James E. Akers is the president of Akers Kennedy & Associates, PO Box 22562, Kansas City, MO 64113, akainckc@aol.com.

James E. Akers

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

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

Although tacitly acknowledged for some time, only during the last few years have both industry experts and regulatory authorities fully acknowledged that humans working in cleanrooms are the chief source of microbial contamination. Because humans are the only significant source of contamination, it follows that they are also the greatest obstacles to the achievement of sterility assurance. It is logical, then, that contamination control technologies currently described as "advanced" focus on enhancing the control of human contamination. 

The two technologies most often referred to as advanced at the present time are isolators and blow–fill–seal systems. These technologies represent two different strategies for contamination control. Isolators rely on what can be termed a 

 in which the human operator cannot access the aseptic work environment directly. Access to the isolated aseptic zone is achieved only through features that allow separated manipulation. Glove and sleeve assemblies and, in some cases, half-suits are used to provide indirect access with a substantially reduced potential for human contamination. 

In the case of blow–fill–seal systems, human-borne contamination control is achieved by not requiring human access into the critical aseptic area during production. Because blow–fill–seal systems usually fully form the container system 

 they do not require interventions by operators to supply components for closure of an open container. Sterilization of the fully assembled product contact pathway in place obviates the need for aseptic connections. The intended result is aseptic production without intervention. 

No barrier separation is used; instead an ISO 5 air shower provides a positively pressurized environment that effectively seals the critical zone against the entry of environmental contamination. The effectiveness of this approach has been established in studies in which high concentrations of contaminants have been released into the surrounding environment with no effect on contamination rates in process simulation studies. 

If isolators and blow–fill–seal systems represent the apogee of current aseptic processing technologies, it seems reasonable to ask where evolutionary development in aseptic processing will take industry in the future. The authors believe that equipment concepts already under development, or even in use, clearly point the direction forward. This article will discuss systems that are already in validated operation and others that are likely to follow in the foreseeable future (1, 2). 

All current aseptic processing environments, including isolators and blow–fill–seal systems, rely on filtered high-efficiency particulate air (HEPA) to provide air that is free of microorganisms to the aseptic zone. Concerns have been raised about the effectiveness of HEPA filters, but experience has shown that in well-designed and properly operated systems, the risk of contamination from HEPA filters is so low that it can be considered insignificant. A frequent criticism of HEPA filters is that they are depth filters rather than the pore–size-controlled membrane filters used for the removal of microorganisms from liquid or gas process streams that come in direct contact with aseptically produced product. HEPA filters have been particularly valuable in human-scale cleanrooms because they are efficient at submicrometer particulate removal and also present a manageable level of backpressure. 

As a result, high flow rates of air that is free of detectable microbial contamination can be supplied with reasonable efficiency. In human-scale cleanrooms, high air-exchange rates of as many as several hundred clean-air changes per hour effectively dilute contaminants derived from personnel. In systems that do not allow direct human interventions (and thus exhibit lower contamination potential), lower air-exchange rates are more than adequate to protect the critical environment. This reduces air-flow requirements, hence air-conditioning and dehumidification systems can be downsized. The resulting energy savings could be significant in an era of rising energy costs. 

The authors do not believe that a scientifically compelling argument can be made at this time for filtering air by means other than HEPA-grade filters. In modern air-handling systems with prefiltration of makeup air, the risk of microbial contamination entering the environment through the air-handling system is negligible. From a risk perspective, significant gains in aseptic processing contamination control can be made only by better control of human contamination. It is obvious from risk-analysis modeling that the best way to control human contamination is the elimination of human interventions of any kind (3). 

The notion of an aseptic processing operation that requires no human intervention may seem an impossible dream to engineers and scientists familiar with current pharmaceutical manufacturing approaches. The authors, however, have experience with aseptic processing systems already in production that do not rely on human interventions during routine manufacturing operations. The authors have worked with aseptic manufacturing systems used to fill beverages into polyethylene terephthalate bottles at throughput rates of as many as 1200 bottles/min that have been validated and have achieved regulatory approval. These systems use isolator enclosures without gloves. Component feeds for containers and caps are accomplished with an automated, continuous hydrogen-peroxide sterilization system that supplies the components directly to the production isolator. 

These systems also use direct process control in the form of gravimetric fill control for each container, with automatic out-of-specification unit rejection. Capping also is controlled in-process with a servo-controlled system that measures the torque on each cap and rejects any bottle–cap assembly that is outside the established specification. The decontamination of the enclosure and all product contact parts also are accomplished automatically. Clean-in-place (CIP) and sterilization-in-place connections also are made automatically.  

The performance of these systems demonstrates the clear advantage of eliminating human interventions completely. More than one billion production containers have been filled without a single report of contamination. Milk products and low-acid beverages filled in these systems are highly susceptible to microbial spoilage because they support the growth of typical environmental organisms. Contamination control at such a high level simply would not be possible if human interventions were required. For this reason, aseptic enclosures or isolators are now standard equipment for the production of these beverage products. In the most advanced of these systems, the routine use of gloves for separated human interventions is not required, thus resulting in exceedingly low contamination risks. 

The level of performance afforded by this technology is an economic benefit to the manufacturer because the elimination of contamination risk results in both high productivity and high customer satisfaction. The authors find it ironic that most aseptically produced healthcare products are not manufactured with a comparably high level of contamination control because they still rely on direct intervention by gowned operators. It seems counterintuitive that beverages are produced in more technologically advanced contamination-controlled environments than many healthcare products (4). 

Aseptic processing of healthcare products: present and future

In the authors' opinion, the lessons learned from the aseptic beverage model are directly applicable to the manufacture of healthcare products. Since 2003, the authors have designed pharmaceutical aseptic processing systems that eliminate human interventions to the greatest possible extent. The ultimate objective is the complete elimination of human interventions through the use of: 					

high-quality components such as glassware and stoppers that eliminate misfeeds;

machine automation and robotics to eliminate all routine and repetitive interventions such as component feeding;

identification and diagnostic systems that eliminate unfilled containers and containers without stoppers and use positive control of containers wherever possible to avoid jams and fallen containers;

the automation of in-process control and environmental monitoring activities, thereby reducing risk from interventions associated with these activities;

complete elimination of aseptic connections, using automated CIP and steam-in-place (SIP) systems and automatic filter-integrity testing (5).

In the authors' view, taking parenteral aseptic processing to a higher level of contamination control performance and manufacturing efficiency requires a second paradigm shift. The authors define the first paradigm shift in aseptic processing as the realization that gowned human operators pose a significant contamination risk. Not long ago, it was common to speak of cleanrooms as "sterile" environments and to believe that a properly gowned operator could intervene with impunity without increasing process risk. The first paradigm shift was the move to separative technologies such as isolators or closed restricted-access barrier systems to control human contamination better. This first paradigm shift, which occurred in the 1990s, resulted in isolator designs that were intervention tolerant. This required a great emphasis on ergonomics because it was accepted that interventions were inevitable. Currently, it is considered acceptable practice to test or even to validate interventions in process simulation tests  Interventions—even those performed within isolators—are considered acceptable in all instances. 

First- and second-generation pharmaceutical isolators might require 20–40 gloves to enable ease of intervention. It has been recognized that the ease of intervention comes at a price. Commonly used, two-piece glove systems may separate at the mounting ring or the wrist. In addition, gloves and sleeves may develop pinhole leaks or tears, some of which have proven difficult to detect. Glove-leak rates in some first- and second-generation isolator systems have been higher than expected and have resulted in the selection of heavier duty gloves made of puncture-resistant materials (

 Hypalon, DuPont Performance Elastomers, Wilmington, DE) and increased use of single-piece glove and sleeve assemblies to reduce the risk of separation. Although these changes have improved performance, they have done so at the price of increased costs and, in some cases, reduced flexibility because some users and members of the regulatory community have frowned upon changing gloves during an aseptic process. 

Therefore, the second paradigm shift in modern aseptic processing must be the realization that interventions no longer can be considered inevitable, even in isolators. The authors believe that the future lies in systems that consistently reduce the need for interventions of any kind, including those performed in isolators. The authors believe that this second paradigm shift will result in different approaches to separative enclosure design and, most importantly, to the equipment enclosed within these isolators. Several technological features that point the way to the second paradigm shift in aseptic processing already are in use. A list of such features includes the following: 					

Smart depyrogenation tunnel systems count containers into rows and do not allow in-feed until there is space available on the conveyor belt. These systems keep a tight pack for efficient heating and do not put unnecessary physical stress on glass when it is being heated and cooled.

Direct measurement of glassware depyrogenation temperatures can ensure that process-control requirements are met consistently, which should lead to reduced validation requirements and minimize excessive heating times.

CIP and SIP can be combined with automatic filter-integrity testing for both process and vent filters, thereby improving process control and reducing the potential for error.

Fill-dosing systems based upon mass-flow metering are highly accurate, easy to clean and sterilize, can be set up remotely, require no in-process adjustment, and eliminate the need for manual weight checking. These systems also minimize the mechanical complexity common to piston or pump filling and obviate the need for complex mechanical systems required for gravimetric fill-volume checks.

Vapor-phase hydrogen peroxide (VPHP)-resistant robotic systems for component feeding are validated and in daily use. In these systems, robots are used to load and unload the sterilizer. Component containers are recycled, thereby eliminating waste management inside the isolator and eliminating another class of interventions. Electronic signals from the component hopper inform the programmable logic control of component level and signal the robot to refill the hopper.

Cappers can be placed in an ISO 5 VPHP-decontaminated isolator. This capping isolator is a separate enclosure downstream of the capping section and operates at a negative pressure relative to the filling–capping isolator. This arrangement prevents aluminum particulate from entering the critical filling and capping zone. The capping isolator, however, is at a positive pressure relative to the surrounding ISO 8 room. This feature also eliminates concerns about the application of aseptic conditions throughout the manufacturing process. In addition, sensors and automatic rejection systems can detect and remove containers with missing stoppers.

Cappers with the ability to measure seal effectiveness directly on each container afford better process control and ensure that only properly sealed containers reach the consumer. These systems have been installed and validated and have proven to be valuable assets in process control.

Automatic environmental-monitoring systems using VPHP-resistant robots located inside the isolator enclosures can handle active air sampling, surface sampling, or, if required, settling plates. These automated environmental-monitoring systems eliminate the need for glove interventions to take samples. Because the robots record the time, location, and sample identification, they reduce the likelihood of error. In addition, because no human handling is required, the possibility of false-positive results is reduced. Other environmental control factors that can be measured and controlled electronically are particulate levels, humidity, enclosure relative pressure, and pressure differentials across filter systems. These data, along with all critical control data, can be read remotely in a control room.

The second paradigm shift in aseptic processing does not involve the separation of interventions, but rather their complete elimination. This shift already is well underway as modern processing systems increasingly rely on automated process control, intelligent machine design, and robots to eliminate the need for human intervention. VPHP-resistant robots already are available and in use. Future generations of robots will be more flexible, and users also will be able to operate them remotely. Thus, for some process steps for which manipulation still is required, remote robotic handling may well prove to be the answer. 

Aseptic systems already are in use that fill containers at extremely high speeds without human intervention. In fact, given the very high mechanical speeds this equipment attains, human intervention is unsafe and cannot be allowed. These installations have set the bar for what is possible in aseptic processing, and the challenge in the coming years is one of applying the lessons already learned. Fortunately, the elimination of interventions in parenteral aseptic processing requires only the application of technologies that already exist. Innovation and creative application of these technologies certainly is required. The authors expect that, in addition to advances in sensor, automation, process control, and robots, there will be parallel improvements in container-closure systems as well. The recent interest in presealed, sterilized vials, and other drug delivery innovations will require more specialized or customized processing solutions. 

The authors understand that the pharmaceutical industry is conservative and that paradigm shifts historically have occurred at a relatively slow pace. The authors are optimistic, however, that the revolution currently underway in information technology, sensor systems, robotics, and the use of electronics to reduce mechanical complexity will result in widespread, intervention-free aseptic processing quicker than many readers may expect (6). Furthermore, the authors believe that parallel developments in advanced aseptic delivery systems will help drive this second paradigm shift in aseptic processing. 

 is the president of Akers Kennedy & Associates, PO Box 22562, Kansas City, MO 64113, 

, is general manager, microbial control department and 

 is senior chief engineer, both at RP System Division, Shibuya Kogyo Co., Ltd. 

*To whom all correspondence should be addressed. 

 1. J. Agalloco, J. Akers, and R. Madsen, "What Is Advanced Aseptic Processing?" 

 2. W. Whyte, "A Cleanroom Contamination Control System," 

 3. J. Akers and J. Agalloco, "Risk Analysis for Aseptic Processing: The Akers–Agalloco Method," 

 4. Y. Oshima, S. Yoshida, and J. Akers, "PasepT: A New Concept in Aseptic Processing," in 

Proceedings of the 36th R3 Nordic Symposium and 5th European Parenteral Conference

 (The Nordic Association for Contamination Control and Cleanrooms, Linkoping, Sweden, 2005) 

 5. J. Agalloco and J. Akers, "Aseptic Processing: A Vision of the Future," 

 6. J.P. Agalloco and J.E. Akers, "A Critical Look at Sterility Assurance," 

The industry has acknowledged only recently the significance of the contamination risk posed by humans. The authors assert that this realization, together with technological advances, will lead to the elimination of human intervention and, hence, improved sterility. 

The Next Generation of Aseptic Processing Equipment

Many in the pharmaceutical industry consider aseptic processing to be among the more difficult activities to execute properly. A substantial number of variables affects the safety of sterile products manufactured aseptically. The design and operation of an aseptic production facility must minimize the risk to the patient. This article presents a new approach to risk assessment for aseptic processing that emphasizes the contributions of personnel. 

In September 2004, the US Food and Drug Administration issued the long awaited guidance on sterile drug products produced by aseptic processing (1). That month, FDA also published 

Pharmaceutical CGMPs for the Twenty-First Century—A Risk-Based Approach

. (2) Each of these documents refers to the other, but there is no substantive information offered in either to assist the practitioner in establishing a risk-based approach for the design and control of aseptic processing operations. 

Numerous pre-existing risk-analysis methods have been adapted for the pharmaceutical industry, including:  					

Hazard Analysis and Critical Control Point (HACCP);

Failure Mode, Effect, and Criticality Analysis (FMECA).

These commonly used methods have proven successful in a variety of applications. None, however, was ever specifically intended for application to aseptic processing, which relies on a large number of variables and has the lowest tolerance for failure of any process in the entire industry. Simply put, failure in aseptic processing is unacceptable, and a suitable risk-analysis method must incorporate all of the factors affecting product sterility and patient safety. The risk-analysis approach used must incorporate the recognition that there is no acceptable level of risk associated with sterile products, regardless of the manufacturing method. The goal is, and must always be, perfection in all elements of sterile product manufacturing, especially when products are made using an aseptic process (3). With this in mind, the authors embarked on an evaluation of previously established risk-evaluation methods as applied to aseptic processing. 

The risk-analysis method that is chosen must focus clearly on the manufacturing activities that are essential for success in aseptic processing. This normally is manifested by a concern for criticality. Not all of the constituent activities of a process being evaluated have an equal effect on the end result. 

 as defined within FMECA, is a multiplication of the criticality of an occurrence by its frequency. This definition seemingly can be applied directly to aseptic processing. Determining what constitutes an "occurrence," however, is generally difficult in aseptic processing. One could argue (perhaps incorrectly) that environmental monitoring results can provide a measurement of occurrences, but this assumption is theoretical at best. Environmental monitoring, especially in the ISO 5 environments commonly used for aseptic processing activities, hardly can be considered a quantitative or fully effective tool. In the typical ISO 5 environment, microbial contamination is detected only rarely, and even the most aggressive sampling plans only sample a limited amount of air or surfaces* (see endnotes). Another drawback of viable environmental monitoring is that results are unavailable in real time when using common growth and recovery methods. Nonviable particle monitoring, which is available in near-real time, has never been correlated sufficiently to viable data. The authors consider it of limited value in aseptic risk assessment. 

The authors believe that there is a straightforward way to analyze risk in aseptic processing. 

is by general agreement a function of the release of human-derived contamination into the operating environment. The full extent of human contamination risk is substantial: a gowned operator may release as many as 10,000 colony-forming units (cfu) or more per hour (4, 5). The estimated value is derived from operators performing controlled and defined movements immediately after donning sterile gowns. These methods merely provide a way to estimate the intensity of the contamination source from activities substantially different from those performed during aseptic processing. Direct application of these data to aseptic processing unfortunately is not possible, given the differing environments and activities. 

Whyte developed a risk-assessment model based on his research on microbial deposition, which was an extension of his earlier efforts with settle plates (5–8). The premise behind this approach is the recognition that personnel are the primary source of contamination. It assumes the mechanism for dispersion of that contamination is airborne deposition. Whyte's model is the following: 

Number of microbes deposited on product = 

 is the concentration of microbes in the source (people), 

 is the quantity of air or material dispersed from a source over time (usually cfu/m

 is the proportion of organisms effectively transferred, 

 is the proportion of organisms that arrive into the product area, and 

 is the time during which microbes could be transferred. 

Whyte also has proposed a slightly simpler deposition model for risk evaluation in which the risk from microbial contamination is defined as: 

 is the microbial contamination on or arising from a source (

 is the proximity of the source to the critical area (one can assume the contamination decreases as the inverse of the square of the distance), and 

 is the effectiveness of the control method (

 isolator, restricted-access barrier systems [RABS], automation, sealed container, intervention frequency). 

Whyte chose five levels of risk for each of the terms in the equation: 0 indicates no risk, 0.5 indicates very low risk, 1 indicates low risk, 1.5 indicates medium risk, and 2 indicates high risk. In the case of factor 

 (effectiveness of control), he suggests 0 for "full barrier control." This means that, should a truly full barrier exist, the overall risk effectively would be zero. Products such as sealed vials that mimic the capabilities of a closed isolator system logically would fall into the "full barrier control" category (9, 10).  

The authors present a new approach to risk assessment for aseptic processing that emphasizes the contributions of personnel.