The results of bioburden testing provide manufacturers with a ‘snapshot’ in time but the problem is that the situation can change without warning. A manufacturer could be presented with a bioburden result that fails the action limit seemingly for no apparent reason. Due to the nature of microbiological testing, the bioburden result may be discovered only after the medical device has been sterilised. So just what are the implications for the manufacturer of a failed medical device bioburden result, given that most sterilisation methods are validated on an overkill approach, having a high level of conservativeness built in?
Bioburden testing determines the microbiological loading of a medical device that is a combination of the microbial content of raw materials, storage conditions, manufacturing environment and cleanliness, manufacturing process steps and personnel. As long as the bioburden result remains consistent and within defined limits, it indicates there has been no change in any aspect that could lead to increased microbial contamination, especially as the bioburden represents the microbial challenge to the sterilisation process. To comply with the standard, ISO 13485 Medical devices – Quality management systems – Requirements for regulatory purposes, all non-conformances must be investigated, and this includes bioburden results. If the issue is serious enough or continual bioburden excursions are noted, this may require converting to a CAPA (Corrective Action and Preventive Action).
Investigating bioburden excursions for medical devices can be problematic as the issue is reactive in nature, on an event that has already occurred. Root cause analysis is dependent on the use of good problem solving tools – for example, the Ishikawa or fishbone diagram in Figure 1 with some suggested topic areas – utilising a multidisciplinary team. The idea is to try and identify if there has been a step change in the process that has led to the bioburden excursion. Namely, is this a one-time random event or is it due to a consistent source of microbial cross-contamination? The investigation will probably require additional testing at incoming goods and all stages of manufacture and this is usually time consuming and costly. Previous risk analysis should have identified typical sources of microbial contamination that require control; even a small change in a process may have a large microbial effect.
The types of micro-organisms prevalent on the medical device are a useful indicator of the potential source of the contamination. For example, Staphylococci or Micrococci are indicative of personnel contamination from a handling perspective, or Gram negative micro-organisms are indicative of water-borne contamination. The types of micro-organisms present may determine the effectiveness of the sterilisation process. Microbiological characterisation of the product bioburden is an essential requirement of ISO 11737-1:2006 Sterilisation of medical devices – Microbiological methods – Part 1: Determination of a population of microorganisms on products. The methods used to characterise the type of micro-organisms present on a medical device are listed in Table 1.
| Table 1: Microbial characterisation methods |
| Staining properties |
| Cell morphology |
| Colony morphology |
| Use of selective culturing |
| Biochemical properties |
| Genetic sequence data |
The first three methods (staining properties, cell morphology and colony morphology) are easy, quick and inexpensive to perform within the microbiology laboratory, providing adequate determination of micro-organism identification to the genus or family level for routine bioburden testing. The three remaining microbial characterisation methods (use of selective culturing, biochemical properties and genetic sequence data) are more time-consuming, require specialist equipment or media and are therefore more expensive. However, they can determine the identification of the micro-organism to species or sub-species level, providing more detailed analysis.
Genetic sequence data relies on a database of microbial species for comparison, to determine the microbial identification. Typically the application for this type of database has been for the medical or food industry. Non-pathogenic or environmental isolates that may be present on medical device bioburden may not be incorporated into the genetic database, rendering the use of this technique of limited value. The specific methods used will be determined by the capability of the microbiology laboratory and the specific risks to the safety and performance of the final medical device (for example, species of Gram negative bacteria liberate endotoxin on their death) or as an aid in investigating bioburden excursions.
The most likely root causes should be addressed through action plans prior to moving to verification and closure
Notified Bodies will expect to see a thorough investigation with appropriate action plans generated to control the bioburden excursion issue if, as typically happens, a root cause cannot be identified. The most likely root causes should be addressed through action plans prior to moving to verification and closure. This can be challenging if the source of the contamination is identified at the supplier level, where additional manufacturing controls may be difficult to assert, as the benefit to the supplier is of little value to them. Alternative methods of controlling the raw material bioburden may be required – for example, decontamination steps prior to use in manufacture.
The final issue from a high bioburden revolves around the impact to sterilisation. There are a number of sterilisation methods utilised by medical device manufacturers. The most common ones are:
- Irradiation (Gamma, Electron Beam, X-ray)
- Ethylene oxide
- Steam (including air ballasted steam)
- Gas plasma
All sterilisation methods require validation to demonstrate process parameters with a specified load configuration, to ensure a minimum sterility assurance level (SAL) of 10-6 according to EN 556-1:2001 Requirements for medical devices to be designated Sterile – part 1: Requirements for terminally sterilised medical devices. For medical device sterilisation, gamma irradiation and ethylene oxide are the predominant sterilisation methods of choice. For ethylene oxide and gas plasma, the most popular method to validate the sterilisation process is by using an overkill approach with biological indicators having a known resistance to the particular sterilisation method under investigation, e.g. Bacillus atrophaeus for ethylene oxide sterilisation.
Validation of steam sterilisation also often uses biological indicators, but sometimes this is accomplished simply through physical parameters. For irradiation sterilisation, once the minimum sterilisation dose has been established, dose audits are required to demonstrate continued compliance to the minimum sterilisation dose as detailed in ISO 11137-2:2013 Sterilisation of health care products – Radiation, Part 2: Establishing the sterilisation dose.
When an increase in the normal medical device bioburden occurs, there is the potential that the number or the types of sterilisation-resistant micro-organisms will also change. Any change could have an impact on the effectiveness of the sterilisation validation. For irradiation sterilisation this could be observed as a dose audit failure but would not necessarily be visible to the other overkill sterilisation methods without additional testing.
Figure 2: An increase in the normal medical device bioburden level means there is a risk that the number (or types) of sterilisation resistant micro-organisms could change too
The additional testing requirement for sterilisation validations includes assessing the medical device natural product bioburden for resistance to the sterilisation process and biological indicators, also known as a sub-lethal cycle where reduced exposure time is used to challenge the medical device and then a sterility test is performed. This is run in conjunction with the biological indicators as utilised in the sterilisation validation. This testing indicates if the bioburden contains more sterilisation-resistant micro-organisms than the biological indicators used in the sterilisation validation. As long as the biological indicator remains more resistant than the medical device bioburden, the sterilisation validation remains valid.
If ethylene oxide is utilised as the terminal sterilisation method then natural product bioburden resistance testing is required to be completed at defined intervals to ensure the continued suitability of the biological indicator, as detailed in ISO 11135-1:2007 Sterilisation of health care products – Ethylene oxide – Part 1: Requirements for development, validation and routine control of a sterilisation process or medical devices. In addition, sterilisation requalification may be necessary due to an increase in product bioburden to demonstrate continued effectiveness.
The (larger) more serious implications for medical devices sterilised by irradiation is where there is a failed dose audit, depending on the number of failures identified. It may require an augmentation of the sterilisation dose to continue sterilising, or even ceasing sterilisation. Of particular concern is where a VDmax dose audit failure occurs, which may require re-establishment of the sterilisation dose by an alternative method, as detailed in the ISO 11137-2:2013 standard. The manufacturer cannot re-establish using VDmax but must choose another method, e.g. method 1 – this has both financial and lead time implications for the manufacturer.
Regulatory authorities will require a medical device manufacturer to assess the impact of bioburden excursions on the final product sterility
Regulatory authorities will require a medical device manufacturer to assess the impact of bioburden excursions on the final product sterility. For medical devices terminally sterilised by irradiation, this has been incorporated into the ISO 11137-2:2013 standard, section 10.4: ‘…the effect of processing product at the sterilisation dose that has failed sterilisation dose audit on the achievement of the specified SAL for previously processed batches of product shall be considered and a risk assessment undertaken on their suitability for use.’
The impact for the medical device manufacturer, particularly if it has reduced its dose audit frequency, could be that a large number of batches and device types could be affected and would therefore require justification for why a voluntary product recall was not instigated for a potential sterility issue.
Medical device manufacturers should recognise the importance of bioburden testing in relation to the overall final safety and performance of the medical device, ignoring results at their peril. Bioburden results indicate the level of control that the manufacturer has achieved over microbial contamination through the materials, manufacturing, personnel and environment. Bioburden excursion investigations can help pinpoint the source of the contamination and identify step changes in processes or materials that may have been thought minor at the time of initiation. Not performing bioburden testing at a suitable frequency can lead to manufacturing supply issues and irreparable customer confidence in a medical device when things go wrong.