By - Robert Westney - Founder and President, Cryologics Inc. - Principal Consultant, Westney & Associates Consulting LLC - Member of the Microbiology Network consortium of consultants
Introduction
“Good practice includes the periodic challenge of prepared media with low levels of microorganisms. This includes USP indicator microorganisms as well as normal flora.”
Guide to Inspections of Microbiological Pharmaceutical Quality Control Laboratories. U.S. Food and Drug Administration. July 1993.
For nearly four decades, the U.S. Food and Drug Administration’s (FDA) expectation has been that pharmaceutical Quality Control (QC) microbiology laboratories include in their media growth promotion testing (GPT) not only type culture (compendial) microorganisms, but also those 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 GPT. 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 inspection guide, many U.S. and international guides support the requirement to include in-house microbial isolates in GPT. In its 2015 sterile drugs inspections guide,(1) the FDA states, “The inspection of [the] microbiology laboratory should evaluate the following: ... Microorganisms (e.g., ATCC) are used for growth promotion tests of media. Microorganisms isolated from environmental monitoring samples can also be used to perform growth promotion test”. In 2012, the World Health Organization published its guide(2) for environmental monitoring (EM) of vaccine manufacturing facilities. The guide states, in part, “Each aseptic manufacturer should consistently evaluate the growth promotion properties of media for a predefined list of microorganisms... This standardized list should include compendial microorganisms and/or environmental isolates ...”. The Pharmaceutical Inspection Convention/ Pharmaceutical Inspection Cooperation Scheme (PIC/S) published a recommendation(3) in 2011 which included a reference to media used for aseptic process simulation, “The medium selected should be capable of supporting a wide range of microorganisms, which might reasonably be encountered and be based also on the in-house flora (e.g., isolates from monitoring etc.)”.
The FDA issued a Warning Letter in 20104 that included, “...your firm does not perform challenge testing to the sterility media with environmental isolates from the environmental monitoring program”. In 1998, the agency issued a Warning Letter(5) that stated, “Growth promotion qualification of the media used for environmental monitoring does not include a challenge with mold isolates”. A 2014 FDA 483 observation(6) stated that “In-house environmental isolates were not included in the growth promotion of ... media used for aseptic process simulations and EM media”. Less than two years earlier, the FDA issued a 4837 that included the observation, “Environmental isolates used during growth promotion testing of prepared and purchased media only include gram positive microorganisms for growth promotion purposes. Environmental isolates do not include gram negative microorganisms, molds, and yeasts”.
While there are myriad other examples of FDA enforcement actions regarding GPT,(8) 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 GPT and have implemented programs to comply.
Selection of in-house isolates for GPT presents many challenges. First, you must formalize your selection approach in a procedure. Not doing so represents a compliance risk. For example, in 2012 the FDA issued a 483,(9) which included the observation, “The procedure ‘Quality Control of Microbiological Media’ ... does not specifically describe how to select environmental isolates to be used in growth promotion”.
Secondly, compendia, guidance, and industry literature often describe different approaches (or none at all), often making the process for selecting in-house microbial isolates confusing. They generally instruct what to do, but not how to do it. For example, USP <1116> (10) refers to “environmental isolates from samples obtained through the monitoring program” for use in GPT of sterility test media but does not provide selection guidance. USP <1115>,(11) which is for manufacturers of non-sterile products, does not refer to GPT at all. The FDA’s Aseptic Processing guidance(12) refers to “environmental monitoring and sterility test isolates” for GPT, but again there is a lack of guidance for selecting EM isolates (NB: reference to sterility test isolates is more specific). As referenced in the Introduction above, the FDA’s microbiology laboratory inspection guide(13) refers to “normal flora” but does not specifically define “normal”. The Parenteral Drug Association’s Technical Report No. 2214 recommends that “environmental microorganisms or those isolated from sterility test positives” be included in GPT. While reference to “sterility test positives” provides specific guidance, reference to “environmental microorganisms” does not. PIC/S’ “Recommendation on Sterility Testing”(15) cites “environmental or fastidious microorganisms” for use in GPT of sterility test media. While the term “fastidious microorganisms” provides specific guidance, again the term “environmental ... microorganisms” is not specific and is consistent with other regulatory and guidance documents.
Many industry thought-leaders have put forth recommendations for including in-house microbial isolates in media GPT. For example, in her 2006 book “Environmental Monitoring for Cleanrooms and Controlled Environments”,16 Anne Marie Dixon stated that “representative isolates identified from environmental monitoring, personal monitoring, and positive sterility test results” should be included in GPT. Again, reference to “positive sterility test results” provides good guidance for selecting isolates, but reference to EM and personnel monitoring is somewhat unspecific. In 2007,17 James Agallaco stated that many companies include “randomly selected environmental isolates” for use in the GPT microbial challenge panel. However, the rationale for this approach is not described.
For aseptic manufacturers, some guidance can be gleaned from regulatory, compendial and industry guidance, namely the use of isolates from sterility test positives for use in sterility test media GPT. This author suggests that this concept be extended to isolates recovered from aseptic processing simulation (“media fill”) failures.
For both aseptic and non-sterile manufacturers, there is essentially no guidance for selecting isolates from EM and Clean Utility monitoring. Reference to “normal”, “common”, “representative”, and “random” appear in the literature. Perhaps these terms are referring to the predominance of in-house microbial isolates. If so, this elicits the question of the scientific validity of this approach. Perhaps the need for demonstrating that the in-house microbial isolates used as a GPT challenge panel are accurately recovered (within normal variability). Regardless, until or unless a scientific rationale is described for “normal” microflora, it is prudent to establish the approach to selection of in-house isolates in a procedure. This will help to avoid compliance enforcement issues. Recall the 2012 483 observation9a described above. The Standard Operating Procedure (SOP) for EM and Clean Utility trend reports is optimum for prescribing this.
This author suggests that atypical microorganisms be a focus for selecting in-house microbial isolates for media GPT. 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 media GPT 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.
Understanding the intended use of a firm’s media is essential for selecting appropriate microorganisms. For example, microorganisms associated with EM and Clean Utility adverse trends are appropriate for inclusion in the GPT challenge panel for the media used for this monitoring. One would not select an in-house mold or spore-forming Gram-positive rod to challenge Reasoner’s 2A agar (R2A). This medium is commonly used to test water samples, and these two groups of microorganisms are not waterborne. It would be inappropriate to challenge Sabouraud Dextrose Agar (SDA) with an in-house Gram-positive coccus, as this medium is selective for fungi. In addition to the previously described use of sterility test positive and media fill failures for GPT of sterility test media and media used for aseptic process simulations, manufacturers of non-sterile products may wish to consider the use of isolates obtained from in-process and finished product test failures. Isolates from raw material test failures may be worth consideration for inclusion in the media GPT for that raw material.
Preparation of microbial suspensions for use in media GPT poses several challenges. Because the microbial concentration is unknown, it must be determined in order to achieve the proper concentration to comply with the USP <61>18 requirement that not more than 100 colony-forming units (CFU) be inoculated. Achieving a very low concentration presents the risk that, for agar media, a small difference (e.g., 1 CFU) between the test and control results leads to nonconformance with the USP <61> requirement: “For solid media, growth obtained must not differ by a factor greater than 2 from the calculated value for a standardized inoculum”. Achieving a very high concentration presents the risk that the number of recovered CFU exceeds 100 due to normal variability.
Another inherent challenge is the nature of the microorganisms themselves. These are “wild-type” microorganisms with frequently unpredictable growth characteristics. Such characteristics may impact the concentration of a microorganism suspension during storage. 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 adverse trend, etc.).
Having microbial suspensions ready to use at all times is another challenge. This requires the continuous need for labor and materials. Preparation of a suspension in response to a demand, such as an emergency medium order, requires that the medium be released at-risk (e.g., planned deviation) while the suspension is being enumerated and standardized prior to GPT.
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 ≤100 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 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. 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. Because the concentration is an estimate, the suspension can be used in a “bracket” manner. The suspension is serially diluted to obtain a set of three dilutions that span the estimated concentration, with the estimated concentration in the middle of the “bracket”. The control plates that recover the appropriate number of CFU are used for determining the results of the corresponding test medium. The remaining two dilution plates and corresponding test medium are discarded. Because the concentration of a filamentous fungi suspension cannot be estimated using a spectrophotometer, a broader “bracket” approach is warranted.
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.
In conclusion, the use of in-house isolates in GPT is a longstanding FDA requirement. Enforcement actions since publication of its 1993 inspection guide1a are clear evidence of this. Noncompliance risks enforcement. While preparation and use of in-house microbial isolates presents many technical challenges, and is time- and labor-consuming, this author has provided guidance for overcoming many of these challenges.
References
- Food and Drug Administration. Compliance Program Guidance Manual. Program 7356.002A, Sterile Drug Inspections. September 11, 2015.
- World Health Organization. Environmental Monitoring of Clean Rooms in Vaccine Manufacturing Facilities. November 2012.
- Pharmaceutical Inspection Convention / Pharmaceutical Inspection Cooperation Scheme (PIC/S). Recommendation on the Validation of Aseptic Processes. 1 January 2011.
- Food and Drug Administration. Warning Letter. CP Pharmaceuticals, Ltd. October 29, 2010.
- Food and Drug Administration. Warning Letter. Centocor, Inc. July 10, 1998.
- Food and Drug Administration. 483 Observation. Jubilant HollisterStier, LLC. April 14, 2014.
- Food and Drug Administration. 483 Observation. Hospira, Inc. August 24, 2012.
- Cryologics bibliography. Accessed on May 8, 2021 at: https://www.cryologics.com/ bibliography.
- Food and Drug Administration. 483 Observation. Alexion Pharmaceuticals, Inc. August 6, 2012.
- United States Pharmacopeia <1116>, Microbiological Control and Monitoring of Aseptic Processing Environments.
- United States Pharmacopeia <1115>, Bioburden Control of Nonsterile Drug Substances and Products.
- Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practice. Food and Drug Administration. September 2004.
- Guide to Inspections of Microbiological Pharmaceutical Quality Control Laboratories. Food and Drug Administration. July 1993.
- Parenteral Drug Association. Technical Report No. 22: Process Simulation for Aseptically Filled Products. December 2011.
- Pharmaceutical Inspection Convention / Pharmaceutical Inspection Cooperation Scheme (PIC/S). Recommendation on Sterility Testing. 25 September 2007.
- Dixon, A.M. (Editor). (2006) Environmental Monitoring for Cleanrooms and Controlled Environments. New York: Informa Healthcare.
- Agallaco, J.P., Carleton, F. (2007) Validation of Pharmaceutical Processes. Florida: CRC Press.
- United States Pharmacopeia <61>, Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests.
Author Biography
Robert Westney is the Founder and President of Cryologics Inc., 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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