Testing laboratory Microbiology
Accredited according to ISO 17025 for the testing of medical devices and pharmaceuticals.
Our microbiological testing laboratory is accredited according to ISO 17025 and has a GMP certification.
We offer comprehensive services in the field of microbiology. Thanks to our many years of experience and competence, we can provide you with complete solutions ranging from the development and validation of product-specific test methods to production monitoring and final testing of the products.
Our microbiologists will advise you on all aspects of sterilisation safety, the microbiological quality of your products, environmental hygiene and validation activities, testing methods, production monitoring and final product testing.
Our offered services cover the following methods:
- microbial count determination / bioburden determination on medical devices and pharmaceuticals according to ISO 11737 or Ph. Eur. 2.6.12 [more]
- Sterility testing of medical devices and sterility testing of pharmaceuticals according to ISO 11737 and Ph. Eur. 2.6.1, respectively [more]
- Validation of the microbiological test methods (microbial count determination and sterility testing) according to the ISO standards or pharmacopoeia methods mentioned above [more]
- microbiological validation of gamma sterilisation according to ISO 11137 [more]
- Validation of further sterilisation methods (e.g. steam sterilisation, non-standardised sterilisation methods according to ISO 17665, ISO 14937, ISO 14160) [more]
- Validation of aseptic production processes according to ISO 13408
- microbial identification [more]
- Test for bacterial endotoxins according to Ph. Eur. 2.6.14 or 5.1.10, according to ANSI AAMI ST 72 and USP Chapters 85 and 161 [more]
- environmental hygiene tests of air and surfaces in clean rooms (also on site) according to ISO 14664, ISO 14698 or EU-GMP Guideline Annex 1 (environmental hygiene) [more]
Microbial count determination / bioburden determination on medical devices and pharmaceuticals according to ISO 11737 or Ph. Eur. 2.6.12
The term microbial count or bioburden refers to the type and number of detectable bacteria and fungi that are present on a product prior to sterilisation.
To detect these, a sample is either dissolved in a suitable test solution or the microorganisms are rinsed off and then cultivated. The result is traditionally a number of grown colonies, which is why the result is expressed in colony-forming units (CFU).
In order to correctly determine the type and number of micro-organisms, a comprehensive validation of the determination method is necessary.
Sterility testing of medical devices and pharmaceuticals according to ISO 11737 and Ph. Eur. 2.6.1
We conduct sterility testing for our customers according to common pharmacopoeia methods for pharmaceuticals or according to ISO 11737 for medical devices. In doing so, we use the following processes:
- Direct immersion
- Membrane filtration
Sterility testing of medical devices is preferably carried out by direct immersion, in which the products to be tested are incubated for 14 days with a medium suitable for microorganisms at certain temperatures.
For sterility testing of pharmaceuticals and medical devices with growth-inhibiting influences, the pharmaceutical in the solution or an extract of the medical device is filtered directly through a membrane filter. This membrane filter is then incubated in a suitable medium for 14 days, as in the direct immersion process.
The visual evaluation of the sterility test for bacterial growth takes place after an incubation period of 14 days.
Validation of microbiological test methods (microbial count determination and sterility testing) according to ISO 11737 or Ph. Eur. 2.6.1 and 2.6.12
According to the intention of the pharmacopoeia, the test methods described therein are already validated. However, these methods must still be validated to see whether they are generally suitable for testing the specific sample.
In the microbiological test methods, this is done by a so-called growth suitability test, often also referred to as bacteriostatic/fungistatic test. It is tested whether certain reference strains show equally good growth reactions in the presence and absence of the product to be tested.
The ISO standard goes far beyond the requirements of the pharmacopoeia, as it does not only require a test of the growth conditions on the basis of reference microorganisms. In addition to the bacteriostatic/fungistatic test, a comparative microbial count is carried out using different nutrient media and incubation conditions to check under which conditions most microorganisms and their species are detected. Since medical devices (in contrast to pharmaceuticals) usually have to be extracted in order to remove the adhering microorganisms and transfer them into the test solution, it is usually necessary to check the microorganism’s removal procedure in order to determine the recovery rate and derive a corrective factor from this.
The test is particularly complex if the samples contain antibiotics, for example, or if they are sponge-like products. These types of products can worsen the process of removing microorganisms immensely. Products containing antibiotics can possibly cause toxic effects on the microorganisms during the detachment process.
Microbiological validation of gamma sterilisation according to ISO 11137
In addition to application-technical validation and dose distribution measurement, gamma sterilisation also requires the validation of microbiological sterilisation.
This is the minimum irradiation dose required to bring the product into the sterile state. The initial microbiological state - number and type of microorganisms - is determined for this purpose. On the basis of the initial state, a certain dose, the verification dose, is determined. The products are irradiated with this verification dose and then tested for sterility. If the verification dose has been successfully verified by the sterility test, the sterilisation dose shall be confirmed. In doing so, it can be guaranteed that the validated sterilisation dose reaches a Sterility Assurance Level (SAL) of 10-6.
The sterilisation validations in our company are based on the following standards: ISO 11137-1, ISO 11137-2, ISO 11737-1, ISO 11737-2 and EN 556.
As far as possible, we recommend the so-called VDmax25 method and method I. The VDmax25 method is suitable for the validation of sterilisation with a minimum dose of 25 kGy (the usual method) and a microbial count of not more than 1000 CFU/product unit.
In addition to the VDmax25 method, method I (dose setting) can also be applied. This method is suitable for determining a minimum irradiation dose necessary for successful sterilisation. With this procedure, lower sterilisation doses can be validated in the case of demonstrably low microbial counts.
Revalidation / dose of gamma sterilisation audits
In addition, we offer our customers sterilisation dose testing to prove consistent efficacy by means of a typically quarterly revalidation (dose audit). Compared to microbiological validation, we only need a fraction of the test sample here. The dose audit consists of a microbial count determination (bioburden determination) and a check of the verification dose determined in the validation.
Special requests regarding gamma sterilisation
At the customer's request, individual batch validations and VDmaxSD methods in accordance with ISO/TS 13004 can be carried out in our company.
Building on our numerous years of expertise, we can advise you comprehensively on the selection of the most suitable validation method for your product at any time.
Validation of further sterilisation processes
At our company, we have extensive experience with
- general sterilisation validation according to ISO 14937
- sterilisation with liquid/chemical sterilisers according to ISO 14160
- validation of steam sterilisation according to ISO 17665
- validation of dry heat sterilisation according to ISO 20857
We will also support you in the development or validation of further new or non-standard sterilisation processes. You can thus benefit from our extensive experience. For example, we can support you in analysing the microorganisms of the product-specific bioburden that are hardest to inactivate and determining their inactivation kinetics. Building on this, the validation of the sterilisation process can then be tackled.
Efficient handling of unusually high or inexplicable contaminations is only possible once the microorganism has been identified correctly.
We offer the following identification methods:
- Testing for anaerobic bacteria
Here the bacteria are cultivated under anaerobic conditions and under aerobic conditions and thus the presence of anaerobic bacteria is excluded or confirmed.
- Microscopic identification
This method includes macroscopic and microscopic assessment, gram staining, spore staining plus mould identification.
- Biochemical identification
In a biochemical identification using the analytical profile index (API), the growth with different substrates is tested and whether certain enzymes are active.
Test for bacterial endotoxins and pyrogens according to Ph. Eur. 2.6.14 or 5.1.10, according to ANSI AAMI ST 72 and USP Chapters 85 and 161 and monocyte activation according to Ph. Eur. 2.6.30
BBF Sterilisationsservice GmbH has many years of experience and proven competence in the detection of endotoxins and pyrogens and carries out these tests for you in accordance with the applicable standards and regulations.
Pyrogens are substances which, even in small concentrations, trigger fever reactions, a drop in blood pressure and life-threatening shock in both humans and animals. The most important group of pyrogens are lipopolysaccharides from the cell membrane of gram-negative bacteria, usually referred to as endotoxins, which are released when the microorganisms die or the cell membrane disintegrates. They are very thermally stable and cannot be removed or inactivated by conventional sterilisation processes.
In addition, there are also a number of other pyrogenic substances, e.g. lipoteichoic acid of gram-positive bacteria, viruses, RNA, etc.
For the endotoxin determination the units are indicated in endotoxin units per ml (EU/ml). For the pyrogen determination the amount is indicated in endotoxin-equivalent units per ml (EE/ml). The determined endotoxin content is compared with the applicable limit value or the one specified by the customer.
We offer the following endotoxin and pyrogen determination methods as limit or quantitative tests:
The Monocyte Activation Test (MAT) detects proinflammatory cytokines with high sensitivity, which are released by monocytes in the blood after contact with pyrogenic substances and then trigger the fever reaction. Compared to the rabbit test, the MAT has the advantage that it works with human blood or primary blood cells or with human monocytic cell lines and therefore does not require test animals. For our test, we use qualified peripheral mononuclear blood cells (PBMCs) from at least 4 donors and check the presence of pyrogens through the release of the cytokine Interleukin-6 (IL-6).
In the context of the pyrogen determination it is possible to indicate the pyrogenicity by the amount of endotoxin-equivalent units per ml (EE/ml). Within the monocyte activation test it is possible to investigate the potency and presence of different pyrogens (e.g. lipopolysaccharides, lipoteichoic acid or non-bacterial endotoxins).
According to EU Directive 2010/63/EU on the protection of animals used for scientific purposes, preference should be given to alternative methods over animal testing methods. For example, according to the European Pharmacopoeia, chapter 2.6.8, the rabbit pyrogen test should be explicitly replaced by the monocyte activation test according to Ph. Eur. 2.6.30.
Since the majority of pyrogens are due to endotoxins, especially lipopolysaccharides (cell wall components of gram-negative bacteria), the Limulus amebocyte lysate test (=LAL test) was developed for their detection. For this test components from the blood of the horseshoe crab are used which coagulate on contact with lipopolysaccharides. The test is carried out according to the Ph. Eur. 2.6.14 and USP chapter 85 as well as in the field of medical devices according to ANSI AAMI ST 72 and USP chapter 161.
We mainly conduct the test as a limit value test. This means our customer defines his limit value, e.g. 20 EU per product unit and we test dilutions below this limit value to ensure the acceptance criteria. The endotoxin content can optionally be determined quantitatively in our laboratory.
In order to produce lysate, the animals must undergo a risky blood sampling procedure. In addition, the lysate reacts not only to lipopolysaccharides but also to beta-glucans. These are polysaccharides found in the cell walls of plants, bacteria and fungi, such as cellulose or chitin.
That is why biotechnologically produced equivalents have been preferred in recent years. See recombinant factor C test in this context.
We offer testing for the detection of bacterial endotoxins by means of the Recombinant Factor C test. According to Chapter 5.1.10 of the European Pharmacopoeia, this is an alternative method to the Limulus amebocyte lysate test.
We offer two systems of the Recombinant Factor C test: the typical Recombinant Factor C test and the ELISA (enzyme-linked immunosorbent assay) based test for the elimination of interfering factors in advance. Both test systems offer the option of quantitatively determining the endotoxin amount.
In the case of the Recombinant Factor C test, the decisive enzyme, the so-called Factor C, which can usually be found in the blood of horseshoe crabs, is biotechnologically produced. The pure enzyme eliminates false-positive results, which may result from beta-glucans, for example.
The advantages and disadvantages of the single detection methods are provided in the following table:
Validation of pyrogen and/or endotoxin determination
For the validation of the pyrogen and/or endotoxin determination, if possible three batches of a product are tested according to chapter 5.1.10 of the European Pharmacopoeia. As per ANSI AAMI ST72 two to ten products per batch are tested and three to ten products are to be inspected as a merge sample according to USP chapter 161.
The interference factor analysis is important for the detection of endotoxins and pyrogens. The goal here is to demonstrate that the detection system is not disturbed in the presence of the product. For this purpose, a known amount of endotoxin is added to the preparation and then checked to see whether this amount can be detected. The recovery should be between 50 and 200 % to be able to rule out interfering factors.
Environmental hygiene tests of air and surfaces (also on site) according to ISO 14644, ISO 14698 or EU-GMP Guideline Annex 1 (environmental hygiene)
Sterile medical devices and pharmaceuticals are manufactured in cleanrooms. The hygiene in cleanrooms plays an important role here and is regulated by the ISO 14644 and ISO 14698 standards as well as in Annex 1 of the EC Guide to Good Manufacturing Practice (EU-GMP).
Various methods, such as air particle measurements, air microbial count or surface contact checks are carried out for the hygienic examination of cleanrooms.
You can carry out the sampling for the microbiological tests yourself and send us the prepared samples for evaluation, or you can commission us to carry out the complete procedure including sampling on site at your company.
Your contact persons
Should you have any questions about our services, please do not hesitate to contact us.