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/)
Posted by Dr. Tim Sandle
