Assessing Microbial Contamination: Best Practices for Pharmaceutical Microbial Data Investigations

Tim Sandle- Head of Compliance and Quality Risk Management, Bio Products Laboratory

Introduction

Assessing microbiological data and undertaking investigations into the origins of contamination, establishing root causes, and setting appropriate preventative actions constitutes a core part of the contamination control strategy.¹ Despite the importance of this process, regulatory findings frequently cite poor-quality microbial investigations.² This can be partly overcome through consistency of practice, driven by the use of proforma and having a good microbiological investigation procedure in place.

This article looks at the main steps involved for conducting investigations and provides some best practice advice for the company microbiologist. The goals here are to help to structure investigations that are:

• Simple: Easy to execute and to understand.
• Effective: Producing the correct result.

The advice is presented as a series of phases. While the phases represent activity blocks, they should not necessarily be thought of as discrete entities for they can run concurrently or may overlap. This is especially useful in order to complete a detailed and thorough investigation within a reasonable timeframe (as with other investigations undertaken within pharmaceuticals, a target of 30 days is often applied).

The article draws on an investigation into microbial contamination of a water system to illustrate the points made.

Limitations of Microbial Data

Like any area of pharmaceutical science, microbiology is data-driven (albeit equally qualitative and quantitative in terms of data types). Unlike analytical processes, microbiological data is relatively imprecise (an outcome heavily influenced by the non-normality of microbial distribution in the environment).³ Hence, most microbiological investigations into out-of-limits results, unless captured by a real-time rapid microbiological method,⁴ are likely to be limited. Limitations include:

• Data is captured after the event, often after several days have elapsed.
• The act of sampling is imprecise, which is a factor of sampling methods and in relation to the uneven distribution of microorganisms in the environment.
• The microbiological condition of the sample is dynamic (and as such any re-test or re-sample may not be indicative of the condition that was initially observed).
• There is inherent variability with microbiological test methods and in relation to the sample itself.
• The state of the microorganism is itself subject to variation depending upon the growth phase.
• Individual results do not always provide a suitable indication of the risk (trend analysis is often preferable).
• Finding a definitive conclusion is not always possible.
• Assessing the root cause and understanding the effectiveness of remediation can require considerable amounts of additional testing.

Furthermore, null data in itself does not provide sufficient evidence that the risk is low. Many microbiological methods are limited by their metrological precision, many organisms are unculturable using conventional methods, and many environments render a level of cell stress that makes recovery difficult. Therefore, microbial control can often only be fully assured by assessing a range of physical data (such as cleanroom pressure differentials, storage temperatures, water flow rates and so on).

First Phase: Assessment of Microbial Test Validity

In putting together a robust microbiological investigation procedure, the initial phases will be similar to the standardized out-of-specification (OOS) approach. Here the objective is to assess laboratory error.⁵ A result can only be invalidated where a definitive error has been demonstrated. The rationale and impact for the invalid test must be clearly documented. In cases where there is no laboratory error, the result is accepted, and the investigation focus is with manufacturing. In cases where the outcome is inconclusive, the balance of evidence must always be towards a process related issue.

A great deal of OOS guidance appears, upon first reading, more applicable to the analytical laboratory. However, much of it is readily translatable to the microbiology laboratory as well.⁶ For example:

1. Assessment of obvious error. To ascertain whether the result is valid, there needs to be a check for ‘obvious error’. This could be, for example, microbial growth not being in the expected area (such as, with a finger dab plate, colonial recovery not being in the location where contact was made). Other examples relate to positive and negative controls not passing. Other factors to consider include:

1. Is the culture media receptacle integral?
2. Was similar contamination present on other tests from the same session?
3. What do control plates indicate? (this is especially important with negative control plates)
4. Was the correct media used?
5. Are dilution errors likely?
6. Is cross-contamination likely?

2. Supervisory assessment. It is additionally important to make sure that the test was conducted according to its procedure, by a trained person, using suitable materials, reagents, and equipment. Aspects to assess include:

1. Could calculation errors have occurred?
2. Results of other samples tested during the same session.
3. Test history and evidence of recurring issues.
4. Analyst training and competency, including the extent of their experience, their history in relation to testing, and their longer-term competency (such as involvement in proficiency testing schemes). It may be necessary to interview the analyst.
5. Control plates.
6. Environmental conditions relating to the test environment.
7. Equipment suitability and calibration status.
8. Cleaning and disinfection of test areas.
9. Incubation conditions.
10. Storage conditions.
11. Sampling technique.
12. Sampling equipment.
13. Environment within which the sample was taken.
14. Environmental monitoring data, when undertaken in the laboratory.
15. Sample expiry times.
16. Pipettors.
17. Culture media suitability and any supplier concerns.
18. Culture media growth promotion results.
19. Suitability of any test reagents.

The two stages of laboratory assessment may help to establish whether a significant and continual problem exists with a test method or practice.

As with standard OOS guidance, samples should be retained until the test has been signed out and where an OOS is suspected, samples should be retained until the investigation is completed and approved (most samples should be held at 2-8°C). Any justification for additional testing needs to be carefully made and based on hypothesis testing. In cases of laboratory error, a ‘no’ result should be recorded. Where a ‘no’ test is recorded, the impact of a missing data point should be determined, and the assessment documented. The absence of the data may in itself prevent batch release if no further sampling can be undertaken. In other cases, other information may be available to assess the microbiological quality of the material, utility, product or process.

Where laboratory error looks unlikely, it is also important to assess all data in its wider context, to determine if the result is signaling a wider problem. Examples here include warning/alert level results or unexpected shifts in trend when data is plotted. Successive warning level excursions or atypical patterns (such as shifts in non-zero values or repeated recoveries of atypical microorganisms) should be sufficient to become actionable events.

Second Phase: Immediate Actions

The primary concern with any investigation is product quality and patient safety. Once the result is confirmed not to be due to laboratory error (a process in itself that should be concluded within a working day), decisions will need to be made as to whether the related batch needs to be placed on hold and as to whether other batches are affected. If any part of a process or a batch is placed on hold, this needs to be effectively communicated. As a minimum, such communications are within the facility, but this may need to extend out to customers and regulatory agencies.

Third Phase: Laboratory-Based Evidence Gathering

Once laboratory error has been concluded, the investigation into the significance of microbial contamination begins.⁸ When starting the investigation, evidence needs to be gathered:

• The test that triggered the failure.
• Whether other test data relates to the failure (either the same test on other samples or other test data).
• The microorganism recovered and related information:

» The type of organism.

» The potential origin or source.

» Where the organism was found in relation to where it is likely to be found.

» Whether the organism has been detected anywhere else?

» Whether the organism could be detected anywhere else? (this is an important consideration, especially where different culture media, test procedures, and incubation conditions are deployed. An organism recovered from a bioburden test, especially one involving an enrichment step, is probably not recoverable from environmental monitoring. Therefore, absence of detection from the environment does not necessarily mean the organism was not present).

» If there is a wider implication from the organism identity, e.g. an association with biofilms?

• An appreciation that microbial counts are imprecise.
• An understanding that the type of microorganism might be more important than the count obtained.

As an example, when considering sampling the following diagram illustrates considerations in relation to sampling pharmaceutical-grade water (Figure 1): With microorganisms, it is useful to take a photograph of the contaminant both in terms of colony morphology (assuming a conventional growth-based method has been used) alongside a ruler and of the Gram-stain. This can be useful for making future comparisons, address some data integrity aspects associated with microbiological data, and provide a source to return to should misidentification be suspected. Important metadata to report incidents: date of subculturing; verification of organism purity; identification test results; and acceptance of the identification. It is of utmost importance that the identification result is verified by an experienced microbiologist to confirm that the organism is typical and likely, not least because all identification methods – even genotypic ones – possess a degree of inaccuracy. For example, an obligate anaerobe could not be recovered from aerobic environmental monitoring; a thermophile would not be recovered from pates incubated at 20-25°C; and a rare ocean dwelling bacterium would not be expected to be found in a cleanroom.

Fourth Phase: Responsibilities and Boundaries

Once the initial information has been collected, the core investigation can begin (ideally using some form of pre-written investigative procedure, supported by lists of information to capture and decision trees). Many investigations will require a multi-disciplinary team, such as microbiologists, engineers, manufacturing personnel, and quality assurance. For large and complex investigations it might be preferable to have an independent facilitator.

Prior to doing so, the process becomes less confusing if it is agreed upfront who will look at what, especially the division of responsibilities between manufacturing and laboratory personnel. It is also useful to set the boundaries of the investigation, and for the investigation to occur within a reasonable timeframe. This can be irrespective of the test (in that a wider remit may be required to fully assess contamination concerns) but it should not be too wide leading to the investigation losing focus and becoming too open-ended.

Fifth Phase: Field Work

Few examinations of confirmed microbiological contamination can be undertaken from within the laboratory (a statement that tallies with the oft-quoted aphorism that the best place for the pharmaceutical microbiologist is in the production facility). In most cases, area visits are required to assess where samples were taken and to look at the related process. Visits should include interviews with personnel (such as manufacturing staff and engineers) and a review of applicable records. Where decisions are made in the area or as part of post-visit review, these should be documented.

When carrying out this form of evidence gathering, it is important not to discount anything and to keep notes and records.

For example, following on from Figure 1, after sampling error has been discounted, then for when investigating high microbial counts from a water system the following schematic can be useful (Figure 2):

Sixth Phase: Root Cause Analysis

Root cause analysis refers to the process of discovering the root causes of an event in order to identify appropriate solutions. This is along the lines that it is more effective to systematically prevent and solve underlying issues instead of simply treating ad-hoc symptoms. The essential steps are to:

• Scope
• Plan
• Execute against the plan
• Capture outcomes
• Interpretation
• Conclusion

Often with microbiological events it may not be possible to determine the exact root cause; instead, the most probable root cause becomes the outcome. In concluding, it is as important to be conclusive about what the root cause is not as much as it is to be as sure as reasonably practical what the root cause is. This is especially important when the root cause is inconclusive.

When reviewing the output of the root cause analysis into microbiological contamination it is important to be able to answer this sequence (as per Figure 3):

In establishing the cause, it is possible there may be more than one point of origin and more than one contributing factor. It is important not to oversimplify the contamination source and vector. For example, the contamination source may not be the ‘root cause’; instead, the vector might be the cause, as with transferring contamination into the facility by inappropriate practices.⁸

It is possible that the root cause might trigger a re-examination of the laboratory test and return the process to a consideration of laboratory error. Caution is required here since simply failing to find evidence within the production area is not in itself sufficient to conclude that the origin was the laboratory. To conclude laboratory error there must be unequivocable evidence of laboratory error.

For the root cause process some useful points to consider are:

• Construct process, personnel, waste and sample flows to help to consider the likely source of the contamination.
• Ask what unusual events occurred and review records.
• Has the event occurred before? (what does past data inform about the current event?)
• Use science-based decisions.
• Consider technical controls.

When working through the potential sources of contamination and vectors, should control weaknesses be identified that are not considered to be related to the event these still need to be addressed as part of continual improvement.

When writing up an investigation, it can be useful to ask whether the argument is logical, based on science, and easy to follow. For major investigations it can be useful to have the report peer reviewed by someone from a different discipline to ensure the report and its core argument is intelligible.

Seventh Phase: Risk Assessment

Once the root cause or most probable root cause has been set, the impact of the event needs to be considered. A risk assessment will need to assess the hazard (in this case, a microorganism) against the likelihood of it contaminating product (this will depend upon the type of sample, whether the result is from direct testing or from associated monitoring such as environmental data) and the severity. This enables a decision to be reached about the batch disposition. All data collected from the original test and from the investigation must be used in the disposition assessment process (unless it has been conclusively determined that the original result is invalid).

When assessing supporting data, such as monitoring result from a cleanroom or from a utility, the risk assessment becomes more challenging when assessing the impact or potential impact upon a product. For example, consider a sterile manufacturing facility and the detection of high numbers of microorganisms in the water system. The assessment, continuing with the example used for Figures 1 and 2, would need to include areas like (as per Figure 4)

This assessment will be dependent upon the process stage and whether any subsequent data is available, such as decreasing microbial counts or bioburden reduction steps. For finished product, there will be no further steps, but the risk may still be acceptable for some non-sterile product based on the potential for an organism to grow or due to the incorporation of a particular preservative with proven antimicrobial activity against the organism. With finished sterile products, the recourse is batch rejection.

Eighth Phase: Setting CAPA

As with other types of non-conformities, the microbial contamination event needs to go through the corrective action-preventative action (CAPA process). In many cases it will not be possible to set a ‘corrective action’, especially where unacceptable levels of microbial contamination gave been confirmed. This places greater emphasis upon preventative actions, based on putting in place measures to prevent a recurrence. Where corrective actions are often appropriate is with addressing laboratory error and where there is a justifiable case for retesting. Both corrective and preventative actions should be prioritized and accompanied by reasonable timelines.⁹

Ninth Phase: Assessing the Investigation

Everything should be documented through the course of the investigation. At the end of the process, the event needs to be written up. For more complicated investigations, or for investigations where the consequence is significant like batch rejection, it can be useful to have the investigation subject to independent review. A non-specialist can be useful for the review as they might well ask the unexpected questions. Whoever is selected, the reviewer should be able to adequately assess the logical flow and scientific basis of the report.¹⁰

Poor Investigations

Weak microbial investigations will not select the correct root cause and therefore the wrong decision about the product disposition will be made and an inappropriate CAPA will be set. Weaknesses arise for investigations that are unplanned and uncoordinated; when goals are unclear and blinkered approaches are adopted, including assuming the outcome of the investigation before the conclusion; when the rationale for the conclusion are not clearly defined; and when the overall report is inadequate.

Summary

This article has outlined some best practices form conducting microbiological investigations. For every microbiological function, out of limits events will occur. For each event, it is best to consider each microbial excursion as a unique event but also to try and follow a documented plan, in order to achieve consistency.

Given that guidance is sometimes limited in this area and because most OOS guidances approach the topic from the perspective of the analytical laboratory, the article set out to provide both a reminder of good practices and to provide a fresh perspective on the topic. For microbial investigations to be effective, it is useful to carry out as much pre-thinking as possible, using appropriate subject matter experts, and to establish a procedure and supporting documents to help to structure the investigation and root cause analysis. The effort required in terms of assessing the laboratory testing and handling should be equal to the level of effort put into the manufacturing side of the process. All decisions reached should be based on scientific principles.

A well-written, thorough investigation will provide confidence to regulators that the risks to a process have been well understood and this will give confidence that the facility is in control.

References

1. Sandle, T. Tracking and tracing to the root cause: case studies in microbial contamination, European Pharmaceutical Review, 2022 at: https://www.europeanpharmaceuticalreview. com/article/170186/tracking-and-tracing-to-the-root-cause-case-studies-in-microbial-contamination/
2. Cundell, T. Microbiology: Mould contamination in pharmaceutical drug products and medical devices, European Pharmaceutical Review 2013: https://www. europeanpharmaceuticalreview.com/article/23351/microbiology-mould-contamination[1]pharmaceutical-drug-products-medical-devices/
3. Ilstrup, D. Statistical Methods in Microbiology. Clin Microbiol Rev, 1990; 3(3):219-226
4. Miller, M.J., Ragheb, S.M. Towards the Real Application of Rapid Microbiological Methods in developing Countries. European Pharmaceutical Review. 2013; 18(5): 13-16
5. Sutton, S. Laboratory Investigations of Microbiological Data Deviations (MDD) In: Sutton, S. (Ed.) Laboratory Design: Establishing the Facility and Management Structure, 2010; DHI Publishers pp. 81-100
6. Moldenhauer, J. Conducting Microbial Investigations, American Pharmaceutical Review, 2015: https://www.americanpharmaceuticalreview.com/Featured-Articles/177313- Conducting-Microbial-Investigations/
7. McCullough, K. and Moldenhauer, J. (2015) Chapter 1 Introduction. McCullough, K. and Moldenhauer, J. (Eds.) Microbial Risk and Investigations. Parenteral Drug Association (PDA) and Davis Healthcare International (DHI). Bethesda, MD
8. Salaman-Byron, A. L. Limitations of Microbial Environmental Monitoring Methods in Cleanrooms, 2018, American Pharmaceutical Review: https://www. americanpharmaceuticalreview.com/Featured-Articles/349192-Limitations-of-Microbial-Environmental-Monitoring-Methods-in-Cleanrooms/
9. Sutton, S. Successful Microbiological Investigations, American Pharmaceutical Review, 2011; 74 (2): https://www.americanpharmaceuticalreview.com/Featured-Articles/37190- Successful-Microbiological-Investigations/
10. Sandle T. (2012): Sterility Test Failure Investigations, Journal of GxP Compliance, 16 (1): 1- 10

Subscribe to our e-Newsletters
Stay up to date with the latest news, articles, and events. Plus, get special
offers from American Pharmaceutical Review delivered to your inbox!
Sign up now!