Opinion Paper | Open Access | Published 7th January 2025
Evaluation and Standardisation of the Approach to Using Environmental Isolates in the AstraZeneca Microbiology Laboratories
K Capper, P Duncanson, A Gravett, A Johnson | EJPPS | 294 (2024) | https://doi.org/10.37521/ejpps29404 | Click to download pdf
Summary
A thorough evaluation and standardisation of the use of environmental isolates (EIs) in AstraZeneca's microbiology laboratories was performed. The study was prompted by the increasing use of EIs in pharmaceutical microbiology laboratories over the past two decades and aimed to ensure robust methods for recovering all types of microorganisms that may pose a risk to the quality of medicines. The cost, time and robustness of the testing performed with environmental isolates was identified as an area for continuous improvement by internal audit of site procedures. The review identified a wide variety of interpretations of where and what to include for EIs in growth promotion tests, methods, and disinfection efficacy studies across AstraZeneca's microbiology laboratories, leading to potential overuse.
The study involved a global cross-site review that utilized a risk assessment process, involving 10 expert pharmaceutical microbiologists with significant industry experience and a risk management subject matter expert. The review assessed various uses of environmental isolates, such as growth promotion testing, disinfectant qualification studies, and method suitability testing, with a focus on regulatory requirements, external audit commitments, and scientific rationale.
Regulatory guidance for the use of environmental isolates was reviewed, and a requirement for their use was noted in specific documents, such as the EU Guidelines for Good Manufacturing Practice for Medicinal Products² and various USP chapters. The literature review provided external viewpoints and aligned with the approach within AstraZeneca, confirming the need for a risk assessment in the use of isolates.
The paper concludes by outlining the scientific rationale for the use of environmental isolates and provides guidance on when and how to select the most appropriate EIs for different test types. The study resulted in the production of a global guidance guideline, allowing sites to implement a standard approach to the use of EIs, leading to a significant reduction in their overuse and a standardisation across the business. The conclusion emphasises the importance of utilising environmental isolates where they add the most value or where they are required by regulations.
This paper provides valuable insights into the evaluation and standardisation of the approach to using environmental isolates in AstraZeneca's microbiology laboratories, offering a foundation for a global standard and a reduction in their overuse.
Key words: Environmental Isolates; Factory Isolates; Growth Promotion
Introduction
The use of environmental isolates in pharmaceutical microbiology laboratories has grown over the last 20 years as a result of various good intentions to ensure that our methods are robust in recovering all types of microorganisms that may be of risk to the quality of our medicines. The microbiology laboratories in AstraZeneca utilise environmental isolates (EIs) in various tests and qualification studies. A review of the use of these isolates was performed in order to standardise the approach across all AstraZeneca (AZ) Microbiology laboratories. It was identified that there was a wide variety of interpretation of where and what to include for EIs in our various growth promotion tests, methods and disinfection efficacy studies. In some cases, there was potential overuse of EIs and it was important to understand the reasons behind this before recommending any change. The approach adopted for this global cross site review used the global risk assessment process¹⁴ and this was documented in the AZ global pharmaceutical quality system.¹ The multi-site workstream that performed this review contained 10 expert pharmaceutical microbiologists with significant industry experience and a risk management subject matter expert.
Risk Assessment
In terms of risk, the Quality Impact assessed what is the impact if we do not use isolates in the study. The various uses of environmental isolates in the microbiology laboratories were identified and assessed separately. The principles used to complete each risk review of their use was as follows:
Regulatory Requirements
External Audit Commitments
Scientific Rationale
Quality Impact
The uses of environmental isolates across the AstraZeneca network were surveyed and summarised below:
Growth Promotion Testing of Approved Certified Ready to Use Media Suppliers.
Growth Promotion Testing of In-House prepared Media.
Growth Promotion Testing of New Unapproved Ready to Use Media Suppliers.
Growth Promotion Testing Post Aseptic Simulation Trials.
Disinfectant Qualification Studies.
Qualification of Test Equipment, including identification systems.
Qualification of new methods for environmental monitoring such as desiccation studies and incubation sequence.
Qualification of Alternative Methods such as Rapid Sterility/Bioburden.
Method Suitability Testing of products, hold studies and in process test methods.
Regulatory Requirements
Regulatory guidance for the use of environmental isolates was reviewed with respect to the test types. A requirement for their use was noted in the following documents.
EU Annex 1²
Environmental isolates are mentioned in section 9.36 vi for aseptic processing simulation (APS): “In developing the APS plan the selected nutrient media should be capable of growing a designated group of reference microorganisms as described by the relevant pharmacopeia and suitably representative local isolates.” ²
“On completion of incubation: Samples of the filled units should undergo positive control by inoculation with a suitable range of reference organisms and suitably representative local isolates.” ²
Environmental isolates are mentioned in section 10.9 vi for environmental monitoring and process simulation trials: “Media used for environmental monitoring and APS should be tested for growth promotion before use, using a scientifically justified and designated group of reference microorganisms and including suitably representative local isolates.” ²
The definition of local isolates in this document is “Suitably representative microorganisms of the site that are frequently recovered through environmental monitoring within the classified zone/areas especially grade A and B areas, personnel monitoring or positive sterility test results.” ¹
Annex 1 indicates that the use of environmental isolates is a requirement for growth promotion testing post aseptic simulation trial incubation and growth promotion testing of new unapproved media suppliers. Once a supplier is qualified with these isolates and certified as part of the quality management systems there is no scientific reason to continue to use environmental isolates on every batch or delivery of those media. The suppliers of triple bagged environmental monitoring media cover a wide variety of environmental organisms in addition to pharmacopeial groups, so changes in media quality will be identified as part of that quality control process. These risks were considered controlled at this point and documented in the global risk register.
Disinfectants
The AstraZeneca Global Guidance⁴ for qualification of disinfectants follows the principles of various regulatory guidance documents.
PIC/S Validation of Aseptic Processes³
Food and Drug Administration Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practice. 2004. US⁴
USP NF Chapter <1072> Disinfectants and Antiseptics⁵
USP NF Chapter <1227> Validation of Microbial Recovery from Pharmaceutical Articles. ⁶
This guidance document states, “The rationale for the selection of each microorganism to be included in the study should be clearly documented and based on scientific rationale.” A minimum number or organisms from groups is recommended. “Selection of a representative for each group may include consideration of factors such as:
Typical flora for the area where the disinfectant will be used.
Typical flora from product bioburden.
Organisms that have been recovered from both the environment and product bioburden.
Organisms that have been associated with an adverse environmental monitoring trend.
Organisms classified locally as objectionable.
Organisms that are known to have reduced sensitivity to the disinfectant under test.” ⁴
The disinfectants that have been qualified by AstraZeneca sites with the selected organisms is detailed in the appendix of this guidance disinfectant document and represents a scientific global approach. The risk was considered controlled and documented in the risk register.
The workstream did not identify any other mandated guidance on the use of local isolates.
LITERATURE REVIEW
A literature review was also completed of recent papers on the application, use and value of using environmental isolates in microbiological tests. The purpose of this review was to understand opinion outside the AZ business and risk assess any emergency trends.
Guilfoyle & Cundell, 2022¹² concluded “In response to regulatory citations for not including plant isolates in method suitability and growth promotion testing of microbiological culture media, the authors make the case that the compendial designated cultures meet the requirements of the official tests and are sufficiently representative of the most frequently identified environmental isolates. It was our conclusion that this compliance request lacks scientific justification. The scope of this review largely directed to the growth promotion and suitability testing requirements for USP <60>, <61>, <62> and <71>.¹⁵⁻¹⁸ Other microbiological tests such as USP <51>¹⁹ Antimicrobial Effective Testing, media fill validation, and water and environmental monitoring are discussed.”
Booth, 2019¹¹ concluded “Guidance documents are not clear regarding the appropriate use of (EIs) in microbiological assays. However, regulatory expectation for the use of EIs in the microbiology laboratory appears to be gaining momentum. Not all microbiologists agree regarding the use of EIs in the growth promotion challenge test. However, most microbiologists agree that EIs do have a place in the laboratory. Proper use of EIs in the laboratory includes adding them to disinfectant efficacy studies, microbial assay validations, antimicrobial effectiveness testing, and sterility testing media growth promotion assays. The rationale for deciding which environmental isolates to include in microbiological assays should be established and documented based on a formal risk assessment.
To remain compliant and avoid regulatory observations, follow the established compendial chapters, regulatory guidelines, and execute assays properly according to established Standard Operating Procedures (SOPs). It is also recommended to stay abreast of growing industry trends and regulatory expectations.”
Salaman-Byron, 2019¹⁰ concluded “The utilization of EIs is considered a good practice to challenge the robustness of microbial methods. Nevertheless, the inherent risk of unexpected outcome due to the unpredicted nature of EIs is present. There is not sustainable data that support regulatory agencies continuing the enforcement of EIs for growth promotion testing (GPT). The fact is EIs usually recovered from environmental monitoring (EM) programs are not representative of the pharmaceutical manufacturing environment bioburden due to the limitation of the EM methods. Well-controlled manufacturing suites are such a hostile environment for organisms to be established. EIs within pharmaceutical suites are influenced by many factors besides the manufacturing process, much of them antimicrobial in nature. The presence of organisms within process areas is incidental and accidental in nature and their residence is transient. In fact, most EIs will be recently dispersed or recovered prior to any stress that might come. However, there are exceptions such as Bacillus spp, capable of surviving and even building up biofilms on surfaces. It is reported that laboratory culture conditions differ markedly from those that exist in natural ecosystems. Over time the organism would undergo a selection process in microbial media when alleles may be lost or mutated due to environmental pressure.”
Guilfoyle & Cundell¹² presented on this topic at the Joint FDA and PDA conference in 2021. Their summary at the end of the presentation was,
There are still potential benefits with using EIs, if carefully applied.
The USP QC microbes are very representative of common EIs.
Media manufacturers perform additional growth promotion testing before release.
Domestication of laboratory EIs may significantly alter both the phenotype and genotype of the stored microorganisms.
The use of Environmental Isolates is not necessary as supplement QC microbes for Media Growth Promotion and Suitability testing for the following USP Microbiology Test methods:
The pharmacopeial chapters ¹⁴⁻¹⁸ were also reviewed, all have no requirement for the use of EIs in method qualification.
In summary the literature review provided confidence that the approach of performing a risk assessment for the use of isolates across the business was appropriate and external viewpoints were aligned with those within AZ.
The literature confirmed the view that the laboratory domestication reduces the value of the challenge of using them. The use of EI’s should be risk assessed and used where they add scientific value.
EXTERNAL AUDIT COMMITMENTS
In order to ensure no historical external audit commitments would be compromised A review was performed at each AZ site using the internal Global Audit Management system. No external audit commitments had been made for the use of EIs. Only one audit observation was noted which related to isolates. In this observation the recommended improvement was to document the choice and reasons for selection of the isolates used for a disinfectant qualification study. This review confirmed the ability to implement a global standard without opening up sites to risks from regulatory inspections.
SCIENTIFIC RATIONALE
It was recognised that several aspects of how EI use has been interpreted has led to applications that do not follow scientific rationale. The AZ risk assessment cross site team documented the following scientific arguments against the use of EIs in routine growth promotion tests.
Once isolated and cultured stressed isolates are no longer difficult to grow and tend to cause high inoculum issues of >100cfu once sub-cultured and used in this way.
Selecting isolates based on high occurring local frequency for use in studies is contrary to selecting isolates that may have issues growing as they are already known to be recovered well.
The use of isolates to qualify new identification equipment is flawed when the libraries for the methods of identification are not equivalent.
Continually qualifying the same media type and supplier with the same environmental isolates which have already grown on that media adds no additional value, once proven, to the standard group of recommended culture collection isolates.
The purpose of isolate use was originally intended to ensure that any stressed environmental organisms could grow or that any local isolates of specific risk could be cultured by the various methods. It was considered by the AZ review team that the use of isolates should follow the scientific need of where they add most value.
To ensure new disinfectants are effective against commonly found flora in the specific facility.
To ensure any isolates that present a specific risk to a product or process can be recovered by routine methods if a change to process, method or facility occurs. Specifically, where these isolates represent a different challenge from those described in the pharmacopeia and may require specific growth conditions.
When comparing equivalency on alternative test methods (rapid methods) or media suppliers to assess suitability for use as a change to a current approved standard.
SITE RECOMMENDATIONS
The scientific rationale was used to develop the guidance and is seen in Table 1 and 2. In Table 1, for each use type the AZ recommendation is stated and the rationale guidance to support discussions. In Table 2, how to select the most appropriate EI to use and the number expected is provided as guidance. These recommendations were implemented gradually across the various sites using the global change control process.
Table 1 Environmental Isolates Required per Test Type
¹Unless used for environmental monitoring purposes
²EU Annex 1 10.9 states Environmental Isolates for Environmental Monitoring and APS media only
³Where specific product risk of an organism has been identified during development or at another site, source the same organism species where possible and include in method suitability testing during technology transfer
CONCLUSION
The conclusion of the technical review work and risk assessment was to utilise environmental isolates where they add the most value or where there was a requirement by regulations. Producing a global guidance guideline to support individual site assessment along with the principles of this review allowed the sites to implement a standard approach. The result was a significant reduction in their overuse at some sites and a standardisation across the business. An improvement in the number of repeat growth promotion tests performed was seen and an ability to justify our approach with documented risk assessment. Individual sites applied these principles without further technical assessment and used the same rationale to support discussions during regulatory audit.
References
01. RSKREG-000274 Global Risk Assessment - Risks associated with not using Environmental Isolates in microbial studies.
02. The Rules Governing Medicinal Products in the European Union Volume 4 EU Guidelines for Good manufacturing Practice for Medicinal Products for Human and Veterinary Use. Annex 1 Manufacture of Sterile Medicinal Products.
03. PIC/S Validation of Aseptic Processes
04. Guidance for the Qualification of Disinfectants for the use in GMP Area 8-P216-CV-M GUID-0005829
Food and Drug Administration Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practice. 2004. US
05. USP NF Chapter <1072> Disinfectants and Antiseptic
06. USP NF Chapter <1227> Validation of Microbial Recovery from Pharmaceutical Articles.
07. Sandle, T. (2022) “Examination of the growth rates of environmental isolates compared with compendial strains,” EJPPS EUROPEAN JOURNAL OF PARENTERAL AND PHARMACEUTICAL SCIENCES [Preprint]. Available at: https://doi.org/10.37521/ejpps.27201.
08. Cundell, A.M., Chatellier, S. and Schumann, P. (2010) “Equivalence of Quality Control Strains of Microorganisms Used in the Compendial Microbiological Tests: Are National Culture Collection Strains Identical?” PDA Journal of Pharmaceutical Science and Technology, (64), pp. 137–155.
09. Guilfoyle, D.E. and Cundell, A.M. (2021) “PDA/FDA Joint Regulatory Conference,” in Do Environmental Isolates (EI) have a Role in Method Suitability and Growth Promotion (GP) Testing in the Microbiology Laboratory?
10. Salaman-Byron, A.L. (2019) “Facts about Environmental Isolates and Growth Promotion Test,” American Pharmaceutical Review, pp. 1–6.
11. Booth, C.M. (2019) “Environmental Isolates: What's The Proper Use Of In-House Cultures?,” Pharmaceutical Online, pp. 1–4
12. Guilfoyle, D.E. and Cundell, A.M. (2022) “Do plant isolates have a role in method suitability and growth promotion testing in the Microbiology Laboratory? is it A matter of science versus compliance?,” PDA Journal of Pharmaceutical Science and Technology, 76(5), pp. 444–460. Available at: https://doi.org/10.5731/pdajpst.2021.012675
13. GUID-0021517 Guidance on the use, preparation and selection of environmental isolates in microbiological testing
14. Global Quality Risk Management Procedure, 1-P57-cv-X Quality Risk Management (QRM). SOP-0034065
15. Chapter <60> Microbiological Examination of Non-Sterile Products: test for Burkholderia cepacia complex
16. Chapter <61> Microbiological Examination of Non-Sterile Products: Microbial Enumeration Tests
17. Chapter <62> Microbiological Examination of Non-Sterile Products: Tests for Specified Microorganisms
18. Chapter <71> Sterility tests
19. USP <51> Antimicrobial Effective Testing
Authors
K Capper¹, P Duncanson¹, A Gravett and A Johnson¹
¹ AstraZeneca, Macclesfield, UK
K Capper, P Duncanson, A Gravett and A Johnson AstraZeneca, Macclesfield, UK
* Corresponding author:
Karen Capper, Senior Director - Microbiology Science & Technology
AstraZeneca,
UK Operations,
Silk Road Business Park,
Macclesfield
Cheshire. SK10 2NA
England
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