Microbial Recovery
When we experience microbial recovery in our components, intermediates, or finished products, the first response may be one of panic. Think of it – a microscopic creature, almost alien, appearing out of nowhere with the potential to kill. What is this thing that I cannot see without the aid of powerful optics? Where did it come from? What does it want? How can I make it leave me alone? Once we get over our initial visceral reaction, we can begin to focus on potential quality and regulatory impacts on our product and company. Lack of information during the initial microbial recovery can induce worry, and to allay fears, we need to obtain more information. Specifically, the organism identification. Having this information puts us in a proactive position and able to effectively assess risk and develop a mitigation plan.
Magic Microbes?
Microbes aren’t magic. They just sometimes seem that way because they’re small. While on a visceral level, it can seem like microbes spontaneously appear in our environment, raw materials, or products, intellectually w,e know that that is not the case. They do not appear magically; they come from somewhere. Yet, when all we see are different colored dots on an agar plate, we sit in ignorance – and within that ignorance, we may become anxious. Historical microbial characterization techniques like the classic Gram stain and basic microscopy provide little actionable information. This lack of information has historically led us to make sweeping generalizations that are not necessarily based on science. Things like “all Gram-negative rods are dangerous” while “all Gram-positive rods are fine.” Or “Gram-positive cocci are okay so long as they are coagulase-negative.” These statements are not founded on good science, but, unfortunately, the lack of specific information regarding the organism puts microbiologists in a weak position.
Naming Organisms
Give an organism a name and take away its power. In truth, Gram reaction and other simple biochemical characterization techniques tell us almost nothing about whether an organism is objectionable. Indeed, what does it mean for an organism to be objectionable? While the term is used plainly in 21 CFR 211.165, in practice, it is often a point of confusion.
“There shall be appropriate laboratory testing, as necessary, of each batch of drug product required to be free of objectionable microorganisms”.1
Objectionable Organisms are those that, by their number or identity, may hurt the product or cause harm to the person using the product. This general definition is spoken to quite directly in the Federal Register:
“Microorganisms could be objectionable by virtue [sic] of their total numbers or their detrimental effect on the product or by their potential for causing illness in the persons ingesting them. A definition of the term is not practical in the regulations, however, because the objectionable nature of a microorganism may develop only about the unique circumstances of a particular formulation, a particular ingredient, a particular method of manufacture, or the conditions found at a particular firm”.2
Most regulatory bodies expect firms to have a process for determining whether organisms are objectionable, and this has become a more focused area of regulatory scrutiny. This is certainly the mindset of the United States Food and Drug Administration (FDA) when performing inspections and assessing risk. There has been a steep uptick in FDA Warning Letters citing firms for insufficient processes for assessing whether recovered microorganisms were objectionable. A company must consider several factors when assessing whether an organism should be considered objectionable. Such factors include, but are not limited to:
1. Organism Survival: What is the potential that the organism may survive in the product? It is crucial to determine the likelihood that the organism can survive in the product. This evaluation often involves reviewing Antimicrobial Effectiveness Testing (e.g., USP ) (AET) data. The goal of AET analysis is to determine if a specified organism is likely to die rapidly or if it may survive (or worse, survive and grow, in the product. It is important to note that the panel of organisms used in AET is limited and does not provide a universal guarantee of product hostility towards all microbes. Species-level organism identification is incredibly valuable when evaluating historical AET data on the organism recovered in a product.
2. Maximum Exposure: What is the maximum exposure the consumer is likely to be exposed to? For dose-limiting drugs, this can be a straightforward calculation. For products that are not dose-limiting, including cosmetics or certain OTC drug products, the maximum exposure assessment may be more abstract. A risk-based decision – typically based on “worst-case scenario” considerations may need to be made.
3. Pathogenicity: Perhaps the most critical question that must be answered during a risk assessment is whether exposure to the organism is likely to cause infection and/or disease. This is rarely a straightforward answer. Often, the organism recovered is not a primary pathogen but may be an opportunistic pathogen. Determining whether such an organism should be considered objectionable requires consideration of several factors. Who is the intended consumer? Might they be immunocompromised? How is the product intended to be used? Topically? Inhaled? Injected? Understanding how the product is used and who will be using it can help determine the risk associated with opportunistic pathogens.
4. Product Defect: Even if it has been determined that an organism is not likely to lead to infection or disease, microbial contamination of a product is considered adulteration. Consideration should be given to whether microbial contamination could cause a product defect, whether that be a negative impact on drug efficacy or consumer experience. Microbial contamination may cause issues such as swelling of primary packaging, off-colors, off-odors, or even visible colony growth (particularly in the case of mold contamination). The potential for these defects should be considered as part of the risk assessment to protect brand integrity and market equity.
5. Regulatory/Recall History: While lack of a formal regulatory position or recalls associated with a particular organism are not in themselves a reason to not deem an organism objectionable, the opposite is quite telling. If there have been several recalls, particularly of similar product types as your product, it indicates what a regulatory agency may consider appropriate. However, even if you find recalls/regulatory statements that suggest that deeming the organism objectionable is appropriate, this does not absolve the firm from conducting a full risk assessment/OOS investigation of its own.
Maintaining Microbial Profiles
The maintenance of robust microbial profiles associated with recoveries in a manufacturing environment is necessary to aid in microbial control of a facility and positioning the microbiology and QA professionals in those facilities to assess risk. It is becoming common for regulatory agencies to ask what organisms were recovered during utility validation (e.g., purified water, environmental air, compressed gases, etc.). Sites are expected to know ‘what is normal’ both quantitatively as well as qualitatively. Biochemical characterization provides little information to assist in profiling in this context. A more useful microbial profile, therefore, would involve identifying all unique morphologies to the species level during validation and profiling the identifications based on the frequency of recovery. Then, a risk assessment can be performed on those organisms most frequently recovered. Ideally, the organisms are assessed to be ‘not objectionable.’ They are simply the ‘normal’ house isolates. It also allows the facility to see qualitative changes in the water, which may indicate an issue.
Take the example of a USP purified water system. Documenting that the organisms recovered from a purified water system are Gram-negative rods tells a site little from a quality perspective. Most recoveries from water will be Gram-negative rods; that is an environment in which they live and thrive. Conversely, getting a microbial identification for a Gram-negative rod that the site has never seen before may be very useful in identifying a negative impact on the system. Or, if the organism is assessed to be objectionable, the site can take appropriate remediation steps and be prepared to effectively assess the remediation – is the specific organism gone or not?
Genetic Sequencing and Proteomic Fingerprinting
Genetic sequencing and proteomic fingerprinting represent two of the most commonly relied upon modern techniques for species-level microbial identification. While automated biochemical testing instruments have existed for years, there is only so much granularity that can be achieved through biochemical characterization alone.
Genetic Sequencing
The “oldest of the modern” microbial identification techniques. This involves nucleic acid amplification of conserved sequences (commonly from the 16s ribosomal subunit for bacteria and Fun ITS for fungi) and comparison of these sequences to large databases for a known match. Because genetic sequencing has a longer history, the comparison databases tend to be larger than those for proteomic fingerprinting, which may result in a higher likelihood of getting species-level resolution. For certain organisms, genetic sequencing databases may even be able to provide strain-level resolution, which can be useful in certain investigations. With the increased demand for microbial identification, as well as advancements in technology, the relative cost of genetic sequencing continues to become more affordable.
Proteomic Fingerprinting
A newer technology involves extracting proteins (typically ribosomal) from the unknown organism and analyzing them via mass spectrometry (commonly MALDI-TOF). Spectra are compared to proteomic databases for a known match (in a similar fashion to genetic sequencing). The workflow for proteomic fingerprinting tends to be simpler than genetic sequencing, making it more approachable for laboratories with minimal molecular biology expertise. Proteomic databases are smaller relative to their genetic counterparts, largely because the technology is newer. This can result in a slightly lower percentage of species-level identifications, especially with more obscure species. Likewise, standard proteomic fingerprinting typically does not provide strain-level resolution. However, newer systems are beginning to offer strain-level resolution for certain organisms with the addition of infrared spectroscopy.
Both genetic sequencing and proteomic fingerprinting are widely used and provide accurate and reliable organism identifications. The cost of onboarding one or both of these microbial identification technologies can be quite high. This includes both the initial capital investment and the ongoing maintenance and operation costs (including having analysts to run them). However, this should not dissuade one from performing microbial identifications.
Outsourcing to a Contract Lab
Microbial identification can be outsourced safely and reliably to a trusted contract laboratory. The increased demand for microbial identifications has made the outsourcing of such work increasingly affordable in recent years. Therefore, a company should not let concerns about bringing microbial identification capabilities “in-house” dissuade them from performing those identifications. Whether your company does not require microbial identification with a frequency to justify in-house identification capabilities or simply does not want to incur ongoing consumable, maintenance, and operational costs, outsourcing microbial identifications is a resource-effective option. When considering a contract partner for your microbial identifications, you should consider their expertise with microbial identification, as well as their quality system. The contract partner should be qualified per your internal procedure, and like all contract partners, drafting a Quality Agreement between your two companies is a best practice (see FDA Guidance Document Contract Manufacturing Arrangements for Drugs: Quality Agreements Guidance for Industry 33).
Species Identification
Routine microbial species identification is a critical part of the future of industrial microbial integrity. Access both physically and financially to robust, species-level microbial identifications has never been easier. Microbial identification is not only a regulatory expectation but also an invaluable scientific tool to help us understand our manufacturing environments and products. Microbial identifications allow us to understand the normal microflora in our environments and raw materials. They allow us to identify changes in dynamic systems. They allow our investigations to be more robust, and our corrective actions are more scientifically driven. We collect mountains of microbial data. Microbial identifications help to ensure that we are making the most of those data.
References
- 21 CFR 211. Current Good Manufacturing Practices for Finished Pharmaceuticals. United States Food and Drug Administration.
- Federal Register VOL 43, No. 190 – Sep. 29th, 1978. United States Food and Drug Administration.
- Contract Manufacturing Arrangement for Drugs: Quality Agreements Guidance for Industry. United States Food and Drug Administration. November 2016.
Author Details
Michael Loewenstein- VP of Scientific Consulting, Q Labs LLC
Publication Details
This article appeared in American Pharmaceutical Review:
Vol. 28, No. 1
Jan/Feb 2025
Pages: 13-15
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