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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.
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.
Disposable equipment development
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 cm2 to 4 m2. 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).
Figure 2: Example of a bag laminate.
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:
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.
Freeze – thaw
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.
Validation and qualification necessities
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:
These tests are conducted under standard settings with standard solutions. The data of these tests are available from the manufacturer.
Because qualification tests run under standard conditions, possible process specific validation requirements must be met. Such validation studies can be supported by the services of the vendor. Process validation studies would, for example, use a model solvent, but the process parameters would be within the end-user's specifications. Leachable testing with a product is commonly not possible because the product would cover any potential peaks. For this reason, model solvents are used that are similar to the solvent used within the product stream. However, tests must be conducted to determine the possible influences by the environmental conditions used in the end-user's processes. These tests will ensure the disposable device performs to the end user's specifications.
The process validation steps vary because the disposable devices have different purposes. Sterilizing-grade filters must undergo a product bacteria challenge test under an end-user's process conditions. If the actual fluid is bactericidal or bacteriostatic, then a placebo solution can be used. In any case, the influence of the process conditions and fluid toward the challenge organisms or separation mechanisms must be determined. Product hold bags or mixing bags do not need to undergo bacteria challenge tests, but they may have to undergo bacteria ingress tests. Both the filter and the bags systems must be tested for leachables or extractables. As mentioned, the end-user should take advantage of the vendor's services, which support the qualification documentation and process validation.
Many disposable devices are already available, most commonly as a single entity, but in some instances already connected (e.g, filter–bag or bioreactor–filter systems). Rapidly developing connectivity will enhance the development of connected, integral systems and potentially total disposable processes, at least in small volume scales. Some developments (e.g., filling lines) are still necessary. The last step in aseptic processing has not reached the level one finds in the intermediate steps.
The benefits of disposability within aseptic processes are obvious. Cleaning deficiencies are a common regulatory observation, which would be eliminated by disposable equipment use. The risk of cross contamination is greatly reduced. Moreover, disposable, aseptic connectivity will reduce the level of end-user manipulation within the process and therefore create higher safety. Disposability is also valuable from economic and environmental standpoints because there is a cost savings in cleaning solutions and copious amounts of water, as well as the high energy levels required to heat the cleaning solutions or steam sterilize reusable equipment.
Maik W. Jornitz* is group vice-president of marketing FT/FRT at Sartorius Stedim, email@example.com. Jean-Marc Cappia is group vice-president of marketing FMT at Sartorius Stedim SA. Theodore H. Meltzer is principle of Capitola Consultancy.
*To whom all correspondence should be addressed.