The product safety of drugs and medical devices has always been the top priority throughout the entire life cycle of pharmaceutical products. For this reason, strict requirements are imposed – adapted to the method of administration (e.g., topical, oral, parenteral) – with regards to microbiological and particulate purity. This inevitably means that, from a microbiological point of view, qualified cleanrooms are necessary for the manufacture of sterile or microbiologically critical dosage forms [1]. These cleanliness zones are designed and operated in such a way that they meet defined physical and microbiological requirements when monitored [2]. 

The highest requirements apply to the manufacture of products that are required to be sterile. Compared to the manufacturing of non-sterile products, sterile production poses the greatest challenges by far and can therefore rightly be described as the premier league [3]. In this premier league, the manufacture of pharmaceuticals that cannot be terminally sterilized but must be produced aseptically takes the top spot in terms of the challenges posed. In order to effectively prevent the entry of living and non-living particles into these products, production takes place in cleanrooms of class A or ISO 5 according to ISO 14644-1. 

The strictest regulations apply to this cleanroom class, which means that only a very small number of non-living particles and no microorganisms whatsoever are permitted. For other less critical products (e.g., oral dosage forms), lower cleanroom classes are sufficient for manufacturing, where the requirements for the occurrence of microorganisms and the presence of non-living particles are significantly lower. The lower the cleanroom class, the more nonliving and living particles are tolerated. 

Microbiological and particulate contaminants can enter the pharmaceutical product from the environment in various ways and then contaminate it. A very good and clear description, which includes a wide range of different sources of contamination, can be found, for example, in the US Pharmacopeia [4]. The most important sources of contamination mentioned here are personnel, the environment (air, surfaces, water), raw materials, auxiliary materials and the manufacturing processes themselves. These sources of contamination must be kept under control. Environmental monitoring is used to check whether the measures taken to achieve this are effective. Monitoring cleanrooms is therefore an important part of the overall contamination control strategy [2]. 

An environmental monitoring program should be established and documented. The purpose of the environmental monitoring program is to [5]: 

Provide assurance that cleanrooms and air handling units continue to provide an environment of appropriate air cleanliness, in accordance with design and regulatory requirements. 

Effectively detect excursions from environmental limits triggering investigation and assessment of risk to product quality. Environmental monitoring should support root cause analysis when contamination occurs and help to ensure that the cause can be eliminated. The results of the identification of microbiological contaminants can then be used to deduce the natural habitat of the microorganism and, in a further step, to determine where the actual source of contamination is to be found. At least, this approach is recommended for most groups of microorganisms. 

However, when Bacillus sp. occurs, this procedure becomes extremely complicated, as these microorganisms are ubiquitous, making it very difficult to narrow down the source of contamination. 

Trending the monitoring data allows early detection of any negative trends developing in the cleanroom, which can then be counteracted in due time. It is recommended that the trending contains all measured sampling points. The great advantage of trending is that slow, gradual changes in the microbial and particulate status of the cleanroom can be detected at an early stage, whereas in routine monitoring these remain under the radar because they are still below the threshold of warning and action values. According to Annex 1, section 10.10., trend data generated for classified areas should be reviewed as part of product batch certification/ release.

In order to prove that a cleanroom complies with the relevant class, the specified limit values must be adhered to for both viable and non-viable particles. Various methods are available for this purpose, the most commonly used are briefly described below. 

It should always be borne in mind that the values obtained for monitoring are not absolute values, but merely a snapshot of the actual condition of the cleanroom [10,11]. Depending on the method used, it should also be taken into account that there may be considerable measurement inaccuracies, e.g., due to the sampling itself. In general, surface monitoring has been found to recover <50 % [6], even when used with relatively high inoculum levels on standardized coupons. In actual production environments where organisms are stressed to varying degrees, recovery rates may be lower. 

In addition, sampling can only detect microorganisms for which the environmental conditions during incubation are actually suitable. For example, the incubation conditions would have to be adjusted for the detection of microaerophilic/anaerobic microorganisms. With the incubation conditions specified for aerobic monitoring, it will not be possible to detect the above mentioned microorganisms. 

By selecting the appropriate culture media and incubation conditions, the operator thus limits the spectrum of microorganisms that can be detected. In many respects, USP <1116> is correct when it states that a negative result in microbiological hygiene monitoring does not necessarily mean that there were no microorganisms in the tested environment. If no microorganisms were detected, this does not mean that none were present, so it can be assumed that there are a corresponding number of unreported cases. 

From a microbiological perspective, blinders are being put on here, as the focus is on a fairly limited spectrum of microorganisms, but at the same time, this small spectrum is being searched for with a magnifying glass. This makes sense, as the regulatory requirements are fully met, but it does not hurt to be aware of this.

Particle counters are used to determine non-viable particles. These actively draw in the prescribed volume of air (at least 28 L/min) and then indicate the corresponding values for particles >5 μm and >0.5 μm. 

Different Strategies for implementation in daily routine
The regulatory requirements described above can be implemented using various strategies. Each user can decide for themselves which strategy to choose, whether to continue with conservative processes or to pursue alternative, progressive, or completely new approaches. This is less relevant for surface sampling, where there have been no significant optimizations in recent years, but the situation is quite different for the monitoring of air. Three options for air monitoring are described below: a conventional, an advanced, and a future-oriented option.

To meet the Annex 1 requirement for continuous monitoring in cleanroom class A, settle plates can be used. The settle plates are replaced with new ones after a maximum of 4 h so that the growth properties are not lost due to the agar plates drying out. Short interruptions in continuous monitoring due to plate changes are acceptable. Regulatory requirements stipulate that active air sampling must also be carried out at predefined times with the usual sampling volume of 1 000 L (tab. 1).

Some manufacturers of devices for active air sampling have launched models that sample with a lower air flow rate than the standard tested 1 000 L per 10 min. With a reduced air flow, the time required to measure a given volume is extended. This makes it possible to meet the requirement for continuous monitoring while conducting active air sampling. Once the required amount of air has been sampled, a new active air sampling with reduced flow is started. There is a wide range of models available on the market, with flow rates of 25, 50, or 100 (standard) L/min being the most common, and even single-use systems are available. The benefit of this implementation option is that extended active air monitoring increases the time where the process is controlled with a quantitative method (tab. 2).

Devices based on alternative and/or rapid microbiological methods (RMM) are significantly more future-oriented and, with the appropriate practical experience, can provide real-time information on the microbiological contamination of the sampled air (tab. 3). The devices currently available on the market contain particle counters that can indicate the number of particles by size using known methods. In addition, the number of microorganisms present in the sampled volume is also indicated, using the biofluorescence method. The laser integrated into the measuring device detects particles which fluoresce, if they are of biological origin and thus contain endogenousfluorophores (e.g., amino acids, proteins, NAD[P]H, flavins). The laser radiation is absorbed by the particle and re-emitted with a longer wavelength [12]. 

The rapid method using biofluorescence presents the pharmaceutical industry with the challenge of accepting a new unit of measurement, moving away from the classic CFU and instead dealing with auto fluorescent units (AFU). This requires a significant shift in mindset. The pharmaceutical industry and their microbiologists have been accustomed to dealing with and evaluating CFU for decades, and Annex 1 also provides a clear definition of this term:

“A microbiological term that describes a single detectable colony that originates from one or more microorganisms” [13].

However, a critical examination of this unit of measurement, which has been in use for over 125 years, reveals that it is more of an estimate than a precise specification [14,15]. Microbiology is known for not being one of the exact sciences (for example, a factor of 2 is used in the evaluation of many analysis results) and for dealing confidently with uncertainties. A logarithmic approach is more meaningful than a consideration of absolute measured values. 

The use of AFU once again presents the pharmaceutical industry with the challenge that this unit covers a significantly wider range of particles that are of biological origin but include not only viable and culturable particles but also non-viable particles (physically damaged cells, viables but non-culturables) [16]. The consequence of this is that the number of particles measured is significantly higher than is the case with conventional measurement. The pharmaceutical industry has already had a similar experience when, a few years ago, a change was made to the Reasoners agar (R2A) culture medium for microbiological testing of water. Here, too, the change in culture medium suddenly led to conspicuous findings in perfectly functioning systems, because in addition to microorganisms that grew well, microorganisms were now also detected that were not detectable with the previously used culture medium. It was necessary to learn how to classify and evaluate the new, more realistic values in a meaningful way. This is exactly what is needed again this time. 

It is perfectly clear that it will not be possible to make a reliable statement about the correlation between CFU and AFU, as there is no separation within the AFUs into culturable and non-culturable particles. The significance of the new AFU values still needs to be defined. In any case, it is crystal clear that the future belongs to rapid methods. Annex 1 in particular, explicitly points out in many text passages that the use of rapid methods should be considered (e.g., 2.1, 2.5, 9.28). It is therefore not only a good idea, but rather a strong expectation to look into rapid methods and consider their use. The following section therefore describes the use of a biofluorescent particle counter (BFPC) using the example of the Rapid-C+ from Plair. 

Practical example: Use of a BFPC for environmental monitoring in aseptic areas 

With a special BFPC like the Rapid-C+, a single device can be used to perform several environmental monitoring measurements in aseptic areas in parallel, continuously, in real time and with exactly the same air: 

• Total particle counts Determination of the number of particles in the size range of 0.5 μm to100 μm. 
• Viable particle counts Microbial air monitoring with a BFPC providing instant result in case of presence of viable particles. 
• CFU counts Active air sampling on agar plates. The same particles as mentioned above are sampled on traditional agar media using a removable aseptic container holding an agar plate under a sieve. 

Continuous air sampling with the presented BFPC eliminates the need for settle plates if these were used to meet the requirement for continuous monitoring. It also eliminates the need for active air sampling of 1 000 L at specific times, as the active air sampling is already performed by the device itself. 

An unbeatable advantage lies in the fact that the same sample of air is used to collect the air for total and viable particle and germ count determination. In aseptically critical areas, space is usually very limited, therefore it is a great advantage to be able to measure multiple parameters simultaneously using the same sampling probe/measurement position. 

The device draws in 5 L of air per min. This has the advantage that continuous measurement can be carried out for more than 4 h without impairing the growth properties of the agar plate used due to moisture loss caused by the air flow. The only disadvantage, however, is that the Annex 1 requirement for a test air volume of at least 28 L/min for continuous particle measurement is not met, and therefore an additional particle counter must currently be installed or there is also a paradigm shift here, and the measurement of 5 L instead of 28 L is regulatorily accepted, provided that the equivalence of both methods is demonstrated.

The measurement data obtained with the aforementioned device and continuously collected from environmental monitoring allow the following conclusions to be drawn:

This results in 2 scenarios for how the rapid method can be used in pharmaceutical manufacturing.

Scenario 1 is well suited for gaining initial experience because comparative data can be collected. Starting with scenario 1 and later switching to scenario 2 can therefore make sense in order to successfully implement a RMM in a riskbased and well-founded manner. The Rapid-C+ can be implemented in both conventional isolators with gloves and in gloveless isolators. Especially in gloveless isolators where the focus is already on innovation and automation the implementation of an RMM brings advantages. The device presented can be built under the base plate of the isolator and exchange of the nutrient medium in the autoclavable holder can be performed by the manufacturing personnel without intervention into the isolator grade A environment. 

Following successful comparability studies all nutrient media plates needed for active and passive air monitoring could be eliminated from the isolator.

Originally published in Pharmind Ind. 88, Nr.2, 146-153 (2026), ECV - Editio Cantor Verlag, Aulendorf