Bioburden Testing 101

August 11, 2023

Bioburden is the term used to describe microbial contamination of a product prior to sterilization. Microorganisms can be introduced at any point during the manufacturing process through raw materials, the environment,humans, or during the final stage of cleaning and packaging. Since the sources of contamination can vary widely, a product's bioburden can fluctuate from batch to batch, routine bioburden testing is recommended [1].

Bioburden testing analyzes a product to quantify viable (living) microorganisms thereby providing a measure of microbial contamination — in colony forming units (CFUs) — within the product [1][2].

Bioburden Testing Applications
Bioburden testing is most commonly performed by the pharmaceutical and medical industries for quality control testing of pharmaceutical and medical products. Bioburden testing ensures that pharmaceutical products and medical devices meet the stringent health and safety regulations required by Title 21 of the Code of Federal Regulations [3] in the USA and by ISO 11737 [4] worldwide [1].

Conducting a Bioburden Test
A bioburden test is conducted using culture media appropriate for the type of microorganism being targeted, and typically involves two primary methods to measure the total viable aerobic count: 1) total aerobic microbial count (TAMC), which is used to measure aerobic mesophilic microbes; and 2) total yeast and mold count (TYMC), which is used to measure aerobic mesophilic fungi [2]. Casein soybean digest and sabouraud dextrose based culture media are recommended for TAMC and TYMC counts respectively [2][5].

Bioburden Testing Methods
In order to estimate the bioburden load of a product, a sample needs to be prepared according to its physical characteristics, i.e. whether the sample is water soluble, fatty or non-fatty, or in aerosol form. Once the sample is prepared, there are two primary methods used to quantify the microbial load: the membrane-filtration method and the plate count method.

When using the membrane-filtration method, the sample is filtered through a membrane with a pore size of ≤ 0.45 micrometers to isolate the microorganisms from the sample. The filter membrane containing the isolated microorganisms is then placed onto the casein-soybean digest agar and incubated for at least five days at 86-95°F (30–35°C) in order to detect bacteria. To detect fungi, use sabouraud dextrose based culture media and incubate for five days at 68-77°F (20–25°C) before counting the number of colonies [1][2].

When using the plate count method to detect bacteria, the sample product together with melted casein-soybean digest agar is poured into a petri dish and allowed to solidify. It is then incubated for five days at 86-95°F (30–35°C) before the TAMC is recorded. For fungi, use sabouraud dextrose agar and incubate for five days at 68-77°F (20–25°C) before the TYMC is recorded [1][2].

A third method, the most probable number (MPN) method, is a statistical method used to estimate the number of viable microorganisms in a sample. It is considered less accurate than the above two methods, but it can be used to test products where the bioburden is determined to be too low to provide reliable counts using the above methods, or when neither of the above methods are available [1][2][6].

The recorded bioburden load is expressed in colony forming units (CFUs), which must be within the established safety guidelines for that particular product. The maximum allowable CFUs safety limits for bioburden testing can vary depending on the specific industry, product, and regulatory standards. In the pharmaceutical industry, bioburden limits for different products and materials are usually specified in pharmacopeial standards such as the United States Pharmacopeia (USP) or the European Pharmacopoeia (Ph. Eur.).[7] These limits dictate the maximum allowable CFU counts for specific products, depending on their intended use and route of administration. For example, injectable products typically have stricter limits compared to topical products. Below are some examples of the maximum CFU safety limits for bioburden testing in the pharmaceutical industry set by the United States Pharmacopeia (USP) [8]:

Pharmaceutical Industry (USP <61> and <62>): In the United States Pharmacopeia (USP), the microbial limits for non-sterile pharmaceutical products are specified in chapter <61>, while the limits for sterile products are specified in chapter <62>.

For example:
Oral and topical non-sterile products: Total Aerobic Microbial Count (TAMC) ≤ 1,000 CFU/g or mL, and Total Yeast and Mold Count (TYMC) ≤ 100 CFU/g or mL
Sterile products: TAMC and TYMC ≤ 10 CFU/100 mL.

It's important to note that these are just examples, and the actual allowable CFU limits for bioburden testing varies from industry to industry, and will depend on the specific product or material being tested, its intended use, and the regulatory requirements in the relevant region. Manufacturers are responsible for determining and complying with the applicable standards and guidelines specific to their industry and product. Consulting the appropriate pharmacopeias, regulatory authorities, and industry-specific standards is essential to ensure compliance with the required safety limits for bioburden testing.

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