GxP-Blog

Evidence of microbiological germs in aseptic or sterile production usually signifies a high risk to the health of the patient. As part of the risk-based approach, it is necessary to assess whether this is a one-time (germ) event or a systemic error that makes recurrence probable. This also gives quick rise to the question of how the germs entered the production facility, the production equipment and, as the case may be, the medicinal product.  A systematic root cause analysis can help to identify the source of the germs. The start of this analysis can be a germ differentiation, supported by the evaluation of an experienced microbiologist. Furthermore, an evaluation of the associated production processes, technical risks, and ambient conditions is just as important as the rapid identification of correlations. With the latter, having a broader knowledge of the possibilities of occurrence of bacterial contamination and effective countermeasures, is helpful. And for some germ findings, there are sometimes surprising explanations.

The sterile production of medicinal products is subject to strict requirements. In order to adequately ensure the sterility of medicinal products, appropriately designed buildings, technologies, manufacturing equipment, suitable and well-defined manufacturing processes, as well as qualified and trained personnel, are required for production. The safety that is required of the medicinal product in terms of sterility depends on the sterility acceptance level, abbreviated SAL (Sterility Assurance Level), that is prescribed for the respective medicinal product`. For example, parenteral nutrition solutions intended for venous infusion have a sterility acceptance level of SAL = 10^-6. This means that a maximum of one in every one million product units is potentially contaminated with germs.

The high level of safety required can be achieved, among other things, with terminal sterilization. In this process, the product solution or substance, which may still contain a bacterial load of up to a defined maximum value, is sterilized at the end of the manufacturing process in a closed container, in a manner that ensures the desired low SAL.

It is usually more complicated for products that are manufactured aseptically and cannot be terminally sterilized. This applies, for example, to products or product ingredients that are thermally unstable. In such manufacturing processes, the product is composed, mixed or filled with already sterile components. This production on the open product must be conducted under very controlled conditions, as the required SAL must also be observed here. One such manufacturing process would be, for example, the filling of sterile injection solutions into ampoules or syringes.

But what should be done if the microbiological checks of the ambient conditions during production, or even of sterility samples taken from the product, show a bacterial load that poses a critical risk to the treatment of patients with the product? What should be done if germs are detected on surfaces in contact with the product or in the immediate vicinity of the open product?

As with any deviation or unexpected event during manufacture, it is also necessary to describe the deviation event and the thus-associated circumstances as precisely as possible. A successful root cause analysis can only be carried out on the basis of this description.

As a rule, bacterial loads arise when either a.) increased numbers of germs are introduced into a production plant from the outside, or b.) germs multiply within a production plant. In the first case, germs are usually carried into a production facility via persons/employees or materials (e.g. production materials). In the second case, liquid residue in production systems, for example, can promote bacterial growth.

Differentiation (microbial determination) of the microorganisms found can provide initial indications of their origin. These include typical human skin germs such as Micrococcus luteus, Staphylococcus epidermidis (90%), and other coagulase-negative staphylococci, Staphylococcus aureus, Corynebacteria and Propionibacterium acnes. Single cell fungi (molds or yeasts) in turn can be interpreted as indicators of insufficient fungicidal disinfection or mold processes.

One should also not be surprised when meeting "Conan the bacterium“. This bacterium, with the scientific name Deinococcus radiodurans, exists comfortably under ionizing radiation. In such a case, the possibility that this bacterium has been introduced into the production process through production materials sterilized by radiation, should not be ruled out.

The presence of typical agricultural micro-organisms has already been observed in rural production facilities. This can be an indication that germs are increasingly being brought into the production area by employees, especially if these employees work part-time on agrarian or livestock farms. Occasionally, certain bacteria species from the agricultural sector are difficult to remove once they have settled into a production plant (e.g. pipes and containers). Early detection in such plants is also extremely important, to be able to take suitable countermeasures in good time.

If causes cannot be directly and clearly identified, an extended root cause analysis must be conducted. Here, the well-known cause-effect diagram according to Ishikawa (fishbone diagram) usually proves to be suitable for use. It graphically lists the main influencing variables and the associated specific causes, and facilitates a systematic approach.

In principle, microbial findings are to be assessed as critical with regard to sterility. However, as explained above, sterility means the probability of occurrence of microbiological germs. The occurrence of a single finding/germ in a production plant cannot be completely ruled out.

Thus, the question of whether this involves a single event, or a systemic event that is likely to recur, will decide whether production should be continued or stopped.

Microbial findings do not always mean that the drug batches produced are actually contaminated with germs. If the findings are based, for example, on incorrect handling during sampling and originate from the sampler itself, this cannot initially be distinguished from a "real bacterial load“. However, in order to "save" the affected batches, a substantive and complete verification must be conducted, showing that the germs found can indeed be traced back to this exact secondary cause.   

In order to provide evidence, it is first necessary to determine whether the microbial findings can be explained in terms of time through the sampling by a specific person. Microbiological verification must also be carried out. Ultimately, it must be demonstrated that the germ spectrum or specific germ (if applicable) found, clearly points to the sampler. A specific DNA analysis of the germ (differentiation by PCR) can be useful. Practical experience also shows, however, that such rigorous proof is not always possible.

With regard to sampling and hygiene monitoring, it is necessary to consider that humans have different skin types and different spectra of skin germs. This means that different numbers of skin particles and germs will appear on different individuals. Seasonal fluctuations can also occur if the skin becomes susceptible to scaling due to winter heating. Employees with an excessively high particle and germ separation are not suited for hygiene monitoring, sampling in sterile production, or generally for working in certain clean room areas. They should perform other type of work. If recurring bacterial findings correlate with the presence of certain persons during sampling, this shows that there could be problems with bacterial loads in individuals.

It is not just humans who can be the source of secondary germs on sterile samples. The materials used for sampling, e.g. contact agar plates, could already contain faulty germs before the sampling process is initiated. There are well-known cases of such a situation, even involving multi-packaged sheets. If there are indications that this is the case, negative controls should be conducted for confirmation purposes through incubation of unsampled plates, and the manufacturer should be notified immediately.

Microbial findings can often be attributed to improper disinfection and sanitization measures. These can affect, for example, the surfaces of the production equipment as well as the surrounding rooms. A prerequisite for appropriate cleaning and disinfection is validation of the respective procedures. It is also often forgotten that the detergents and disinfectants used must be validated in relation to the relevant surface materials. However, this alone is not enough. Those employees who perform these procedures must be qualified and well-trained. Incorrect preparation of a solution for room disinfection can have far-reaching consequences for the hygiene conditions of an entire production plant, especially if this error repeats itself continuously.

In those cases in which the origin of the germs cannot be clearly established, boosted cleaning and disinfection does not help much as it cannot be performed in a targeted manner. It is important to identify the source or cause of the germs or bacterial accumulations, and to identify the path of transmission of the germs. Only in such cases can effective measures be established. First, the causes of the germs should be eliminated to the extent possible, thereby reducing the likelihood of recurrence and the bacterial load. In addition, it is important to decide whether changes to the cleaning and disinfection procedures or a change in the frequency of disinfection are sensible and necessary.

However, a proper check for suitability must be carried out before changing the cleaning and disinfecting agents. Ultimately, it is also important to assess not just the disinfecting effect, but also the surface compatibility.

All essential areas of production must be examined in terms of their effect on the hygiene and sterility of the product, and should have measures implemented accordingly. The hygiene concept contains a summary of these observations and the measures derived from them. A well thought-out hygiene concept is therefore the basis for avoiding microbial findings in sterile production.

In summary, it should be said that all essential and relevant hygiene aspects are summarized in a structured manner in the hygiene concept. What does this mean in the event that bacterial events are monitored anyway?

In such a case, it is necessary to ask whether the hygiene concept was not implemented or applied as prescribed. If this question can be clearly answered in the negative, a revision of the hygiene concept with regard to necessary changes and/or additional measures, should be conducted at least in the event of recurring bacterial events. 

It is not enough to treat individual symptoms or to implement measures (e.g. disinfecting more frequently) in the event of microbial findings in sterile production. A systematic and risk-based approach is required for processing the associated deviations and root cause analyses. Initially, bacterial detection can help to identify the origin and source of the finding. If the bacterial event has a multi-causal origin, expertise and experience are then required. Furthermore, it is necessary to assess and prove whether a singular event or potentially a systemic problem is involved, making a repetition probable. This is the only way to decide whether production should continue. For the release of individual batches, it is necessary to individually assess where the germs appeared locally, and their level of criticality with regard to the sterility of the batch. This also includes deciding whether the germ actually originates from the tested material or whether it entered the sampling material in a secondary manner. A basic prerequisite for avoiding "sterility problems" is a solid hygiene concept. This concept should be continuously refined and reviewed in relation to any critical bacterial events that have occurred.