The aim of this sterilization validation is to determine the dose required to achieve a sterility assurance of 106. The probability
of sterilization is commonly assessed by bioburden determination (determination of the bacterial and fungal load), as recommended
by ISO 11137. This involves the following four steps.
Determination of the bioburden of the product. This is normally done by taking 10 units per batch from three different batches
of product, post-irradiation. In doing so, it is important to be sure that the product that was subjected to gamma radiation
was representative of the product normally manufactured. The manufacturer of the product normally carries out the bioburden
determination. It is important to ensure that the bioburden recovery method is accurate because insufficient recovery of microorganisms
during bioburden tests would result in an underestimation of the true bioburden of the product and lead to an inadequate sterilization
dose applied to the product.
The bioburden of the product is dependent upon several factors. These include the nature and source of the raw material, the
components used in manufacturing, the product design and size, the manufacturing process, the manufacturing equipment, and
the manufacturing environment (e.g., the type of cleanroom used). For products manufactured using approved suppliers and assembled
within cleanrooms certified as ISO 14644 class 7 under localized unidirectional airflow protection, the expected bioburden
would be relatively low (e.g., not more than 10 CFU per device). Additional data relating to the risk from the manufacturing
environment can be provided through microbiological environmental monitoring.
There are different methods for bioburden determination. One of the most common methods is the repetitive (exhaustive) recovery
method. This method involves washing the sample product repeatedly until it is estimated that no further microorganisms will
be recovered. The washing process can include the addition of sterile glass beads or ultrasonication to facilitate microbial
recovery. The eluent from the washing should be tested using an appropriate TVC test method (where membrane filtration is
the method of choice, followed by the pour plate technique).
The microbial counts from all washes are compared in order to assess the total bioburden. Such extraction methods require
validating. Method validation involves deliberately inoculating a sterile disposable item with a known number of microorganisms
and then assessing the number recovered from the washing steps to the theoretical inoculum challenge. A valid method should
recover the microbial challenge as per the guidance in USP <1227> for microbial method validation. Where the challenge cannot be recovered, one factor may be the variation with the
process of drying the microbial challenge organism onto the plastic item prior to washing (23).
Calculation of appropriate dose based on the resistance of an identified microbial population. This is based on the total
number of bacteria and fungi isolated and an assessment of the types of species recovered, as characterized using microbiological
identification techniques. This is an important distinction as it is not simply the total numbers recovered as some microorganisms
have a theoretically greater resistance to gamma radiation processes than others, most notably Streptococcus faecium and Micrococcus
Radiation dose assignment. To select the appropriate radiation dose in relation to the theoretical resistance of the microorganism,
ISO 1137 provides a guidance table.
Validation of calculated dose. As part of the validation, the calculated dose is verified by selecting an appropriate sample
size (normally 100 units of the product) to determine if the dose is efficacious. The test is undertaken by placing individual
units of product into sterile bottles containing microbiological culture media (e.g., soybean casein digest medium) and incubated
for 14 d. This is the "sterility test," although it bears some similarities to the direct inoculation sterility test described
in the US or European pharmacopoeias, it is not equivalent (25). Any bottle of media that exhibits microbial growth (turbidity)
is indicative of the product not being sterile and that the sterilization cycle is inappropriate for the product.
As with the bioburden determination method, the test for sterility requires validation. The object of the validation is to
show that the product material does not inhibit the growth of microorganisms (a method suitability test). Inhibition of microorganisms
would lead to the risk of a false negative result occurring. The method suitability determination involves using the same
type and volume of culture media used for the sterility test. The product is inoculated with known numbers of a bacteria culture
and a fungal culture. The inoculated product is then incubated. Any product that shows no growth or slowed growth is considered
bacteriostatic or fungistatic and the method declared unsuitable (26). Within the medical device industry, an acceptance criterion
of a sterility assurance level of 102 has been used (i.e., two units could fail the sterility test and results could be deemed
as acceptable). This level of assurance is unacceptable for single-use technologies used in conjunction with aseptically-manufactured
products. In such cases, further modification of the method is required, such as increasing the volume of culture media or
using culture media with an added neutralizer.
There are complications with the testing of large products for the bioburden and sterility tests. To carry out tests as commonly
practiced, disassembly (i.e., "sectioning") would be required so that the product can be immersed into microbiological culture
media (i.e., broth). This is an activity that could result in adventitious contamination due to the need for personnel to
manipulate the product. With such devices, an alternative approach is more often adopted that involves sterility of a product's
fluid path (i.e., passing a sterile buffer through the product and examining it for microbial growth). The test is assessed
by incubating the media in the presence of the product (or product rinse) for 14 days and examining the broth, post-incubation,
for microbial growth.
When assessing the data to determine the final sterilization dose for routine batches of single-use products, it is normal
to use the highest dose (Dmax) established in the validation in order to set a level of overkill. This provides additional
assurance should the product bioburden shift upwards or should more resistant strains appear.
A further important consideration is that the bioburden test and sterility test are often carried out by the product manufacturer
and gamma irradiation plant, respectively. In order that the data are comparable, it is important that similar test methods,
microbiological culture media, and test incubation parameters are employed. If this is not done, then the reporting of a sterility
test as zero microorganisms may be incorrect if the sterility test method is not capable of detecting all of the microorganisms
detected in the bioburden test. This can arise if different microbiological culture media are employed or if the medium is
incubated at a different temperature for a shorter time period. The sterility test result may then be due to the inability
of the organism to grow under the test conditions rather than an indication that the organism has been destroyed by the sterilization
Variations to the bioburden method are permitted within the ISO 11137 standard, such as the VDmax25, which permits fewer units
of product to be tested and for similar types of product items to be grouped together for the validation (for 25 kGy doses).
This variation can only be used where it has been established that the bioburden level is relatively low (at less than 1000
CFU per device). When considering whether different products can be grouped together, account must be made of the plastic
materials, construction processes, surface area and handling.