In November 2008, the US Food and Drug Administration issued a much needed document titled, 

Draft Guidance for Industry on Process Validation: General Principles and Practices

. The draft guidance clarifies the quality-by-design (QbD) approach to processing human and veterinary drugs, including biologics, active pharmaceutical ingredients (APIs), and medical devices. Although the document may have been better titled "process design," it has addressed many industry concerns regarding how to take a life-cycle approach and how to meet regulatory expectations with regard to validation. Many questions still remain, however, and industry must grasp and integrate the proposed guidance concepts along with the International Conference on Harmonization's guidelines Q8 

Individuals working in pharmaceutical sterile development and manufacturing will undoubtedly be the most affected by regulations associated with QbD because of the unique associated technologies, processes, and products currently in use and development (see sidebar, "Quality-by-Design Overview"). Many of these technologies require new manufacturing processes that support existing traditional fill-finish operations. Many facilities, utilities, processes, and equipment may need to be modified (e.g., improving controls to reduce variability). This article provides an overview of FDA's draft guidance on process validation and QbD's impact on sterile manufacturing operations.  

The following sections discuss how the draft guidance may influence the manufacturing process and product development. Processes addressed include manufacturing, aseptic manufacturing, lyophilization (freeze drying), and others that depend on multiple-unit operations (see Figure 1).  

Figure 1. (ALL FIGURES ARE COURTESY OF THE AUTHOR) 

Process design is typically the biggest challenge for a company's process development team and has tremendous impact on the success of the product. Sterile manufacturing has become increasingly more complex because of the increase in the number of poorly stable compounds, new technologies, unit operations, and controls. Numerous biotechnology companies, for example, use unique freeze–thaw systems to support dispensing and bulk-drug storage at temperatures often ≤ –80 °C that provide complete control of bulk-drug thawing, dispensing , freezing, and storage. 

FDA's draft guidance suggests that the process development team design a process suitable for routine commercial manufacturing that can consistently deliver a product that meets its critical quality attributes (CQAs). The team's objectives are to: understand the sources of variation, detect presence and degree of variations, understand the impact of variation on the process and product attributes, and control variation in a manner that is commensurate and proportionate to the risks presented to the process and product. FDA further recommends an integrated team approach, with members representing multiple disciplines. The team should have solid project-management skills and archiving capabilities, be able to capture scientific knowledge and maintain project plans, and have full senior-management support. The latter may involve regular reports or presentations to senior management (3, 4).  

In addition, the draft guidance calls for process decisions and justifications of control to be documented, internally reviewed, and preserved for later use in the product life cycle. Verification and mapping of the process design through to commercial documentation is required and planned changes should be documented and justified. Finally, after the performance qualification (PQ), a report documenting and cross-referencing results, data, issues, nonconformances, corrective actions, and overall conclusions regarding whether the process is in an adequate state of control must undergo management review and approval. 

 Design of experiment (DOE) and risk analysis are recommended in the draft guidance to provide data that support process design. The emphasis should be to reveal relationships between variable inputs (e.g., component characteristics, processing parameters) and resulting outputs (e.g., in-process material, intermediates, or the final product).  

The draft guidance states "early process design experiments do not need to be performed under CGMP conditions." Emphasis during early stage development are verification rather than validation. "Decisions and justifications of the controls should be sufficiently documented and internally reviewed," according to the draft guidance. Some examples of early-stage product assurance are outlined below (4, 5). 

Viral and impurity clearance studies are required because they have a direct impact on product quality

Cleaning verification would replace validation during the early stages of development 

Sterility must be ensured but may not be completely validated (e.g., if performing sterile filtration on a water-based product, a filter integrity test may be performed using water-for-injection to demonstrate that the filter still meets its manufacturer's integrity test value)

Preliminary specifications, tests, acceptance criteria, and limits should be operational.

Overall, the further along a company is in its product development, the more verification and validation are expected. Process controls that address variability can help to ensure the product's quality. 

 "It is important to understand the degree to which models represent the commercial process including differences that may exist," states the guidance (4). The significance of understanding the process increases with a model that properly reflects all the variants of the process and product. A good model can be used as a tool for process and equipment design, process control development, estimating variablity, and training personnel. 

Process analytical technology and qualification.

 Process analytical technology (PAT) "uses timely analysis and control loops to adjust processing conditions so that the output remains constant," states the guidance (4). As a result, there is a higher degree of process control. It is unclear, however, whether maintaining the process within the design space using PAT reduces process-validation testing. The draft guidance places added emphasis on design (qualification = design plus verification). There are requirements, for example, to challenge the process under load, to test interventions, and to test stoppage and start-up routines as expected for production. 

The common industry practice of testing three batches for PQ may no longer apply. Specific requirements regarding when batches can be released and the information needed to begin commercial distribution are included in the draft guidance. PQ must have a higher level of sampling, testing, and scrutiny of process performance to confirm the product's quality during batch processing. 

According to the draft guidance, "In the case of PAT strategy, the approach to process qualification will be different from that for other process designs,"  but the agency did not explain how the process will be different (4). This difference is important because additional use of PAT leads to additional validation to verify the technology's capability. Industy will assume this means an easier validation approach because conformation of control would be readily available, but there are no examples provided in the draft guidance. 

With regard to continued process verification, the goal is to ensure the process remains in a state of control during commercial operations (3, 4). The draft guidance recommends manufacturers include intra-batch as well as inter-batch variation as part of their continued verification program. PQ sampling levels should continue until variability is assessed. 

The draft guidance suggests that a statistician be involved in the development of the company's data collection plan and in the selection of statistical methods to evaluate process stability and capability. Included in this review should be quantitative statistical methods where feasible. Process flow diagrams for commercial manufacturing, for example, should be completed as follows:   

Describe each unit operation and placement in the process

Describe process monitoring and control points

Describe components and other processing material inputs

Describe expected outputs (e.g., in-process and finished products). 

In addition, the flow diagrams should preserve a life-cycle approach to facilitate comparison and decision-making regarding their comparability. 

Assuming that the manufacturing process design space has been properly developed and CQAs and DOEs have been verified, technology transfer, as well as scale-up, should go smoothly. As with most typical API-manufacturing operations, process design and controls ensure a state of control. When these approaches and technologies are applied to a batch-based system, they may, in the future, lead to more continuous sterile processing due to the increased level of control. 

The following sterile-process technologies are well defined in the literature: sterile filtration (F), autoclaves (A), steam cycles, dry-heat (DH) ovens and tunnels, gas sterilization (ETO), chlorine dioxide (ClO

), ionization radiation (ebeam, gamma), and terminal sterilization (steam, gamma radiation). These technologies must be validated with the specific component, drug product, or commodity.  

Sterilization technology review. Sterile filtration.

 Filter validation is normally done by filter manufacturers or outside laboratories but the responsibility remains with the drug manufacturer (5). 

 Steam sterilization (i.e., autoclaves, sterilize-in-place [SIP]), is the method of choice whenever possible due to data available and capability to assure sterility. This method is used for transfers into the aseptic area, process sterilization, SIP of the product delivery system, and product contact-component sterilization. Steam sterilization is limited by its temperature and pressure impact. Many plastic items, therefore, require other methods of sterilization. Control is typically ≥121.1 °C. Product contact components must have had prior pyrogen removal steps to ensure expectation of a minimum 3-log reduction. 

 The DH approach is mainly used for glass components because of the temperature during processing (a dry-heat oven is typically controlled ≥200–250 °F and dry-heat tunnels are typically controlled at 275–350 °F). Dry-heat sterilization can also be validated for a 3-log or greater pyrogen reduction. 

  Gas sterilization or ETO, is used for product contact plastics and commodity transfers. The method is not used in processes and operations due to safety issues. Product contact components require ETO degassing after the cycle is completed. ETO is a toxic and hazardous chemical. Cycle control includes ETO concentration, humidity, and pressure and similar to steam sterilization, requires prior pyrogen removal steps. 

 Ebeam is easy to define and makes it easy to control sterilization of the surface and, to some extent, the  depth of exposure and microbial kill. Recently, the ebeam method has been used to sterilize the lids of syringe bulk containers before filling. Because ebeam is not currently used for product contact components, pyrogens are not an issue. The sterilization dose for radiation processes (i.e., ebeam and gamma) are 25 kGy (2.5 Mrad). 

  VHP is currently the method of choice for isolator decontamination. Sterility is sometimes claimed. Because VHP is not a true gas, it can be affected by cold spots. VHP also can be used to sanitize transfer items into aseptic-filling operations, but cannot be used to sterilize or depyrogenate components. Cycle requires control of concentration of H

, relative humidity, and temperature. Circulation fans within an isolator are often used to help provide constant conditions within the isolator (5). 

 CD is currently the least used method throughout industry for sterilization, but provides significant opportunities because it is a true gas and can be validated for sterility. Areas of opportunity include isolators, transfers to the aseptic area, and processing equipment. CD is widely used in the food industry for sanitization and disinfection. Concentration can be monitored and controlled. Aeration is repeatable. Passivation of stainless steel may be required. Controls consists of concentration, humidity, temperature, and pressure.  

Other sterilization technologies include gamma radiation, which is used for product terminal sterilization and component sterilization by contract manufacturing organizations (CMOs). Gamma-radiated presterilized syringes are a common usage of this technology in the industry. Transfer of these types of presterilized components to aseptic-filling areas can be troublesome, however, without a defined transfer sterilization method. Recently, some equipment suppliers have included ebeam sterilization to improve this transfer. Ozone (O

) technology has potential as a sterilization method as well, but is not currently commercially available. Peracetic acid as a sterilization technology may still be used by a few companies, but most have switched to VHP. Other forms of sterilization or environmental control have limited support data and therefore represent an increased risk requiring in-house technical knowledge (e.g., high-intensity light, ultraviolet light).  

Environmental testing is designed to check facility, process, personnel and environmental cleaning methods to determine: (A) that after being in a state of shutdown that includes relaxed gowning, the facility and equipment can be cleaned, sanitized, and in a state of microbial control appropriate for pharmaceutical operations; and (B) that the facility can maintain a level of environmental control during normal processing operations. 

Critical testing required before startup include: high-efficiency particulate air (HEPA) filter testing, room differential verifications (e.g., air flows, alarms), cleanroom smoke studies, and environmental mapping during static and dynamic conditions (i.e., nonoperational and operational conditions). Note that any physical change(s) made in aseptic processing (e.g., facilities, equipment, layout) when adding a new process requires revalidation. 

Aseptic processing includes sterile manufacturing, aseptic filling, lyophilization, stoppering, sealing, and spray-drying. Examples of facility and processing-type CQAs that enhance overall process control from outside influence are outlined below.  

Aseptic operations should be protected from potential leaks (e.g., roof leaks and process leaks, including condensation from heat, ventilation, and air conditioning [HVAC] systems). Some processing equipment (e.g., restricted access barrier systems [RABS]) may also contain HVAC or refrigeration that create moisture issues. 

Critical operations should be visible without environmental impact. The objective is to keep personnel out of the critical processing areas while still being able to view operations for documentation and training.

Gowning areas, including barriers such as the step-over for applying booties, are typically dirty due to employee activity. Consider airflow turns higher than normally recommended (at least 80 air turns is preferred). 

Cleanroom airlocks should be designed at the same classification as the area they support. Consider having sweeping airflow from the clean side to the dirty side with the return close to the dirty side's entry door. 

Cleanrooms should be designed to take up just one classification level, thereby eliminating confusion (e.g., no Class A (ISO 5] and Class B [ISO 6] in the same room).

Filling lines and critical processes should be physically separated from operators. Equipment within isolators or RABS can help to significantly control contamination.

), consider the location of HVAC room inlets and returns to avoid potential cooling impact on the sterilization process.

Articles

The author reviews the draft guidance on process validation, its QbD applications, and its potential impact on sterile manufacturing operations.

Applying Quality by Design to Sterile Manufacturing Processes

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

A visit to a pharmaceutical aseptic processing operation in the 1970s would have some automation on display, but certainly nothing like what is possible today. Just three decades ago, automation consisted primarily of filling, stoppering, and capping of vial products, or filling and heat-sealing of ampules. Filling of syringes, to the extent it was done at all, involved extensive manual intervention. Lyophilization required completely manual transport of filled and partially stoppered vials from the end of the filling line to the lyophilizer. Once the containers arrived at the lyophilizer, the unit had to be completely loaded by hand, starting generally from the top shelf and working to the bottom, with the operators often ascending and descending ladders to reach the top shelves of the large-production lyophilizers. Of course even this task was a major advance over what one would have seen in the 1940s, when aseptic processing was predominantly a manual operation using rudimentary clean booths or glove boxes.  

By the late 1970s, there was a greater awareness emerging regarding the impact of gowned operators in aseptic processing, and some industry scientists began to state this fact clearly and publicly. A 1988 article entitled, "A Review of Current Technology in Parenteral Manufacturing," written by members of the PDA research committee, stated forcefully that the principal contamination concern in aseptic processing was personnel-related contamination. This article noted that emerging technologies were already mitigating contamination risk. It was in that same decade that the authors became fully aware of the impact of personnel-borne contamination and took part in projects in which the processing equipment was specifically designed to mitigate that risk through the application of automation (1).  

Early experiences in automation as a risk-mitigating factor

In 1981, one of us (Akers) took a position in which he was responsible for environmental monitoring and media fill. The aseptic processing operation in this facility was quite diverse and consisted of vials, ampuls, drop-dose products (both otics and ophtalmics), powders, and ointments. The basic mode of production was generally the same in all cases. Glass or plastic containers as well as caps, stoppers, dropper tips, and ointment tubes were sterilized by dry heat, ethylene oxide gas, or moist heat. After sterilization in bags or trays, these components were manually transferred to the filling rooms and then loaded by operator interventions onto accumulators or into parts hoppers. Process filters were also autoclaved and connected to the wetted path aseptically by operators; in fact, all setup activities were manual. In addition, all on-line sampling, weight checking, and equipment adjustment during processing were fully manual and required near-constant intervention.  

It is both important and interesting to note that the practice of process simulation was at that time in its infancy as an absolute requirement and in fact had not yet become a standard practice for ointments. Typical media fill acceptance criteria were no more than one contaminant per each 1000 units filled. In our facility, successful media fills (i.e., those that met the acceptance criteria) were in the majority, but perfect media fills were a rarity. It should be noted as well that no media fill required more than a 3000 unit sample size. Typical line speeds were in the 40–60 unit/min range.  

It was obvious as we studied media-fill outcomes that ampul processes provided the best results. Interestingly, ampul lines consisted of only one component and generally required fewer people and fewer interventions. Plastics, tubes and–to a lesser extent–vials were more challenging and required more personnel and a higher level of intervention intensity. However, the media fills that caused the most concern were those that involved lyophilization, which not surprisingly required more personnel and more interventions because the lyophilizer chambers had to be loaded manually. Thus, we knew empirically in the early 1980s what subsequent risk analysis studies have confirmed; namely, that manual lyophilizer loading was (and in some cases still is) a significant aseptic risk factor.  

After a few years, a new vial line was installed that for the first time introduced automatic vial washing, continuous dry-heat tunnel for sterilization, and depyrogenation of glass vials. This far more advanced processing line operated at up to 300 vials/min and required only two operators, while the older vial lines it replaced required as many as six people working constantly. This processing line also featured far better stopper feeding and needed far fewer line interventions. The results were dramatic. As a result of the higher speed, media-fill tests expanded in sample size to 5000 units or more, and zero contamination results became far more common (2). In addition, although the cleanroom design and gowning conditions remained unchanged, environmental monitoring results were quite obviously improved in every facet. This fact was attributed to the lower personnel population in the filling room and the less vigorous work required because vial supply, previously a laborious task, had been fully automated. 

Not long after the introduction of this improved and more automated vial line, a continuous ampul line was introduced and the improved performance was even more striking. Because of the very high speed operation and changing regulatory requirements, management made the decision to conduct media fills that lasted one full hour. As a result, media-fill sample size increased to approximately 25,000 units. Only one operator was required to run the line, and by and large they were only required to observe the line and to correct infrequent jams. An important lesson was learned and a lasting impression was made: Automation and the elimination of interventions reduced risk, not just theoretically but in practice as the results emphatically demonstrated (3, 4).  

During the late 1980s, Shibuya Kogyo (Kanazawa, Japan) was selected to design, build, and install a state-of-the-art vial processing line at E.R. Squibb (New Brunswick, NJ). This vial production line embodied what were a number of automated features that are now rather common but were quite unusual two decades ago. One of us (Izumi) was directly involved in this project and the other (Akers) was fortunate enough to visit the facility not long after validation was completed and eventually became a technical consultant to Shibuya Kogyo. This facility not only had a fully automated vial-washing and depyrogenation system but also included automated weight checking, vial-handling advancements designed to prevent misfeeds, automated clean-in-place and steam-in-place systems to eliminate aseptic connections in set-up, and automated lyophilizer loading of three large production lyophilizers. Although the data processing and acquisition systems available at that time were far less sophisticated than what is available today, this filling line was arguably the most sophisticated aseptic production line in use in the pharmaceutical industry at the time of its installation. Automation had eliminated even the highly risk intensive aseptic set-up and lyophilization loading activities.  

The ability of this system to run with minimal line stoppages and infrequent interventions pointed the way to the future in another way. It seemed that a marriage of automation and isolator technology, which was also an emerging and exciting new concept in pharmaceutical aseptic processing, would be a logical way forward. This has proved to be the case and the remainder of this article will be devoted to an overview of a few projects undertaken over the past decade and a half and will explain the role played by automation in solving sometimes difficult production challenges. 

 defines automation as "The technique, method, or system of operating or controlling a process by highly automatic means, as by electronic devices, reducing human intervention to a minimum." This is obviously and appropriately a very broad definition that includes what might be called machine automation as well as the field of robotics. Over the years, we have been involved in several projects that use automation and robotics, usually within the same process with the objective of as 

 says, "reducing human intervention to a minimum." However, the objectives in these pharmaceutical manufacturing projects are not merely to relieve human operators of repetitive and often boring tasks undertaken in difficult or uncomfortable environments but also to reduce end user or patient risk from contamination. Experience has shown that automation and robotics can achieve both of these goals. In cases of high pharmacological activity and allergenic, cytotoxic, and radiological products, the benefit of increased operator safety also can be added (5).  

Combination products, automation, and separative technologies

The first project began in 1994, when Shibuya Kogyo designed several filling lines for Nipro Corporation (Osaka, Japan) (6). These aseptic fill lines were for beta-lactam antibiotics and involved both automatic parts feeding and assembly. The first filling line involved no less than 13 isolators, a blow–fill–seal bag filling process, terminal sterilization with automatic feed and unload, and two semicontinuous vapor-phase hydrogen peroxide (VPHP) surface decontamination systems for component in feed. The finished product consisted of a terminally sterilized bag aseptically assembled to a double-ended linkage into which a prefilled standard glass vial was fit. The user activated the product and initiated reconstitution by simply twisting the linkage that contained a device with double-ended needles.  

A fully safe liquid pathway was then created to mitigate risk that inevitably arises from product admixture. Nipro designed and built the main assembly system that incorporated robotic assembly stations. Shibuya was responsible for the isolators, the VPHP tunnels, the parts feeders, and total system integration. The largest isolator was the autoclave interface, which contained the conveying apparatus for loading specially designed bag positioning pallets into the autoclave. This isolator had an enclosed volume of more than 1800 ft

, in fact the both the number and total enclosed isolator volume of this project still exceed that of any aseptic processing line build anywhere in the world. 

Given the size, scope, and complexity of this system, success would simply not have been possible without an extensive use of automation. In some cases, the materials supply requirements were such that undertaking them by human intervention would have been impossible in isolator technology.  

Shibuya Kogyo and Nipro have collaborated on several other filling lines for dual-container products in which two separate bags are assembled together and reconstitution occurs by the opening of a pealable seal between the two bags. These products require both a highly automated liquid fill line and a powder fill line as well as an assembly station. Restricted access barrier systems (RABS), isolators, automation, and terminal sterilization are used to achieve the most reliable outcome in terms of sterility assurance while retaining desired production reliability and therefore consistent throughput (4) 

Radiopharmaceuticals are not only aseptically filled but also have human exposure considerations. For this application, an isolator was the most effective choice for the filling environment, while robots were used for product packaging, labeling, and inspection. This system produces radiopharmaceuticals in both vials and syringes, which underscores another important feature of robotics: positive container handling with easy adaptability without the change parts normally associated with format changes.  

Robotics has the further advantage of avoiding the need for extensive lead shielding because the system is designed to operate without intervention. This design saves both equipment and facility costs and makes access simpler when the facility is undergoing periodic maintenance. The flexibility of the robots also facilitated the incorporation of visual systems for the inspection of each syringe and vial and the verification of proper labeling information. The system has automatic alarm and rejection should a problem be detected. This production system proved again that a variety of advanced technologies, including isolators, automatic inspection systems, automated filling systems, and robotics could be brought together to ensure a high level of aseptic process control with enhanced overall production reliability. 

A few years ago, Shibuya Kogyo undertook the design and manufacture of a high-speed vial line for vaccines that was delivered to Handai-Biken (Kagawa, Japan) (7). Commercial production from this facility commenced in 2005. All product filling, lyophilization, and stoppering are performed in unidirectional airflow isolators that comply with ISO 14644 Class 5 requirements. The system consists of five isolator sections:  

Depyrogenation tunnel and filler interface

The total enclosed volume for this isolator network is 47.3 m

, with the lyophilizer loading and unloading and rubber stopper feed isolators each comprising about 17 m

Both machine automation and robotics play central roles in the operation of this production line. The depyrogenation tunnel has push-button setup and fully automatic sterilization of the cooling zone. It also has an automatic vial-counting system to ensure that each row of glass entering the tunnel is carefully arranged and introduced without crashing the vials into the previously loaded row. This automated feature reduces the likelihood of vial breakage.  

The filling system has two weight-control features. The first is an automated mass flowmeter filling system in which all fill data are digitized and electronically stored. The second system is a gravimetric weight-check system that uses load cells to take empty and filled vial weights. The gravimetric weight-check data are also digitized and stored electronically. Both systems automatically alarm and reject out-of-specification containers. Actual fill-weight accuracies attained with the mass flow system are measured within ± 0.5% of the target. 

The rubber-stopper supply system is fully automatic and uses the first VPHP-compatible robots installed and validated in the world. One such robot unloads sterilized containers of rubber stoppers from the autoclave, while a second robot lifts these cans and replenishes the load of stoppers in the feed hopper when signaled to do so by a level sensor. Thus, human operators are not involved in transporting containers of stoppers nor do they play any role in the feeding of stoppers into the feed hoppers. 

Like the previously described vial line installed in the United States, the Handai-Biken system has fully automated lyophilizer loading and unloading. In this case however, the loading system is not only fully automatic, it is also VPHP decontaminated and operates within an isolator enclosure. This filling line is an excellent example of the marriage of machine automation and robotics with modern isolator technology. The use of isolators and VPHP decontamination requires that all components be resistant to long-term exposure to H

This article provides only a glimpse into the history of automation in aseptic processing and its development during the past three decades (8). Although our experience is extensive, we realize that there are many other examples of the use of automation in aseptic processing and the marriage of automation with modern separative environments such as isolators. We do not see the evolution in aseptic technology described in this article coming to an end; rather we believe it will follow an accelerating trajectory onward and upward. The future of aseptic technology lies in the continued implementation of automation with the ultimate goal being the complete elimination of interventions altogether. We have glimpsed this future with the design and installation of high speed "interventionless" aseptic processing systems operating at throughputs as high as 1200 containers/min.  

Robotics continue to evolve as well, and VPHP-compatible six-axis robots are being designed for cytotherapy products among other potential production, research, and testing applications. These robots can be operated either under full electronic programmed control, or by humans controlling them by remote control devices called "Manipens." Current software allows the operator to quite literally train the robot to perform a specific task. These robots can interface well with single-use technologies (disposables) and are well-suited for aseptic compounding operations as well.  

The future of automation and robotics in aseptic processing is an exciting one. Thousands of industrial robots are undertaking a wide range of manufacturing tasks in facilities throughout the world. Our industry has actually been relatively slow to adopt robots, but we believe change is underway and that the future of aseptic processing is one in which human contamination is no longer a risk (9). 

 is executive vice-president at Shibuya Hoppmann Co., a subsidiary of Shibuya Kogyo, Co. Ltd. 

 is president of Akers Kennedy Associates and technical consultant to Shibuya Kogyo, Co. Ltd., 

*To whom all correspondence should be addressed. 

 1. J. Akers et al., "A Review of Current Technology in Parenteral Manufacturing," 

Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing

 3. J. Akers and J. Agalloco, "Aseptic Processing: A Current Perspective," in 

, R. Morrisey and G.B. Phillips, Ed. (Van Nostrand Reinhold, New York, NY, 1993). 

 4. J. Agalloco, J. Akers, and R. Madsen, "Current Practices in the Validation of Aseptic Processing–2001," 

PDA Technical Report #36, PDA J. Pharm. Sci. Technol. 

 5. J. Agalloco and J. Akers, "Risk Analysis for Aseptic Processing: The Akers-Agalloco Method," 

 6. J. Akers et al., "Manufacture of a Dual Chamber 'Kit' Product Utilizing a Combination of Aseptic Fill in Isolators and Terminal Sterilization," 

Proceedings of the ISPE Barrier Isolator Technology Conference, 

 7. J.E. Akers, M. Kokubo, and Y. Oshima, "The Next Generation of Aseptic Processing Equipment," Aseptic Processing Supplement to 

 8. J. Akers, K. Tanimoto, and M. Kawata, "Aseptic Processing the Japanese Way," 

 9. W. Morris and E. Hough, "Risky Business-Aseptic Processing," 

The authors review the role of automation in aseptic processing and describe their experience in implementing advanced technologies, including the use of isolators and robotics.

The Impact of Automation on Aseptic Processing

I dentification of fungi, especially filamentous fungi, has been a very difficult task. Because of the amount of experience required to accurately identify filamentous fungi to the species level, it has become acceptable to either identify these organisms to the genus level or, in some cases, simply identify them as "molds." Over time, there have been numerous attempts to automate biochemical tests. However, because eukaryotic organisms exhibit far less metabolic diversity than prokaryotic organisms, these systems tended not to have the resolution required to differentiate fungi at the species level. 

During the manufacture of sterile and nonsterile pharmaceuticals, the presence of molds in the manufacturing area is often a cause for concern. A well-designed environmental monitoring (EM) program should detect the presence of molds before they can contaminate the product, but on occasion, fungal isolates will be recovered from sterility or media-fill failures. In these cases, it is extremely important to identify the contaminating organism at the species and, possibly, the strain level to track its origin. 

A recent review of the US Food and Drug Administration's product recalls illustrates the inability to identify fungal samples at the species or, in many cases, even to the genus level (1). At the conclusion of FDA's investigations, of the 12 fungal isolates responsible for sterile pharmaceutical product recalls, none was identified at the species level, only four were identified at the genus level, and eight were simply designated as mold or yeast. In nonsterile FDA product recalls, 32 products were contaminated with fungi. Of these, three were identified at the species level, none was identified at the genus level, and 29 were identified as mold or yeast.  

Although any type of fungal contamination is cause for concern, a species-level identification is needed to provide a definitive root cause as part of an investigation. Fungal identification, especially mold identification, has not been held to the same standards as microbial identification regarding the investigation of contaminated pharmaceutical products. This identification has been done primarily because it is difficult to identify fungi at the species level, not because the identity is not important. The availability of molecular-based identification for fungi—both yeasts and molds—to all pharmaceutical microbiology laboratories has led to the virtual disappearance of these limitations. 

For many decades, ribosomal DNA (rDNA) sequences have been used for organismal taxonomic classification. More recently, ribosomal genes have been used to study the phylogenetic relationships of fungi, with some surprising results. The first came in 1993 when Baldauf and Palmer discovered that fungi are more closely related to animals than they are to plants (2), as had been assumed to be the case based on morphology and other phenotypic characteristics. Another realization was that many species of fungi are synonymous with one another, with many names used to describe the same organism. 

, for example, has 173 synonyms, many of which have been in common usage during different periods of time (3).  

Another reason for taxonomic confusion is the fungal life cycle. Many ascomycetes, basidiomycetes, and zygomycetes go through three stages. In each stage, they may have different microscopic and macroscopic characteristics. In the anamorph (or asexual) stage, the cells are haploid, meaning they contain only one copy of each gene. Organisms in this stage can continue to reproduce through mitosis. A special structure called a conidium or sporangiospore is formed that can split from the cell and form a new organism. During the teleomorph (or sexual) stage, the cells are diploid and contain two copies of each gene. The spores created from this type of sexual reproduction are also diploid, having been formed by meiosis from the nuclei of two different organisms. The last stage (dikaryotic) results when sexual reproduction occurs, but the two parent nuclei do not fuse; rather, they coexist within the cell. It is these differences brought on by the particular sexual state that led taxonomists to consider these organisms different species. 

Today, through molecular phylogenetics we understand that although an organism may appear and behave differently during different phases of its life cycle, it is still fundamentally the same species—not unlike how a caterpillar eventually turns into a butterfly. The same genetic information reveals itself in very different phenotypic characteristics. 

Although fungal taxonomists have used phylogenetic analysis to characterize, classify, and reclassify fungi for many years, this approach has only recently gained popularity for routine identification of fungi in the pharmaceutical manufacturing environment. Phylogenetic analysis has been the preferred approach because,  although the technology existed, applications developed that are compliant with current good manufacturing practice (CGMP) and can be validated have only recently been made available through products and contract-service laboratories. Now that the technology is available, the expectation is that it will become more widely adopted as an accepted approach.  

During the past several years, DNA sequencing-based fungal identification systems have developed rapidly. The introduction of MicroSeq (Applied Biosystems, Foster City, CA) as well as the use of DNA sequencing for fungal identification have assured pharmaceutical microbiologists that the technology has the required level of support to provide high quality, accurate, and compliant answers. DNA sequencing provides data that are much more accurate and reproducible than visual characteristics and phenotypic properties. It is independent of growth stage, cultural medium and age of the sample. As a result, companies can get more accurate and reproducible identification results. In fact, FDA recommended the use of genetic methods in its 2004 update to its 

Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing—Current Good Manufacturing Practice

Genotypic methods have been shown to be more accurate and precise than traditional biochemical and phenotypic techniques. These methods are especially valuable for investigations into failures (e.g., sterility test; media fill contamination)....     Sterility test isolates should be identified to the species level. Microbiological monitoring data should be reviewed to determine if the organism is also found in the laboratory and production environments, personnel, or product bioburden. Advanced identification methods (e.g., nucleic-acid based) are valuable for investigational purposes. When comparing results from environmental monitoring and sterility positives, both identifications should be performed using the same methodology.

Despite overwhelming acceptance and support from the scientific community, pharmaceutical microbiologists, and regulatory agencies, commercially available identification systems can still be improved. The first limitation is the coverage of the database of known fungal species. This coverage should not be confused with the size of the databases; a database filled with hundreds, or even thousands, of species not encountered in the pharmaceutical manufacturing environment adds no value to the identification system. This point has been addressed in recent literature by Rozynek et al., and Hall et al. (4, 5). In both publications, the technology is well accepted, but the coverage of the database reduces the effectiveness of current technology to provide meaningful identifications. 

A second limitation of commercially available systems is the choice of gene target with which to build the DNA sequence library. This is a limitation of the application rather than the technology. A gene target is appropriate for phylogenetic analysis when it undergoes enough genetic mutation for there to be observable differences in the DNA sequences of similar but different species. The rate of accumulated nucleotide differences, however, should not be so great that truly related species appear to be more dissimilar than they actually are. This confusion can pose a great challenge in choosing the appropriate target and, in many instances, becomes a case of trial and error. The D2 expansion segment of the Large Subunit (LSU) of the ribosomal gene, as used in the MicroSeq system, typically does a very good job of placing an unknown fungal isolate into the appropriate taxum. However, because of limitations in the resolution of the D2 segment, closely related organisms may have identical, or very similar, DNA sequences.  

The D2 expansion segment of the LSU is effective at linking together higher level taxa (e.g., genus, family, order) and can differentiate many species acceptably, but not in all cases. One common example of the inability of D2 to differentiate close, but distinct, species is 

, is an organism frequently used in biological drug production. Because of the amount of genetic manipulation required to introduce genes of interest into this species—as well as all the eventual monitoring involved in scaling up the fermentation from a starter culture—it is extremely important to ensure the same organism is used in each process step. The easiest way to do this is through identification. Unfortunately, the D2 DNA sequence of 

, as only one nucleotide separates the sequences of 

from the other two species. This is just one of many examples where the D2 DNA sequence was unable to fully identify an isolate to the species level. 

More recently, ribosomal internal transcribed spacer (ITS) regions have been used for fungal systematics and classification. There are two ITS regions in the DNA genes encoding the fungal ribosomal RNA (rRNA) "operon." The first, ITS1, is found between the 18S and the 5.8S rRNA genes. The second, ITS2, is found between the 5.8S and the 28S rRNA genes. The entire rRNA "operon" is transcribed, but, after transcription, the two ITS sequences are excised and, therefore, not used for any functional purpose. 

Because ITS sequences are important enough as spacer regions to be maintained by the cell, but not used for any functional purpose, they are allowed to accumulate mutations at a faster rate than the 5.8S, 18S, and 28S rRNA genes. It is this slightly increased rate of accumulated mutations that allows the ITS sequences to provide an improved level of resolution compared with the D2 sequence.  

It is generally accepted to sequence the entire stretch of ITS1-5.8S–ITS2 for use in fungal classification. However, for the purposes of routine identification, our laboratory has found that ITS2 alone is usually sufficient for species-level identification. 

As an example of the increased resolution of ITS2 sequences compared to D2 sequences, we will revisit the 

problem. When comparing ITS2 DNA sequences of the three 

 species, we see many more nucleotide differences. There are 16 differences between 

 This increased resolution offers a much more confident identification and allows for small strain-to-strain variability to ensure correct identification. 

Phylogenetic analysis has offered fungal taxonomists the ability to reclassify thousands of species to create a more accurate taxonomic system. A huge advantage of DNA sequencing is its ability to classify an isolate based on DNA sequence alone, regardless of the accepted name of the organism or its official name. 

 is a newly described species that was, in part, created by the transfer of several existing 

 (6). Most significant in the creation of this new species to pharmaceutical microbiologists was the inclusion of 

 ATCC 16404; this organism is cited in several 

 chapters as a quality control (QC) organism, including 

 General Chapter <61> Microbial Limits Test—Enumeration and 

 General Chapter <71> Sterility Test. Because of the number of pharmaceutical companies performing these tests, it is very important to correctly identify the QC organism. Unfortunately, this is another example where virtually all phenotypic tests, as well as D2 DNA sequencing, are unable to differentiate the two species. Phenotypically, it has always been difficult to differentiate between 

strains because of lack of diversity in morphological features, unstable phenotypic characters, and the significant influence of culture conditions on the phenotype (7). Furthermore, D2 sequences add no additional information because the DNA sequences for all observed 

A recent study by Houseknecht et al., looked at the justification for this recent classification (8). The study concluded that although 

are very similar, there are ways to differentiate isolates of these species: through evaluation of conidia morphology under high magnification of an electron microscope (the differences are not observable using a light microscope), or through ITS DNA sequencing. The entire ITS1–5.8S–ITS2 DNA sequence shows five differences between the strains of 

. When comparing ITS2 sequence alone, there is only one nucleotide difference between the species, but this difference has been shown to be extremely reproducible and is, therefore, considered a diagnostic indicator of the species. 

The ability to accurately and reproducibility identify fungi—both yeasts and molds—has been greatly enhanced through comparative DNA sequencing. Fungal taxonomists have used DNA sequences for many years as a basis for reclassification of all fungal taxa and have more recently moved to ITS sequencing as the "gold standard." With the ability to use these powerful technologies as part of a comprehensive EM program, pharmaceutical microbiologists are given one more tool to ensure product safety. As made clear by FDA as part of its 

Pharmaceutical cGMPs for the 21st Century 

and process analytical technology initiatives, the agency encourages industry to use new rapid, and accurate technologies. 

 is vice-president of business development at Accugenix, 223 Lake Drive, Newark, DE 19702, tel. 302.292.8888, 

 1.  L. Jimenez, "Microbial Diversity in Pharmaceutical Product Recalls and Environments," 

 2.  S. L. Baldauf and J. D. Palmer, "Animals and Fungi Are Each Other's Closest Relatives: Congruent Evidence From Multiple Proteins," 

 3.  S.A. Meyer, R.W. Payne, and D. Yarrow, "Candida" in 

 C. P. Kurtzman and J. W. Fell, Eds (Elsevier Science B.V., Amsterdam, 1988) pp. 476–477. 

 4.  P. Rozynek et al., "Quality Test of the MicroSeq D2 LSU Fungal Sequencing Kit for the Identification of Fungi," 

 5.  L. Hall, S. Wohlfiel, and G. D. Roberts, "Experience with the MicroSeq D2 Large-Subunit Ribosomal DNA Sequencing Kit for Identification of Filamentous Fungi Encountered in the Clinical Laboratory," 

. Nov., ABiseriate Black Aspergillus Species with World-Wide Distribution," 

 7.  E. Rinyu, J. Varga, and L. Ferenczy, "Phenotypic and Genotypic Analysis of Variability in 

 8.  J. Houseknecht et al., "Reclassification of ATCC 16404 and ATCC 9642 as 

Identification of fungi, especially filamentous fungi, has been a very difficult task. Because of the amount of experience required to accurately identify filamentous fungi to the species level, it has become acceptable to either identify these organisms to the genus level or, in some cases, simply identify them as "molds."

Identification of Fungi Using Ribosomal Internal Transcribed Space DNA Sequences

) was recast from its traditional focus of how to make medicines to the role it would eventually take as a book that describes the safe making of medicines. The US Pharmacopeial Convention resolved to "append assay processes to as many of the potent drugs and preparations made therefrom as may be found possible, provided that the processes of assay are reasonably simple and lead to fairly uniform results in different hands" (1). This publication was followed shortly by the Federal Food and Drugs Act of 1906, which elevated the position of USP and increased its authority dramatically. It was against this background that the first informational chapter about sterilization appeared. 

 The original chapter on sterilization appeared in 

 IX, which became official in September 1916 (2). Although this chapter was sparse on technical detail, it provided a background discussing the need to sterilize various medicines, containers, and stoppers with recommendations as to how to achieve this sterilization. Part of this early emphasis stems from USP's focus at that time on the practicing pharmacist and the need to put procedures in place that were accessible to the practitioner (1).  

Bear in mind, that this chapter was published more than 20 years before the passage of the Food Drug & Cosmetic (FD&C) Act of 1938, which gave the US Food and Drug Administration the authority to require proof of a product's safety before marketing, and almost 50 years before the first good manufacturing practice (GMP) was finalized (3–5). As an aside, the importance of sterilization and sterility assurance continues to be a grave issue to this day. It was the 1972 Devonport incident involving lax control of a sterilization process and the resultant damage that drove modern concepts of sterility assurance as an operational imperative (6, 7). 

 The 1916 informational chapter was extensively revised in 1947 (and increased in length by approximately sevenfold) to provide a great more detail about many types of sterilization methods, several of which are no longer in use (8). This version provided information about what would probably be called "compendial conditions" for sterilization by dry heat (>1 h at >170 °C), for "steam under pressure" (115.5 °C for 30 min, 121.5 °C for 20 min, or 126.5 °C for 15 min), and for "free-flowing steam." This last technique was used in a series of exposures to kill vegetative cells with respites to allow spore germination before repeated exposure to steam. Also known as tyndallization, this method of sterilization is no longer recommended (9). The informational chapter also included a discussion of "fractional moist-heat sterilization at low temperatures" (inspissation), in which the medicine is heated at 60–80 °C for 4–7 days, as well as a discussion of sterilization by boiling in an oil bath. Interesting methods all, although on the whole this author is glad that validation and demonstration of efficacy has become the expectation. This 1947 chapter has some specific recommendations for sterilization by filtration and aseptic processing (along with a direction that the label is to state "Prepared by Aseptic Manipulation").  

 The title of the chapter was changed back to "Sterilization" for 

 XVI (10). This version was a complete rewrite of the chapter. It included a special note that while the "fractional sterilization" methods might work well for bacterial growth media, it is not appropriate for pharmaceutical preparations because spores may not germinate in them (not mentioned is the issue of metabolic byproducts created by the germinating spores if the process works).  

This version includes a greatly expanded section about filtration and aseptic manufacturing, including an emphasis on the control of the fill environment. A strong suggestion is included to the pharmacist and operators to perform aseptic manipulations under hoods or shields in an area protected from visitors. A new section was added to the chapter on the need to monitor the environment, with settle plates and media fills recommended. Finally, the chapter states that making sure the janitorial staff cleans the area after the shift. leaving a full overnight period before the next day's fill, is considered very important. This chapter was rewritten for 

 XVII (11). Although most changes were editorial, specific instructions for types of sterilization methodologies were included as separate sections and new methods–sterilization by ethylene oxide and sterilization by irradiation–were included for the first time. 

 The chapter "Sterilization" was amended again for 

 XVIII (12). In addition to extensive editorial changes, two important new sections were added. The first section dealt with biological indicators and the need for appropriate selection of the indicator (one for heat may not be a suitable species for irradiation) as well as the need for competent manufacture of the indicator itself to maintain its relevant properties (e.g., heat resistance or similar property). The second new section dealt with the sterility test. The opening paragraph of this section is so entertaining that I have to share it: 

The significance to be attached to a demonstration by test that a drug or device has been rendered sterile is determined largely by the extent of the control exerted during the manufacturing and sterilization processes. The object of the sterilization process is to make the article safe for use, but the tests may be expected to reveal only that living organisms have been removed or destroyed to the extent where they no longer multiply in appropriate culture media under favorable conditions. Interpretations of the results of sterility tests must allow for the possibility that the degree of contamination is of a low order of magnitude. Confidence in the results of the tests with respect to a given lot of articles is based upon knowledge that the lot has been subjected to a sterilization procedure of proven effectiveness. Where feasible, the effectiveness of the process should be demonstrated each time it is carried out by including marked, intentionally contaminated controls, or indicators, which are examined under the conditions of the tests.

This passage is somewhat ironic because speakers to this day insist that 

 created a flawed test for sterility that should have been fixed long ago. Although this argument is undoubtedly true, this test was first entered in the 

 of 1932 with no significant improvements in the intervening 77 years. One would think someone would have come up with a better assay by this time.  

The author provides a history of the information chapter USP â€¹1211â€º "Sterilization and Sterility Assurance of Compendial Articles," from the early 1900s to the current version.

Pharmaceutical Technology-05-01-2009

Applying Quality by Design to Sterile Manufacturing Processes

Disposable Components in Aseptic Processing

The Impact of Automation on Aseptic Processing

Identification of Fungi Using Ribosomal Internal Transcribed Space DNA Sequences

USP <1211>: The Compendial Informational Chapter on Sterility Assurance

Trend Analysis for Sterile Manufacturing

FDA's Draft Guidance for Process Validation: Can It Be Applied Universally?