By Paula Peacos, ValSource, Inc.
Microbial data deviation investigations (MDDIs) are notoriously difficult to perform. They are time consuming and labor intensive. For even the most experienced microbiologists, determining the definitive root cause is often challenging. In most cases, the microbiologist is investigating a past event — sometimes by as much as a week or two. Evidence to support conclusions and decisions takes time to collect, and the available evidence is usually circumstantial and often scanty. Direct evidence (i.e., the “smoking gun”) is rare. The microbiologist is often under intense pressure to complete the MDDI as quickly as possible, as manufacturing operations are continuing and, therefore, additional batches could be impacted and possibly subjected to rejection.
Any production-related MDDI also requires a batch impact assessment. This is arguably the most critical part of the investigation. It is the determination of whether the impacted batch should be released or rejected based on risk to the patient and whether or not quality attributes have been compromised. This assessment must address not only the batch in which the deviation occurred, but also any other batches that could potentially be impacted through use of common raw materials, components, pieces of equipment, similar processing lines, common processing rooms, etc.
Most MDDIs consist of at least two parts: the laboratory investigation and the manufacturing investigation. The laboratory investigation is performed to confirm the validity of the deviation and to characterize any microorganisms involved to facilitate the root cause analysis and batch impact assessment, which are generally performed in the subsequent manufacturing investigation. The information contained in the laboratory investigation, particularly the organism characterization, is critical to the success of the root cause analysis and batch impact assessment, yet many laboratory investigations fail to provide as much information as they could.
Laboratory investigations, in general, include the identification of the organism(s) to an appropriate level depending on the type of product or process in question. For example, a microbial deviation occurring in a traditional nonsterile oral solid dosage operation may require an identification to the genus and/or species level, while a deviation occurring in a biological or aseptic process usually requires identification to the species level and, sometimes, the strain level. The natural habitat of the organism and other general information is also provided to aid in finding the source of the deviation. Information regarding the pathogenicity of the organism is also usually included.
However, many laboratory investigations fail to characterize the organism further, which is unfortunate because the more detail provided, the more useful the lab investigation will be when performing the root cause analysis and particularly the batch impact assessment. Not only will the additional information strongly support the eventual batch disposition, but it can greatly reduce the amount of time it takes to determine that disposition.
There are additional factors regarding the microorganism(s) that should be evaluated and addressed by the microbiologist. The criticality or applicability of these factors will of course vary based on the specific combination of product and microorganism etc. Nevertheless, they should be considered. These factors include, but are not limited to:
Characteristics Of The Organism
Is the organism a known pathogen, either obligate or opportunistic?
It is important to remember that almost any organism can be pathogenic under the right circumstances. At the very least, they will have the potential to be pyrogenic. If the organism is a pathogen, it can be helpful to include the infectious dose. The infectious dose, often expressed in colony forming units or CFUs, is the amount of the organism that generally needs to be present to cause infection or disease. A highly pathogenic organism will have a low infectious dose, and vice-versa.
Is it a toxin producer?
Toxins produced by microorganisms can be extremely detrimental, even in in the absence of the organism. Endotoxin, for example, is released from Gram-negative bacteria, especially when they die. Endotoxin can suppress the immune response and aid in disease progression. It can cause such symptoms as fever, septic shock, and severe diarrhea, among others. Escherichia coli, Bordetella pertussis (whooping cough), and Vibrio cholerae (cholera) are all good examples of organisms that produce dangerous endotoxins.
Endotoxin is not the only toxin of concern. Exotoxins are substances that are secreted by living bacterial cells. These toxins are also capable of causing serious complications. Examples include Bacillus anthracis (anthrax), Bacillus cereus (food poisoning), and Staphylococcus aureus.
In 1978, a new tampon design that allowed women to wear them for a longer period of time also allowed for the overgrowth of a particular toxin-producing strain of Staphylococcus aureus. This resulted in in what is now commonly known as toxic shock syndrome.1 In 1980 there were 652 reported cases, 63 of those resulting in death.2
Aflatoxins and mycotoxins are poisonous and carcinogenic substances that are produced by fungi. They are thought by some to be involved in food allergies. These toxins can also cause sickness and death in humans and animals. Examples include Aspergillus species (aspergillosis) and Penicillium species (penicillin).
If further processing is to occur, can you remove the toxin?
Toxins are very difficult to remove once they are present in a product. In general, removal requires advanced technologies such as ultrafiltration. The efficacy of removal, or the ability to remove the toxin at all, depends on factors such as the molecular size of the toxin and amount present. The chemical and physical nature of the product is also an issue. For example, not all products are filterable.
Do you test for that particular toxin?
Endotoxin can be detected with the Limulus amoebocyte lysate test. Exotoxins can be detected using biological and immunological assays and nucleic acid probes. Aflatoxins can be detected using assays such as ELISA (enzyme-linked immunosorbent assay), mass spectroscopy, TLC (thin layer chromatography), or HPLC (high performance liquid chromatography).
Does the microorganism have known resistance to antibiotics?
More and more microorganisms are emerging that are resistant to existing antibiotics, Known as “superbugs,” examples include, but are not limited to, methicillin resistant Staphylococcus aureus (MRSA), vancomycin resistant Enterococcus (VRE), and carbapenem resistant Pseudomonas aeruginosa. These organisms are of growing concern in hospital environments, as infections by these organisms are becoming increasingly difficult to treat.
Characteristics Of The Product And Dosage Form
What is the dosage form/preparation type and route of administration?
The dosage form and route of administration have a huge impact on the level of risk associated with a particular microorganism. For example, oral solid dosage forms such as tablets will generally not support the proliferation of a microorganism if the water activity is below 0.75aw, as per USP <1112> Application of Water Activity Determination to Nonsterile Pharmaceutical Products.3 If the water activity is below 0.60aw, even the most xerophilic microorganisms will not grow. The extreme forces inherent in the compression process also tend to destroy any microorganisms present. In contrast, nonsterile liquid preparations often utilize preservatives/antimicrobials to prevent the proliferation of any organisms present.
The preparation type also affects what the route of entry for the organism would or could be. Common preparation types include, but are not limited to, injectables, topicals, oral dosage forms, rectal and vaginal preparations, and inhalants. Routes of entry include injection, ingestion, wounds or sutures, eyes and other mucous membranes, and the bloodstream.
For example, the very acidic pH of the stomach will destroy most vegetative bacterial forms present in an oral preparation if ingested. However, it may not destroy toxins that may also be ingested (e.g., Clostridium botulinum, which causes botulism).
Similarly, Micrococcus luteus is a very common human skin organism. In most cases, it is considered to be generally harmless. However, M. luteus is an opportunistic pathogen known to infect wounds and sutures. So, while M. luteus might not be a particular issue in an oral preparation as it is not a toxin producer and would be easily destroyed in the stomach, it could certainly cause a problem in a topical preparation used in wound dressings. If the patient is immunocompromised, this risk increases. There are times when an organism is not known to be a human pathogen until it is introduced into the human body through an artificial means such as injection. Such an occurrence happened in 2012, when a fungal meningitis outbreak occurred after patients were injected with a preservative-free steroid preparation made by the New England Compounding Center in Framingham, MA. The Centers for Disease Control and Prevention (CDC) and state health agencies identified over 750 cases in 19 states, and 64 patients died.4. In most cases, the causal organism was Exserohilum rostratum, a common saprophytic soil-borne fungus and plant pathogen with no prior known history of human pathogenicity. However, when injected into the patients, it colonized the bloodstream and the spinal cord, causing serious fungal infections resulting in meningitis, strokes, and other central nervous system-related disorders. Many patients required invasive procedures and treatments.5
Are preservatives/antimicrobial agents present in the product? Is the organism likely to proliferate either in the product or in/on the patient?
Biological preparations, especially if they are protein-based, are often required to be biologically neutral and contain no antimicrobials or preservatives. They also cannot be terminally sterilized. Microorganisms, if present, can often proliferate in this type of environment.
In contrast, it is common for nonsterile preparations to contain some level of antimicrobial substances or preservatives intended to prevent microbial proliferation in the product. However, it should be verified that the antimicrobials and preservatives have been tested against the microorganisms in question. This is important, as microorganisms differ in levels of resistance to different compounds.
Proliferation is not the only issue of concern when living microorganisms are present in the product. The organisms will feed on any sugars and carbohydrate sources, etc., in the product and release waste products and metabolites. This has the potential to eventually result in adulteration of the product.
What is the target patient population (e.g., pediatric, healthy adults, geriatric, immunocompromised, etc.)?
The target patient population is also a critical consideration. If the intended population is immunocompromised, pediatric, or geriatric, the risk that they will be adversely impacted is greater than that of a target population of healthy adults. These individuals are more susceptible to infection due to the weakened states of their immune systems.
How much of the organism is present, or potentially present, in the batch?
This is not easily quantifiable. The amount of the organism detected in the test sample or through environmental monitoring only provides an estimate of the total amount of the organism that could actually be present in the batch. In relation to overall batch size, the test sample size is usually exceedingly small. Furthermore, microbiological samples are not homogeneous, so the microbiologist must consider the possibility that the concentration in other parts of the batch could be higher than that found in the test sample.
How likely is the target organism to persist in the environment (or your product)?
Some organisms do not survive for long periods of time on inanimate surfaces, especially in the harsh environment of the production area. However, others can persist for extended periods. Bacillus species and most fungal species produce spore forms that are highly resistant to desiccation and temperature fluctuations, etc. They are also more resistant to commonly used cleaning and disinfectant agents than are vegetative cells. Similarly, if a product contains nutrients, lacks preservatives and antimicrobial agents, and provides a hospitable environment for microorganisms to exist or thrive in (favorable storage conditions, pH, osmotic conditions, etc.), the possibility of persistence should be evaluated.
The Manufacturing Process
When during the production process did the organism most likely enter?
The point in the process where the microorganism was detected is also a critical factor. In general, microorganisms detected early in the process, especially during setup, can have a bigger impact because they are more likely to have been spread to other parts of the process. For example, consider the following scenarios:
A single colony of a microorganism such as Staphylococcus epidermidis is found inside the stopper bowl on an aseptic filling line. The immediate concern would be that the organism could be transferred to one or more stoppers, which would then be inserted, contaminating filled vials. Conversely, consider the same organism being found on the vial off-feed turntable after the capping process has been completed. The risk is much lower in that scenario, because the vials were already stoppered and sealed, so ingress would be highly unlikely unless the container was damaged.
Will additional processing, filtration, or terminal sterilization occur? Compression or desiccation?
Sterile filtration (0.22 mm) is usually sufficient to remove bacterial colonies from a liquid preparation, providing the bioburden level is within the retentive capacity of the filter. However, sterile filtration alone will not remove any residual toxins present. Terminal sterilization will likely kill any microorganisms present, but, again, residual toxins may be an issue.
As mentioned earlier, processes such as compression and desiccation in combination with very low water activity will often destroy any microorganisms present or, at a minimum, prohibit them from reproducing, but residual toxins can be an issue in these products as well, depending at which point the organisms entered the production process.
Is the product marketed in an area where there is easy access to appropriate medical care?
This is an important consideration, and one that is often overlooked. In some areas of the world, medical care and services are not readily available. If a patient should experience an adverse event, what is the likelihood that they could obtain adequate care in a timely manner? In some places it may be three to four days (or three to four weeks) before medical help is available. They may also have to travel a great distance to get it.
The microbiologist can compile and analyze this information to produce a robust, risk- and science-based organism impact statement and recommendation for batch disposition based on the sum of the evidence. If this degree of detail is contained in the laboratory investigation, the document immediately becomes a much more valuable and useful tool for assessing batch impact and determining the disposition. It provides much of the supporting scientific rationale and justification for those important decisions.
Is a formal medical assessment is required?
If after reviewing all collected data there is still a question as to what the batch disposition should be based on risk to the patient, consider obtaining a formal medical assessment from the firm’s Medical Department or equivalent authority. A qualified physician possesses the necessary expertise to weigh the evidence and make a proper recommendation.
Compiling this amount of information may appear daunting to a microbiologist under pressure to close the investigation as quickly as possible, but the benefits provided and the time and labor it could save in terms of increasing the quality of the informed decision-making process are clearly something to consider.
While it does take a bit of time and diligence to incorporate this information into a lab investigation, it is usually not necessary to repeat the exercise more than once for a given organism. A catalog of completed organism assessments can be created and the completed assessment applied to other deviation investigations involving the same organism. If such a catalog is created, however, it must be periodically reviewed to ensure currency of the information. Furthermore, if a facility is operating in a state of control, there should not be so many MDDIs to complete that this should become overly burdensome.
About The Author:
Paula Peacos is a senior consultant with ValSource, Inc. She has over 25 years of industry experience as a microbiologist, working for contract manufacturing organizations as well as small, midsize, and large pharmaceutical organizations. Peacos has extensive experience in aseptic processing, API/drug substance manufacturing, cell therapies, nonsterile production (both clinical and commercial), microbiological laboratory management, and performing compliance audits internationally. She is an experienced trainer and has developed and implemented customized developmental and remedial programs. Peacos has also published articles and delivered presentations at industry meetings on topics such as risk assessment and using microbial recovery rates for trending analysis. You can contact her at email@example.com.