Production conditions

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The term bioburden refers to the microbial contamination of a medical device before its sterilisation. The bioburden load is the sum of all micro-organisms from different sources, including raw materials, the production of components, the assembly processes, the environment during production, the tools for assembly and manufacture, the purification process and the packaging of the finished product. Therefore, the bioburden determination of medical devices provides an opportunity to verify and evaluate the efficacy of all production hygiene steps on the final product.
It is not possible to describe accurately the microbial contamination in numbers, as many factors can affect the determination of the bioburden. In practice, a determination of the bioburden is carried out according to a documented procedure, which, in addition to the quantification or estimation of the microorganisms, also includes a characterisation of the population. Due to the large variety of materials used for the manufacture and design of medical devices and the associated different surface adhesion of bacteria, DIN EN ISO 11737-1 does not define a single method for the determination of the bioburden, but actually presents different process parameters.
The same applies to the vast number of different microorganisms which may be found on the medical devices. DIN EN ISO 11737-1 also provides a wide range of appropriate rinsing solutions and culture media, including incubation conditions in different combinations, to enable the application of the optimum conditions for the medical devices. The method used by GfPS consideres the different needs of the micro-organisms: Slow-growing fungi must be cultivated differently than fast-growing bacteria. Anaerobically growing bacteria can grow only under the exclusion of atmospheric oxygen.
One focus of DIN EN ISO 11737-1 is on the microbiological characterisation of the bacterial load. Hereby, changes in the microflora of the product can be detected, which may have an effect on the sterilisation validations. Using the GfPS method, the characterisation is routinely carried out during the quantification of the bacterial load.

The bioburden limits are determined by the manufacturer. Here, DIN EN ISO 11737-1 supports to define the alert limits. This first requires the determination of the mean and the standard deviation for each product group. A bioburden alert limit (or action limit) results by the sum of the mean and the double (or triple) standard deviation.

According to DIN EN ISO 11737-1, the validation of the bioburden method is mandatory. Two different methods to validate the bioburden recovery rate are presented in the norm:

  1. Validation using the exhaustive method repetitive recovery method (with a relatively high natural microbial contamination of the product)
    Three samples are repeatedly subjected to the selected bioburden method. A recovery rate can be determined from the ratio between the number of colonies after the first application and the total number of colonies.
  2. Validation using inoculated product materials (with a low natural rate of contamination of the product)
    Five (sterile) samples are contaminated with a defined spore count of e. g. bacillus atrophaeus and then subjected to the selected bioburden method after drying. The ratio between the number of spores detached from the product and the total spore count (with which the product was contaminated) then enables the determination of the recovery rate.
    A correction factor is determined from both described methods, which is multiplied by the bacterial counts of the routinely performed bioburden and results in the estimated values of the microbial load.
    A bioburden revalidation or new validation must be carried out in fixed time intervals according to DIN EN ISO 11737-1. The extent to which revalidation is to be undertaken shall be determined. This may also mean that a theoretical consideration is sufficient. Changes of the product and/or manufacturing processes must be reviewed and evaluated and may then lead to a revalidation.

The LAL test is conducted for all medical devices that are declared to be pyrogen-free. These are mostly medical devices that come into contact with blood or spinal fluid (= intrathecal administration). The LAL test is used for the qualitative and/or quantitative detection of endotoxins. The test is highly specific and very sensitive (approx. 100 times more sensitive than the rabbit fever test), so that no other groups of pyrogens can be detected. Therefore, an examination for the presence of pyrogens (via LAL test) after sterilization is a corresponding release criterion if the product is to be declared as sterile and pyrogen-free. The implementation of the LAL test is described in the Ph.Eur. in chapter 2.6.14 “Test for bacterial endotoxins” and in the USP in chapter <85> “Bacterial Endotoxins Test”.
Pyrogens are defined as fever-causing substances. Pyrogenic substances are heat resistant, dialysable (i.e. very small) substances from non-pathogenic and pathogenic bacteria, fungi and viruses. These are effective in very small quantities: approx. 0.2 µg / kg body weight cause fever in higher animals and in humans after intravenous injections.
Endotoxins are a group of pyrogens that have the highest efficacy. These are cell wall components of gram-negative bacteria (e.g. enterobacteria and pseudomonads). These are high molecular complexes consisting of a polysaccharide, protein and lipid component. The detection of endotoxins follows via animal testing (rabbit fever test) or biochemical testing (LAL test). As animal testing should be avoided if possible, a debate is currently ongoing as to whether the rabbit fever test should not be replaced entirely by the LAL test. However, the LAL test cannot always be implemented due to disturbance factors caused by certain materials that have inhibitory or enhancing properties. Therefore, validation of the LAL test is essential.
The number of samples for the LAL validation is described in the specifications of the withdrawn FDA guideline and the ANSI/AAMI ST72. The number of samples to be tested depends on the batch size:
Batch size / samples per batch

<30 / 2

30 – 100 / 3

> 100 / maximum of 10


However, at routinely conducted pool tests (= multiple items are pooled and tested in an LAL test), care should be taken that all the products have been considered in the validation. A total of three validation runs should be performed (for three different batches). As part of the validation test, the test on inhibition and enhancement is also carried out. At this, a comparison between an endotoxin dilution series in water and an endotoxin dilution series of the test solution (eluate or extract, dilution below the maximum valid dilution) is carried out. The sample is deemed to have no inhibition and enhancement if the results of the endotoxin-dilution series of the test solution do not differ from the endotoxin-dilution series in water by more than one dilution level. Otherwise, the interfering (disrupting) factors of the test for inhibition and enhancement have to be removed (neutralisation, filtration, heat treatment, further dilution, etc.).

GfPS carries out sterility tests according to ISO 11737-2 or the American Pharmacopoeia USP or the European Pharmacopoeia Ph. Eur. The number of samples is not defined for medical products in the Ph. Eur. According to USP, 2 × 20 samples (in a pool) should be examined. The ISO 11737-2 does not specify, but test sample numbers of 10 pieces are recommended, which are to be examined in a single test. The implementation of the sterility test is carried out according to the requirements defined in the pharmacopoeias (ISO 11737-2 refers to the USP).

For sterility testing of medical devices, the inoculation method (direct inoculation method) is normally used: The sample or samples to be examined are transferred to the liquid nutrient media and then incubated. For sterility testing of liquid products the membrane filtration method is normally used: The product or products to be examined are first membrane-filtered. After that, the filter is transferred to liquid growth media and then incubated. If the products show antimicrobial properties, the filter can be washed up to five times with 100 ml of rinsing solution each to wash out residues of the product before transferring to the nutrient solution.

For the pharmacopoeia tests two media are used: Tryptic soy broth (TSB) and thioglycollate broth (THB). The media themselves are checked prior to performing the sterility test for sterility and growth of certain test organisms. The culture media are incubated for at least 14 days at 30–35 °C (thioglycollate broth) or 20–25 °C (tryptic soy broth) respectively.

Tryptic soy broth (TSB) is used for sterility testing according to ISO 11737-2. This medium is also checked for sterility and growth of certain test organisms before performing the sterility test. The incubation then follows for 14 days at 30 +/- 2 °C.
During the 14-day incubation period, the vessels with the media are checked every weekday for macroscopically visible growth of micro-organisms. If the vessels show positive results before the 14th day, the incubation may be cancelled.
The Method Suitability Test is carried out to validate the test method after the end of the incubation or parallel to the sterility test. As the evaluation of the Method Suitability Test is based on the comparison of the growth behaviour of the test micro-organisms, a test is absolutely necessary in presence and absence of the product. Defined germ concentrations of < 100 CFU are used for the Method Suitability Test, the qualitative assessment follows by visual growth control. This test is always to be conducted when new products are to be examined or when a change has occurred in the experimental conditions. The incubation period of the Method Suitability Test is set at a maximum of 5 days.

Contact plates are suited for the determination of the microbial count on plain, dry surfaces as well as for personnel hygiene (textiles and gloves or hands).
The culture dishes for the contact plates have an inside diameter of 55 mm. The agar surface arches over the upper edge of the culture dish bottom. The culture dishes have a grid on the bottom with counting squares of 1 cm² in size, which can be used for later analysis.