Approaching bioburden testing: Pointers for a successful approach

 

Bioburden refers to the microbial content of a material (or on the surface). It can also refer to the microbial levels within the environment. For non-sterile products, understanding the level and types of microorganisms together with the route of administration and intended patient population is of great importance. For sterile pharmaceutical products knowing the bioburden during manufacturing, from the cleanroom environment, and immediately prior to sterilisation are of importance in order to have confidence that the sterilisation step will be effective or that aseptic filling begins with an unadulterated bulk. This article considers the scientific basis to bioburden testing.

By Tim Sandle

In relation to assessing the level of microbial content during processing, bioburden testing refers to an estimation of the numbers of bacteria and fungi present in a liquid sample. This is commonly assessed using the Total Viable Count (TVC) method, whereby a portion of the material is plated out onto agar or mixed with agar and the incubated to assess microbial growth. 

 

To assess growth the common TVC methods are membrane filtration (where a portion of material or a rinse of the material is passed through a microbially retentive membrane filter); pour plate; and spread plate. Due to the larger size of material that can be tested and due to the relative ease of eliminating any antimicrobial activity, membrane filtration is the method of choice and the other methods are deployed only where a material cannot be filtered. With the advent of rapid microbiological methods, alternatives are available to some of these traditional growth-based methods.

 

With the conventional methods, the method of reporting a result from a test is by colony forming units (CFU) per unit of testing (typically CFU per mL or per 100 mL). A colony forming unit does not necessarily equate with a number of microorganisms present in the sample but it does provide an indicator of the bioburden load. This is because a colony forming unit (CFU) is an estimate of one or more microbial cells which, on the introduction of microbial growth media, can form macro-colonies under the conditions of the test. One colony forming unit is expressed as 1 CFU.

 

In relation to pharmaceutical processing, bioburden testing is implemented in order to assess the quality of the starting materials and to track process hygiene as the product is being manufactured. With non-sterile products a bioburden assessment is additionally required of the final product. Here it is expected that such products will contain a level of bioburden, therefore what matters is how many microorganisms are present and which species are present. With sterile products an assessment is required up until the point where a product is sterilised (either terminally sterilised or filtered and then aseptically filled).

 

In drawing up a bioburden test regime a number of factors need to be considered. These include:

 

• The time of sampling (in order to assess process hold times, sampling normally occurs at the end of the process in order to assess any growth of microorganisms).
• Sample homogeneity (where possible, samples are taken from a homogenous bulk. This means, that where there is a process mixing step, samples are taken post-mixing).
• Sampling handling and aseptic technique.
• The use of sterile sampling containers.
• Selection of test methods and verification of method suitability (this includes the appropriate test agar and incubation times).
• The expiry time of the sample (what is the last point in time when the sample can be tested and a ‘valid’ result be obtained?)
• Assignment of alert and action levels.

 

In relation to the environment, this is assessed through microbiological monitoring using active air-samplers, settle plates and contact plates, supported by particle counting.

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)