Cleanroom manufacturing rules use Colony-Forming Units per cubic meter (CFU/m3) to measure active air sampling findings.
In grade A cleanroom environments, EU GMP Vol. 4 Annex 1 (2022 Revision) demands continuous active air monitoring during crucial processes, including the assembly/ set-up phase. A similar technique should be developed for Grade B situations.1
9.24 Continuous viable air monitoring in grade A (e.g. air sampling or settle plates) should be undertaken for the full duration of critical processing, including equipment (aseptic set-up) assembly and critical processing. A similar approach should be considered for grade B cleanrooms based on the risk of impact on the aseptic processing.
EU GMP Annex 1: 2022
As a result, the volume of sampled air in these places - which is strongly related to duration and flow rate - may exceed one cubic meter during the monitoring period.
It is not recommended to normalize air monitoring results to one m3 when the duration of a production process exceeds the time needed to sample one m3 of air. This applies to both viable and total particle counts. Specifically, for viable active air sampling, it is crucial to consider three significant parameters:
1. Contaminants are not uniformly distributed over time or in space
The environmental monitoring results represent a snapshot of circumstances at a specific time and are not repeatable.
Air flow, people movement, door openings, manufacturing activity, and local turbulence are all potential sources of brief contamination surges. Mathematically adjusting data to one m3 does not adequately reflect microbiological risk during sampling.
2. Colony Forming Units (CFUs) are distinct, non-fractionable units
Normalizing data to one m3 may result in non-whole values, which is contradictory with microorganisms as whole units (see point 3).
3. In grade A environments (most critical from the product point of view), the expected limit is ‘no growth.’
In grade A conditions, the goal is not to achieve 0 CFU/m3, but to confirm the lack of growth. The key issue is to determine whether there is any growth, not how many colonies are in one m3. According to Annex 1 (Table 6, at paragraph 9.30), the criterion is non-detectable colonies:
Source: Particle Measuring Systems
| Grade |
Air sample CFU /m3 |
| A |
No growth(c) |
(c) It should be noted that for grade A, any growth should result in an investigation.
Thus, normalized results with units of CFU/m3 are primarily useful for investigative purposes (to assess an impact on product quality) rather than routine monitoring evaluation, because the fractionalized result is unimportant in terms of SOP and regulatory requirements when conducting microbial investigations:
9.31 Microorganisms detected in the grade A and grade B areas should be identified to species level, and the potential impact of such microorganisms on product quality (for each batch implicated) and overall state of control should be evaluated.
EU GMP Annex 1, 2022
Recommended approach
Instead of normalizing to one m3, the absolute number of CFUs identified alongside the sample volume should be reported.
This contextualizes the results, taking the sampling length and flow rate into account. This should be followed by an interpretative remark to back up the qualitative assessment of compliance, as quantitative compliance may not be applicable in such situations.
Note: Users should round up to the nearest whole number because a microbe (viable presence) cannot be fractionated, especially when determining its potential impact on product quality.
Examples
These samples in grades A and B reflect routine continuous sampling for four hours (240 minutes) at 25 LPM.
Grade A
- Flow Rate: 25 L/min
- Duration: four hours (240 min)
- 1000 Liter (L) = 1 m3 (conversion factor for L to m3 = 1000)
- Sampled volume = (Flow Rate x Duration) / (conversion factor) = (25 L/ min x 240 min) / (1000) = six m3
- Result: two CFU on the plate after incubation
Recommended approach for data interpretation: 2 CFU in 6 m3
Qualitative assessment: investigation is necessary
Quality investigation:
- Microbial identification is mandatory.
- Mathematical calculation: 0.3 CFU/ m3 (2 CFU/ six m3) ≅ 1 CFU/ m3 (growth)
- OUT OF ACTION LIMIT = DEVIATION (= process investigation for batch impact assessment
Grade B
First and foremost, in grade B regions, it is critical to assess the potential for influencing aseptic processing before starting continuous monitoring. The Annex 1 guidelines state:
“A similar approach should be considered for grade B cleanrooms based on the risk of impact on the aseptic processing.” (Ref. EU GMP Annex 1, 2022)
Continuous monitoring is not required in grade B areas; alternative options, such as 1 m3 sampling (for example, 40 min at 25 LPM) with a scheduled and process-related sampling frequency, may be adequate to cover the risk (such as the beginning, middle, and/ or end of manufacturing activities or during specific interventions/ transient events).
A risk assessment document is recommended to gather all essential information and develop a tailored sampling plan based on process requirements. Otherwise, in the event of continuous monitoring sampling, an alternative strategy might be:
- Flow Rate: 25 Liter/ min
- Duration: four hours (240 min)
- 1000 Liter (L) = 1 m3 (conversion factor for L to m3 = 1000)
- Sampled volume = (Flow Rate x Duration) / (conversion factor) = (25 L/min x 240 min) / (1000) = six m3
- Result: 11 CFU on the plate after incubation
Recommended approach for data interpretation: 11 CFU in 6 m3
Qualitative assessment: investigation is necessary
Quality investigation:
- Microbial identification is mandatory.
- Mathematical calculation: 1.8 CFU/m3 (11 CFU/ six m3) ≅ 2 CFU/m3 (lower than 10 CFU/m3). This limit is in accordance with Annex 1 (Table 6, paragraph 9.30).
- OUT OF ALARM LIMIT: deviation is not necessary at this phase, but process investigation is highly recommended.
- This strategy requires assessment in accordance with the customer policy on deviations and the company’s contamination control strategies.
- It is recommended to establish the same time frame for any deviation event because if an anomalous event (relevant to biologics or manufacturing) is highlighted, the occurred out-of-alert limit must be scaled up to a deviation (batch impact).
A note on alternatives to CFU/m3 in grade B environments
When using an alternative technique to CFU/m3 sampling (e.g., continuous monitoring), it is critical to remember that out-of-alarm limit investigations should occur only occasionally. Microbial excursions in grade B environments should be minimal if a strong contamination control program is in place. Such excursions include effective cleaning, disinfection, maintenance, and monitoring procedures.
Normalizing data to per cubic meter (m3) is therefore not necessary under controlled settings. Effective trend analysis is vital to avoid the onset of more serious product quality issues.
As a result, monitoring procedures should include a trend analysis approach to evaluate, as a minimum, the following scenarios: expanding numbers of excursions from action limits or alert levels, recurrent excursions from alert levels, frequent but isolated excursions from action limits that may have a common reason (e.g. single excursions that consistently follow planned preventative maintenance), variations in microbial flora type, and quantity and dominance of specific organisms (refer to Annex 1, paragraph 9.11).
An adverse, or undesired, trend is defined as a sequence of data points that show an upward drift above the alert level and approach or exceed the action level compared to previous data.
Conclusions (PMS perspective)
- The non-homogeneous distribution of microorganisms, both in space and over time, raises concerns surrounding the use of only normalized data (CFU/m3), particularly in grade A environments.
- In Grade B environments, conducting a risk assessment is recommended to determine possible effects on aseptic processing.
- Normalized findings (CFU/ m3) can be useful for trend analysis, but lack microbiological relevance for routine monitoring. Microbial identification is usually required.
- The most thorough and robust technique is presenting both the absolute CFU count and the normalized number, along with a technical explanation of their biological significance and regulatory implications.
- Using trend analysis can help to prevent out-of-control incidents and improve product quality. Sporadic out-of-alarm occurrences may occur, although they are typically documented as indicators of a general control system drift. If a comprehensive contamination management strategy is in place, such excursions should be rare in Grade A and B workplaces. As a result, normalizing figures to per m3 should not be the major focus.
- Effective process knowledge, preventative maintenance, adequate monitoring, and frequent reviews are essential for success. This gives customers assurance that the product’s quality will be maintained and guaranteed throughout time.
References
- European Commission (2022). The Rules Governing Medicinal Products in the European Union. Vol. 4 Good Manufacturing Practice Medicinal Products for Human and Veterinary Use – Annex 1 Manufacture of Sterile Medicinal Products. (online) European Commission. Available at: https://health.ec.europa.eu/medicinal-products/eudralex/eudralex-volume-4_en.
Acknowledgments
Produced using materials originally authored by Rosa Laronga, Giulia Paternò, and Serena Steidl, Advisory Specialists at Particle Measuring Systems.
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