1. The different applications of bioburden assessment

The term bioburden is often used in different contexts and for different applications :

• Checking the bioburden before sterilization or aseptic filling for a sterile pharmaceutical product
• Checking the bioburden during production (In Process Control)
• Checking the bioburden before sterilization or in the context of the periodic validation of a sterilization process for medical devices
• Checking the bioburden of raw materials for pharmaceutical use
• Performance of bacterial counts for non-sterile products

In my view, the best definition of this test is a quantification of the total microbiological flora which includes :

• Total Viable Aerobic Count (TVAC)
• Total Viable Anaerobic Count (TVAnaC)
• Total Yeast and Mold Count (TYMC)

The search for anaerobic bacteria may be questionable. In all cases, a risk analysis should performed in order to define the flora to be researched.

2. Regulations relating to bioburden Several texts, guidelines or regulations frame the expectations of the evaluation of the bioburden (see bibliography). All these regulations define a framework to limit and control the maximum microbial load before sterilization. The main sources of contamination are :

• Raw materials
• The production environment
• The manufacturing process: impact of the presence of humans
• Pharmaceutical water

3. Bioburden limits In accordance with Annex 1 of the GMP: : “The bioburden (microbial contamination) must be checked before sterilization. A limit value must be set for microbial contamination immediately before sterilization, a limit which depends on the effectiveness of the method used. The determination of the bioburden must be carried out on each batch whether it is aseptically produced or sterilized in its final packaging.” The bioburden limit is therefore to be defined for each product and production. In the Note for guidance on manufacture of the finished product CPHMP/QWP/486/95 1996 two limit values are indicated :

• 10 CFU/100mL in the context of sterilizing filtration
• 100 CFU/100mL or g for heat sterilization

The revision of this guidance (EMA/CHMP/QWP/245074/2015) in 2017 indicates that the limits must be adjusted depending on the product.

4. Method for assessing the bioburden There is no specific chapter for the bioburden test. Generally the bioburden tests are carried out according to chapter 2.6.12 “Microbiological control of non-sterile products: microbial counting tests.” of the European Pharmacopoeia. The membrane filtration method allows the analysis of 100mL of product. In case the filtration of 100mL is not possible an analysis of a minimum of 30mL is possible with justification. After filtration, the filter is deposited on culture medium with casein and soy peptones, then incubated in the presence of air for DGATs and anaerobically for DGanaTs for 5 days at 30-35 ° C. For yeasts and molds three options are possible:

• Performance of the analysis in combination with the TVAC if the method has been validated in this context.
• Performance of the analysis in combination with the TVAC with incubation at 30-35°C then at 20-25°C if the method has been validated in this context.
• Performance of another filtration with filter being deposited on Sabouraud medium with incubation at 5-7 °C for 20 to 25 days.

5. Validation of the method By abuse of language, we speak of method validation for the bioburden test. In reality, it is an applicability test. In the applicability test of the method, reference microorganisms are added to verify that the method makes them visible. As a basis, the following reference microorganisms tested :

• Staphylococcus aureus
• Escherichia coli
• Bacillus subtilis
• Candida albicans
• Aspergillus brasiliensis
• Clostridium sporogenes (if anaerobic bacteria are being researched)

In addition to the reference microorganisms, it is recommended to test for endogenous microorganisms. These microorganisms generally arise from production (obtained during monitoring) or have triggered an out of specification result.

6. Bioburden assessment a test with shades of gray For bioburden analyses, the term assessment takes on its full meaning. There are indeed limits depending on applications or stages of manufacture, but bioburden is not a solely quantitative test. The identification of the bioburden is as important, as it helps to :

• Recognize the microbial flora
• Define the pyrogenic risk
• Define the risk of resistance to sterilization

7. Recognizing the microbial flora Bioburden assessment allows for better recognition and control of the microbial flora of the product. During the bioburden analysis of the first production batches, it is recommended that all microorganisms that are detected are identified. A complete identification will allow the normal flora of the product to be controlled and defined. The initial tests also allow a warning limit to be established. Periodic re-evaluation of the nature of the bioburden also allows for checks for changes in the latter and helps to define endogenous microorganisms.

8. Bioburden and pyrogenic risk Since the update of chapter 5.1.10 “Recommendations for carrying out the test of bacterial endotoxins” it is necessary to assess the risk relating to all pyrogenic substances. Microorganisms are a major source of pyrogens. The qualitative analysis of the bioburden helps to assess the pyrogenic risk. The presence of gram negative bacteria (e.g. Pseudomonas ou Enterobacteriaceae) may indicate a risk relating to endotoxins. The presence of Gram-positive bacteria (for example Staphylococcaceae, ou Bacillaceae), yeasts or molds may indicate a risk of non-endotoxin pyrogens (NEP).

9. Resistance to sterilization Microorganisms have varying resistance to sterilization. This resistance is characterized by the value of D “time required under defined conditions to reduce the population of microorganisms present by a factor of 10 (1 log)”. Spores of bacteria such as those of Bacillus generally have a high D value (greater than 1 min) while it is low or even very low for non-sporulating forms. The value of D in the moist heat of a microorganism can be evaluated using equipment of the BIER VESSEL type. Qualitative knowledge of the bioburden therefore also makes it possible to assess the risk before sterilization.