1、 Regulatory requirements

GMP testing methods for the pharmaceutical industry need to refer to ISO testing methods, while ISO 14698-1 Microbial Pollution Control Regulation mentions that microbial monitoring requires evaluating collection efficiency. Sampling methods and equipment with high collection efficiency can obtain more representative data. For environments with low biological load, such as Class A and B environments, more active sampling of planktonic bacteria should be used instead of passive sampling of settling bacteria.

Guidelines for evaluating the collection efficiency of microbial samplers have been proposed. currentThe 2003 version of ISO14698 divides collection efficiency into physical collection efficiency and biological collection efficiency. Physical collection efficiency is more concerned with whether the sampling method has the ability to collect airborne bacterial particles, while biological collection efficiency is more concerned with whether the sampler or sampling method can ensure the survival rate of the bacterial particles while collecting them.

2、 Sampling efficiency of sedimentation bacteria

The passive sampling method based on gravity can obtainThe collection efficiency of active particles with a particle size of 12 microns settling on a 9cm sampling plate is 0.106 CFU/h, and the collection efficiency of settling on a 14cm sampling plate is 0.256 CFU/h. The collection efficiency for active particles with a particle size of 1 micron settling on a 9cm sampling dish is 0.008 CFU/h. If the maximum concentration limit of the zone is 1CFU/m3, then a 9cm petri dish needs to be exposed to zone sampling for 1250 hours (equivalent to 52 days) to detect a meaningful data (one CFU) in a low biological pollution load environment.

So: sedimentation bacteriaIt is a sampling method that is too insensitive, and sedimentation bacteria better reflect the sedimentation effect of bacterial particles, only more sensitive when sampling from larger petri dishes (such as 14cm) or multiple sampling petri dishes. The collection efficiency of the active sampling method for collecting particles of 1 micron is 2250 times higher than that of the passive collection method for settling bacteria of 9cm. For microbial sampling in laminar flow environments, it is recommended to use more sensitive sampling methods.

Sampling methods such as sedimentation bacteria for non sterile processes with relatively high microbial loadsC. D-level environment can serve as a supplement to microbial sampling and can be used in conjunction with planktonic bacterial sampling methods. For A and B level environments, the planktonic bacteria sampling method is more suitable as a more efficient sampling method.

3、 Risks in daily monitoring

As we all know, the European UnionThe draft of Appendix I for GMP aseptic production was released on December 24, 2017, with significant changes including:

a) Microorganisms and related terms have doubled (23 times mentioned in the original appendix and 68 times mentioned in the new draft)

b) The mention of risk has increased nearly fourfold (20 times mentioned in the original appendix and 92 times mentioned in the new draft).

Therefore, it can be understood that regulatory authorities have placed an unprecedented level of emphasis on microbiology and risk requirements. For daily monitoring, the new appendixThe monitoring location for active particles (microorganisms) mentioned in Article 9.7 should be at the risk of contamination in sterile production, such as sterile surfaces, open container stoppers, etc. As mentioned in Article 9.29, the sampling method for active particles (microorganisms) should not pose any risk of contamination to pharmaceutical production (Reference 2). Therefore, we can see the thoughts of regulatory authorities, who both believe that microbial monitoring is very important and that using inappropriate sampling methods will have a negative impact on the production environment.

This is a dilemma for our industry. Previously, many companies were concerned that frequent replacement of petri dishes for planktonic bacteria sampling methods could cause pollution to the pharmaceutical environment due to the inability to find a suitable microbial method. Therefore, they decided not to conduct active microbial (planktonic bacteria) monitoring. With the development of modern pharmaceutical industry, we have realized the need to improve our risk assessment and identification of microbial sampling methods.

The sedimentation bacteria method may cause pollution to the environment. We know that the surface of sedimentation bacteria contains a large amount of nutrients and waterBetween 10 microns and 20 microns, when the settling bacteria are exposed to critical production environments, these nutrients and moisture are likely to be blown onto sterile surfaces, medicine bottles, containers, and drug solutions, causing contamination and cross contamination.

Due to the easy drying of the culture medium, modern sampling flow rates are designed for low flow rate sampling, which can prolong the coverage and production time of planktonic bacteria sampling as much as possible, and avoid frequent dish replacement. Usually consideredThe purpose of airflow protection in laminar flow environment is to design isokinetic sampling for planktonic bacteria sampling, ensuring that the air above the sampler is sampled at a constant velocity and does not cause pollution to the site.

The traditional microbial sampling method involves placing the entire sampler insideThe environment, and modern aseptic process control methods require that any incoming materials and equipment be fully sterilized. The traditional method involves placing the sampler inside, but due to the inability to sterilize the electrical components of the circuit board inside the sampler, companies usually use VHP or alcohol to disinfect the surface of the sampler, which cannot achieve the desired sterilization effect. The WHO sterile process control requirements suggest that pharmaceutical companies should adopt a split microbial sampling method, which separates the sampling head and sampler. The sampling head should be placed and can be pre sterilized with high temperature or chemical sterilization, while the sampler should be operated in a B-level background environment. In this way, the impact of the sampler's inability to sterilize can be avoided.

Conclusion

In summary, the efficiency validated planktonic bacteria sampling method has a higher collection efficiency compared to the settling bacteria sampling method, and is suitable for use in key, low bioburden sterile production environments. The settling bacteria sampling method can be used as a supplementary method for sampling planktonic bacteria in the production process, and can be applied to relatively non sterile environments with high biological loads. When using it, it is necessary to avoid the risk of contamination caused by improper placement or operational errors. Regardless of the method used, the location, sampling frequency, sampling coverage time, and sampling flow rate should be determined based on the risk assessment of the production process.