The Myth Called "Sterility"

Background
A common perspective underlying regulatory documents that call for a "proof" of sterility is the belief that industry can somehow use microbiological analysis and other select and, often-subjective, tests to prove that sterility has been attained. Such proof does not technically exist and is not scientifically possible. There are dangers implicit in regulatory authorities requiring industry to attempt to prove the unprovable. These misguided efforts create circumstances in which industry can never truly accomplish the intended objective and, as such, can always be found to have made insufficient efforts to support sterility-assurance programs.
Users of isolation technology, for example, have been asked to increase environmental monitoring (EM) to extreme levels because existing monitoring programs established for manned cleanrooms cannot detect contamination. Scientifically and legally, these standards have left industry with both feet firmly planted in mid-air. The result, as evidenced from recent inspections, is that if an inspector wishes to use these documents to insist that a firm lacks "sterility assurance," then there is virtually no way the manufacturing firm can defend itself.
It is always possible to start an inspection report with the following statement, "The firm failed to demonstrate sterility assurance in that...." It's impossible to objectively prove or disprove this allegation. Sterility is an absolute concept, and its presence cannot be proven, regardless of the effort to do so.
Examples of unprovable regulatory citations include claims of inadequate air visualization (smoke studies), claims regarding the conduct of media fills, the acceptability (or not) of a specific aseptic intervention, and charges regarding the adequacy of environmental monitoring. Metrics for smoke-test success are absent. This test is strictly an eye-ball exercise in which one party may see one thing and another sees something quite different. Although smoke tests are valuable for fine-tuning certain elements of critical-zone performance, they rarely lead to real performance improvements.
Airflow is another example. Airflow in cleanrooms is some-times incorrectly called "laminar," but in practice, laminarity cannot be achieved. No matter how well-designed or qualified an isolator or cleanroom is, there always will be turbulence and eddy currents. There is no objective standard for the point at which adequacy no longer exists or at which turbulence might affect sterility assurance, if it ever does.
Media-fill conduct is yet another issue without an endpoint. In recent years, regulators have required larger and longer media fills and placed an increased emphasis on using media fills as long as the longest production run. However, change does not enable proof of sterility. No media fill, no matter how large or intensive, can ever prove sterility. New conditions can always be added to a media-fill program, even if those conditions are atypical. Recently, FDA has expected that the production and filter sterilization of media parallel the compounding and filtration of product. Yet, media and product are two very different materials with different attributes. The most obvious of these differences is that media will amplify the presence of contamination, which the majority of products will not do. Also, media may contain insoluble particu-late in quantities not seen with most aqueous formulations which means prefiltration is a must. There is nothing to be gained from ever larger media fill tests with activities that really don't relate to process simulation being required
Another prevailing notion is that some aseptic processing interventions are inherently bad. There's no question that heroic efforts during aseptic processing should be avoided. But what makes a particular intervention good or bad? We have no useful metrics, to assess the difference, yet such categorization is all too swift and final. Furthermore, media-fill tests, no matter how intensive, cannot reliably demonstrate (or validate) that an intervention is low-risk, nor can they unequivocally prove that an intervention is bad. Neither media-fill or eyeball tests are inherently sound arbiters of sterility assurance. In absolute terms, they are both inadequate for such a task. Destroying a batch because an intervention is arbitrarily assumed to be "bad" is not much different than accepting a different batch made with a series of "good" interventions. EM has been increasing as a consequence of regulatory pressure for at least 20 years, but there is never any consideration of diminishing return or even patient risk arising from EM-related interventions. Quite simply, it is not possible to monitor quality into product (something we've known since the very origins of validation in the 1970s) and it will never be possible to use EM to prove sterility. EM is neither sensitive nor accurate enough to pinpoint when intervention might put a patient at risk. EM has significant and inherent, technical limitations and we have likely passed the point of diminishing return on it within even manned cleanrooms. This is an acknowledged fact that's never contemplated in current regulation.
The need foranewdirection. The absolutist thinking regarding sterility assurance plays a significant role in standards development. Both industry and regulatory authorities would benefit from a serious dialogue about the nature of aseptic processing regulation. Intensity and length of effort cannot alone ensure sterility. Monitoring, even if continuous, and smoke tests, even if comprehensive, cannot ensure sterility. Unfortunately, subjective evaluation of such data can result in regulatory observations that are not pertinent and may be irrelevant.
The authors are raising this issue now because we believe that evolving aseptic-processing technology has rendered the traditional evaluative methods less useful. These more advanced processes consistently operate below the limits of detection for the presently available microbiological assays.
As processes have improved and "zero" results have become the norm, the regulatory reaction has been to multiply the test and in-process workload which, while intuitively logical, is scientifically inappropriate and, unfortunately, valueless. We suggest that rapid microbiology, drawing increasing attention by industry, only provides the same imprecise information about the aseptic process we already have, albeit somewhat sooner. The use of that "information" is what the authors are concerned about, not the time it takes to obtain it. Rapid microbiology is very useful technology, but it cannot overcome the sampling limitations that exist. No analytical method (microbiological, chemical, or physical), regardless of how advanced and sensitive, can measure the complete absence of something. Sterility is, by definition, the complete absence of viable contamination.
The authors seek to highlight the increasingly arduous regulatory spiral into which we have been drawn. The most practical way forward is to carry out honest and detailed dialogue between all stakeholders. In many technical endeavors, there's a time at which paradigm shifts are required. Discipline of aseptic processing is now at such a point. Continuing to follow the same path of the past two decades will neither improve end-user safety nor the economics of manufacture.
A process-centric approach for superior performance
To successfully manufacture sterile products by aseptic means, it's necessary to redefine the process controls essential for success. Central to the authors' suggested approach is the use of the Akers-Agalloco (A-A) method for aseptic-processing risk analysis to support the evaluation and selection of the specific means for aseptic-process design and execution (1,2). Our preference for this over other methodologies is based upon the absence of inference from EM results. Katayama et al, in their review of aseptic-processing risk models, identified the A-A method as having the closest correlation to the operational performance evidenced for a variety of different installations (3).

Figure 1: Influences on aseptic processing (Adapted from L. Mastrandrea, Ref. 9).

Central to aseptic processing is the understanding that there are numerous factors that can influence the outcome (see Figure 1). The authors believe it's the decisions, selections, and approaches—made with respect to each of the factors depicted in Figure 1—that have the greatest effect on results. Poor choices, regardless of the monitoring outcomes associated with them, must be acknowledged as unsound. Our approach differs from those derived from EM expectations because of our focus on personnel and their impact on contamination levels. The rankings in the A-A method devolve from a singular focus on the operator. From that perspective, the authors established the following basic precepts to the A-A risk method and the recommendations outlined below (4, 5):

• Interventions are to be avoided at all times in aseptic processing
• Interventions always mean increased risk to the patient
• There is no truly safe intervention
• The "perfect" intervention is the one that doesn't happen.

In turn, these steps should be followed with respect to aseptic processing: separate personnel from the aseptic environment; limit employees' interaction with sterile materials; where possible, entirely remove personnel from the aseptic environment; and combinations of the above. The means for accomplishing these goals are embodied in following methodologies (6):

• The use of automation technology to reduce or eliminate personnel interventions and, thus, personnel-borne contamination
• The use of separative technologies to minimize the impact of personnel-derived contamination.

These methods are central to our recommendations for the supportive elements of aseptic processing. In defining these elements, the authors are adapting a quality-by-design (QbD) approach as defined in recent regulatory documents (7,8). The details for QbD in aseptic processing are somewhat different from the applications of this concept in the typical formulation or synthesis process. As we outlined in the first half of this paper, the establishment of direct linkage between a monitored condition and the outcome, with respect to an aseptic process, is uncertain. The situation, with respect to the definition of physical-design elements, is very similar. Contemporary aseptic-processing facility and process design include several seemingly rigorous design expectations for performance, including such precepts as:

• Air velocities of 90 FPM (0.45 m/sec) ± 20% for ISO 5 air in critical environments
• Air changes of > 150 per hour in critical environments
• Pressure differentials of NLT 0.05" water column between different classifications.

These expectations, and others like them, should be considered suggestions rather than definitive requirements because they have less correspondence to the process outcome than EM. The authors' recommendations for QbD, with respect to aseptic processing, are non-numeric because it is our strong belief that there are no ready means to demonstrate their suitability. Instead, we suggest that QbD for aseptic processing be driven toward eliminating the impact of personnel on the process. The means to accomplish this vary depending on the particular aspect of the overall process under consideration.
The following recommendations for various aspects of the aseptic processing facility adhere to our central premise of reducing the potential adverse impact of personnel on the core aseptic process. They are not intended to be inclusive—other suggestions could be added.
Facilities:

• Facilities should be designed for easy sanitization/decontami-nation through proper use of construction materials, ease of access, and design details that facilitate cleaning
• Facility layouts should minimize the potential for mixups and cross-contamination
• Air system should provide adequate air pressurization to preclude the ingress of contamination from surrounding less-clean environments
• Airflow patterns should facilitate the removal and exclusion of contamination from critical environments
• Air systems should be supplied with HEPA filters that are periodically integrity tested
• Differential pressure should be monitored and alarmed to demonstrate continuous integrity of the core aseptic area
• Temperature and humidity should be controlled to maximize personnel comfort during operations consistent with product stability/safety requirements
• Interlocks should be used to prevent pressure reversal
• Advanced aseptic-processing designs such as closed restricted access barrier systems (RABS) and isolators, should be given preference in selection of processing environments
• All facility and environmental surfaces should be resistant to the potential corrosive action of sanitizing and decontamination agents
• The aseptic portion of the facility should be maintained in clean state at all times and periodically sanitized or decontaminated
• Isolators and closed RABS should be treated with sporicidal agents on a periodic basis.
• A minimum of materials should be retained in the aseptic portion of the facility
• Smoke studies, air-velocity measurements, expectations for unidirectional airflow, absence of eddy's, and other subjective expectations imposed on aseptic-processing HVAC systems should be recognized as useful, but non-definitive means for assessing aseptic processing environmental performance.

Equipment and utensils:

• Product-contact surfaces of equipment must be sterilized using a validated method (vibratory feed systems may be exempted from this requirement provided they are high level decontaminated with a sporicidal agent in-situ)
• Sterilization-in-place and clean-in-place should be used wherever possible
• Equipment should be assembled to the fullest extent possible prior to sterilization
• Equipment and utensils should be sterilized in sealed containers (the use of paper and tape is not recommended)
• Equipment and utensils should be introduced in a manner that retains at least one layer of sterilized-protective covering or wrap until entry into the critical zone
• Equipment and utensils should be sterilized/depyrogenated using a just-in-time approach
• Inventories of materials in the aseptic environment should be minimized
• Equipment should be selected for high reliability, ease of changeover, and adjustment
• Remote adjustment of equipment should be utilized where possible
• Tool free change over from one format to another should be possible
• Equipment should be tolerant of container-closure miss-feeds, jams, and other problems to minimize the need for interventions
• Equipment (and to some extent facility) should use process analytical technology and other feedback systems for ease of control, operation, and documentation
• Non-product contact portions of the equipment should be easily decontaminated and non-invasive of the critical zone
• All equipment surfaces should be resistant to the potential corrosive action of sanitizing and decontamination agents.

Containersand closures:

• Containers and closures must be prepared and sterilized/de-pyrogenated using a validated process
• Containers and closures should be introduced in a manner that retains at least one layer of sterilized-protective covering or wrap until entry into the critical zone
• Containers and closures should be selected for reliability of handling in the processing equipment
• Containers and closures should be suitable quality for their intended use because higher acceptable quality levels for defects can result in a reduction in the need for interventions
• Containers and closures should be sterilized/depyrogenated using a just-in-time approach.

Product:

• Product materials must be prepared and sterilized using validated methods
• Product delivery should be made directly into the critical zone
• Where product is supplied to the critical zone in sterile container (e.g., sterile powders), it should be introduced in a manner that retains at least one layer of sterilized-protective covering or wrap until entry into the critical zone.

Personnel:

• Personnel must receive initial and periodic training in current good manufacturing practice, aseptic processing, microbiology, aseptic gowning, and job specific tasks
• Where appropriate, personnel should be initially and, periodically thereafter, assessed for their proficiency in aseptic gowning
• Personnel should be initially and, periodically thereafter, assessed for their proficiency in aseptic technique
• Personnel should be monitored upon each exit from the aseptic core (gloves on enclosures should be monitored at the end of the batch or campaign)
• Gown materials should be cleaned and sterilized using validated methods
• Gloves on enclosures should be replaced periodically, sterilized, and integrity tested
• Personnel should be trained and diligent in their adherence to aseptic techniques
• Manual filling by aseptically-gowned personnel should be recognized as an anachronistic throwback to an earlier time and no longer used for aseptic processing.

Procedures:

• Procedures should be reviewed to eliminate unnecessary work steps and simplify aseptic processes
• Interventions should be designed for minimal risk of contaminating sterile materials
• Interventions performed during aseptic processing must be recognized as increasing the risk of contamination dissemination
• All interventions should be performed using sterilized tools whenever possible
• Intervention procedures should be established in detail for all inherent interventions and, more broadly, for corrective interventions (where some flexibility is necessary due to their greater diversity).

Monitoring:

• Despite the limitations, with respect to its performance noted in the first part of this effort, monitoring of aseptic processing should be performed.
• Monitoring of any type must not subject the product to increased risk of contamination. No monitoring is preferable to monitoring that risks contamination of sterile materials.
• EM must be recognized as interventions and subject to the similar constraints and expectations.
• Monitoring must be recognized as subject to adventitious contamination unrelated to the environment, material, or surface being sampled.
• Viable monitoring should not be considered an in-process sterility test.
• EM results should not be considered as 'proof' of either sterility or non-sterility.
• Microbial monitoring can never recover all microorganisms present in an environment, nor on a surface.
• The absence or presence of microorganisms in an environmental sample is not confirmation of asepsis, nor is it indicative of process inadequacy.
• Significant excursions from the routine microbial prolife should be investigated.
• Detection of low numbers of microorganisms within the critical zones of manned cleanrooms should be considered a rare event. Such a finding does not correlate to a loss of process control, since it is within the normal range of observations.
• Investigations into recoveries of low numbers of microorganisms in manned cleanrooms should be recognized as predominantly make-work exercises.
• Process simulation are indicators of capability, but cannot definitely establish the sterility of any material.
• Process simulations in excess of 5-10,000 units are of relatively limited value; their greatest utility is in the evaluation of aseptic set-up practices.

Conclusion
The first section of this work addresses the limitations of monitoring tools used for aseptic processing. The current methods cannot prove sterility (or its absence). It is the authors' contention that to achieve success with aseptic processing, the practitioner must properly address the relevant issues outlined in the latter half of this work. There is nothing industry can do to provide proof of sterility. The authors believe, however, that adherence to the recommendations herein will make aseptically-produced products as safe as currently possible.
The methods proposed are largely incompatible with existing aseptic processing guidance, regulatory, and pharmacopeial doctrine because the authors have, essentially, deconstructed monitoring as a means for defining or accepting aseptic-processing activities and endeavored to outline a comprehensive QbD approach for establishing it more appropriately. If industry is to continue to improve aseptic processing beyond its current capabilities and, even to proper control contemporary aseptic-processing operations, the authors believe that greater attention should be focused on the design elements. We offer this work as an opening statement in what we hope will be a continued dialogue through which sterile products can be manufactured by—and controlled—in the safest means possible.
James Agalloco* is president of Agalloco & Associates and a member of Pharmaceutical Technology's Editorial Advisory Board, 908.874.7558,
. James Akers is president of Akers Kennedy & Associates.
*To whom all correspondence should be addressed.
References
1. J. Agalloco and J. Akers, Pharm. Technol., 29 (11), 74-88 (2005).
2. J. Agalloco and J. Akers, Pharm. Technol., 30 (7), 60-76 (2006).
3. H. Katayama et al., PDA J Pharm Sci and Tech., 62 (4), 235-243 (2008).
4. J. Agalloco and J. Akers, Pharm. Technol., 30 (7) (2006) 60-76.
5. J. Agalloco and J. Akers, supplement to Pharm. Technol., Aseptic Processing, 31, s8-11 (2007).
6. J. Agalloco and J. Akers, supplement to Pharm. Technol., Aseptic Processing, 29, s16-23 (2005).
7. M. Nasr, "Quality by Design (QbD)-A Modern System Approach to Pharmaceutical Development and Manufacturing-FDA Perspective," presentation at FDA Quality Initiative Workshop at ISPE meeting (Bethesda, MD, February 2007).
8. ICH Q8(R2) Pharmaceutical Development (ICH, Geneva, 2009).
9. L. Mestrandrea, presentation at the 4th Annual PDA Global Conference on Pharmaceutical Microbiology (Bethesda, MD, Oct. 2009).