Particle Measurement and Microbial Monitoring

In the aseptic manufacturing of pharmaceuticals, maintaining the cleanliness of the manufacturing environment is critically important for ensuring product quality and safety. However, many beginners wonder about a fundamental question: “How can we truly confirm that there are no microorganisms in a sterile environment?” In reality, with current technology, it is impossible to measure the number of viable microorganisms in real time, and as an alternative measure, particle measurement is widely employed.

Limitations of Real-Time Microbial Measurement

The current standard method for detecting microorganisms is the culture method. Specifically, this involves collecting a sample, inoculating it onto a culture medium, incubating it at an appropriate temperature for a certain period (typically 24 to 72 hours, or longer for certain organisms), and then counting the colonies that form. While this method is highly reliable, it takes time to obtain results and is therefore not suitable for real-time control of manufacturing processes.

At present, no practical technology exists that can instantly detect the number of viable microorganisms in the air. This is because microorganisms are extremely small (most bacteria range from approximately 0.5 to 5 μm), and it is technically difficult to instantaneously determine whether they are alive or not. Even when microorganisms are detected, distinguishing viable organisms from dead cells or spores remains a significant challenge.

Indirect Monitoring Through Particle Measurement

To overcome this technical limitation, the pharmaceutical industry has adopted an indirect method called “particle measurement.” Using particle counters, the number of particles 0.5 μm and larger suspended in the air is measured in real time.

The rationale behind this approach is based on the following principles:

Microorganisms always exist as particles (all microorganisms are particles, but not all particles are microorganisms). Microorganisms rarely exist as single cells in the air; they typically attach to or are carried by larger particles such as dust, skin flakes, or respiratory droplets. There is a certain correlation between particle count and microbial count. As the number of particles increases, the risk of microbial contamination increases proportionally.

For example, ISO 14644-1:2015 and GMP guidelines (EU GMP Annex 1 revised 2022, PIC/S GMP Annex 1) establish upper limits for particle counts for each cleanroom grade, and meeting these values serves as one indicator of ensuring sterility.

Cleanroom Classification and Particle Limits

Current regulatory standards define cleanroom grades based on particle concentration. The following table shows the particle limits for pharmaceutical manufacturing areas according to EU GMP Annex 1 (2022) and ISO 14644-1:2015:

Grade	At Rest – 0.5 μm	At Rest – 5.0 μm	In Operation – 0.5 μm	In Operation – 5.0 μm
A	3,520 per m³	20 per m³	3,520 per m³	20 per m³
B	3,520 per m³	29 per m³	352,000 per m³	2,900 per m³
C	352,000 per m³	2,900 per m³	3,520,000 per m³	29,000 per m³
D	3,520,000 per m³	29,000 per m³	Not defined	Not defined

It is important to note that “at rest” refers to the condition where the cleanroom is complete with all equipment installed and operating, but with no personnel present. “In operation” refers to the condition where the room is functioning in the defined operating mode with the specified number of personnel present.

Correlation and Limitations

Critically important is the fact that there is no absolute correlation between particle count and microbial count. The majority of particles are of non-biological origin (dust, fibers, chemical crystals, etc.), and actual microorganisms constitute only a small fraction of these.

To express this numerically, for example, in a Grade A environment (the most stringent sterile area), 3,520 particles of 0.5 μm or larger per cubic meter are permitted, but for microorganisms, less than 1 CFU (Colony Forming Unit) per cubic meter is required during operation. These numbers clearly demonstrate how small the proportion of particles that are actually microorganisms is.

The following table compares particle limits with microbial limits according to EU GMP Annex 1:

Grade	Particle Limit (0.5 μm)	Microbial Limit (Air Sample)	Microbial Limit (Settle Plates)
A	3,520 per m³	<1 CFU/m³	<1 CFU/4 hours
B	352,000 per m³ (in operation)	10 CFU/m³	5 CFU/4 hours
C	3,520,000 per m³ (in operation)	100 CFU/m³	50 CFU/4 hours
D	Not defined	200 CFU/m³	100 CFU/4 hours

This stark difference between particle and microbial limits underscores why particle counting serves as a sensitive early warning system rather than a direct measure of bioburden.

Comprehensive Environmental Monitoring Strategy

Due to these limitations, management of aseptic manufacturing environments employs a comprehensive approach that combines multiple complementary methods, including the following:

Real-time particle monitoring: Continuously checks environmental cleanliness. This provides immediate feedback on changes in environmental conditions and can detect breaches in containment or failures in air handling systems before microbial contamination occurs.

Periodic microbial sampling: Includes settle plate testing (passive air sampling), active air sampling using volumetric samplers, surface sampling using contact plates or swabs, and personnel monitoring. These methods provide direct evidence of microbial presence but with delayed results.

Environmental parameter monitoring: Includes temperature, humidity, differential pressure (pressure cascades between different grade areas), airflow patterns and velocity, air changes per hour (ACH), and recovery time studies. Maintaining proper environmental parameters is essential for the effectiveness of the cleanroom system.

Process simulation: Media fill tests (also known as aseptic process simulation or APS) are performed to validate that the entire aseptic process, including human interventions, can be conducted without introducing contamination. These are considered the most important validation tool for aseptic processes.

By combining these methods, it becomes possible to compensate for the limitations of real-time measurement while ensuring the sterility of the manufacturing environment with a high degree of confidence.

Current Status of Rapid Microbiological Methods (RMM)

Real-time microbial detection technology is rapidly developing. Various Rapid Microbiological Methods (RMM) are being researched and, in some cases, implemented in the pharmaceutical industry. These include:

ATP (Adenosine Triphosphate) bioluminescence: Measures ATP present in living cells to provide rapid indication of microbial presence. While this method can provide results within minutes, it cannot distinguish between different types of microorganisms and may give false positives from non-microbial sources of ATP.

Flow cytometry: Uses fluorescent dyes and laser detection to count and analyze cells in real time. This method can differentiate between viable and non-viable cells and provides results much faster than traditional culture methods.

Fluorescence-based methods: Various techniques use fluorescent markers that bind to specific cellular components or metabolic activities to detect microorganisms.

Biosensor technology: Utilizes biological recognition elements combined with physical transducers to detect specific microorganisms or their metabolic products.

Laser-induced fluorescence (LIF): Some advanced particle counters incorporate LIF technology to distinguish biological particles from non-biological ones in real time.

However, regulatory acceptance of these technologies is still evolving. The FDA, EMA, and other regulatory authorities have published guidance documents (such as FDA’s “Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing — Current Good Manufacturing Practice” September 2004, and subsequent updates) that acknowledge RMM but emphasize that these methods should complement, not replace, traditional culture-based methods. The revised EU GMP Annex 1 (2022) explicitly recognizes the potential of RMM while maintaining traditional methods as the standard.

Currently, no fully established real-time microbial detection technology has completely replaced traditional methods, and the combination of particle measurement and conventional microbial testing remains the industry standard. The primary challenges include:

• Inability to distinguish between viable and non-viable microorganisms in real time
• Difficulty in detecting all types of microorganisms with a single method
• Need for extensive validation to demonstrate equivalence to traditional culture methods
• Regulatory acceptance and harmonization across different jurisdictions
• Cost-effectiveness compared to established methods

Future Prospects

The future of environmental monitoring in pharmaceutical manufacturing will likely involve a hybrid approach. As RMM technologies mature and gain broader regulatory acceptance, they will increasingly be integrated into routine monitoring programs alongside traditional methods. The goal is not to replace culture-based methods entirely but to provide faster feedback, enabling more responsive contamination control and trending.

Advances in artificial intelligence and machine learning may also enable better interpretation of particle monitoring data, potentially improving the predictive value of particle counts for microbial contamination risk. Integration of multiple data streams (particle counts, environmental parameters, personnel movements, and microbial results) through advanced data analytics platforms may provide a more holistic understanding of contamination control.

Conclusion

In the aseptic manufacturing of pharmaceuticals, because real-time detection of microbial contamination is technically difficult, an indirect method called particle measurement is widely used. While this method has limitations, by combining it with other microbiological tests, it becomes possible to ensure the sterility of the manufacturing environment at a high level.

Professionals in the pharmaceutical industry face this “invisible risk” daily and, through a comprehensive, scientifically based approach, achieve the production of safe, high-quality pharmaceuticals for patients. The regulatory landscape continues to evolve, with increasing emphasis on risk-based approaches to contamination control, as reflected in the revised EU GMP Annex 1 (2022) and ICH Q9 (Quality Risk Management).

Understanding the relationship between particle measurement and microbial monitoring, along with their respective strengths and limitations, is essential for anyone involved in aseptic pharmaceutical manufacturing. While we await the development and validation of more advanced real-time microbial detection technologies, the current multi-faceted approach to environmental monitoring remains the gold standard for ensuring product sterility and patient safety.