For at least 20 years, the global parenteral industry has recognized that personnel are the dominant risk relative to microbial contamination in aseptically produced sterile products (1). The contamination source strength of the gowned aseptic processing operator has been estimated in several research studies. Concomitant with this awareness, we have witnessed a series of technological advances that have endeavored to mitigate this contamination risk. These advances can be roughly categorized as follows: 									

separate personnel from the aseptic environment;

limit personnel interaction with sterile materials;

remove personnel from the aseptic environment;

Each of these approaches provides some added measure of security to the process by increasing the degree of separation provided between personnel and the sterilized materials, components, and product contact surfaces required for the aseptic assembly of the sterile product from its individual elements. 

To the extent that these approaches are effective, they can reduce contamination risk in aseptic processing. Designs that most effectively eliminate human-borne contamination have been identified as providing "advanced aseptic processing." The term 

 was perhaps first used at the USP Open Conference on Microbiology in May 2002 (2). During that conference, only two aseptic technologies were discussed under the heading of "advanced": isolators and blow–fill–seal. Over the intervening years, we have witnessed the term 

 come into ever wider use. Considering the goals of aseptic processing, that other technologies intended to meet the high expectations for sterile product manufacturing aseptically should emerge is unremarkable.  

Several technical features distinguish advanced from conventional aseptic processing. We believe the following condition should be met for an aseptic processing technology to be considered advanced: no interventions should be conducted during aseptic operations by gowned employees. In other words, all interventions must be conducted using separative features such as isolator gloves or half-suits. Obviously, this condition also could be met if manned interventions of any type were not required—as can be the case in blow–fill–seal or gloveless isolators. The contamination potential from the human operator, even under the most ideal conditions, is such that the aseptic process may be compromised by even a single manipulation by gowned personnel in proximity to the sterile materials. At a minimum, the allowance of limited human interventions creates risk uncertainty that we believe is incompatible with the concept of advanced aseptic processing.  

A brief historical review of the environmental systems used for aseptic processing is useful to understand the genesis of today's processing technologies (see Figure 1). 

 Figure 1: Aseptic processing family tree.			

Perhaps little known to many current practitioners is the previous use of gloveboxes for aseptic processing before the introduction of cleanroom technology. Clearly, the early practitioners of aseptic processing understood the risk from human-borne contamination and used physical separation between the operator and the so-called sterile field to provide greater control over microorganisms.  

The emergence of the HEPA filter in the mid 1950s changed facility designs and operating practices dramatically. It was now possible to position equipment inside a room and, using gowned personnel, produce larger numbers of units with less human manipulation. This design has continued to evolve to the more advanced barrier designs we see today. Gloveboxes never became obsolete, but labor requirements, throughput limitations, decontamination constraints, and other factors limited their application in aseptic processing. 

The cleanroom dominated the industry as the preferred choice for aseptic processing because it was amenable to high-speed filling, inclusion of processing equipment, and easy adaptation for various applications. Later designs included partial barriers to provide greater separation between operators and sterile materials.  

In the early 1980s, isolation technology was reintroduced to the sterile-products industry as an alternative to cleanrooms. Many practitioners recognized it as an improvement in contamination control relative to even the most sophisticated cleanroom designs then available. Some in the industry were so enthusiastic as to claim that sterility assurance equivalent to terminal sterilization would be possible with this new technology. As with any new technology, its implementation took time, and missteps were made. Although some firms implementing isolation technology experienced difficulties, there were more successes than failures (3).  

Frustrated perhaps by the difficulties they encountered with isolators—particularly relating to decontamination, leak testing, ergonomics, and flexibility of access—several firms endeavored to find a means to obviate the perceived inflexibility of isolators. Thus the restricted access barrier systems (RABS) concept was developed. RABS endeavor to provide the sterility assurance benefits of the isolator with fewer complications. At first, there was no standard definition for RABS and, as a result, there was no standardized minimum performance criterion. Fortunately, this shortcoming has been fully recognized, and a more standardized description of a RABS has been developed recently (4).  

Chronologically, RABS emerged more or less as an offshoot of efforts to implement isolator technology in the mid-1990s. In our view, RABS were conceived not because of contamination-control shortcomings in isolators, but rather in hopes of solving validation and, perhaps most important, to allow more flexible access for repairing and adjusting equipment where necessary.  

Shortcomings and perceived benefits of RABS

From the standpoint of contamination control, no claims have been made that the performance of RABS is superior to that of isolators, but rather that RABS may be easier to implement and more flexible in the manner in which it allows operator access. Of far greater importance is whether RABS represent an improvement relative to patient safety over earlier aseptic technologies.  

Limitations and comparison with isolators.

 Clearly, RABS have the potential to improve contamination control over what might be termed 

 which have been used in cleanrooms for years. It is our opinion, however, that RABS that allow some open interventions by gowned operators fall well short of the ideal of advanced aseptic processing (see sidebar, "Advanced aseptic processing requirements"). 

Advanced aseptic processing requirements			

Moreover, we believe RABS designs are less capable than isolators relative to their ability to exclude microorganisms for several reasons. First, isolators provide a measurable pressure differential between the enclosed environment and the operator. In contrast, RABS rely on air overspill to exclude contamination from the surrounding environment in which the aseptically gowned operator is located. 

Second, isolators are subjected to a reproducible decontamination process (in some cases, this is a sterilization process) performed by a microprocessor-controlled system delivering a sporicidal agent in a consistent manner each time. The effectiveness of that process can be supported by the multiple-log kill of resistant microorganisms. Although it is true that RABS and the rooms surrounding them also can be reproducibly decontaminated by microprocessor-controlled equipment, this advantage is immediately lost if gowned operators are given free access to the critical zone at any time during production. Of course, should manual disinfection be required after such gowned interventions, comparable levels of control would be impossible to demonstrate. This treatment might be as effective as what is performed on the isolator from a chemical lethality perspective, but reliance on humans for execution makes it susceptible to occasional error or omission. Moreover, because this activity requires the operator to access the interior of RABS, there is always the contamination potential associated with any aseptic intervention. 

Third, the product contact surfaces such as feeder bowls and stoppering equipment inside the isolator can be preinstalled and treated with a sporicidal agent with the isolator in the same process used to decontaminate the isolator. RABS designs allow these items to be sterilized remotely, transferred to the RABS, aseptically installed, and readied for operation. This represents a clear risk of microbial contamination that cannot be easily avoided. 

Fourth, by the very nature of the RABS design, there may be areas of the installation that personnel cannot reach easily that require treatment. Consider for example a RABS installation with a large lyophilizer. The ability of the gowned operator to disinfect the interior of RABS adequately without physically entering the system is remote. This presents a potential microbial insult to RABS of such magnitude that a claim for this practice or design as being an advanced aseptic process cannot be easily supported. 

Fifth, aseptic processing isolators operating under positive internal pressure are the preferred means for the filling of sterile cytotoxic products. RABS designs cannot be used for this type of product because the level of containment is minimal. 

Finally, the suggestion that the doors to RABS can be opened for a major intervention, and then filling resumed after a "high-level disinfection" process seems tenuous at best. As noted previously, "high-level disinfection" by gowned personnel may not be truly reproducible nor without risk. Any intervention that requires the doors of the RABS to be opened is unacceptable within the context of advanced aseptic processing. We would have the same objection were this to occur in an isolator, but under those circumstances, no one would consider the system acceptable for continued use without a full decontamination comparable with the initial treatment. 

 Some perceived advantages of RABS designs have been identified as follows: 					

flexibility of access—but not without potential open intervention risk;

lower cost—lower initial capital investment is probable, but because full aseptic core zoning and gowning are required, operating costs are likely higher;

shorter validation timeframe—this may be true, but there is not enough of a track record to know for certain;

more conventional in terms of validation requirements—no performance standards for RABS have been defined, however, and when they are defined, it is uncertain whether they will be based upon manned cleanrooms or isolation technology.

It is our strong belief that although RABS may be superior to manned cleanrooms, they cannot attain the certainty of performance demonstrated by present-day isolators. Perhaps the most appropriate application for RABS would be as retrofits to existing aseptic processing facilities. In this situation, the existing infrastructure of cleanrooms, corridors, and gowning rooms would remain largely unchanged, with RABS concepts applied solely to the filling lines. We remain highly skeptical relative to the utility of RABS in installations with large or multiple lyophilizers, given the access limitations they impose. These installations, however, while representing a potentially valuable contamination-control improvement over conventional cleanrooms, fall well short of advanced aseptic processing. 

Ideal RABS for advanced aseptic processing			

Improving RABS for advanced aseptic processing

We can define a RABS design that meets industry expectations for advanced aseptic processing (5). To realize that designation we believe the RABS design must possess specific attributes (see sidebar, "Ideal RABS for advanced aseptic processing"). To our knowledge, this type of design has not yet been used, but some operations have come very close to this ideal, and newer technologies may make it feasible. With this type of design, we would also impose the following additional requirements: 					

Internal decontamination requirements would be comparable to isolators.

Media fill and environmental monitoring requirements in the critical zone are comparable with those for isolators. Monitoring in the surrounding environment also is required.

Glove-leak testing (where gloves are required) would be required.

Personnel accessing the environment require full aseptic gown certification.

If lyophilization is required, then automated loading and unloading must be provided with freedom from personnel access.

Environmental monitoring should be performed in the same manner as isolators.

The use of RABS for extended campaigns must be explored, and the operational conditions that must be met must be determined.

Future considerations for RABS implementation

If RABS are to become a useful technology in our industry, what will be required and what can we expect to occur? First, the fundamental characteristics of RABS for advanced aseptic operations as we have endeavored to define must be broadly accepted. Also, it is quite likely that the validation of RABS designs will be fairly similar to isolator validation. 

As in all forms of aseptic processing, the elimination of interventions must be paramount. Advanced aseptic processing cannot exist when personnel have the ability to intervene at will. In addition, RABS designs probably offer the most direct and cost-effective route to upgrading existing aseptic facilities. Finally, elements of RABS may be an appropriate solution to some of the most common interventions in manned aseptic processing (

We doubt that any aseptic processing system, including RABS, which is tolerant of open-gowned aseptic interventions, will have a long shelf life based upon current and developing technology. We believe aseptic processing technology will continue to evolve toward the complete elimination of operator interventions of all kinds. In fact, gloveless isolators already exist and have been in use in some aseptic processing industries for about 10 years.  

Therefore, technologies that are designed to allow easy intervention will lack staying power as machine automation and robotics replace operators. In our view, RABS' principal utility will be in the reduction of risk in existing facilities, but it will not be the technology of choice for new facilities. Of course, it is equally possible that separative enclosures such as isolators will suffer the fate of obsolescence as well. The authors have no technology preference regarding the elimination of human interventions and hence human contamination risk. Given the rapid advancement currently underway in flexible robotics, equipment self-diagnostics and self-correction, information technology, in-process control, and so forth, it is quite possible that human intervention can be eliminated by means other than barriers or separative enclosures such as isolators in the near future. In fact, systems that came very close to this ideal have been in operation since the late 1980s.  

Validation of advanced aseptic processing is perceived to be substantially more difficult than conventional aseptic processing. Witness the claimed difficulties with isolation technology at some firms. A significant number of advanced aseptic processes, however, have been successfully validated. Risk- and science-based regulation should favor the systems that are best are reducing risk. Recently, FDA has sought to ease the validation requirements for advanced aseptic systems by reducing validation expectations where justified by risk management. We believe this is a great step forward. It seems logical to us that the greatest validation benefits should come to processes that truly eliminate human interventions and to those with the most capability for in-process control. We look forward to further definition from FDA so that industry has a clear understanding of what is possible in terms of reduced validation activity as a function of risk mitigation.  

We believe that a definition for advanced aseptic processing much like that proposed at the USP Open Conference in 2002 is most appropriate. Thus, advanced aseptic processes are those that eliminate direct intervention by personnel. For RABS to be an appropriate technological solution, it must be very isolator-like. Systems that tolerate any form of direct intervention by gowned personnel are nothing more than highly evolved conventional cleanrooms with gowned personnel.  

Articles

Any aseptic processing technology that allows intervention by gowned personnel during operation cannot be considered an advanced technology. Although a standardized definition of restricted access barrier systems has been developed, these systems fall well short of being classfied as advanced technologies. 

RABS and Advanced Aseptic Processing

Particularly in the fields of cleanroom technology and sterile products manufacturing, good manufacturing practices (GMPs) regulations and guidances and technical cleanroom standards are mutually dependent on each other. The classification of air cleanliness specified in the International Organization for Standardization (ISO) 14644-1:1999 (1, 2) is quoted as a tool in Annex 1 of the European Union (EU) GMPs (3) and in the US Food and Drug Administration's 2004 aseptic processing guidance (4). The mutual dependence of GMPs and cleanroom standards is clear in that the ISO cleanroom standards are not application specific. The standards don't advise which levels of cleanliness are applicable to specific processes. Similarly, the EU GMPs do not explain how to measure airborne contamination or how to classify a controlled environment. 

In May 2003, the European Medicines Agency (EMEA) published a revision to Annex 1 of the EU GMPs that was implemented in September 2003 (5). The 2003 version of the sterile products requirements was created in response to the publication of ISO 14644-1:1999, the internationally accepted standard for airborne-contamination control. In particular, this version of the EU guidance tried to clarify a perceived requirement to control ≥5.0-μm airborne particles at very low levels with the ability to measure such low concentrations reliably. The net effect of the September 2003 changes was to frustrate and confuse industry with requirements that were written poorly in some areas or were plain bad science in others. In response to industry's negative reaction, the EMEA initiated another revision to the Annex 1 document. Proposals for the further changes were published on the Eudralex Web site in November 2004, with a closing date of April 30, 2006 for public comments (6). 

The EU GMPs have a much greater impact than on just EU nations and those supplying them with medicines. The complete EU GMPs are adopted by the Pharmaceutical Inspection Convention and the Pharmaceutical Inspection Co-operation Scheme and are referred to as the PIC/S GMPs (7). In turn, many nations adopt this document as their GMPs. Thus, the effect of poorly written documents with elements of poor science could reverberate around the world and lead to confusion and unnecessary environmental control, monitoring, and deviation investigation work. 

At present, industry has the opportunity to correct these problems and move forward in a positive way by ensuring that it makes significant improvements to Annex 1 of the EU GMPs. Failure to improve this document could endorse bad science. 

Table I: Proposed number of airborne particles in environmental grades AâD (per the November 2005 draft proposals).			

Grade limits for airborne particles: classification

The following list includes the effects of the November 2005 draft proposals for airborne particles (see Table I) and associated requirements for classification and monitoring:  					

Industry wants and expects to have excellent control of ≥0.5-μm particles and expects negligible concentrations of ≥5.0-μm particles in Grades A (at rest and operational) and B (at rest).

Industry wants to avoid extremely large sample sizes for formal classification that would be required by specifying a very low limit. 

  samples must be taken at each location for a formal classification of Grade A zones at rest and in operation, which is much more demanding than the current requirement to collect a total of 1 m

The sample size for Grade B at rest is not provided in the guidance. It might be thought that the 1-m

  sample size applies, however. If this is the case, then industry will bear a huge burden of airborne particle counting with limited value. It is for this reason that a formal classification of ≥5.0-μm particles in Grade B at rest should be avoided.

Industry must avoid triggering classification or monitoring failures because airborne particles measurements could be in a range for which real and false counts might be confused. In formal classification, the proposed acceptance of a limit of 20 particles/m

A full classification of "in operation" conditions is required. This process would require an invasive testing regime not demanded previously. It always has been accepted that adequate confidence can be obtained from the "at rest" classification combined with operational monitoring. The guidance proposals suggest this "operational" classification could be carried out during media fills. Many parties will find it is an inappropriate time to add challenge to the process environment.

Airborne particle concentration is perhaps the best known and the quickest way of determining the cleanliness of a working environment and detecting deviations from target values in real time. The EU GMP Annex 1 defines airborne particle control requirements for the working environment. Tables I–V show the evolution of airborne particle limits related to the environmental Grades A–D. Note that both the "at rest"and "in operation" conditions are defined in the EU GMPs. This is an important difference from the requirements in the equivalent US guidance. 

The "0" requirements in Table II were both unrealistic and inappropriate to use as a limit for airborne particle counting for classification or monitoring. 

Table II: Number of airborne particles in environmental grades AâD (before the May 2003 guidelines).			

Both regulators and users of clean-air devices complying with the EU GMPs guidance accept that information relating to "at rest" and "in operation" conditions are appropriate. From the viewpoint of cleanroom or clean-air device procurement, it is important to have a performance benchmark to differentiate the basic technical performance of the equipment from the operating conditions that might prevail. 

Table III: Number of airborne particles in environmental grades AâD (per the May 2003 guidelines, if they had been fully aligned with EN ISO 14644-1:1999).			

The requirement to consider ≥5.0-μm airborne particles continues to be somewhat more contentious because the current limit for Grades A and B "at rest," set at 1 particle/m

  (see Table III), can be less than the false count rate of typical particle counters. Although arguments for and against the consideration of ≥5.0-μm particles are likely to continue, it is more important to set meaningful and measurable levels. 

Table IV: Number of airborne particles related in environmental grades AâD (per the published May 2003 guidelines, when submitted to industry).			

Close analysis of the air sample size calculation in EN–ISO 14644-1:1999 shows that the sample volume required at each location for classification is defined as the air volume required to obtain a count of a minimum of 20 particles at the class limit for the largest particle size considered. The equation is 

 is the number of particles at the class limit. If this algorithm is applied to the various class limits previously mentioned, we would find the following sample sizes. (It must be remembered that the minimum sample size is 2 L and the minimum sample time is 1 min.) 					

  indicates a sample size of 690 L. The collection time would be nearly 25 min at a sample rate of 28.3 L/min.

  indicates a sample size of 1000 L. The collection time would be just more than 35 min at a sample rate of 28.3 L/min.

. Collection would take nearly 12 h at a sample rate of 28.3 L/min. Because this sample is impractically large, the Annex 1 proposals recommend a sample size of 1000 L for Grade A and B areas. This testing might be practical and acceptable in Grade A critical core areas but would present a huge, unnecessary burden in Grade B areas. This sample size would be adequate for a class limit of 20 particles/m

, which is from where the suggested practical limit of 20 particles/m

  is derived. Therefore, if one were to rationalize this approach, the table might be adjusted as shown in Table V. 

Table V: Suggested number of airborne particles in environmental grades AâD to resolve the problem of 5.0-Î¼m particles more practically.						

Grade limits for airborne particles: monitoring

Real-time particle monitoring also is important, and the acceptance criteria will be based on Table I. Once again, the challenge is to accomplish real-time monitoring with ≥5.0-μm particles because there is the potential that false counts may be greater than real counts at such low concentrations. When looking at frequent zeros and occasional ones or twos it is better to look at the frequency of events rather than extrapolating short-term data. Remember, 1 particle/ft

The following requirements are found in the draft guidance: 					

Automated systems are expected for Grade A and are desirable for Grade B zones. Such systems are clearly intended to be used to monitor for excursions and events. The guidance offers no information about their use in Grade B zones.

Grade A zones must be monitored for ≥0.5- and ≥5.0-μm particles. The guidance goes on to suggest that both manifold and local counters can be used. This is erroneous because manifold and tube multipoint systems will always suffer from an unacceptable loss (dropout) of particles.

The guidance accepts the possibility of occasional counts that exceed the published limits, but it doesn't effectively explain how to deal with them. What is needed is clear guidance about how, in principle, to examine the average performance and identify unsatisfactory events. Many applications of real-time systems evaluate small samples taken frequently and consider cumulative count over a greater time. When considering ≥5.0-μm particle counts, three sequential samples of 28.3 L with counts of two or three particles could be considered an event, whereas a single isolated sample with one or two counts could be ignored as being insignificant.

Unidirectional airflow (UDAF), formerly referred to as laminar flow, is a key method of protecting the critical aseptic core and other clean zones through the delivery of a controlled airflow of defined particulate quality. 

In recognition of the vast range of various clean-air devices, EN–ISO 14644-4 simply specifies that the velocity should be >0.2 m/s for unidirectional airflow systems (8). The standard emphasizes that demonstrating the effectiveness of the airflow by determining the airflow uniformity is more critical than choosing only a specific velocity. This focus on the demonstrable effectiveness of the clean-air system also is reflected by the current text in Annex 1. There is confusion about the range of velocities given and where to measure it. EN–ISO 14644-3 clearly differentiates between the filter-face velocity and the system's working level velocity (9). 

For most UDAF systems, the well-established value of 0.45 m/s (±20%) will be appropriate. Higher values of approximately 0.6 m/s and lower values of approximately 0.3 m/s may be needed to protect hot bodies and isolator internals, respectively. The current Annex 1 also is not clear about the acceptability of using non-unidirectional airflow in devices such as isolators. The text should be revised to endorse the use of such systems where appropriate. 

New requirements for handling stoppered vials

The text proposals recognize the advantage of segregating particulate-generating processes such as vial capping from other more-sensitive activities. Progressively, industry has developed a preferred practice to over-seal vials outside the aseptic area. This practice is now threatened by the proposed text that seems to define the over-sealing as an aseptic process. Industry believes that the following are reasonable expectations: 					

Over-seals can be applied outside the aseptic processing area.

The transport route (conveyor) from the exit of the filling machine's stopper insertion zone to the boundary of the Grade B aseptic processing area should be within a Grade A environment.

The transport route to and the capping station should be protected by UDAF ISO Class 5 air.

The protected capping station can be located in a Grade D environment.

Additional security can be gained by introducing stopper position detection with automated rejection of unsecured units.

At a time of significant change to the European Union good manufacturing practices (EU GMPs) requirements for sterile products, there is still a need for a symbiotic working relationship between GMPs and the technical cleanroom standards. The standards, for example, can be considered as the basic tools and the GMPs' specifications as the requirements and principles.  

The current proposals for a revision of Annex 1 of the EU GMPs are an opportunity to get it right in the world of risk-based decision making. The authors of the EU GMPs have a great responsibility because the guidance will be incorporated as part of the Pharmaceutical Inspection Convention and the Pharmaceutical Inspection Co-operation Scheme (PIC/S) GMPs. 

is principal consultant at Bovis Lend Lease Technology, Tanshire House, Shackleford Road, Elstead, Surrey, GU8 6LB, United Kingdom, tel. +44 1252 703663, fax +44 1252 703684, 

 1. International Organization for Standardization (ISO) 14644-1, 

Cleanrooms and Associated Controlled Environments, Part 1: Classification of Air Cleanliness 

Cleanrooms and Associated Controlled Environments, Part 2: Specifications for Testing and Monitoring to Prove Continued Compliance with ISO 14644-1

The author explores the importance of the proposals to revise Annex 1 of the EU GMPs in the context of the desire for science-based, internationally respected GMPs. Commentary also is provided about the relationship between this annex and CEN–ISO cleanroom standards. 

Effects of the 2006 Proposed Revisions to Annex 1 of the European Union Good Manufacturing Practices

There are advantages and disadvantages to using model microorganisms in the validation of sterilizing filtration. Although filtration validation studies may demonstrate the removal of the challenge organisms (whatever they may be), those studies can only imply that any particular filter used to filter a batch of drug product will yield a sterile filtrate. Successfully passing a bubble-point test (1) (or multipoint diffusive-flow integrity test), the results of which have been correlated to successful microbial challenge testing and validation studies, provides additional assurance that a sterile filtrate has been obtained. The filter manufacturer's quality-management system and production-control program further support the validity of the sterilizing filtration process. All three elements must be present to predict the successful outcome of a particular sterile filtration event (2). 

Perhaps the best method of determining whether a filter can yield sterile effluent is to subject the drug product to a proper microbial challenge and to analyze the filtrate for its microorganism content. This method of direct measurement would seem to be the most reliable way of characterizing a filter's sterilizing ability. Even though an individual filter can be characterized by a direct microbial challenge, however, the tested filter may not then be used in a processing operation because it has been contaminated by the test organisms. 

In practice, the measurement of a filter's sterilizing capability is derived from a correlation of its degree of bacterial retention with a characterization of its largest pores, essentially in terms of their width. The latter is obtained by means of the bubble-point integrity test method (3). It is necessary to confirm this correlative relationship because to assume it exists could prove a mistake. For instance, two qualities or properties of a filter may so constantly occur together as to be assumed to be correlated. Actually, they may in fact be two independent but parallel events that are not correlated (1). For example, single-point air diffusion rates of a filter measured at 5 psi had formerly been thought to correlate with a bubble point of some 50 psi for that filter, but it was discovered that it was necessary to determine the bubble point by measurements made at the actual bubble-point pressure. It did not necessarily correlate with the extension of the 5 psi single-point air diffusion test reading in all cases. So what had seemed to be a correlation turned out to be a case of parallel events. As a result, the use of single-point diffusive airflows measured at 5 psi was abandoned. In the present instance, extensive examination by many investigators has established the validity of correlating the bubble point with microbial retention (4). 

A filter can be characterized by direct microbial challenge as being capable of producing a sterile effluent. Extrapolating from the properties of the tested filter to other "identical" filters would, however, involve assumptions related to the uniformity of the filters' manufacture. To investigate this aspect of the filtration picture to a statistically meaningful level would involve a large number of correlated studies. Moreover, it might well necessitate the disclosure of proprietary information in a competitive market. This would prove highly impractical. From this one consideration alone, it becomes evident that a fundamental assumption must be made, namely, that the filter manufacturers can be depended upon to control their manufacturing processes so that "identical" filters achieve the same level of retention performance. 

Reliance upon the filter manufacturer is one necessary factor in this action. Identifying the requirements does not ensure the attainment of sterile effluent. 

Until rather recently, it was believed that the sterilization of liquids could be achieved by their filtration through a sterilizing membrane whose proper and pertinent identity was confirmed by its pore-size rating, which was itself determined by integrity testing. The pore-size rating, plotted against organism size, indicated an inverse correlation. However arranged, organism removal by a filter was seen as resulting from the mechanism of size exclusion, also known as sieve retention, wherein the organism is retained because it is larger in size and shape than the filter's pores. The sizes of various organisms are available from listings in the literature, and the size of a filter's largest pores derives from its bubble-point value. Therefore, it was possible to accommodate the size of the restraining pore to the size of the organism to be retained. In the interest of obtaining a maximum flow rate consonant with complete organism removal, the 0.2/0.22-μm-rated membranes were widely accepted as being sterilizing filters. 

The sterilizing filter was defined in 1987 by the US Food and Drug Administration on the basis of its retaining a minimum of 1 × 10

(at that time taxonomically identified as 

) per square centimeter of effective filtration area (5). 

The situation is complicated in its application by the absence of pore-size rating standards. The pore sizes may not be assumed to have been rated in the same fashion by the various filter manufacturers. Thus, while filters may bear the same pore-size designation, they may not be identical in this regard. Nevertheless, such filters may prove interchangeable in filtration processing operations. Manipulative adaptations often make it possible to substitute one such filter for another. 

Although the individual rating methods differ, bubble-point measurements performed in as similar a manner as possible enable comparisons to be made among the various types of filters. Given the importance of gauging the sizes of the largest pores, the integrity testing should be as accurate and reproducible as possible. Accordingly, using automated integrity test instruments is recommended, because their use eliminates the subjectivity inherent in manual integrity testing (6). For the same reason, the bubble point should be identified by the straight line plotted from multipoint diffusion measurements extended through and beyond it to a robust flow of air. Single-point diffusive airflow measurements are not sufficiently reliable for this purpose (7). 

There are numerous types and sizes of microorganisms. To explore the sterilizing capabilities of the available filters with each of these different organisms would not be practical and might not even be meaningful from the standpoint of assessing patient risk. Given the large number of organism types and their differences in size, it would be advantageous to select for testing an organism that could serve as a model for all the others, or at least for those commonly encountered in pharmaceutical operations. The smaller the test organism, the more its removal by a filter would ensure the sieve retention of larger organisms. Properties other than size, however, also are important in the selection of the model microbe. Ease and safety in cultivation and handling are real considerations. The likelihood of the selected organisms' being encountered in pharmaceutical operations is another influence on its selection. 

In the mid-1960s, Frances Bowman of FDA identified an organism that would penetrate the 0.45-μm-rated membranes that typically were used for sterilizing filtration at that time (8). The organism, currently known as 

 American Type Culture Collection 19146, had the ability to distinguish between 0.22- and 0.45-μm-rated membranes (it readily penetrated the latter) and, at concentrations higher than about 1 × 10

, it penetrated 0.22-μm-rated membranes, demonstrating that because penetration was concentration-dependent, mechanisms other than sieve retention were active. Although not the smallest organism known (smaller organisms had been known for decades), 

generally was considered small enough to represent whatever smaller organisms were likely to be present in pharmaceutical preparations. ASTM standard F 838, "Standard Test Method for Determining Bacterial Retention of Membrane Filters Utilized for Liquid Filtration," is based on the use of 

 as the challenge organism. The standard was originally approved in 1983 and was designed to determine the bacterial retention characteristics of membrane filters for liquid filtration. 

Since then, however, some 25 cases have been noted wherein the 0.2/0.22-μm-rated membranes qualified by withstanding the requisite 

 challenge did not yield sterile effluent (9, 10). Obviously, 

 does not serve as a universal model for all organisms. This finding has led some to advocate that the choice of the model organism whereby a filter will be qualified for use as a sterilizing filter should be an organism native to the drug preparation. In this view, 

 suffers from the demerit of not being part of the drug preparation's bioburden. Be that as it may, selecting a component of the bioburden as the model organism is not enough. The test organism must be amenable to identification and cultivation. Its life stages must allow amply for its management in the necessary test manipulations. Above all, its susceptibility to size diminution by contact with a given drug preparation will have to be investigated, and the kinetics and direction of the morphological changes, if any, will require elucidation. 

As previously stated, it had been believed that a sterilizing filter could be defined by way of its pore-size designation as identified by integrity testing. Developments in filtration practices showed this belief to be too simplistic. What had once seemed simple now is recognized as being quite complex. It was discovered that the conclusions based on pore-size ratings were subject to modification by the physicochemical specificity of the organism-suspending fluid, the individuality of the organism type in its size-changing response to the fluid, the possible change in pore size induced by the fluid, and the adsorptive qualities of the filter resulting from its particular polymeric composition, all influenced by the filtration conditions in their numerous varieties, but especially by the transmembrane pressure (11, 12). 

				A filter may not sterilize the same preparation under different filtration conditions, especially under dissimilar differential pressures (13). A given membrane may or may not retain a particular organism type suspended in a different drug vehicle (8). The organism type need not remain constant in size, but may alter in response to its suspending fluid (14–16). The effect of the vehicle upon the polymeric membrane may cause a change in its pore size (17). 			

The certainty of obtaining sterile effluent requires far more than the identification of a sterilizing filter by a pore-size rating. The variety of influences governing the outcome of an intended sterilizing filtration necessitates a careful validation of the process, including the filter. The very drug preparation of interest, the exact membrane type, the precise filtration conditions, and the prefiltration bioburden, including specific organism type(s) and number, should be used in the necessary validation. 

Given the complexity of the organism removal operation, it is doubtful whether a universal sterilizing filter can be devised. Certainly, there is no known absolute filter, one that will retain all organisms under all conditions, especially if viruses are included. Therefore, the successful attainment of a sterile filtration with regard to specified organisms of interest must in every individual filtration sterilization be corroborated by the documented experimental evidence that constitutes validation. The specifics of such operations are discussed in the Parenteral Drug Association's "Technical Report No. 26." Adherence to the teachings of this report is a second factor that is essential to attaining sterile effluent. 

Obviously, the many organisms whose presence in pharmaceutical contexts deserves consideration may differ from one another so greatly as to compel individual techniques for their identification and cultivation. Conclusions cannot be made regarding the sterile filtration of microorganisms unless methods of quantifying them by culturing and counting are available. Organisms such as the L-forms, nanobacteria, and "viable but nonculturable" entities may not be amenable to such analyses. Concerns about their presence may be justified, but without the means to cultivate and count them, it is impossible to attest to their complete absence. It follows that a sterilizing filter can be judged only by its performance in the removal of identifiable and culturable organisms known to be present in the drug preparation (18). 

Model organisms, although perhaps necessitated at least as an opening gambit, will not truly serve their intended, more general use as surrogates for most other organisms. In the present circumstance, however, 

 can serve as a model as well as any bioburden organism. Albeit with surprise to some and consternation to others, 

 did fail to serve as a filtration model in some specific circumstances. Nevertheless, the failures were relatively few. Moreover, they were the result of organism size reductions occasioned for particular organisms by contact with particular drug preparations. Many involved in pharmaceutical filtration were unaware that an organism's morphology could reflect its manner of cultivation, as well as the nutritional value of its suspending fluid, but those facts are known to microbiologists. 

It was not, nor is it as yet, sufficiently recognized within the pharmaceutical industry that organisms penetrating the 0.2/0.22-μm-rated membranes were likely to have undergone size reductions occasioned by exposures to particular drug preparations (

Ralstonia pickettii, Burkholderia cepacia,

 in particular). The dependability of the exclusion mechanism was predicated on the stability of the organism and pore sizes as measured in certain specific situations. It does require modification when organisms or pore sizes do not remain constant. Each may undergo change upon contact with particular drugs. At present, little is known about the kinetics of these size alterations. 

There is no universal sterilizing filter. Sterile effluent results from a combination of many influences, not solely from the use of filters of any particular pore size. Each filtration is an independent event that arises from a proper combination of several stated requirements.

Sterility must be defined in terms of specific organisms that are targeted in a given filtration. The ability to identify and cultivate the organism of interest is indispensable.

 as the model organism likely to represent the bioburden of the drug preparations presented for aseptic processing by sterilizing filtration is limited to those microbes that do not undergo shrinkage upon contact with the drug preparations. Also, it is clear from considerable experience that risks arising from the use of the 

 model are small enough in the vast majority of cases to be considered completely insignificant to public health.

Choosing an actual bioburden organism to replace 

 as a filtration model is unlikely to improve the situation. The substituting organism will replace the present problems with those peculiar to itself. It is questionable whether a universal model is at all possible.

 has served its model role adequately, except for those organisms like itself that undergo size diminution during their cultivation or that manifest size shrinkage through contact with drug preparations that are nutritionally poor for them. Thus far, these have been limited to relatively few, 

 These Gram-negative rods can be kept to extremely low numbers in prefiltration bioburden with reasonable and effective bioburden control measures.

Bacterial challenge studies should be conducted under process conditions by means of inoculating the product to be evaluated with 

  and evaluating the filtrate for the presence of the challenge organism. In the event that 

 is not viable in the product formulation, precondition the filter by recirculating product through it to simulate process conditions and follow this with a microbial challenge by modifying the process to ensure the viability of the challenge organism (

 change temperature), modifying the formulation to ensure the viability of the challenge organism (

 adjust pH, remove bactericidal component), reducing the exposure time to ensure the challenge organism remains viable, or change from 

 to a microorganism that has been isolated from the formulation. It is important to use the product formulation if possible, because there have been instances in which 

 has penetrated a sterilizing filter in contact with the product formulation but has been retained by the same filter when inoculated into a surrogate fluid. 

The ability to predict the successful outcome of a particular sterile filtration event requires a filter manufacturing enterprise that is highly capable and consistent, a dependable and accurate method to conduct integrity testing, and possession of the understanding and ability to implement effective filtration validation strategies. 

is president of the Williamsburg Group, LLC, 18907 Lindenhouse Rd., Gaithersburg, MD 20879, tel. 301.869. 5016, 

is president of Akers Kennedy and Associates. 

is group vice-president of global product management, bioprocess of Sartorius North America. 

is a consultant for Capitola Consulting. Madsen, Akers, and Meltzer also are members of 

*To whom all correspondence should be addressed.

 (DHI Publishing, River Grove, IL, 1st ed., 2003). 

 2. Parenteral Drug Association, "Technical Report No. 26: Sterilizing Filtration of Liquids," 

 3. P.R. Johnston and T.H. Meltzer, "Comments on Organism Challenge Levels in Sterilizing-Filter Efficiency Testing," 

 (Marcel Dekker, New York, NY, 1st ed., 2001), p. 623. 

Guideline on Sterile Drug Products Produced by Aseptic Processing

 6. M.W. Jornitz, "Integrity Testing," in 

M.W. Jornitz, Ed. (Springer-Verlag, Heidelberg, Germany, 2006), pp. 143–180. 

 "Considerations in Sterile Filtrations, Part I: The Changed Role of Filter Integrity Testing," 

 8. F. Bowman, M.P. Calhoun, and M. White, "Microbiological Methods for Quality Control of Membrane Filters," 

 "An Application of Membrane Filtration for Removal of Diminutive Bioburden Organisms in Pharma Products and Processes," 

 10. M.S. Cooper, "Microbial Retentive Filtration," 

 11. R.E. Madsen, "Filter Validation," in 

 M.W. Jornitz, Ed. (Springer-Verlag, Heidelberg, Germany, 2006), pp. 125–141. 

Model organisms are useful when validating sterile filtration, but successful retention of the model organism does not always guarantee that effluent is sterile. The authors explore the various factors that influence sterile filtration. 

The Use of Model Organisms in Sterilizing Filtration

Particulate and microbiological tests conducted on selected clothing systems in a dispersal chamber installed at KTH (1–3) have shown that the particle levels reach higher values after 25 washing and sterilizing cycles than after 50 cycles (see Table I). This result might be explained by the fact that after a certain number of washing and sterilizing cycles, the fabric releases particles but with time, the released particles seem to be washed away from the fabrics. With this information from performed studies of the source strength, it is possible to calculate predicted contamination levels from people dressed in clothing systems washed several times while in cleanrooms with turbulent mixing ventilation and in cleanrooms with vertical unidirectional airflow. 

If the contamination sources and the design of the ventilation system are known, a mathematical model can be built of the level of airborne contaminants in a cleanroom having completely turbulent mixing air. (Descriptions of such ventilation systems are provided in various textbooks.) With the assumptions of no leakage into the room and of close to 100% efficiency from the final filters (

 HEPA-filters), the simplest possible expression describing the concentration 

 is source strength—outward particle flow (numbers/s) and bacteria-carrying particles (colony-forming units [cfu]/s)—and 

/s). This equation provides estimations of contamination levels in the following examples. 

  and 20 air changes per hour, the only contamination sources are the cleanroom-dressed operators. How many operators are allowed in the filling room if FDA 2004 (5) requirements for US Customary Class 10,000 (ISO Class 7) shall be fulfilled? 

To find the answer to this question, use the following limit values of FDA 2004 (5), US Customary Class 10,000 (ISO Class 7) in a dynamic state:  					

number of particles ≥0.5 µm = 352,000/m

Using the data provided in the example, the total airflow 

/s. The calculation procedure is as follows: 					

Use Equation 1 to calculate the concentration in the cleanroom when one person is present: find the source strength for one person (data from Table I) and divide by the total air volume flow. 

The maximum allowed number of people in the cleanroom will be the limit value (or recommended value) divided with the above calculated concentration for one person.

Table I: Comparison of the source strength (mean values) for people dressed in various clothing systems after various wash and sterilization cycles (4).													

According to Table I, the maximum particle-source strength for a high-quality cleanroom clothing system occurs around 25 washing and sterilizing cycles. For particles ≥0.5 µm, the value of 

 is 3950 particles/s. The concentration becomes: 

Concentration (particles ≥0.5 μm) = 7900 particles/m

The maximum allowed number of people in the filling room depending on particle size will be the limit value of the FDA Class 10,000 divided by the calculated concentration. Therefore, for particles ≥0.5 μm, the maximum allowed number of operators within the filling room is 44. 

The source strength for cfu increases with the number of washing and sterilizing cycles (see Table I). Following the same calculation steps and using the initial data from Table I for cfu and high-quality cleanroom clothing systems, results have been summarized in Table II. 

Table II: Source strengths for high-quality cleanroom clothing systems and estimated number of operators allowed.			

Table II shows—with FDA US Customary Class 10,000 requirements—that after 50 washing and sterilizing cycles, the allowed maximum number of operators is four. Should the FDA 2004 (5) with Clean Area Classification US Customary Class 1000 be used instead, the microbiological limit is set to 7 cfu/m

 , and the maximum number of operators allowed would be only three. 

This example shows that when the operators are the only contamination source, the value for cfu presents a stricter requirement than that of particles. On the other hand, various production machinery also generate particles, which could be a reason for the larger ranges for particles ≥0.5 μm. 

If unidirectional flow units with HEPA-filtered air are used, the situation improves. The airflow through the units should be added to the room airflow. For example, if in the filling room a unidirectional flow unit with HEPA-filtered air is installed with the same airflow as the room airflow, the total airflow is 1.0 m

/s. Therefore, the maximum number of people allowed in the filling room is about twice the value estimated in the above examples. 

Calculations show the predicted contamination levels in cleanrooms with turbulent mixing air and with vertical unidirectional airflow when people are dressed in modern cleanroom clothing systems. Comparisons are made between operation theatres and cleanrooms. 

Pharmaceutical Technology-05-01-2006

RABS and Advanced Aseptic Processing

Effects of the 2006 Proposed Revisions to Annex 1 of the European Union Good Manufacturing Practices

The Use of Model Organisms in Sterilizing Filtration

Predicted Contamination Levels in Cleanrooms When Cleanroom-Dressed People Are the Contamination Source

The Next Generation of Aseptic Processing Equipment

Thinking Inside the Box: The Application of Isolation Technology for Aseptic Processing