By: Robert Westney - Founder and President, Cryologics Inc. - Principal Consultant, Westney & Associates Consulting LLC - Member of the Microbiology Network consortium of consultants
Introduction
“Routinely used disinfectants should be effective against the normal microbial vegetative flora recovered from the facility... If indicated, microorganisms associated with adverse trends can be investigated as to their sensitivity to the disinfectants employed in the cleanroom in which the microorganisms were isolated.”
Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practice. Food and Drug Administration. September 2004.
For nearly two decades, the U.S. Food and Drug Administration’s (FDA) expectation has been that pharmaceutical Quality Control (QC) microbiology laboratories include in their disinfectant efficacy testing (DET) microorganisms isolated from the firm’s manufacturing facility. The QC Microbiologist is tasked with selecting which specific microbial isolates are suitable for this testing. This has presented several technical challenges, including criteria for selection, and microbial suspension preparation and use.
This article will discuss the U.S. and international regulatory guidance and compendial history of the use of in-house isolates in DET. This will be followed by some examples of FDA enforcement actions. Guides to the methods for selecting the appropriate in-house microbial isolates will be discussed. Finally, technical challenges, including recommended solutions, will be presented.
Discussion
In addition to the above reference to FDA’s Guidance for Industry, many U.S. and international guides support the requirement to include in-house microbial isolates in DET. In its 2011 questions and answers on current good manufacturing practices for drug product containers and closures,1 the FDA states, “Designing an effective cleaning program involves ... selecting the right disinfecting agents to inactivate isolates that may be in the product or in the environment”. In 2020, the American Society for Testing and Materials (ASTM) stated in its guide for evaluation of cleanroom disinfectants,2 “Regulatory authorities expect evidence of the effectiveness of disinfection agents against environmental microorganisms isolated from the facility”. The United States Pharmacopeia (USP) provides excellent guidance in chapter <1072>, Disinfectant and Antiseptics:3 “To demonstrate the efficacy of a disinfectant within a pharmaceutical manufacturing environment, it may be deemed necessary to conduct the following tests: (1) use-dilution tests (screening disinfectants for their efficacy at various concentrations and contact times against a wide range of standard test organisms and environmental isolates); (2) surface challenge tests (using standard test microorganisms and microorganisms that are typical environmental isolates ...)”. The Parenteral Drug Administration (PDA) also provides guidance for both new and existing manufacturing facilities in its Technical Report No. 70,4 stating, “Selection of organisms should be based on the type of environmental isolates recovered from the facility (environmental isolates are preferred); however, if facility isolates are not available ATCC cultures ... representing facility isolates are acceptable until facility isolates can be obtained”. In addition to this Technical Report, the PDA advises in its Technical Report No. 13,5 “It is recommended to periodically review challenge testing of the selected sanitizers, disinfectants, and sporicides if representative new isolates are routinely recovered in the environmental monitoring program. This supports the effectiveness of the sanitizer, disinfectant, or sporicide on new contaminants discovered in operations”. (NB: These approaches can be extrapolated to non-sterile manufacturing facilities.)
The FDA issued a Warning Letter in 2012 that included, “Your response states that a supplemental disinfectant efficacy study, using mold spores of in-house isolates on various surfaces, will be performed and completed by [redacted]. We suggest that this proposed study be conducted as soon as possible”. The “suggestion” on the part of the FDA was clearly intended to compel the firm to expedite the study, highlighting its criticality. Four years earlier, the FDA issued a Warning Letter7 to an aseptic manufacturer that stated, “Disinfectant effectiveness studies against representative microorganisms and/ or specific in-house isolates were not conducted for cleaning agents used in your facility to disinfect production areas, including aseptic areas”. A 2013 FDA 483 observation8 stated that “The validation study to evaluate sanitizers ... is inadequate in that ... corporate studies did not evaluate isolates from the (redacted) site”. That same year, the FDA issued another 483 observation9 stating, “The firm has not evaluated if their current cleaning and sanitizing methods/practices are effective against in house isolates...”.
While there are myriad other examples of FDA enforcement actions regarding DET,10 these actions have waned during the past few years. In this author’s experience, this may be due to one or both of two developments. First, it is possible that the compliance enforcement focus of the FDA has shifted (although the requirement remains in place). Recall historical enforcement actions with regard to 21 CFR Part 11 compliance, and the current focus on data integrity, to name two examples. Secondly, perhaps most companies have come to accept the requirement to include in-house isolates in DET and have implemented programs to comply.
As with media growth promotion testing (GPT) described in this author’s previous publication in American Pharmaceutical Review (APR),11 selection of in-house isolates for DET presents some challenges. However, unlike GPT, compendia, guidances and industry literature provide clearer guidance for the selection of in-house isolates. For example, USP <1072>3 states, “... the most frequently isolated microorganisms from an environmental monitoring program may be periodically subjected to use-dilution testing with the agents used in the disinfection program to confirm their susceptibility, as there are real differences among different species in resistance to the lethal effects of different sanitizers”. The PDA’s Technical Report No. 704 states, “Representative organisms should be preserved and included in the panel of organisms in efficacy testing of antimicrobial agents used in the facility”.
Many industry thought-leaders have put forth recommendations for including in-house microbial isolates in DET. In 2019, APR published an article12 by Dr. Ratul Saha, who stated, “the panel of microorganisms selected for the DE study plays a very critical role in assessing the efficacy of the disinfectant. Apart from using standard AOAC challenge organisms, the value is in using typical environmental/facility isolates”. Dr. Tim Sandle and Ravikrishna Satyada published an article in a 2015 issue of the European Journal of Parenteral & Pharmaceutical Sciences13 stating, “Similar levels of recovery of the challenge organisms were observed across the three time points. While this part of the study was satisfactory and the results consistent, further studies could be attempted using more environmental isolates”. Dr. Scott Sutton described in a 2013 APR article,14 “... approximately 37% of the respondents were using additional organisms in the AET. These respondents cited environment monitoring isolates and isolates from products as the sources of the additional organisms”. In his presentation15 to the PDA Southeast chapter in 2013, Dr. Marc Rogers affirmed, “USP (ATCC or USDA) challenge organisms may also be considered but environmental isolates are the most critical”.
The literature cites a frequent theme of “predominance”. A manufacturing firm needs to demonstrate that its predominant microflora is being kept in check as part of its disinfection regimen, in part by generating in vitro laboratory DET data. Environmental monitoring (EM) trend reports are an important tool for identifying predominant facility isolates. Trend reports should identify these and address any shifts (particularly spore-forming organisms). For shifts in microflora, the trend reports should commit to repeating DET using the new organism(s). Organisms associated with adverse trends identified during investigations of EM excursions are ideal for inclusion in DET, for the purpose of demonstrating effective corrective/preventive action. Lastly, a firm should not rely on the claims of the disinfectant/ sporicide manufacturer. These are based upon internal study results using compendial (e.g., ATCC) organisms (most likely using a few of the most common materials of construction [MOC]), and do not reflect the specific in-house isolates and all the MOCs of the purchasing firm.
This author suggests that atypical microorganisms also be a focus for selecting in-house microbial isolates for DET. While it is debatable whether an “objectionable microorganism” list be established, particularly for non-sterile manufacturers, microbiological expertise is invaluable for identifying microorganisms that are unusual, whether the risk of their recoveries is known. For example, the recovery of a yeast species in a Grade A aseptic manufacturing environment is highly unusual, and worthy of inclusion in a DET challenge panel. It is inadvisable that a clinical approach be taken with regards to any pathogenicity and/or virulence of a recovered microorganism unless a firm’s clinician contributes to the rationale for such selection.
Preparation of microbial suspensions for use in media DET poses some challenges. Because the microbial concentration is unknown, it must be determined in order to achieve the proper concentration to comply with the USP <1072>3 requirement that a sufficient organism concentration be inoculated onto coupons of materials of concentration in order to demonstrate the required spore and vegetative log reductions. Current USP <1072> states that “... sufficient organisms need to be inoculated on a 2-inch × 2-inch square of the surface being decontaminated, i.e., a coupon, to demonstrate at least a 2 (for bacterial spores) to 3 (for vegetative bacteria) log reduction during a predetermined contact time”. The currently proposed revision will include clarification, in part, that this spore log reduction requirement also pertains to fungal spores.
An inherent challenge in preparing the organism suspension is the nature of the microorganisms themselves. These are “wild-type” microorganisms with frequently unpredictable growth (phenotypic) characteristics, very much unlike type-culture organisms, which have a long history of predictable phenotypic and genotypic characteristics. Such “wild-type” characteristics may impact the concentration of a microorganism suspension during storage while awaiting results of enumeration testing. This challenge is compounded when an in-house isolate challenge panel changes (e.g., as a result of a conclusion in a trend report, investigation of an EM adverse trend, etc.). The general approach to preparation of a microbial suspension is to first create the culture, then to harvest it and suspend it in a physiologically suitable diluent such as 0.1% peptone or 0.85% NaCl. This is followed by serial 10-fold dilutions with aliquots (e.g., 100 μL) onto a nutrient agar surface, which is then incubated. This initial suspension is stored during the incubation of the serial dilution plates. After incubation, the dilution representing a countable number of CFU is used to calculate the suspension concentration. Using straightforward math, the initial suspension is diluted down to the required CFU per challenge aliquot, followed by 2-8 ̊C storage when not in use.
During storage of the initial suspension while awaiting results of the serial dilutions, the concentration often changes in unpredictable ways. It may become reduced, such has been seen with some filamentous fungi. It may increase, due to cell aggregates becoming dissociated, such has been seen with Gram-positive cocci. The concentration of the suspension at the end of the serial dilution incubation period may not be the same as at the beginning.
This author proposes a solution to this challenge. Studies may be performed in order to ascertain the stability of the suspension’s microbial concentration. Results from such studies may be used to anticipate any change in concentration during storage. These results can be used to extrapolate results (using a “correction factor”) of the serial dilutions performed of the initial suspension, as well as to assign an expiration date to a standardized suspension. Such activities are labor- and time-consuming and need to be repeated whenever a new in-house isolate is added to a microbial challenge panel.
A second approach to using a prepared microbial suspension is to use it immediately. This requires that a fresh culture always be available. This is easily resolved by including preparation of the in-house isolate cultures in the laboratory operations routine schedule, followed by appropriate culturing methods in advance of preparing a microbial suspension. Because the concentration at the time of preparation is unknown, the use of a spectrophotometer may be employed. Studies may be performed to estimate the concentration at a particular absorbance/optical density. Unfortunately, this approach is applicable only to bacteria and yeast, and not to filamentous fungi. For filamentous fungi, a very dense suspension may be prepared, so that the USP <1072> two-log reduction criterion can be met (or not) using a concentration that is far above a 102 cfu challenge.
Lastly, there exists a small number of companies worldwide that can prepare ready-to-use preparations of customers’ in-house isolates. A cost-benefit analysis should be performed, including delivery lead time. The internal costs associated with labor and materials can be used as a basis for comparison to the cost of using one of these service providers.
Conclusion
The use of in-house isolates in DET is a longstanding FDA requirement. Enforcement actions since publication of its 2004 aseptic processing guidance are clear evidence of this. Noncompliance risks enforcement. While preparation and use of in-house microbial isolates presents many technical challenges, and is time-, material- and labor-consuming, this author has provided guidance for overcoming many of these challenges.
References
- Questions and Answers on Current Good Manufacturing Practices, Good Guidance Practices, Level 2 Guidance – Control of Components and Drug Product Containers and Closures. Food and Drug Administration. July 5, 2011.
- Standard Guide for Evaluation of Cleanroom Disinfectants. ASTM E2614 - 15(2020)e1. 2020.
- United States Pharmacopeia <1072>, Disinfectant and Antiseptics.
- Parenteral Drug Association. Technical Report No. 70, Fundamentals of Cleaning and Disinfection Programs for Aseptic Manufacturing Facilities. 2015.
- Parenteral Drug Association. Technical Report No. 13 (revised), Fundamentals of an Environmental Monitoring Program. 2014.
- Food and Drug Administration. Warning Letter. Sanofi Pasteur. July 12, 2012.
- Food and Drug Administration. Warning Letter. Catalent Pharma Solutions. March 28, 2008.
- Food and Drug Administration. 483 Observation. Hospira, Inc. March 1, 2013.
- Food and Drug Administration. 483 Observation. Central Admixture Pharmacy Services. February 19, 2013.
- Cryologics bibliography. Accessed on July 10, 2021, at: https://www.cryologics.com/bibliography.
- Westney, R. “The Use of In-House Microbial Isolates in Media Growth Promotion Testing: Challenges and Solutions”. American Pharmaceutical Review. Vol. 4, Issue 24. May/June 2021.
- Saha, R., PhD. “Disinfectant Efficacy: How Can We Make It Effective?”. American Pharmaceutical Review. August 28, 2019.
- Sandle, T. and S. Ravikrishna. “Assessment of the disinfection of impaction air sampler heads using 70% IPA, as part of cleanroom environmental monitoring”. European Journal of Parenteral & Pharmaceutical Sciences. November 2015.
- Sutton, S., PhD. “The Antimicrobial Efficacy Test, GMP and Investigations”. American Pharmaceutical Review. August 21, 2013.
- Rogers, M., PhD. “Disinfectant Efficacy Testing for Critical Environments”. Steris Life Sciences. PDA SE Chapter. April 18, 2013.
Author Biography
Robert Westney is the Founder and President of Cryologics Inc., which specializes in preparation of in-house isolates for use in compendial testing. He is Principal Consultant for Westney & Associates Consulting LLC, and a member of the Microbiology Network consortium of consultants. He has more than 30 years of experience in the GMP industry, including Quality Control Microbiology, Quality Assurance and Regulatory Affairs. He holds a Master of Science degree from Temple University in Quality Assurance / Regulatory Affairs. He is Regulatory Affairs Certified (RAC) and is a Certified Manager of Quality / Organizational Excellence (CMQ / OE). He is a member of the Pharmaceutical Microbiology Forum (PMF), the American Society for Microbiology (ASM), the Parenteral Drug Association (PDA), the Regulatory Affairs Professional Society (RAPS), and a Senior Member of the American Society for Quality (ASQ).
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