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

James E. Akers

Cleanroom monitoring and classification are vital to operations but are still separate functions. This article discusses the differences between the two concepts and emphasizes the need for the two to remain independent. As they write, “A one-size-fits-all approach to cleanroom monitoring will not reflect the diversity of practices” within the industry. In addition, they write, classification values should not be used as monitoring targets.

Biologics and Sterile Drug Manufacturing 2019

	When referring to this article, please cite it as J. Agalloco, R. Madsen, and J. Akers, “Distinguishing Between Cleanroom Classification and Monitoring," 

Pharmaceutical Technology Biologics and Sterile Drug Manufacturing 

Articles

A one-size-fits-all approach to monitoring practices and results is never appropriate, given the diversity of practice within the pharmaceutical industry.

Distinguishing Between Cleanroom Classification and Monitoring

In this extended op-ed, aseptic processing consultants James Agalloco, Russell Madsen, and James Akers discuss the current regulatory emphasis on environmental monitoring requirements. Based on the mistaken belief that monitoring is an exact measurement of reality, many current requirements emphasize monitoring when the real focus should be on minimizing operator contact with the process, they write. 

Pharmaceutical Technology’s Biologics and Sterile Drug Manufacturing 2018

 Biologics and Sterile Drug Manufacturing 2018

	When referring to this article, please cite it as J. Agalloco, R. Madsen, and J. Akers," Lies That Environmental Monitoring Systems Tell," 

Pharmaceutical Technology Biologics and Sterile Drug Manufacturing 2018 

Environmental monitoring, which operates below the limit of detection, is little more than a rote exercise that provides limited real value. So why does it figure so prominently in current regulatory requirements for aseptic manufacturing?

Lies That Environmental Monitoring Systems Tell

 presented difficulties with the revised European guidelines on sterile manufacturing (1).  The authors included a brief summary of the comments developed on the draft document.  This article expands upon that summary, outlines the authors’ rationale, and highlights the most difficult aspects of the revision draft.

Volume 4, EU Guidelines to Good Manufacturing Practice Medicinal Products for Human and Veterinary Use

, Annex 1, Manufacture of Sterile Medicinal Products (2), when finalized, will be legally binding. Non-compliance could result in regulatory action, refusal, or revocation of marketing authorizations, and civil and criminal penalties. Given the revised Annex 1’s legal standing, it is vital that the document delineates appropriate regulatory expectations and accepts contemporary manufacturing and control procedures and practices.

	The European Medicines Agency (EMA) has put forward Annex 1 for comment, but the document is so badly written that understanding it, much less commenting on it, is far more difficult than it should be. As it stands, this version of Annex 1 will inevitably generate thousands of comments that will be difficult for EMA to reconcile within the framework of the current document. Preliminary review of the document indicates that it is poorly worded, ambiguous, and technically incorrect in many areas. The following comments represent a fraction of those identified during the authors’ review. They are categorized by general areas of concern.

	1. The document is inconsistent in its use of references; they are present in some sections and absent in others. The document should include a comprehensive set of reference documents to support the points made in the text. The inclusion of appropriate references is especially important for those aspects of the draft that are most contentious.

	2. The document presents a view of sterile product manufacture inconsistent with that developed elsewhere, as codified in regulations, international standards, and pharmaceutical compendia, for example:

	a. US and Japanese guidance on sterile product manufacturing differ markedly from what is presented.

	b. The cleanroom content in the draft does not conform to International Organization for Standardization (ISO) 14644 and perpetuates the myths that cleanrooms can be classified microbiologically and that microbiological testing can enhance sterility assurance (3).

) chapters <1211>, <1228>, and <1229> provide more contemporary and appropriate guidance for sterile product preparation (4–6).

	d. There are also conflicts with current EMA guidance (e.g., the water for injection [WFI] Q&A paper and Guidelines on Good Manufacturing Practice specific to Advanced Therapy Medicinal Products) (7-8).

	3. The revised Annex 1 does not provide separate consideration of conventional cleanrooms, restricted access barrier systems (RABS), and isolators; they are demonstratively different in many ways, and considering them together denigrates isolator performance, elevates RABS capabilities, and does not consider the limitations of the far less capable barrier-equipped conventional cleanroom.

	4. EMA’s illogical and arbitrary insistence upon maintaining its Grade A/B/C/D system should be abandoned and the ISO 5 classification system used throughout the document. We have addressed these in some instances, but other citations to Grades A/B/C/D may remain as well as individual citations to Grade A, Grade B, and the unclear Grade A/B. Grades A, B, C, and D should be replaced with the corresponding classes of ISO 5, 6, 7, and 8.  All references to the arbitrary and confusing Grade B, and the totally undefined Grade A/B, should be removed.

	5. Classification of controlled environments is limited to non-viable particle monitoring as described in ISO 14644. There are no means to classify cleanrooms based upon the microbial enumeration. Any suggestion that classification includes a microbial population requirement should be removed from the document.

	6. The document has a perspective on microbial monitoring that is inconsistent with scientific reality. The limit of detection for microbial testing is substantially higher than one colony forming unit (CFU), a level which is used throughout the document. This erroneous belief leads to numerous misconceptions and overstatements regarding what value environmental monitoring has in the preparation of sterile medicinal products.

	7. The document frequently asks for testing of materials, containers, and surfaces, with the expectation that the testing can somehow assure quality. The founding principle of validation is that it can assure confidence in the reliability and appropriateness of the process in ways that testing can never provide.

	8. The document needs better organization to place related content together (e.g., environmental monitoring, terminal sterilization, barrier system, and lyophilization content can be found in multiple locations). In addition, there is non-exact redundancy of the content (i.e., environmental monitoring, terminal sterilization), which only causes additional confusion.

	The use and application of barrier systems, isolation technology, and environmental and personnel monitoring are incorrectly described or applied, as follows:

	m) Monitoring systems-including an assessment of the feasibility of the introduction of scientifically sound, modern methods that optimize the detection of environmental contamination.

 This bullet item suggests that there are available means to demonstrate ‘sterility’ or ‘asepsis’ through the use of expanded monitoring. While these methods can be of value, expanding the application of these methods to enable detection of contaminants not currently found is scientifically incorrect because of the analytical and statistical limitations inherent in them. The environments used for sterile (aseptic) operations need not be, and by in large cannot be, ‘sterile’. The conflation of the condition of asepsis with the concept of sterility is both semantically and scientifically erroneous. It is impossible to test sterility in a product environment using any analytical technology. In addition, monitoring must never be invasive to the production processes in a manner that increases risk by adding activity in the critical zone. The increased interventional frequency that would result from the undefined practice of continuous monitoring (entails excessive risk for no additional useful information gained.

 Monitoring activities must not increase the risk of contamination ingress due to their proximity to critical activities.

	This monitoring should take place immediately after completion of a critical intervention and upon each exit from the cleanroom.

 Monitoring of personnel after each “critical intervention” increases risk of contamination by allowing personnel to reenter the critical zone after monitoring. Furthermore, there is no description provided in the document as to what constitutes a “critical intervention,” which would be required to interpret this statement. Monitoring after interventions attempts to do the impossible, which is to link contamination to specific interventions, and it implies that detection of contamination means sterile products will be contaminated. Neither of these objectives is analytically possible. Monitoring is an intervention subject to microbial contamination independent of any process-related interventional activity. Environmental monitoring in aseptic processing is already done far more intensely than is analytically or statistically warranted. Additional monitoring is not justifiable, as the actual limit of detection of the method is in the range of 10–100 CFU. Industry is already operating below the limit of detection of the method, and additional environmental monitoring is unwarranted as it has no quantitative or qualitative meaning.

  Grade A personnel monitoring should be performed in Grade B adjacent to the Grade A environment. For personnel working exclusively in Grade B, monitoring should be performed upon exit from the aseptic core.

	Normally, such conditions are provided by a localized air flow protection, such as laminar air flow work stations or isolators.

The use of unidirectional air flow in isolators provides no operational advantage over turbulent air. Years of experience in sterility test isolators where extensive manual activity is required have not demonstrated any contamination problem that could be resolved by the use of unidirectional air flow. Laminar air flow is a misnomer, as truly laminar air flow does not exist in any cleanroom or separative technology environment. The authors are establishing expectations that cannot be objectively evaluated or established.

 Normally, such conditions are provided by a localized air flow protection, such as provided in unidirectional air flow work stations.

	Unidirectional air flow systems should provide a homogeneous air speed in a range of 0.36–0.54 m/s (guidance value), the point at which the air speed measurement is taken should be clearly justified in the protocol. During initial qualification and requalification air speeds may be measured either close to the terminal air filter face or at the working height.

There are no reliable and reproducible means to measure air velocity at work height. The most appropriate means is to measure the air velocity proximate to the filter face. Airflow near the work surface is typically impeded by the presence of equipment or machines oriented perpendicular to the general direction of air movement. These conditions render air velocity measurements close to the work surface non-reproducible and thus meaningless. See Mason, W., et al, “Working Height Velocity Measurement in Conventional Cleanrooms, 

. Isolators because of the completeness of their separation do not require the same air velocities as conventional cleanrooms or RABS designs. Air velocities in isolators can be substantially less and non-unidirectional.

 Unidirectional air flow systems should provide a homogeneous air flow over exposed sterile materials. Air velocity measurements should be taken to define the operating condition, however in isolator enclosures or other low volume work areas air velocities can be considerably lower than is customary for manned clean rooms. Unidirectional air is not required in isolators.

	5. Premises–Barrier Technologies, Line 433 (and elsewhere)

 The document uses the term barrier in a manner that fails to distinguish between highly capable technologies such as isolators, which are among the most capable of current aseptic processing technologies, and less capable designs such as open RABS and simple curtain/partial barriers designs. Open RABS systems allow similar intervention strategies to those found in cleanrooms and are substantially less effective in achieving full personnel separation as compared to isolators or closed RABS. To identify these varying capability systems together as is done throughout the document is to equate their performance, which is substantially different. The text could be substantially improved by separate treatment of isolation technology and RABS (open and closed) technologies and by making a distinction between those designs and ordinary manned cleaned rooms, the majority of which rely on some form of minimally effective barrier.

 Provide separate consideration of conventional cleanrooms, RABS, and isolators because they are demonstratively different in many ways and considering them together denigrates isolator performance, elevates RABS capabilities (especially for open RABS designs), and implies that the far less capable barrier designs used in conventional cleanrooms are their equal.

	5. Premises–Clean Room and Clean Air Device Classification–Note, Lines 493-495 

	Note: Classification is a method of assessing the level of air cleanliness against a specification for a cleanroom or clean area device by measuring the airborne particle concentration. The classification is part of the qualification of a clean area.

The content is correct; however, its placement immediately after content that discusses ‘environmental cleanliness level in the operational state’ suggests that classification can be accomplished in the operational state. Classification of environments is restricted to ‘at rest’ or static conditions as stated in ISO 14644. It is not scientifically possible to classify an environment microbiologically; the growth-based methods used for such purposes have a limit of detection between 10–100 CFU and are statistically and analytically incapable of classifying an environment.

 Relocate this content to follow lines 497-499.

	Note: Classification is a method of assessing the level of air cleanliness against a specification for a cleanroom by measuring the airborne particle concentration. The classification is part of the qualification of a clean area and is performed under ‘at rest’ or static conditions.

	Section 9–Viable and non-viable environment & process monitoring–Section 9.13–Note 2, Line 1659–1662

	Note 2: With regards to the monitoring of 5.0 μm, the limit of 20 is selected due to the limitations of monitoring equipment. It should be noted that alert limits should also be set based on historical and qualification data, such that frequent sustained recoveries below the action limit should also trigger an investigation.

 The inability to accurately count low numbers of non-viable particles with current technology precludes the imposition of limits for 5 μm particles in ISO 5. This objection has been raised innumerable times by numerous individuals and organizations since the first issue of Annex 1 in 1999. Maintaining the façade that this is a statistically meaningful measurement is inappropriate as it perpetuates a technical fallacy. There is no scientific justification for retaining a monitoring requirement for 5 μm particles in this guidance.

 Delete the columns relating to 5 μm particles in Table 5 and Note 2.

	Delete content relating to 5 μm particles from the document.

	Technical errors dot the pages of the document. The following are some of the most egregious:

	5. Premises–Section 5.8, Line 357–359

	Floor drains in lower grade rooms should be fitted with traps or water seals to prevent back flow and should be regularly cleaned and disinfected.

 “Traps”, which are intended to allow air and condensate exit from a system while retaining steam, cannot prevent back flow.

 Floor drains in lower-grade rooms should be designed to prevent back flow and should be regularly cleaned and disinfected.

	Materials liable to generate fibers should not be permitted in clean areas.

 This sentence would essentially eliminate the presence of gowned personnel, who, studies have shown, continuously shed particles and fibers. Also, there are almost no aseptic gowns that are not made of fibers.

	8. Production and specific technologies–Section 8.26, Lines 931–932

	Critical defects should not be identified during any subsequent sampling of acceptable containers as it indicates a failure of the original inspection process.

 Inspection of containers for defects is not an absolute process even if automated. The potential for a defective unit to be accepted is always present.

: Critical defects should not be identified during any subsequent sampling of acceptable containers as it indicates a possible failure of the original inspection process.

	8. Production and specific technologies–Section 8.30, Lines 957–960

	Where it is not possible for a product to undergo a sterilization, consideration should be given to using terminal bioburden reduction steps, such as heat treatments (pasteurization), combined with aseptic processing to give improved sterility assurance.

 This sentence adds no value to the document. The included and correct definition of terminal sterilization embraces a wide range of processes when it is understood that the target of the sterilization process is the bioburden present and not the biological indicator (see 

 <1229>). Sterilization processes are intended to destroy the bioburden present (as opposed to reducing its population) and thus, the conditions necessary can be less rigorous than those previously utilized, which are fixated on killing high populations of a resistant biological indicator. The false expectation that mandates biological indicator destruction reduces the use of terminal sterilization rather than increasing it. Reduction of biological indicator population actually supports extremely safe terminal processes for the substantially less resistant bioburden.

	8. Production and specific technologies–Section 8.51, Lines 1086–1087

	Chemical or biological indicators may also be used, but should not take the place of physical measurements.

 Physical measurements rely upon mathematical models from microbiological data, which estimate the lethal effect a process asserts on microorganisms. As such, physical measurements are substantially less reliable (though easier to acquire) than the biological results they attempt to mimic. They should never be given preference over the results of a microbiological challenge study. Additionally, there are locations within items requiring sterilization where physical measurements are taken so remotely that their ability to “demonstrate” lethality is either extremely limited or non-existent.

The results from biological indicators and physical measurements must be considered in validation activities.

	8. Production and specific technologies–Section 8.78, Lines 1251–1253

	If a liquid product cannot be terminally sterilized by a microbiocidal process, it should be sterilized by filtration through a sterile, sterilizing grade filter …

The draft expects that a liquid product is to be “terminally sterilized by a microbiocidal process” an undefined process instead of simply stating that “a terminal sterilization process” be used.

If a liquid product cannot be terminally sterilized, it should be sterilized by filtration through a sterile, sterilizing grade filter …

	8. Production and specific technologies–Section 8.78, Lines 1287–1289

	f) Permit in-place integrity testing, preferably as a closed system, prior to filtration as necessary. In-place integrity testing methods should be selected to avoid any adverse impact on the quality of the product.

 Pre-use post-sterilization integrity tests (PUPSIT) of sterilizing filters should only be performed where a closed system is present throughout. Requiring PUPSIT where the system is not closed creates a greater risk than any value the integrity test can provide. This same expectation is repeated at lines 1331–1340. At the very least, content on the same subject should be together in the document.

	9. Viable and non-viable environment & process monitoring

	Although monitoring of â§ 5.0 μm particles are not required for room qualification and classification purposes, it is required for routine monitoring purposes as they are an important diagnostic tool for early detection of machine, equipment, and HVAC failure.

 See the earlier comment on lines 1659-1662. Further, the suggestion that larger particles would serve as a superior “important diagnostic tool” than smaller particles, which are more accurately counted, is not supported by documented evidence. Persistence in including this “requirement” in the face of objective scientific evidence to the contrary is completely without justification. 

  Delete all content relating to 5 μm particles from the document.

	There is a misdirected focus toward factors and suggested activities that do not increase patient safety or relate to the safety, identity, strength, quality, or purity of a drug product beyond the official or other established requirement.  In some cases, the recommendations add to patient risk.

	4. Personnel–Section 4.4, Lines 205–206

The location of the monitoring is not specified at this point in the document. Table 6 includes expected levels for Grades A/B with different values for each. This implies that Grade A monitoring of personnel take place in Grade A. This is both impractical for the sites identified (see lines 199–200) and excessively risky. Monitoring of personnel in Grade A increases the potential for contamination ingress into that environment because a) personnel (the major source of contamination) are present in Grade A for longer periods, b) monitoring in Grade A increases activity in close proximity to sterile materials, and c) added activity in Grade A increases contamination risk. Monitoring cannot confirm “sterility” or “asepsis” in any event, but there is no benefit to having this sampling being performed in Grade A. Insistence on the Grade A personnel monitoring and consideration of risk should allow for that sampling to occur in Grade B adjacent to the Grade A environment with the Table 6 limits. For personnel working exclusively in Grade B, that monitoring can be performed upon exit from the aseptic core. Personnel monitoring should never be performed in close proximity to sterilized materials.

 Grade A personnel monitoring should be performed in Grade B adjacent to the Grade A environment. For personnel working exclusively in Grade B, monitoring should be performed upon exit from the aseptic core.

	8. Production and specific Technologies–Section 8.18, Lines 883–886

	Containers closed by fusion (e.g., form-fill-seal small volume parenteral (SVP) and large volume parenteral (LVP) bags, glass or plastic ampoules) should be subject to 100% integrity testing.

This is nothing more than testing quality into the product as opposed to validating the sealing process and running confirmatory process checks.  Annex 1 references quality by design, statistical process control, and process validation approaches and then undercuts the asserted value of these well-recognized quality management tools with a poorly considered focus on end-product testing.

	8. Production and specific Technologies–Section 8.18, Lines 1331–1334

	The integrity of the sterilized filter assembly should be verified by testing before use, in case of damage and loss of integrity caused by processing, and should be verified by on-line testing immediately after use by an appropriate method such as a bubble point, diffusive flow, water intrusion, or pressure hold test.

 The document incorrectly applies risk analysis (e.g., integrity testing of a sterilized filter assembly before use [PUPSIT] and on-line integrity testing immediately after use).  PUPSIT adds risk by potentially requiring manipulations downstream of sterilized filters.

A previously published article presented difficulties with the revised European guidelines on sterile manufacturing.  The authors included a brief summary of the comments developed on the draft document.  This article expands upon that summary, outlines the authors' rationale, and highlights the most difficult aspects of the revision draft. 

Annex 1 Misses the Mark–Expanded Version

, Annex 1, Manufacture of Sterile Medicinal Products (1), when finalized, will be legally binding. Non-compliance could result in regulatory action, refusal, or revocation of marketing authorizations and civil and criminal penalties. Given the revised Annex 1’s legal standing, it is vital that the document delineates appropriate regulatory expectations and accepts contemporary manufacturing and control procedures and practices.

	The European Medicines Agency (EMA) has put forward Annex 1 for comment, but the document is so badly written that understanding it, much less commenting on it, is far more difficult than it should be. As it stands, this version of Annex 1 will inevitably generate thousands of comments that will be difficult for EMA to reconcile within the framework of the current document. Preliminary review of the document indicates that it is poorly worded, ambiguous, and technically incorrect in many areas. The following comments represent a fraction of those identified during our review. They are categorized by general areas of concern.

	Inconsistent use of and undefined terminology

	There are many instances where inconsistent use of terms or wording results in ambiguity and the inability to determine or demonstrate compliance, such as listed in the following:

		The terms “medicines” and “medicinal products” are used interchangeably and neither is defined.

		Line 188 instructs that “All personnel . . . in such areas should receive regular training,” but it is unclear to which areas this refers.

		Line 207 mentions a “continuous monitoring program for personnel” without explaining what this is, or how continuous personnel monitoring is feasible, useful, or even safe.

		Line 230 instructs that “Staff who have been engaged in the processing of human or animal tissue materials” should not enter “sterile product areas,” but none of these terms are defined.

		“Grade A/B” used throughout the document is undefined.

		At line 861, “time between the start of the preparation of a solution and its sterilization,” the word “solution” does not reflect the full range of medicinal products that should be subject to pre-sterilization time limits.

	The use and application of airlocks, barrier systems, isolation technology, and environmental and personnel monitoring systems are incorrectly described or applied, such as:

		Airlocks and pass-throughs vary in sophistication and design, but the document limits flexibility and informs at line 365 that “the final stage of the airlock should . . . be the same grade as the area into which it leads,” which is inappropriate for exit airlocks.

		Barrier technologies, other than true closed restricted-access barrier systems (RABS), are substantially less capable than isolators, yet the document considers them near equal in performance.

		Monitoring of large particles (≥ 5 µm) lacks accuracy and is inherently less sensitive than monitoring of smaller particles. Also, the prevalence of such particles in grade A/B areas is too low to make sampling for them statistically useful. And there is no evidence particles of this size range predict microbial risk. EMA should harmonize with International Organization for Standardization (ISO) 14644.

		Personnel monitoring should never be performed in grade A as instructed at line 205.  Such monitoring increases the biological load in critical environments with no product safety benefit.

	The following technical errors dot the pages of the document:

		At line 1251, a liquid product is to be “terminally sterilized by a microbiocidal process” instead of “terminally sterilized.”

		The use of traps for back flow prevention is specified at line 358, even though “traps” do not prevent back flow.

		At line 351, “Materials liable to generate fibres should not be permitted in clean areas,” which would eliminate gowned personnel, who, studies have shown, continuously shed particles and fibers.

		At line 839, the document instructs “The transfer of partially closed containers to a lyophilizer, should be done under grade A conditions (e.g., HEPA filtered positive pressure) at all times . . .” which is contradictory because grade A requires unidirectional airflow.

	There is a misdirected focus toward factors that do not increase patient safety or relate to the safety, identity, strength, quality, or purity of a drug product beyond the official or other established requirement:

		The document incorrectly applies risk analysis (e.g., line 1331, integrity testing of a sterilized filter assembly before use [pre-use post-sterilization integrity testing, PUPSIT] and on-line integrity testing immediately after use).  PUPSIT adds risk by potentially requiring manipulations downstream of sterilized filters.

		Routine and intervention-related monitoring of personnel in grade A adds activity and increases contamination risk, line 205.

		At line 883, “Containers closed by fusion, e.g., Form-Fill-Seal Small Volume Parenteral (SVP) & Large Volume Parenteral (LVP) bags, glass or plastic ampoules, should be subject to 100% integrity testing.”  This is akin to testing quality into the product as opposed to validating the sealing process and running confirmatory process checks.  Annex 1 references quality by design, statistical process control, and process validation approaches and then undercuts the asserted value of these well-recognized quality management tools with a poorly considered focus on end-product testing. 

	Unusual concept of sterile product manufacture

	The document presents a view of sterile product manufacture inconsistent with that developed elsewhere, as codified in regulations, international standards, and pharmaceutical compendia, such as:

		US and Japanese guidance on sterile product manufacturing differ markedly from what is presented.

		The cleanroom content in the draft does not conform to ISO 14644 and perpetuates the myths that cleanrooms can be classified microbiologically and that microbiological testing can enhance sterility assurance.

) chapters <1211>, <1228>, and <1229> provide more contemporary and appropriate guidance for sterile product preparation (2–4).

		There are also conflicts with current EMA guidance (e.g., the water for injections Q&A paper and 

Guidelines on Good Manufacturing Practice specific to Advanced Therapy Medicinal Products).

	EMA should withdraw this draft of Annex 1. It should be significantly revised to include more contemporary and global content on sterile product manufacture and control and reissued for comment once its many faults have been corrected.

	The cited examples represent only a fraction of the more than 130 issues we identified in the document and we plan to submit a comprehensive response to EMA by the comment deadline of March 20, 2018 in the event that the draft is not withdrawn.

, Annex 1, Manufacture of Sterile Medicinal Products, December 2017.

<1211> Sterilization and Sterility Assurance of Compendial Articles

<1229> Sterilization of Compendial Articles

	Russell Madsen is president of the The Williamsburg Group, LLC,  James Agalloco is president of Agalloco and Associates, and Jim Akers is president of Akers Kennedy & Associates.

The revised Annex 1 on sterile manufacturing includes incorrect and ambiguous statements that must be fixed before implementation. 

Annex 1 Misses the Mark

Aseptic processing is understood to be among the more complex processes in the pharmaceutical industry and one whose proper execution is essential to patient safety. One of the keys to success in aseptic processing is defining the operational details properly so that contamination risk can be minimized. Reduction of patient risk is understood to be the primary consideration, and it is intrinsically linked to managing contamination risk in products manufactured aseptically.

	The past 100 years have witnessed an evolution of technologies for aseptic processing that have led to dramatically improved process capability. The evolution of practice was accomplished pragmatically, but without any formal consideration of risk.  The principles of aseptic processing developed and largely codified by FDA, the European Medicines Agency (EMA), the US Pharmacopeial Convention (USP), the Parenteral Drug Association (PDA), the International Society for Pharmaceutical Engineering (ISPE), and others evolved in a similar fashion, again without formalized risk assessment. Thus, aseptic processing practices as they exist today are the result of a series of predominantly subjective decisions about which design, practices, and measurements are most appropriate.

	This absence of formal risk assessment began to change in 2004, when FDA issued an important document outlining its desire to encourage a more formal risk analysis-centered approach to CGMP compliance (1). Subsequent to that publication, the first attempts at development of risk assessment methodologies for aseptic processing were developed (2, 3). It was in this time frame that the authors first developed their risk assessment models (4, 5). These publications focused on the contamination risk associated with personnel activity, which arose from the universal understanding that it was the primary source of contamination, and hence risk, in aseptic processing activities (6). The first publication of the Akers-Agalloco (A&A) method (4) included a complex method for assessing aseptic risk and some activities that included too much redundancy in practice. The second publication (5) corrected these errors  and has been successfully used by several firms (7). To best use this risk method, the rationale provided in the original publication has to be combined with simpler calculations provided in the second publication. Based upon empirical data accrued over many years of practical experience in aseptic processing, the authors knew that, in application, it was possible to differentiate processes that had a comparatively high risk of product contamination from those that had the capacity to ensure lower contamination risk.  

	In the years since the original publications, interactions with clients led to reconsideration of some of the core assumptions made in the earlier publications. There were many queries regarding the arbitrariness of the risk factors used in the interventional portion of the original work, and as explained in the following discussion, the authors replaced the subjective risk factors with a time-based scoring and changed the scoring methodology to use an inverse-square risk factor for proximity, which largely eliminates the authors’ personal bias in that part of the risk assessment.

	In using their risk assessment methodology, the authors discovered that it was actually two independent risk-assessment methods combined into one larger and more complex tool. The first part of the original method was intervention based, while the second focused on process design. Each portion of the method can be used separately from the other, or they can be used together in series as originally published. The authors have had many more opportunities to use the individual components for assessing aseptic risk as compared to their combination. This article will explain how the submethods can be used separately. 

	Because the accepted source of contamination in aseptic processing is personnel, it makes sense to develop a risk model that places human intervention at the very core. The authors had hypothesized that improvement of aseptic processing was primarily a matter of reducing reliance on gowned personnel in aseptic processing, in agreement with the suggestion of FDA’s Hank Avallone that “It is useful to assume that the operator is always contaminated while operating in the aseptic area. If the procedures are viewed from this perspective, those practices which are exposing the product to contamination are more easily identified” (6).  

	The authors had learned from research using aseptic processing in cell-culture work that gowning and aerodynamic separation of humans from aseptic activities resulted in much lower contamination rates. Further research showed that humans were not merely “contaminated” as Avallone had thought, but were actually contamination generators. Published studies describe the rates at which humans who are gowned for aseptic work generate microbial and non-viable particulate contamination (8, 9). The rate of release of these contaminants into the environment depends on the types of gowning materials used as well as level of activity their work involved. The authors observed that ventilation played an essential role in removing the contamination emitted by the contamination generators (e.g., personnel) in aseptic cleanrooms. As advanced separative technologies came into wider use, the elimination of personnel had a profound impact on contamination rates, confirming that human-released contamination constitutes the greatest risk. 

	The intervention aspect of the A&A method incorporates the concept of the human operator as a mobile contamination source directly with considerations of criticality, frequency, and proximity, which relate directly to human-derived microbial contamination in aseptic processing. Human activity is variable in aseptic processing whereas the design of the cleanroom and the operation of its ventilation system is effectively a constant. The following sections discuss these three variables. 

 The type of interventional manipulation being performed is linked to the contamination potential. Inherent interventions that are required parts of the aseptic process and cannot be easily eliminated must be designed to have the lowest risk contamination (10). Corrective interventions involving the removal of items from the operation have a greater level of risk because they vary substantially as to location and complexity. What the A&A method terms as critical interventions are corrective interventions of greater complexity that, in many cases, require the replacement of an item on the line. As such, they resemble the initial set-up of the line and represent the highest level of risk. A hierarchy of intervention risk associated with aseptic processing including some additional interventional categories is shown in 

	Separate consideration of interventions as either inherent or corrective is necessary because the design approaches, execution details, risk mitigation means, and operational perspectives remain different. Inherent interventions require determination of a preferred methodology and training of personnel in their execution. Corrective interventions will always rely on operator expertise and aseptic technique because of their more variable nature. The goal of eliminating all corrective interventions, especially the critical correctives, remains, regardless of the ease with which some of the less invasive corrective actions might be undertaken.

	The designation of interventions as either inherent or corrective, while useful in understanding their design and execution, is less useful in risk assessment. Consider that the replacement of environmental monitoring plates in an air sampler (an inherent intervention) is both more difficult and time consuming than removal of a single fallen vial using a pair of forceps (a corrective intervention). It seems logical to consider duration of the intervention (seconds within the critical zone) as a superior alternative to intervention type in the estimation of risk. A lengthy inherent intervention would thus be considered more risky than a simple corrective one. Time serves as a more definitive and less subjective means to assess the potential impact of an intervention compared to type of intervention. 

. The objective in all aseptic processes is to continuously reduce the frequency of interventions regardless of their type: inherent, corrective, or critical corrective. For example, the frequency of weight checks should be based upon the performance of the filling equipment and not an arbitrary time interval. If an intervention cannot be completely eliminated, reducing its frequency reduces the contamination risk. The “perfect” intervention is one that has been eliminated from the aseptic process (11). 

 The distance between where the intervention is performed and the nearest exposed sterile item affects risk. Logically, the shorter the distance between sterile items and the personnel performing the intervention, the greater the potential is for microbial transfer across that distance. An arbitrary distance scale was used in the initial A&A method for the sake of simplicity. William Whyte, an early proponent of formal aseptic risk assessment, suggested the use of the inverse-square rule for estimating the dispersion of microorganisms from a source (2, 3). This rule relies upon two assumptions: the existence of a point source of microorganisms that disseminates them much like a light bulb casts light and that microorganisms disseminated from that source would uniformly populate the surrounding environment. Although the inverse square rule is appropriate for gravity, energy, sound, and other physical phenomena, it should not be considered a wholly accurate model for microbial dispersion in an environment. Among the reasons for its limitations in this instance are:

		There is no “point” source for microorganisms in aseptic operations; they can be shed from any part of the operator’s gown or gloves.

		Aseptic processing is carried out in ISO 5 environments supplied with unidirectional air, which is specifically provided to suppress the distribution of microorganisms by air movement or dilution. However, other factors, including electrostatic charges, can alter the direction in which airborne contaminants move.

		Microorganisms have mass, and settling will occur even in the absence of air movement.

	The use of the inverse square rule, even though it may not be precisely quantifiable, places greater weight than the original A&A method on interventional activities performed closer to exposed sterile materials. For this reason alone, the authors adapted the A&A method to incorporate this, albeit flawed, more objective means for considering the effect of distance in risk estimation. 

 explains how data gathered from observation of a filling line can be used in the evaluation of interventional impact on an aseptic filling process. For a meaningful assessment, at least one hour of the process should be monitored. Ideally, the assessment should extend to the full process duration. The time for execution of the various interventions is most accurately determined by recording it during each intervention; however, for interventions that are more frequent and similar in execution an average value based upon multiple observations could be used.

	The raw score considers only the intervention contribution and ignores other elements. Normalization of the score by dividing by the number of units produced is employed to refine the assessment. The fewer units produced over the time period, the greater the contamination risk to any individual unit; a lower throughput is more likely to be associated with sterile items being exposed to contamination for a longer period of time with the process environment. When this calculation is made, the determined value is the intervention risk (IR). The goal in risk mitigation is to reduce the IR for the aseptic process.

	Determination of the IR for an aseptic process can be used as a risk assessment tool without additional effort. The IR can be used in risk assessment and, ultimately, for risk mitigation in the following ways:

		Assessing of the performance of a filling line with different set-up personnel

		Assessing line performance with different operators or shifts 

		Assessing line performance with different combinations of components (i.e., stopper and vial)

		Assessing line performance over equipment life/length of the fill

		Assessing line performance with different formulations/fill volumes/operating speeds

		Identifying the “worst-case” selection of conditions and components for use in process simulations

		Supporting the process simulation as “worst-case” relative to routine production

		Evaluation of line performance in conjunction with changes in equipment, personnel, procedures, etc.

		Evaluation of performance changes with different lots of components.

	Clearly, an intervention-focused risk assessment has a variety of uses. Importantly, the absence of subjectivity in the accumulation of the data used in the assessment eliminates the most common deficiency in other risk methods. The IR reported should be the same regardless of the observer. This feature is perhaps the method’s greatest value in that it eliminates bias in the assessment of risk.

	The other component of the initial A&A method was the design-focus element that provided for the assessment of aseptic manufacturing, aseptic filling (i.e., individually addressing aseptic set-up and filling), and lyophilization. The subcomponents differ, reflecting the unique elements of aseptic risk associated each of them. The risk factors in each of these were established with consideration of how the design choices influenced the extent of operator activity with sterile materials. As a consequence, this portion of the risk-assessment model can be used in isolation from the interventional element. 

	Although many clients requested that manual aseptic filling be included in the A&A method, it is already included under the term aseptic compounding; this portion of the document is readily adapted to individual use. The aseptic compounding, filling, and lyophilization components of the method work independently of each other, and it is a simple matter to use only that portion of the design component risk method that is of interest. The authors agree conceptually with adding other aseptic processes to the design component of the risk method; lacking sufficient experience with these less common situations, however, the authors are loath to do so. The authors would encourage others to develop design-based risk assessments for aseptic process elements that the authors did not consider.  

	Consideration of the risk associated with aseptic process design is not new. The evolution of aseptic processing from unclassified environments with minimally gowned personnel to present day isolation technology was the result of informal consideration of the contamination potential. The introduction of separative means, high-efficiency particulate air (HEPA) filters, full aseptic gowning, and automation to aseptic operations were a result of largely subjective evaluation of risk. In the design-focused portion of the A&A method, the authors ranked the various process and facility design elements according to the extent of human intervention required. This portion did not independently consider severity, frequency, or detectability, but rather developed an arbitrary risk ranking based upon what the authors believed was the least to most risky process or design feature.

	In the initial publications, the authors included some examples based upon facilities both had visited; the authors’ clients, however, have afforded them substantially better opportunities to apply this risk tool. In one group exercise, teams of participants were asked to evaluate the risk associated with the filling of the same lyophilized product configuration in multiple facilities using the prevailing aseptic practices at various sites. The facilities chosen for the analysis were the smallest, oldest, newest, and largest that the firm operated. The calculations involved followed the design component of the simplified risk model from 2006 (5). The results of this exercise are presented in 

	The results of this exercise revealed the utility of this form of risk assessment, with the following results:

		The filling risk contribution was essentially the same at all sites except the oldest site. After presenting their risk result, the team from the oldest site was able to review the aspects of their operations that raised their score by comparing their process/facility design to the other facilities.

		The lyophilization risk varied widely across the sites, and the practice/design of the newest site became a model for the remaining operations.

		Most importantly, the participants became aware that objective consideration of aseptic risk could reveal improvements to what were already considered fully compliant aseptic operations.

	Over the past 10–15 years, there have been numerous risk assessment methods published. The majority of these entail some adaptation of failure mode and effects analysis (FMEA) or other subjective tools for risk assessment. The authors often encounter aseptic operations where aseptic process improvement opportunities are either not obvious or inherently limited. The firms operating these facilities fixate on the performance metrics they use, predominantly associated with environmental monitoring, without adequate consideration of the human impact. Environmental monitoring, process simulation, and sterility testing are largely inadequate for assessing the performance of contemporary aseptic operations (12). They rely on growth-based microbiological analysis which is limited statistically as well as analytically. The accepted limit of detection (LOD) of these methods is considered to be 10–100 colony forming units. These methods are being improperly credited with providing evidence of sterility assurance when in reality they are simply unable to provide useful information below their LOD. 

	These methods also suffer from restricted sensitivity as they are unable to recover a number of potentially pathogenic organisms. As a result, the absence of contamination measured as no growth may be an illusion. It must be understood that in microbiology, zero recovery only means that nothing grew and never under any circumstances should it be considered proof of sterility. The authors believe that the A&A risk tools they have developed, and now refined, provide a more insightful means for evaluating and improving aseptic processing. 

	There are two aspects of aseptic risk that are still troubling. First, too much is written and said about risk assessment and too little about risk mitigation. The purpose of understanding aseptic risk in the first place is to take action to reduce it. The knowledge that it has started to rain outside is useless, unless one makes the decision to open an umbrella. It’s not enough to measure risk: active steps need to be taken to minimize it. The more objective the means for assessing the risk, the more likely it will be to be able to determine what measures are best to mitigate that risk. Regardless of the risk level, actions must be taken to reduce it further despite any belief that the aseptic process it is already sufficiently safe. Continuous improvement should be the order of the day.     

	The second concern is the continued use of microbiological analytical data to determine risk, establish “sterility assurance,” and “validate” processes. This industry has never come to grips with the reality that microbiological methods lack sensitivity. The methods have far higher LODs than prevailing standards and guidance documents imply, and they have substantial variability as well. Microbiology is a logarithmic science, and attempting to measure sterility using environmental monitoring is not just difficult; it is impossible. There are far better means of assessing process control and proactive steps that can be implemented to prevent contamination.  

	It is easy to fall in love with an approach to cleanroom assessment that gives positive feedback, and in the authors’ experience, more than 99% of all environmental monitoring samples taken in ISO 5 environments don’t find contamination. This result is superficially reassuring until one realizes that given the LOD of the method, the one thing it doesn’t mean is that the environment is sterile. To those who aren’t microbiologists, using environmental monitoring or media fill data in an effort to assign a risk probability number or assert assurance of sterility seems logical, but it’s actually not. Despite the perceived lower LOD of some alternate methods, limitations in sampling mean that all environmental methods are inadequate. Sterility, or more correctly asepsis, cannot be tested in and thus can never be assumed to be present.   

Pharmaceutical CGMPS for the 21st Century–A Risk-based Approach

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	Vol. 41, No. 11
	November 2017
	Pages: 32–39

	When referring to this article, please cite it as J. Agalloco and J. Akers, "A Revised Aseptic Risk Assessment and Mitigation Methodology," 

 is president of Agalloco & Associates, P.O. Box 899, Belle Mead, NJ 08502, tel. 908.874.7558, jagalloco@aol.com. He is also a member of 

 is president of Akers Kennedy & Associates.

The revised Akers-Agalloco aseptic risk assessment and mitigation method includes two sub-methods that can be used independently for risk assessment.

A Revised Aseptic Risk Assessment and Mitigation Methodology

	Peer-Reviewed:
	Submitted: Feb. 9, 2015. Accepted: May 29, 2015.

Validating sterile product manufacturing processes requires the use of biological indicators to demonstrate the lethality of sterilization techniques. The indicator organisms are there merely to measure the effect of the sterilization process. Today, too much focus on lethality to biological markers results in use of extreme sterilization conditions; insisting on excessive lethality requirements in sterile process validation threatens patient safety, the authors believe. Not only does the practice subject process equipment and raw materials to damaging conditions, it can lead drug manufacturers to opt for aseptic processing in cases where terminal sterilization would be the best and safest choice. Companies should be free to choose the best way to ensure a safe process, whether that is aseptic processing, terminal sterilization, or a combination of the two.

Microorganism lethality requirements for process validation must always be balanced with the need to protect product integrity and patient safety.