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. The current 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 achieve a collection efficiency of 0.106 CFU/h for active particles with a particle size of 12 microns settling on a 9cm sampling plate and 0.256 CFU/h settling on a 14cm sampling plate. 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 in the A-grade area is 1 CFU/m3, then a 9cm petri dish needs to be exposed to sampling in the A-grade area for 1250 hours (equivalent to 52 days) in order to detect a meaningful data (one CFU) in a low biological pollution load environment.
So: sedimentation bacteria "is a too insensitive sampling method", 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 in Class A areas, it is recommended to use more sensitive sampling methods.
Sedimentation bacteria and other sampling methods can be used as a supplement to microbial sampling in C and D grade environments with relatively high microbial loads in non sterile processes, and can be used together with planktonic bacteria 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 draft of Appendix I for EU 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 Appendix 9.7 mentions that the monitoring location for active particles (microorganisms) in the A-level area should be the location with the highest 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 environmental pollution. We know that the surface of sedimentation bacteria contains a large amount of nutrient water, ranging from 10 microns to 20 microns. When sedimentation bacteria are exposed to critical A-level production environments, this nutrient water is likely to be blown onto sterile surfaces, medicine bottles, containers, and drug solutions, causing pollution 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. We usually consider the purpose of airflow protection in Class A laminar flow environments, and we also need to design isokinetic sampling for planktonic bacteria sampling to ensure that the air above the sampler is sampled at a constant velocity and will not cause pollution to the site.
The traditional microbial sampling method involves placing the entire sampler in an A-level environment, while modern aseptic process control methods require that any material or equipment entering the A-level environment be fully sterilized. The traditional method places the sampler in Class A, but due to the inability to thoroughly 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 complete sterilization. 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 in Class A and can be pre sterilized with high temperature or chemical sterilization, while the sampler should be operated in Class B background environment. In this way, the impact of incomplete sterilization of the sampler on the A-level area 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.