Expectations for Microbial Environmental Monitoring Investigations for Sterile Manufacturing Critical Areas

Angel L. Salaman -Byron

Janssen Biotech Inc.

Abstract: Microbial environmental monitoring (EM) is a semi-quantitative assessment that is extensively limited by method, sample size, technical variables, and biological variables. EM action level or out of trend excursions should be investigated and the analysis should be thoroughly documented. This article reviews the expectations for environmental monitoring excursion investigations for critical sterile manufacturing areas. 

Keywords: Environmental Monitoring, Microbial contamination, investigation, cleanroom, environmental control, Grade A, microbial limit.

Introduction

ISO 14644-1 (2015) establishes the classification of air cleanliness specifically in terms of concentration of airborne particles in cleanrooms and clean zones.¹  The viable (microbial) and non-viable air particles limits (i.e., levels) shall be assessed to ensure the engineering controls, administration procedures, and aseptic behavior maintain the required cleanliness of the cleanroom.^2-4 Environmental Monitoring program (EMP) is a system to plan, organize and implement all the activities to achieve and maintain the required levels of air and surface cleanliness in the manufacturing areas.²  The intent is to manufacture aseptic pharmaceutical products at a high quality, by foreseeing deterioration of environments in manufacturing areas, preventing bad influence on the quality of products, and performing appropriate cleanliness control through a proper monitoring of the manufacturing environment. An EMP should provide accurate and reliable information of the manufacturing environment to demonstrate against action and alert limits that the manufacturing environment process is safe.² Microbial EM action limit excursions and alert limit adverse trends should be documented, verified, and investigated.^5-10

The real purpose of a microbiological EMP is to confirm environmental conditions, but the industry has been addressed out of EMP action limit excursions investigation at the same level and expectations as Out-Of-Specifications (OOS) investigations. OOS investigations are triggered by sample test results performed on a bulk product, final product, manufacturing raw materials and/or manufacturing equipment.^11,12 While microbial EM results are obtained from manufacturing cleanroom air or non-product contact surfaces such as walls, floor, personnel and, workbench.

This article reviews the reasonable expectations from environmental monitoring excursions investigations in critical aseptic process areas.

Out Of Specification Concept

An item specification is defined as a list of tests, references to analytical procedures, and appropriate acceptance criteria for raw materials, packaging components, labeling, testing, and performance specifications which are numerical limits, ranges, or other test criteria. The tests are described for the item filed with the relevant Regulatory Authority. According to International Council on Harmonization (ICH), Q6A (2000) Specifications “Test Procedures and Acceptance Criteria for New Drug Substances and New Drug Products: Chemical Substances” “...it establishes a set of criteria to which a drug substance or drug product should conform to be considered acceptable for its intended use”.¹³ FDA defines (OOS) as “all test results that fall outside the specifications or acceptance criteria established in drug applications, drug master files (DMFs), official compendia, or by the manufacturer”.¹¹ Based on this definition, “OOS” is not an applicable concept for environmental monitoring results unless the test includes assaying components of the product (i.e., medical devices, sterile surgical instruments, etc.). Specifications are part of a total control strategy for the drug, pharmaceutical article, or medical device to ensure quality, consistency, and adherence to Good Manufacturing Practices, “e.g., suitable facilities, a validated manufacturing process, validated test procedure, raw material testing, in-process testing, stability testing,  etc. If any of the specification requirement is not met, an Out-of-specification (OOS) occurs.^11,12

The term OOS was established in the Barr decision (1993) and started to identify the procedures and criteria to be used for the production and release of drug products. Differences in definition by different country regulations and changing regulatory opinions since the ruling have led to confusion and indecision when handling microbiological contamination investigations. The intent of many companies to satisfy regulatory expectations lead to an improper use of the term and overwhelmed processes which is what has happened with environmental monitoring of cleanrooms.¹⁴

Out of Trend (OOT) is generally a concept for when a result (i.e., product test data, for example product stability result) does not follow the expected results, either in comparison with other product batches or with respect to previous results collected.¹¹ Test results of starting materials and in-process samples may also yield out of trend data. OOT reveals that there may be a problem with the analysis or the production process. The result is not necessarily an OOS but does not look like a typical data point.¹²

A single result or several results that do not follow the expected trend, for a particular batch or series of batches, either in comparison with historical data is considered an Out of Trend (OOT) result. A product‘s adverse trend must be monitored closely. Additional time points may be required before the next scheduled time point to further confirm the trend. When a trend is regarded as adverse it is important to determine if the reason for the possible OOT is regarded to a planned change, for example, change in raw material supplier, manufacturing process, new equipment and/or equipment component.¹¹ Once identified as OOT, it is determined whether the adverse trend is isolated to one batch or is affecting many batches. In either case, an investigation must be initiated. A detailed investigation must document the review and assessment of the test data, the statistical models chosen, and follow-up actions as required. For example, the registered specifications may require review to ensure that the established limits are set at an appropriate level or if changes are required because of the investigation. Actions may also include but not limited to requesting an investigation to determine process, formulation or testing changes, new analyst, equipment or instrument changes, or deviations associated with the batch.^11,12

Therefore, the purpose of the OOS and OOT investigations is to determine the cause of the non-compliance result or adverse trend. The root cause of the out-of-compliance result is used to determine the corrective action. The expectancy is avoiding the re-occurrence of the event.¹¹

Microbial Environmental Monitoring Investigations

21 Code of Federal Regulations (CFR) Part 211.42 establish the requirement for “A system for monitoring environmental conditions”.¹⁵ FDA Aseptic Processing Guidance states:

“This program provides meaningful information on the quality of the aseptic processing environment (e.g., when a given batch is being manufactured) as well as environmental trends of ancillary clean areas”.

There is a misconception in the industry that a robust microbial EM program can detect nearly all aerobic bacteria and fungi present within environmentally controlled areas. Any manufacturing process where personnel are required, the recovery of microorganisms at some level is inevitable.^5-10,16 Other common sources of microbial contamination include raw materials, the air, and inanimate items such as surfaces and water. Microorganisms may be transferred directly (e.g., touching a surface with a contaminated object) or indirectly (e.g., distributed through the air). There is no single microbial medium or practical combination of media and incubation conditions that can reliably cultivate all possible microbes that may occur.^8,9 In fact, the amount of air and surfaces routinely sampled within process cleanrooms is extremely small compared with the total volume of air supplied and the surface area present.^5,7,8 In addition, the lack of precision of enumeration methods and the endogenous variability of biological results is characteristic of bioburden recovery testing. Harmonized pharmacopeia “Microbiological Examination of Non-Sterile Products (Total Viable Aerobic Count)” recognizes the inherent limitations of the enumeration methods and allows a test value exceeding the monograph limit (or acceptance criterion) by not more than a factor of five.^18-20 Similarly, USP <51>, “Antimicrobial Effectiveness Tests,” notes that variations in test values may exist when multiple samples are collected over time and allows count variability in logarithmic intervals (0.5 log10) for selected results.²¹ In the case of microbiological assays, the USP prefers the use of averages because of the innate variability of the biological test system.

Microorganisms are not distributed evenly in the air and surfaces which leading to more variability. In fact, any microbiological EM sampling plan cannot prove the absence of microbial contamination, even when no microbial contamination is recovered. The absence of growth on a microbiological sample means only that growth was not detected; it does not mean that the environment is free of contamination.^16,17 Microorganisms’ recovery methods rely on the appearance of visible colonies containing one or more clonal bacterial cell strains. The establishment of A Microbial Environmental Monitoring Program (EMP) alert and action limits relies on the ability of microorganisms to make colony forming units.¹⁷ Therefore, the microbial EMP is considered a semi quantitative exercise as a complete quantification of microorganisms is not possible.^5-10

Every EM excursion shall be investigated initially to discount an assignable cause of Laboratory Error. Therefore, the first phase of such an investigation includes an assessment of the accuracy of the laboratory’s data. The manufacturing side must have an EM event plan to make the investigative process consistent. The plan shall provide a clear differentiation between Alert Limits and Action Limits, guidance for the identification of organisms, list of the items to be evaluated, EM historical data and excursions analysis to identify adverse trends and/or similar occurrences. Finally, the EM event plan will provide guidance on notification to production, resampling and following up testing requirements, and timely closure of the investigation. The Microbial EM investigation outcome will be evaluated to assess the quality of the product batch or batches and the evaluation documented. Considering that “Monitoring or testing alone does not give assurance of sterility [Annex 1 (2022) section 2.2.], Annex 1 (2022) say “Where aseptic operations are performed monitoring should be frequent using methods such as settle plates, volumetric air and surface sampling (e.g., swabs and contact plates). Sampling methods used in operation should not interfere with zone protection. Results from monitoring should be considered when reviewing batch documentation for finished product release. Surfaces and personnel should be monitored after critical operations. Additional microbiological monitoring is also required outside production operations, e.g., after validation of systems, cleaning and sanitization.”³

Discussion

As stated before, the scope of OOS has gone beyond the ICH definition and has been applied to more than just product specifications. Regulatory bodies have clear expectations for the total particulate and microbiological control levels in aseptic processing and aseptic preparation environments. However, different aseptic processes have different levels of risk relating to biocontamination. For example, there are more contamination risks when the product or ingredients are opened and exposed in a Grade A/ISO 5 than those that were added through closed transfers. In ‘closed systems’ the risks are limited to the sterile interfaces and aseptic connections/disconnections. Eudralex Annex 1 (2022) guidelines for clean-areas classification, in a Class-A environment recommend limits for microbial contamination of 0 CFU/m3 in air sample, 0 CFU/4 hours in settle plates (diameter 90 mm) and, 0 CFU/plate in contact plates (diameter 55 mm).³ Sharp and coworkers (2010) reported that ISO 5/Grade A zones (clean and correctly designed with unidirectional airflow) at rest conditions, shown zero counts of ≥ 0.5 μm and ≥ 5.0 μm particles, i.e., particles were absent at all; and in operation, there were zero counts, even when an operator in a cleanroom was shaking a hand slightly at a distance of approximately 10 cm aside the sampling probe at the same level (height).²² The evidence and documentation around an isolate excursion in Grade A would not provide enough information to determine a root cause as Grade A data is mostly 0 CFU. Therefore, many samples (and a large volume of air or surface testing) would be required to demonstrate an adverse trend or a recurrent breach in environmental controls. In fact, a more meaningful interpretation can be made by evaluating the portion of samples that yield any growth.¹⁷ The outcome of an investigation would be expected to identify at least the most probable cause if the actual cause cannot be fully identified. Only when data are collected that relate time or location to multiple microbiological observations a meaningful conclusion can be drawn. Similarly, when a single EM excursion occurs, it should be noted and adequately catalogued to enable a comparison when other anomalies occur. This type of analysis is suited for atypical isolates (qualitative data), so rational data storage and retrieval systems are needed to enable this system to work. Statistic Environmental Monitoring data systems coupled with artificial intelligence software may offer an excellent tool to determine adverse trends.

There is also a misconception about entitled Grade A classified areas in low bioburden manufacturing (e.g., Biologics). The Grade A EM limits requirements would be difficult to meet where human intervention is needed. Therefore, a high number of investigations may occur in the absence of a thorough assessment of potential microbial contamination risks. The Grade A area is a zone for sterile product manufacturing. It is widely dedicated to high-risk sterile operations such as fill/finish (i.e., sterile filling of vials and syringes), stopper bowls, open ampoules, vials, and making aseptic connections. The expectation of zero contamination at all Grade A locations during every aseptic processing operation is likely unrealistic in the presence of human intervention. In practice low bioburden manufacturing should not have Grade A areas. Grade A requirements could be applied for processes in unidirectional airflow units within ISO 7 areas for cell-culture process, for example²³ to mitigate microbial contamination to the product (e.g., cell culture).

Grade A microbial in-process excursion is considered the worst case for microbial contamination, as the presence of contamination was detected. It is a breach in the environmental control that occurred within the critical zone where the product and components were exposed to the environment. For this reason, every Grade A microbial excursion must be promoted and investigated. For example, a group of five test results with 1 CFU each one may have more significance (i.e., possibly indicating an adverse trend) in a Grade A area than a single 5 CFU result out of five samples (i.e., one result is 5 CFUs while other 4 samples are 0 CFUs).

Microbiological EM limits must be reasonable in terms of the capability of the recovery method. This leads directly to the question of the linear range of plate counts. USP relies heavily on the established scientific literature in its discussion of this range of countable colonies on a plate to note that colonies have a lower limit of quantification of approximately 25 colonies per plate.^24,25 This is opposed to the level of less than one CFU per plate.³  EM alert and action levels between 1-10 CFU range is of questionable accuracy.^26,27 In that instance, it is highly suggested the verification of plates by a second technician and document the outcome.

Because of the relative rarity of microbial EM action limit events in Grade A, the investigation of EM action limit excursions often proves to be identified as a likely preventable event. For example, the loss of glove integrity or the accidental introduction of material into a closed Restricted Access Barrier Systems (RABS) or Isolator that has not been previously decontaminated are among the most common root causes of microbial contamination excursions.

Finally, viable limit excursions in ISO 7 or ISO 8 aseptic manufacturing supporting areas at rest conditions (i.e., Grade C and Grade D) are less likely to be amenable. Investigations should be addressed to identify adverse impact to critical processes with higher quality air areas. Support areas are less likely to be a thread to the manufacturing environment but may contribute to increase the ingress of undesirable contamination in critical areas. Therefore, the impact assessment of the event must be addressed about the possible adverse impact on critical areas and/or aseptic manufacturer processes. It is not suggested to consider CFU-count-based alert and action levels as one-size-fits-all to assess environmental control conditions. The risk to the manufacturing process or product must be considered case-by-case. For Grade C and Grade D an evaluation of contamination recovery rates in addition to EM limits will help to understand the excursion event around the EM event.

Engineering Controls

Airborne microbial contamination in isolators has always been an exceptionally rare event, and this is true even of the flexible wall turbulent airflow isolators used in sterility testing. As isolators eliminate direct contact between human operators and products,  any aseptic manipulations within the isolator are made with half-suits or glove ports which allow the manipulation within the isolator. The greatest risk of contamination in isolators has been thought to arise from glove tears, separations, or pinhole leaks.^26,27 Sterilization-in-place processes decontaminate other isolator surfaces with steam and chemical treatments to prevent microbial growth. Isolator systems are either “open” or “closed.” Microbiological sampling of surfaces that have been decontaminated with Vapor-Phase Hydrogen Peroxide (VPHP) is unlikely to be positive. RABS  are a type of sterile processing environment for non-sterile and sterile manufacturing. RABS are built inside ISO 5-7 clean rooms. They provide ISO 5 unidirectional air inside the barrier and prevent contamination with an air overspill system from within the barrier. Open RABS have specialized barrier openings to enable human intervention. Closed RABS do not allow human intervention and operate with the same operator restrictions as isolators. Closed RABS operate with positive or negative pressure, like isolator systems. Sterile items are manipulated in RABS using glove ports. Materials are transferred aseptically without opening the system. A RABS, like other regulated cleanrooms, requires decontamination before use. RABS designs are less capable than isolators relative to their ability to exclude microorganisms. Some activities require the operator to access the interior of RABS, increasing the likelihood of the contamination associated with the aseptic intervention. On the other hand the use of RABS requires process items to be sterilized remotely, transferred to the RABS, aseptically installed, and set for operation. This represents a further risk of microbial contamination that cannot be avoided.²⁸

Real-time viable particle detectors have been added to Isolators and RABS.²⁹ Real-time viable detectors use optical techniques to determine particle viability on a particle-by-particle basis. Real-time viable particle detectors must differentiate between viable particles and non-viable particles. False positive results occur when non-viable particles are classified as viable particles. False positives can occur due to the non-specific nature of the laser induced fluorescence (LIF) technique. Non-viable particles such as pollens, skin flakes, and paper dust have fluorescence properties and create optical signals that must be addressed during instrument design. Typically, higher sensitivities result in higher levels of false positives. These false positive results are known as “noise”. For example, if Grade A is placed on a result of 1 cfu it is highly recommended to perform a data analysis for establishing deviations that are not random perturbations of the system. If the investigation reveals a real-time viable particle detector issue, the data shall show an unusual pattern (from the baseline) even if they were considered “noise”. There, the search for assignable causes of non-random data should be emphasized for use in identifying needs for process improvement.

The use of Vertical Horizontal Unidirectional Air Units, Biological Safety Cabinets and Gloveboxes increase the likelihood of microbial contamination. Aseptic manipulations in those units do not have the protections from the operator like isolator and RABS. Most processes would need assistance that should be located nearby to transfer material in and out the Grade A area. All these transfers are considered interventions and increase the likelihood of contamination even more. Materials must be sanitized thoroughly prior to entering the aseptic perimeter. The aseptic perimeter should be disinfected before and after processes. Batches most be worked one at a time. Because the in-process EM sampling is handled by the operator, the possibility of contamination may occur leading to false positive results during every step on the process including the delivery of samples to the QC laboratory. Therefore, Aseptic Behavior is key.²³ The following items are recommended to evaluate the production room and primary and secondary engineering controls.

• Classified Room (Refer to Annex 1):
  • Is the area visually clean?
  • Is there peeling paint, chipped drywall, acoustic ceiling tiles with cutout holes, rusty stainless steel or other breaches in the walls or ceilings?
  • Is the aseptic workbench organized as instructed on site procedures?
  • Is there any new equipment additions, room layout changes and/or any other process changes that may not have been evaluated for airflow?
  • Are return vent(s) unclean or presence of foreign material.
  • Are HEPA filters caulked around each perimeter to seal them to the support frame.
  • Are classified areas used for other activities not essential for manufacturing?
  • Are access doors and path thrus functioning and closing as intended?
• Engineering Controls (Refer to Annex 1). In addition:
  • Is the HVAC of the Isolator/RABS unit on 24/7 or is there a shutdown period?
  • If there is an “off” period, is there a cleaning prior to start of the unit?
  • Are system alarms reviewed for loss of pressure, are pressure differentials maintained?
  • Was a visual inspection performed? Is there presence of dirt, soils, debris, leaks or condensation?
  • Turn on the system: Is there any unusual vibration or noise from the HEPA filter or equipment?
  • Is there any issue with the Grade A area during the process? Are there any issues that occurred during the process around the Grade A area?
  • Was there any intervention during the process? Was the intervention documented and described?
  • Was a smoke study performed and evaluated within the unit?
  • Include the advice of outside expert on the matter.
  • Determine a microbial contamination risk-reduction plan.
  • Include an effectivity check.

The Human Factor

Operators, even when carefully and correctly gowned, continuously leach skin particles that may have microorganisms into the cleanroom environment.^5-10 Cleanroom operators, particularly those engaged in aseptic processing, must strive to maintain suitable environmental quality, and must work toward continuous improvement of personnel operations and environmental control. Japanese “Guidance on the Manufacture of Sterile Pharmaceutical Products by Aseptic Processing” (2011) states that “Deviations from the action level specifications should be investigated for cause(s) prior to shipment of final products manufactured through the process where the deviation occurred, and corrective measures should be taken. The validity of corrective measures taken should be verified to confirm the recovery of acceptable environmental conditions, as needed. The recovery may be readily confirmed in some instances. For example, counting particulate matter, but not reproducible in other instances, such as with bacteria adherence to gowns. If the cause(s) cannot be traced, recovery should be established by general approaches including prohibition of personnel entry for a certain period, retraining of personnel, and reviewing assigned tasks.”²

Features to be considered during a microbial EM excursion investigation within Grade A areas would be the occurrence of unusually high number of colonies recovered, if this incident is isolated or can be correlated with other recoveries and the identity of the organism recovered. Excursions beyond approximately 15 CFU recovered from a single sample, whether airborne, surface or personnel should happen very infrequently in aseptic processing environments. However, when such occurrences do occur, they may be indicative of a significant loss of control, particularly when they occur within the ISO 5 critical zone near product and components. It is advised that any excursion >15 CFU should be the subject of a careful and thorough investigation (USP 1116). An investigation for an isolated single excursion, establishing a definitive cause probably will not be possible, and only general corrective measures can be considered. It is never wise to suggest a root cause for which there is no solid scientific evidence. Therefore, it is likely that any microbial EM investigations of an isolated viable action limit does not provide enough robust evidence that the cleaning and sanitization performance, gown usage or operator aseptic behavior is a definitive root cause of the event. Sub-optimal cleaning and sanitation, and process conditions are the most common contributor of Grade A events in the presence of operators. The most common inadvertent error in aseptic techniques is the unrecognized transfer of microorganisms with no aseptic technique error involved. The most likely source of the contaminated samples is touching contamination. However, the author recognize that contaminated samples can also result from airborne sources³⁰ Such events are rare or simply do not happen when the human presence is drastically reduced or removed from production.

In general, the fewer personnel involved in aseptic processing and monitoring, will reduce the risk of microbial contamination. In Grade A areas within an aseptic processing operation, the microbial recovery should be less than 1% of the EM samples.⁶ The risk of microbial contamination during sterile product preparation would be practically non-existent were people not involved in the process. For technologies such as isolators or closed RABS, the recovery rate must always approach zero.⁵  Robotic arms and robot systems remove human intervention during sterile compounding will reduce EM excursions dramatically.^31,32

• Human Factor:
  • What activities were being performed at the time the viable EM sample was being collected (material transfer, aseptic manipulation, etc.)? Was the product exposed at the time of the sampling?
  • Oversight activities for Aseptic Behavior.
  • Operator interview using a standardize checklist. Include observations in the investigation text.
  • Determine and list potential “at-risk behavior”. Connect action with consequence.
  • Identify system errors that could result in repeat events but not acting upon them.
  • Assess human factors or human error.
  • Identify areas where the human error has been mitigated in previous investigations to minimize repeat events.
• Procedural Controls:
  • Evaluate recent changes in manufacturing procedures; Is there personnel, material, or process flow change?
  • Is there any recent change in gown supplier or material?
  • Is there any outstanding number of personnel in the room at the time of the event?
  • Review most recent gloved fingertip/out of room personnel microbial sample results.
  • Review last Aseptic Process Simulation report.
  • Review cleaning and sanitation procedures/documentation.
  • Review cleaning and disinfection agents
  • Review personnel qualifications
  • Review environmental sampling procedures and bioburden recovery methods; confirm recovery methods are qualified.

Contamination Recovery Rate Analysis

USP <1116> defines Recovery Rates as “...the rate at which environmental samples are found to contain any level of contamination. For example, an incident rate of 1% would mean that only 1% of the samples taken have any contamination regardless of colony number. “contamination recovery rate as the percentage of plates that show any microbial recovery irrespective of number of CFU. “⁶

A microbial EM program should be able to detect changes in the microorganism’s recovery rates that may be indicative of breaches in the state of control of the room or facility.^2-4 Microorganism’s EM program results are compared against established action levels provided by United States Pharmacopeia (USP) and European Union Pharmacopeia (EU), current good manufacturing practice (cGMP), Food and Drug Administration (FDA) among other guidelines. Microbial EM levels should be threshold values to balance between an adequate control but without triggering toward unsafe environment conditions for manufacturing. Microbial EM programs are intended to assess the in-process environmental controls intended by design and cleaning/sanitization programs to maintain cleanliness conditions of the manufacturing-controlled environments. The reality is that Microbial EM programs are not validated processes and an EM action level excursion of the manufacturing environment is not a direct testing to assess product quality attributes. In fact, a microbial EM action level excursion is not considered an out-of-control incident of the manufacturing environment.⁶  Microbial EM action and alert limits are not considered control measures. Therefore, tightening of the EM levels or increase EM sampling frequency are not considered corrective actions.

When contamination recovery rates or number of action limits events increase from an established norm a process for determining the possible source should be initiated.^34-36 For example, a group of five test results with 1 CFU represent 100% contamination rate while a single 5 CFU result out of five samples (i.e., one result is 5 CFUs while other four samples are 0 CFUs) would be 25% contamination rate.

EM investigations may differ depending on the criticality of process step, the quality attributes of the product, and area where the product is manufactured such as a cleanroom, Unidirectional Air Flow area, RABS, or Isolator. The investigation should include a review of area maintenance documentation; visual inspection of the compounding area, sanitization/decontamination documentation; the occurrence of non-routine events; the inherent physical or operational parameters, such as changes in environmental temperature and relative humidity; and the training status of personnel. Refer to PDA Technical Report No. 88, Microbial Data Deviation Investigations in the Pharmaceutical Industry has an excellent guidance for Microbial EM investigations.³⁴

EM Adverse Trend investigations

Trending environmental monitoring (EM) data is a regulatory requirement.^5-10,16,38 Microbial EM data deviations or non-conformances periodical assessment for the presence of adverse trends may occur.³ In addition, EM report on OOT, OOS, Corrective and Preventive Actions (CAPA) and effectivity checks. However, unlike chemical analytical deviations, all microbial non-conformances to include OOS and CAPA investigations, may require months to complete. Therefore, studies-based protocols and additional laboratory tests may be required for correction effectivity checks.

Adverse trend issues must be carefully considered. Variation in microbial counts is an expected phenomenon. The reliability of environmental results resides in validated methods and appropriate procedures that apply. Operator sampling technique variability as well as potential mishandling of samples and laboratory errors (i.e., some degree of subjectivity during colony counting) are unavoidable factors that will make it difficult to ascertain the determination of assignable cause. As stated above, it is unlikely that the cause of the Microbial EM excursion would be determined. Previous occurrences, sample type and location, proximity to the exposed product process and/or product contact surfaces, and other factors must be considered.

An isolated EM action limit excursion does not provide information about a possible control breach or does not identify a possible risk. It is only when data is pulled in the same framework and appropriate timeframe is it suitable to identify a potential risk. Therefore, Microbial EM alert and action level excursions must be evaluated every time new data is generated in an ongoing basis to identify runs of data that indicate a potential adverse trend, whereby an investigation is initiated if an adverse trend is identified. Once the adverse trends have been identified the environment must be monitored closely. Additional time points may be required before the next scheduled time point to further confirm the trend. The data analysis should determine if the adverse trend is isolated to one area/room or other adjacent areas/ rooms. In either case, an investigation must be initiated.

Every EM program must include quantitative/qualitative assessment to identify an adverse trend. In case a potential mold adverse trend is confirmed based on site criteria, a full Quality investigation must be initiated to confirm such an adverse trend, identify the root cause, assess product quality impact, and define corrective actions to restore baseline values in the process/facility. Mold adverse trends do not necessarily indicate that product quality has been compromised but do indicate the need to identify a possible breach in environmental controls. A risk assessment should be performed to evaluate if the manufacturing process should be halted pending resolution of the issue and completion of a “return to service” plan. The EM plan must specify which actions (e.g., investigation and/or preventive measures) need to be taken in case of an adverse trend. However, mold isolation events below total microbial count alert levels may not require specific actions or investigations given limited risk of these events. Nevertheless, a procedure should establish in what cases additional/ special cleaning including sporicidal agents and, verify the absence of the mold in the impacted area/s (i.e., to verify effectiveness of cleaning procedures for removal of the organism) must be considered or performed.

A statistically robust set of rules is proposed for trending excursions in environmental monitoring data. These rules should be designed to minimize false alarms when the process is in control but signal quickly when the process goes out of control. An adverse trend is an early warning that the system is drifting from normal operating conditions. Prompt action may prevent further deterioration and avoid costly out-of-specification events. Adverse trends should be defined by site procedures and followed. Systems such as 98th percentile/95th percentile, provide a common ground to compare historical data even if they are not strictly associated. These percentiles were chosen because they are functional equivalents of control limits and warning limits used in statistical process control charting, which are set at three and two standard deviations above the mean, respectively. In addition, the USP <1116> recommended microbial recovery rates should also be implemented as trend metrics for microbial environmental monitoring of aseptic processing facilities. Occasional isolated alert level excursions may occur even if the process remains in a state of control. However, repeated alert level excursions occurring at a rate greater than 2.5% indicate the process is changing and the system is drifting from normal operating conditions. An adverse trend of alert level excursions should be investigated for root cause. It is critical to determine if an alert level excursion, at its onset, triggers an adverse trend. Rationale for choosing these rules must be justified.

EM trending must be performed for many reasons, including:

• Regulatory compliance
• Ensuring a state of control of the facility
• The ability to be proactive before a problem gets out of hand
• To provide a graphical representation of the data
• To determine any problem areas in the facility
• To determine if the cleaning and disinfection program is working as expected
• Monitoring the microbial flora of the facility and seasonal trends
• Providing a simpler means of communication of the EM data to management
• Identifying sources of microbial contamination.
• Establishing alert and action levels

Root Cause Analysis

The root cause analysis tool or methodology suggested to determine assignable/root cause should be the Fishbone Diagram, also called: cause-and-effect diagram or Ishikawa diagram. This cause analysis tool is considered one of the seven basic quality tools. The fishbone diagram identifies many possible causes for an effect or problem. It can be used to structure a brainstorming session. The major categories of causes suggested to assess during the investigation are the following:

• Methods: procedures, work instructions for process flow, cleaning and sanitation, gowning, aseptic behavior, others. 
• Machines (equipment): stand alone or room engineering controls and/or other equipment used during the impacted process. 
• People (manpower): interview outcome of manufacturing personnel and those who handle samples as applicable/training and qualifications. The use of a questionnaire and/or check list is highly recommended. 
• Materials: materials used for the sampling. 
• Measurement: system used to measure the non-conformance 
• Environment: manufacturing environment assessment at the time of the sampling 
• Mother nature: described as surrounding conditions around the manufacturing process that cannot be controlled and/or predicted.

Every category has multiple possible contributors to the event. The analysis will continue until the root cause(s) or possible root cause of the excursion have been identified. For each cause, supporting information must be provided and documented. Some areas would need some sort of questioning methods, such as the 5 Why’s.

Conclusion

Microbial EMP is a semi-quantitative methodology limited by method, size, operator technique, and biological variables. The EMP  ensures the manufacturing environment is within viable and non-viable counts requirements. There is a misconception that has led to an inappropriate use of microbial environmental monitoring results of critical areas in sterile manufacturing as a surrogate for product release criteria. This is especially troublesome when establishing alert or action levels at very low quantitative levels and assignable root cause is hard to be identified.

Microbial EM sample results are historically variable and depend on the method. However, any action level or out of trend should be defined and the analysis should be documented. There are different strategies that can be established by procedure to follow up alert and action limits excursions and adverse trending to assess the environment quality. Remember that sterile products do not consider manufacturing EM limits as a product specification.

Disclaimer

The scenarios discussed in this article were created by the author to establish his point of view. The views and opinions of the author expressed herein do not necessarily state or reflect those of Janssen Biotech Inc. and the Johnson and Johnson Family of companies.

Conflict of Interest Declaration

The authors declare that they have no competing interests.

References

1. ISO 14644-1(2015) Cleanrooms and associated controlled environments — Part 1: Classification of air cleanliness by particle concentration. 
2. Japanese Health Authority (2006) “Guidance on the Manufacture of Sterile Pharmaceutical Products by Aseptic Processing” https://www.gmp-compliance.org/files/guidemgr/ Aseptic_Guide_Japan.pdf Accessed 06JUL2023. 
3. EudraLex – The rules governing medicinal products in the European Union, Vol. 4 – EU guidelines to good manufacturing practice Medicinal products for human and veterinary use, Annex 1 – Manufacture of sterile medicinal products (2022). Chapter 6. Accessed on Accessed on 05SEP2021. 
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Publication Detail

This article appeared in American Pharmaceutical Review:
Vol. 26, No. 8
Nov/Dec 2023
Pages: 70-77

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