreased and high-level production areas could be reduced a well due to relocation of the respective process steps into lower-classified or nonclassified areas. 

Any discussion of the environmental impart of single-use technologies needs to consider that landfill and incineration are still the common method for plastic waste treatment, with energy savings realized through cogeneration techniques for the production of heat or electricity. Recycling of single-use assemblies is limited due to multilayer films of bags and different materials of additional single-use components (e.g., sensors, filters, connectors, and tubing).  

The overall carbon footprint balance between stainless-steel and single-use facilities has been described by Sinclair et al as approximately 25% in favor of single-use facilities (9). However, a thorough analysis of example processes is still pending. For this reason, vendors are still calculating and evaluating the environmental differences between the two setups.  

The biotechnology industry—like the pharmaceutical industry—has to continuously and rapidly reinvent itself. The patent-expiration cliff, increasing competition, and decreasing development pipelines pose indisputable threats. Single-use technology can help reduce this burden. The technology has already improved specific process steps (e.g., buffer and media prep and hold) and new trends in standardized, single-use unit operations promise even more cost reductions and enhanced product safety. 

Although single-use technologies are not a silver bullet, they do open the door to new, innovative process and facility designs. Initiatives such as QbD and PAT are supported by closed, single-use systems and might accelerate their implementation. The environmental aspect also bodes well for single-use technologies. 

With single-use unit operations surfacing, a complete, single-use process might not be far-fetched for the biopharmaceutical-manufacturing industry, certainly not for development processes with lower volume needs. The future for such processes appears bright and innovative. 

 is global program manager of FlexACT,and 

 is director of sales and marketing for Europe/Asia, all at Sartorius Stedim Biotech GmbH in Goettingen, Germany. 

 is global director of sales engineering at Sartorius Stedim Systems GmbH in Melsungen, Germany, and 

 is vice-president of marketing FT/FRT, at Sartorius Stedim North America, 5 Orville Dr., Bohemia, NY 11949. 

To whom all correspondence should be addressed. 

 1.  E. Cronin and et al., "Sustainability Single Use Technologies, Environmental Impact and Waste Management," presentation, 

www.pharmamanufacturing.com/articles/2006/163.html

http://americanpharmaceuticalreview.com/ViewArticle.aspx?ContentID=48

Harmonized Tripartite Guideline on Pharmaceutical Development

 5.  C. Jimenez-Gonzalez and et al., "An Executive Guide to Pharmaceutical Manufacturing Efficiency and the Effect of Environmental Legislation," (Rockwell Automation, SSB-WP001A-EN-E  December 2009). 

The authors propose increased use of single-use technologies in biopharmaceutical manufacturing to achieve operational excellence without compromising product quality.

A Detail View of Single-Use Equipment Opportunities

Aseptic processing is of great importance within the biotechnology industry because cold sterilization by membrane filtration is the only method of sterilizing biological solutions. Any heat treatment would destructively affect the drug product and target protein. To ensure sterile filtered products maintain their sterile state, an increasing number of companies are using disposable process solutions to process or store the resulting filtrate. Modern disposable connectivity allows joining the filtrate hold bag with the filling line. If the product must be transported to another facility, then the filtrate could be frozen and thawed after it reaches the filling facility. A controlled freeze–thaw process ensures protein degradation is kept to a minimum as a result of the lack of fluid movements and/or uneven protein concentration within the frozen volume. 

In addition, biologics are commonly difficult to remove from fixed, stainless-steel equipment. The cleaning and cleaning qualification process is tedious and must be exceptionally thorough to prevent residues and potential cross-contamination. The cleaning and sterilization down-times for stainless steel vessels, transfer lines, or filter housings might require 8–10 hours and copious amounts of cleaning solutions and water-for-injection (WFI). Calculations have shown that the cleaning and sterilization process of a 1000-L vessel can cost as much as $5000, depending on the WFI costs, whereby a bag of this size is priced at $200–300. The need for shorter cycle times and reduced process-material pushed the development of disposable solutions, which require no set-up times or cleaning. Disposable mixing systems can be connected to capsule membrane filters and a hold bag. These interconnected disposable systems are gamma sterilized and ready to use.  

Another benefit of disposable equipment is the fact that the end-user does not come in contact with the product. Therefore, disposable equipment has increasing popularity in cytotoxic drug applications. The drug product is processed from one disposable process step to the next without the need to either dismantle a filter from a filter housing or clean a vessel by hand. Any time a drug product has a potential to harm the user, disposable processes are the equipment of choice.  

The first disposable units were probably filter capsule devices, which could filter small volumes without the need of a filter housing and required cleaning. These filter capsules incorporated a plastic filter housing and filter cartridge into a one-piece disposable unit (see Figure 1). Capsules nowadays are available in sizes from 100 cm

. These capsules can be assembled to other equipment and autoclaved, or the capsules are delivered presterilized by the filter manufacturer, including equipment connections (e.g., to a filtrate hold bag).  

Figure 1: Small-scale filter capsule devices. (ALL PHOTOS ARE COURTESY OF THE AUTHORS.)			

The next development was single-use sterile bags to replace glass bottles, plastic carboys, or stainless-steel containers for small-volume storage, transport of biological solutions, and growth media. Originally, blood and parenteral solution bags were adapted for process use. Because the mechanical stability had to be strengthened, specific polymeric bag films were developed. These films are laminated in various configurations to gain the mechanical strength for large-volume bags, to reduce any leachable release into the product, and to create an oxygen barrier (see Figure 2).  

The laminate combinations can vary depending on the bag design, manufacturer, and application served. Currently, bag designs are manifold and vary in volume from 20 mL to 3000 L. The assemblies are not restricted to only a bag and a filter. They can be highly complex and engineered to fit specific requirements of the end-user. For example, buffer hold bag systems are commonly a manifold of multiple bags of the same volume, which can be disconnected by tube sealers. These assemblies also contain a sample bag for quality assurance purposes. The filtered volumes can be stored or transferred to individual process steps within a facility. Large-volume systems require storage with trays, tote, or pallet systems to prevent the large liquid volume from moving. The bags within such storage systems require unfolding in a very specific manner, otherwise pressure folds will occur and could damage the polymeric film. The design of the filling and connectivity of any outlet from the bag are essential because the hold-up volume of a hold bag must be minimal.  

As mentioned, disposable systems are no longer restricted to a bag and a filter. Various equipment components are available that can be combined into a single-use system or process step. Such equipment components include: 					

Mixing systems (various designs and volumes)

Bioreactors (many designs, volumes, and agitation technologies) 

Membrane chromatography (e.g., ion exchange)

Viral clearance (e.g., filters and UV inactivation)

Other equipment solutions will soon follow as the requirements for single-use systems by the industry are more pressing and innovative. For example, work is underway to design disposable valves, pump heads, and filling systems. 

Disposable mixing systems are available from 5 to 1000 L and are most commonly used for buffer and media mixing, product compounding, or final formulation purposes. A more recent application is viral inactivation by pH shift. Such mixing bags use disposable sensor patches to determine the necessary pH levels during the inactivation period.  

Mixing methods include recirculation, pulsation of the mixing bag; use of magnetic impellers, stirrer bars, or pads; and use of a levitation mixer. The latter is unique to all others because the mixer does not come in contact with the bag material, and it floats on a magnetic field. This configuration avoids any friction of the bag material and provides fast and thorough mixing results. The various mixing technologies are useful because the applications and complexities of mixing vary. Liquid–liquid mixing has easy mixing requirements, but liquid–powder mixing can be difficult and may require careful design and mixing mode observations.  

Current single-use bioreactor systems are divided into two main categories: rocking motion and cylindrical-tank reactors with various agitation modes. Rocking motion reactors were the first to enter the biopharmaceutical industry. In this design, the cell culture is moved back and forth by a rocking platform, which allows mixing of nutrients and gas input into the liquid phase. These systems produce excellent results for shear-sensitive cell cultures and have been especially established in seed reactor applications. Rocking systems are available as a basic system without sophisticated controls. Separate systems can be connected to a control unit to measure the dissolved oxygen and pH by means of a disposable sensor. The control system can also run the gas and feed strategies, which is important when the system is used as a perfusion reactor. The working volumes of rocking reactors range from 0.2 to 500 L. Recent bioreactor developments have focused on cylindrical tank systems, which use multiple agitation methods, including sleeved or fully polymeric stirrers, vibro-mixing, orbital shaking, or gas mixing. The agitation methods and cell culture requirements determine the design for these reactors. Typical capacity volumes of these cylindrical disposable bioreactor systems range from 10 to 2000 L. These units are most commonly used with the control tower systems of reusable fermentation systems. These control towers create appropriate feed and gassing environments for the cell culture.  

Bulk raw materials are often shipped over long distances, and production processes may require a product-hold step because of downstream equipment bottlenecks. In both instances. solutions are required to prevent any protein degradation, which may occur as a result of enzymatic attack and temperature, pH, concentration, or gas conditions within the hold or transport step. To avoid such yield losses, the industry has resolved to freeze steps to keep the product stabile over a period of time. However, commonly used blast-freeze steps are uncontrolled and can result in freeze concentration, pH shifts, aggregation, ice crystals within the frozen material, or bag damages. To avoid such damaging conditions, disposable controlled freeze–thaw devices have been established. These devices contain a hold bag within a frame and a freeze–thaw module that uses heat-exchanger plates, which ensure a unified controlled freeze and thaw process. The product hold bag with the frame is transferred into the freeze–thaw module, and the heat-exchanger plates move in position, pressing against the hold bag. The heat exchanger plates therefore do not only ensure the temperature transfer, but also the uniform distribution of the liquid over the entire bag design. These units are available as small-scale trial devices with a volume of 30 mL and process scale up to 16 L. 

During the past 10 years, disposable technology innovations surfaced rapidly, and the nondisposable gaps within a biopharmaceutical process are now closing. There are still process steps or specific equipment parts that either will evolve into disposable systems or will be kept and integrated into a hybrid state. How total processes will look like in the future are only predictions, but developments of new disposable process components show the trend toward a total disposable process, at least in the small-scale volume streams (see Figure 3.)  

Figure 3: Possible disposable process stream.			

Such a process would require careful planning because the volumes within a process can change drastically, process steps require proper timing, and essential connectivity requires being qualified. In some instances, radiofrequency identification (RFID) can be used to either to track equipment or process units or to connect the required process units together. These tags will can also create appropriate and necessary shelf-life information, because gamma-irradiated polymers have limited shelf lives.  

The major items still not available as disposable units  are valves and filling lines, which are able to handle fluid volumes at high speeds. There have been attempts to design filling systems, but these have not penetrated the industry as much as the above described disposable technologies. It is only a matter of time when such disposable filling equipment will be made available.  

Innovative developments in disposable equipment enhance the safety in aseptic processing. Furthermore, these developments might create the possibility of a disposable factory. The benefits of complete disposability are relevant to all scales of bioprocesses, especially within the start-up phase. In early development, disposability reduces the need for major capital investments. As these technologies continue to develop critical factors such as drug cost, production cycle times, new product development time, and facility flexibility, all aseptic processing will be affected. 

Because most disposable devices are gamma irradiated, between 25 and 50 kGy short- and long-term, stability studies with the irradiated devices must be performed. Irradiation typically reduces the shelf life of such devices, and it must be determined what the limits are. Furthermore, the irradiation step could accelerate the degradation of the polymeric substances used, which can result in increased leachable and extractable levels. To determine the effects of irradiation and the stability of the polymer used, manufacturers subject these devices to a considerable regime of qualification tests before the device is commercialized. The qualification tests serve as a guidance by the end-user and commonly encompass, but are not limited to, the following tests: 					

Mechanical properties (tensile strength, elongation at break, seal strength, air leak test)

Gas transmission properties (ASTM D3985: oxygen, ASTM F1249: water vapor)

E.P. 3.1.7.: EVA for containers and tubing

Extractable and leachable tests with standard solutions

Gamma irradiation sterilization validation

These tests are conducted under standard settings with standard solutions. The data of these tests are available from the manufacturer.  

The authors discuss current and future disposable technologies and outline the validation and qualification steps that would be required for a possible disposable process stream.

Disposable Components in Aseptic Processing

Where possible, sterility of medicinal products is assured by heat sterilization. However, this cannot be used with every pharmaceutical product as it can alter or degrade the drug — particularly biologicals. In other instances, the gram-negative microbial bioburden level within the product is too high, meaning that heat sterilization would cause an undesirable concentration of endotoxin. 

Microporous membrane filters were invented in 1918 and came to use in the early 1930s.

  These filters either created a sterile effluent or a bioburden level reduction to a point of no concern in regard to any potential endotoxin contamination. Membrane filters in those days could not be standardized or reliably compared in consideration of retention performances. Instead, filter manufacturers performed microbial tests under different challenge conditions, and with organisms grown under diverse conditions and with different culture media. The lack of a standard made it impossible for the filter user and regulatory authorities to determine which filter should be designated the sterilizing grade filter.  

True comparability and performance analysis could only be performed when a standard microbial challenge test was defined that detailed: 					

The first steps towards establishing a standard were made with a document from the Health Industry Manufacturers Association (HIMA),

  which was later developed into ASTM standard F 838-83 and is now ASTM standard F 838-05.

  The focus on this standard for microbial testing led the industry to optimize sterile drug processing using filtration sterilization. The present biopharmaceutical interventions of fermentation-based drugs in medical treatments are the most recent consequences of ASTM standard F 838-05. 

A quarter of a century later, the standard still serves as the bedrock of organism quantification by which the efficacy of drug sterilization by filtration is determined. 

The first sterilizing grade filters were rated 0.45 μ and were used to remove yeast, mould and bacteria from beverages and pharmaceutical fluids. The definition of pore size rating commonly used is not derived from direct measurements, but from the rates of flow of fluids, air or water, through the filters. In filtration practices, however, emphasis is so heavily on particulate retention that it is sometimes popularly, but erroneously, believed that filters are classified according to the actual physical dimensions of their pores.  

Much ingenuity has been exercised in developing methods for measuring the size of filter pores. Flow measurements of both air and water have been widely used and resulted in an ASTM standard procedure (Figure 1).

  Other tests including mercury and/or air intrusion under high pressure have been employed and,

 more recently, vapour pressure analysis.

  New mathematical insights have also been brought to bear.

A first step towards a challenge test definition was occasioned by an incident of bacterial filter penetration. In 1967, Bowman 

. published the discovery of a smaller, water-born organism,

  which penetrated the 0.45 μ-rated filters that were then considered the sterilizing grade membranes. This finding resulted in the development and universal use of 0.2 (0.22) μ-rated membrane filters that could retain the smaller organism. The organism was identified as 

As with 0.45 μ-rated filters, the 0.2 μ rating was not standardized and multiple different challenge tests were again published by filter manufacturers. 

Sterilizing grade filters and their pore size ratings became critical tools within the industry and were, therefore, required to be tested accordingly. Any microbial breakthrough caused by an ill-defined rating could be detrimental to patient safety. Such occurrences were guarded against by assays enabled by the ASTM standard. 

The industry took action against the lack of a standard challenge test for sterilizing grade filters by forming, in combination with the filter manufacturers, HIMA. It established a standard microbial challenge test for sterilizing grade filters rated at 0.2 (0.22) μ. This was the first time the challenge organism, challenge level, growth media and filtration conditions, as well as challenge conditions, were defined. Another beneficial outcome of the efforts of this group was the term 'log reduction value' (LRV) as a description for the microbial removal efficiency of a filter. A filter with an LRV of seven is capable of reducing the organisms in the feed stream by seven orders of magnitude.

From that point onwards, the HIMA test methodology was used by filter manufacturers as the challenge test for 0.2 (0.22) μ-rated sterilizing grade filters, and as a verification method attesting that the filter will retain the challenge organisms at a level of 10

  of filtration area. Nevertheless, it only became an official standard when ASTM used its committees and membership to refine and convert the HIMA standard into ASTM standard F 838-83 in 1983, creating a solid, scientific benchmark. This standard was reviewed in 2005 with the consequent reissue of the standard as ASTM F 838-05.  

Integrity testing of filters is one of a series of interdependent activities for the preparation of sterile drugs. The key requirement of an integrity test is to establish a quantitative correlation between a property or characteristic of the filter of interest and its organism retention capabilities. This would, for example, address the needs of sterilizing filtration practitioners to identify filters of a particular pore size rating that have the necessary bacterial retention capability. 

FDA has defined a sterilizing filter as one that retains the classic challenge of at least 1×10

  of effective filtration area at pressures up to 30 psi (2 bar). This is best assessed by direct confrontation of the membrane by a proper organism challenge. However, once the filter has been tested it is contaminated by the organisms and, therefore, may not be employed as a process filter. Therefore, a surrogate test is used that is nondestructive to filters, and is based on correlation of pore size with organism retention. 

The recognition of a pertinent correlation involves more than the parallelling of two events; the relationship of the two actions that are seemingly correlated must accord to a technical logic as being the resultants of a common origin. The classical integrity test value or number correlates a filter's pore structure to its ability to completely retain challenges of a stipulated number of organisms per filter area. The significance of the integrity test is that it relates to its performance function. 

In the present practice, the membrane's largest pore size is probed by the bubble point test. Its value can be correlated with the membrane's ability to sustain the 1×10

 confrontation made by the use of the standard ASTM F 838-05 microbiological challenge procedure. On this basis, such membranes are experimentally qualified to be of the 'sterilizing grade,' and are, thus, labelled as being of the 0.2 (0.22) μ-rated pore size. They may then be selected for trials in actual product filtration sterilizations to ascertain their suitability to perform in given filtration contexts. 

Filter manufacturers have measured and correlated bubble point/diffusive flow values and LRVs for commercially available membranes. It is this correlation that serves as a nondestructive test for determining a given filter's organism retention capabilities. It is ASTM standard F 838-05 that makes possible the industry wide definition of LRV that is essential to this correlation. This is the information required to ensure the proper choice of membrane for use in filtrative sterilizations. 

Because of ASTM standard F 838-05, filtration with 0.2 (0.22) μ-rated filters became dependable instead of a gamble as to whether the so-rated filter was what it claimed. The FDA Aseptic Guidance document of 1987 was the first to define a sterilizing grade filter based on the ASTM standard description.

  The ASTM standard has also been adopted by many different agencies; for example, ISO's 13408-2 and PDA Technical Report 26.

This ASTM standard correlates the nondestructive integrity tests with the 

 retention characteristics of specific pore sizes. It is employed in defining the test limits of sterilizing grade filters. FDA recommends all sterilizing grade membrane filters to be integrity tested before and after utilization. As the filter user is unable to directly challenge every single filter used with organisms, filter manufacturers developed nondestructive integrity test methods, such as bubble point or diffusive flow, to serve as surrogates. However, these integrity tests must be correlated to the ASTM F 838-05 organism challenge test to determine their compliance with maximum allowable diffusive flow or minimum allowable bubble point limits (Figure 2). Such correlation tests are performed by filter manufacturers within their development laboratories and are published in validation or instruction documents. 

Furthermore, the standard became a cornerstone for the development of new filters in the 0.2 (0.22) μ-rated product area. Every time filtration products are improved or redesigned, these filters undergo standard bacteria challenge tests to verify the desired pore size rating. Moreover, while the ASTM F 838-05 standard defines the challenge test for 0.2 (0.22) μ-rated membrane filters, its guidance is of such value for filter manufacturers and users that its challenge conditions are also employed for other pore size ratings, except with different challenge organisms; for example, 0.45 μ-rated filters are challenged under ASTM test conditions with 

 being used as the challenge organism. For 0.1 μ-rated filters the commonly used organism is 

. New standards for challenge tests of different pore sizes could, potentially, be based on ASTM F 838-05, as the test method is already 'unofficially' used by many filter manufacturers to verify pore size ratings. 

In 1995, FDA employed the ASTM standard to further enhance sterility assurance by filtration. Most challenge tests performed before that point were commonly carried out on a water or broth basis under the teaching of FDA's challenge standard. However, the agency required verification of the sterile filtration performance of the filter under actual process conditions governing the filter's use and, if possible, with the actual drug product being the challenge fluid. The challenge level and the organism type stayed the same, and the challenge conditions and fluid stream utilized were adapted to production process reality. Again, this requirement has been prescribed by multiple organizations and guidances. A specific decision matrix has been established to define which actions should be taken when the challenge fluid is incompatible with the challenge organism (Figure 3). 

So successful were the 0.2 (0.22) μ-rated membranes in producing sterile effluent that the identification of a filter's pore size rating by way of integrity testing was taken to ensure that its use would yield a sterile effluent. The guiding assumption, however, was dependent upon sieve retention or size exclusion being the organism retention mechanism and was based on 

With time, it was found that the 0.2 (0.22) μ-rated membranes did not necessarily result in a sterile effluent. It would seem that the key correlation based on the ASTM developed standard was not acceptable.

Further investigation showed that the organisms that escaped capture by the 'sterilizing' 0.2 (0.22) μ-rated membranes had undergone size diminution following immersion in liquid vehicles of limited nutritional value. Far from proving inadequate, the correlation demonstrated its applicability for retaining smaller organisms by the use of membranes of lower pore size ratings, namely, the so-called 0.1 μ-rated filters. 

Filtration practitioners' understanding that the sizes of organisms were capable of undergoing change was heightened by Leahy and Sullivan who found that the size of the 

 microbes depended upon the method of cultivation.

  This emphasized the need to standardize the procedure whereby the very organisms used to confront the filter pores would repeatedly and dependably be cultivated to match in size. The operational protocol set forth in ASTM standard F 838-05 made this possible, eliminating the issue of organism size variability. The standard satisfied the need for organisms of sufficiently similar size to serve as standard size particles. Consequently, they were suitable for the assaying of their retention by pores of different sizes. This legitimized comparisons among competitive filters on technical grounds as the standard eliminated challenge test variability. 

To increase effectivity of the sterilizing filter, the substitution of the 0.1 for the 0.2 (0.22) μ-rated membrane was suggested. However, of the seven 0.1 μ-rated filters tested, only four were found efficacious. The more generalized use of 0.1 μ-rated filters was, thus, unwarranted.  

Correlation of LRVs with the assaying of organism counts could not be applied to a pore size classification that lacked industry identification. A suitable test organism whereby the ASTM standard F 803-05 test could provide the necessary correlation had first to be agreed upon. 

In the circumstances, the use of the tighter filters was limited by practical considerations such as lower rates of flow. The substitution of 0.1 for the 0.2 (0.22) μ-rated filters is recommended where organism shrinkage is possible — especially for filtrations of long duration. The use of the tighter membrane awaits its LRV correlation to the native bioburden organism colony counts of its effluents. The native microbe appropriate to the inquiry requires identification. The ASTM standard creates a basis to perform such investigations. 

The success of filtration sterilization applications encouraged competition between filter manufacturers that some believe bordered on marketing rather than technical considerations. 

In the circumstances, the fact that ASTM standard F 838-05 existed empowered the pharmaceutical industry to secure scientific-based correlation data that muted commercial concerns. Beneficially, the ASTM standard devoted to technical matters, in quieting commercial considerations, attained a laudable goal. Society and the patient were the beneficiaries of an ASTM standard-setting effort. 

Bioburden studies, integrity testing and process validation are the building blocks of filtration sterilization exercises. The contributions of ASTM standard F 838-05 to bioburden studies and to filter integrity testing have been discussed, along with its influence on fulfiling validation requirements. It created a well-defined testing range and activity flow for the validation efforts essential for verification of the filtrative sterilization operation. Without this industry standardization on behalf of a specific and acceptable test range, individual efforts would be confined to limited goals. The basis for the enhanced challenge definitions, and for the adoption of a true process validation approach was ASTM F 838-83 (now 838-05).

Although realized in 1983, ASTM's standrad test method for determining bacterial retention of membrane filters used for liquid filtration is still hugely beneficial to today's pharmaceutical industry.