Facts about Environmental Isolates and Growth Promotion Test

• Malvern Panalytical

Background

Pharmaceutical companies have been engaging the use of environmental isolates (EI) for tests for media growth promotion. Currently, Regulatory Authorities expect evidence of the use of EI in growth promotion testing (GPT). Regulatory enforcement of these expectations often resulted in observations from health authorities around the world.

• FDA Guidance for Industry for Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practice (September 2004) says “The QC laboratory should determine if USP indicator organisms sufficiently represent production-related isolates. Environmental monitoring and sterility test isolates can be substituted (as appropriate) or added to the growth promotion challenge.¹
• FDA Guidance for Industry concerning Validation of Growth Based Rapid Microbiological Methods for Sterility Testing of Cellular and Gene Therapy Products (draft guidance, February, 2008) suggests in relation to selecting a panel of appropriate challenge microorganisms for validating an RMM, the inclusion of “isolates detected in starting materials, isolates detected by in-process testing or during preliminary product testing, isolates detected by environmental monitoring of your facility, [and] isolates from your production areas which represent low nutrient or high stress environments…” ²
• World Health Organization Points to Consider, Environmental Monitoring of Clean Rooms in Vaccine Manufacturing Facilities (November 2012) states in Section 3.3.4 Growth Promotion Testing that each aseptic manufacturer should consistently evaluate the growth promotion properties of media for a predefined list of organisms… this standardized list should include compendial organisms and/or environmental isolates and should represent a reasonable range of “representative” microorganisms that could be encountered in manufacturing environments.”³
• World Health Organization, Presentation by Nanjing (November 2009) Manufacture of Sterile Medicines – Advanced workshop for SFDA on slide 15, Growth Promotion Testing “containers with media should be inoculated with 10 -100 CFU of organisms such as B. subtilis, S. aureus, C, albicans and A. niger. Environmental isolates should also be used.⁴
• The Japanese Pharmacopoeia (XV, General Information section 11.4.1 Media Fill Tests) says in relation to selection of growth promotion, testing organisms which are frequently isolated in environmental monitoring should be used.”⁵
• Canadian Health Authority Good Manufacturing Practices (GMP) Questions and Answers. Q: “Does the Inspectorate encourage the use of environmental isolates for preservative testing”⁶ A: “While the use of environmental isolates in addition to the specified compendia cultures is acceptable, the use of environmental isolates alone is not acceptable.”

Growth Promotion and EI

USP <61> states that “seed-lot culture maintenance techniques (seedlot systems) are used so that the viable microorganisms used for inoculation are not more than five passages removed from the original master seed-lot”.⁷ Therein microorganisms used in growth-promotion testing may be based on the manufacturer’s recommendation for a particular medium or may include representative EI but these latter are not to be interpreted as a compendial requirement. The USP <61> recommends the use of specified strains for compendial tests as they need to be standardized. Irrespective of the origin of these strain cultures, these QC organisms have a utility of a long history of use by both media manufacturers and lab users; the ability to source them from national culture collections or prepared commercial inoculum derived from those cultures and is GPT requirements for media in general test methods. The challenge should be with <100 CFU and the inoculum level used must be verified and recorded for each test performed as acceptance criteria of the test.⁷

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

Growth promotion testing for media intended for testing pharmaceutical articles and products is of importance. Pharmaceutical companies consistently evaluate the growth promotion properties of media for a predefined list of organisms and must be able to prove that their microbial media are suitable to consistently recover environmental contaminates (assuming they would be present). This standardized list should include compendial organisms, but it has been recommended the list also include EI representatives of the manufacturing environment; Gram positive rods; Gram positive coccus; filamentous mold or yeast; Gram negative rods. However, there is no compendial or health authority regulation that requires the use of EI for the routine growth promotion of microbial media used for viable EM programs. In fact, the EIs used for GPT are initially recovered mainly from EM programs.

It is widely accepted that most environmental microorganisms cannot be grown by traditional techniques. Microbial within pharmaceutical manufacturing are likely to be transient; accidental occurrences of microorganisms possibly introduced into the area by an assortment of external sources.^8,9 The possibility for adaption to the possible pharmaceutical manufacturing environment is unlikely. The range and types of microorganisms able to be recovered from EM samples within cleanrooms is very limited. The parameters that contribute to the ability to recover these microorganisms include the microorganism’s physiology, environmental conditions (stress factors), and nutrient composition of the culture medium and the selected incubation conditions used to recover them. Due to the described limitations of the EM methods currently available for pharmaceutical manufacturing monitoring, it is unlikely to obtain all the microorganisms that may occur in the clean rooms that employ the need for human intervention. When manufacturing is performed in the presence of people, most likely the microorganisms recovered will be human source Gram positive, mesophilic aerobic or facultative aerobic bacteria. The establishment of any microorganism within the manufacturing area shall be considered as a control breach and must be investigated and eradicated.⁹

Wild Type Strains and Phenotype

The pharmaceutical industry has questioned the value added of including EI in media growth promotion of various media types. There is support that EI are the best challenge to media used for sterility testing as they may be more representative of the facility. However, there is a concern that as soon as an EM isolate is subcultured onto laboratory growth media, it is no longer an “environmental” isolate meaning that its growth characteristics/properties have been altered by growth in enriched media. Is it reasonable to infer that in-house isolates may become physiologically more “robust” upon culturing in a laboratory medium? It is also unknown if a limit of 5 passages in laboratory medium would be sufficiently limiting for the organism to retain its inherent “wild type” genetic characteristics. Anderson et al (2007) showed that from 18 isolates of Stenotrophomonas maltophilia from one single patient over a 4-year period, only 15 showed similar growth and colony size pattern.¹⁰ There has been no published empirical evidence that an in-house microbial isolate either does or does not lose its “wild-type” traits upon culturing nor has there been any definition or description of exactly what “wild type” characteristics would be important to retain with the various EI. For example, Grosso-Becerra et al (2014) reported that despite the high degree of genomic conservation between distinct isolates of Pseudomonas aeruginosa from diverse environments, including human tissues, they showed diverse phenotypic characteristics.¹¹ Cundell and colleagues, 2010, reported that strains of Salmonella enterica subsp. enterica serotype abony showed distinct differences at both the genotypic and the phenotypic level, suggesting that the strains sourced from the different culture collections were not identical strains, or that they have undergone detectable genetic shift from the time they were derived.¹² Aspergillus niger exhibits great diversity in its phenotype even between distinct established culture collection included culture collections.¹³ Therefore, it is difficult to justify the science behind the use of EIs for GPT as these organisms might stop behaving as “wild type” organisms and will not be distinct over purchased, standardized microorganisms.

Wild type (WT) refers to the phenotype of the typical system functionality of a bacterium species as it occurs in nature. At the genetic level the most frequent allele within a gene population is defined as the wild type allele. As the definition suggest, that an organism carrying all wild type allele genes should be defined as the wild type organism. This is maybe true within a very local, strain related species. It is the opinion of this author, however, that there is no such thing as a true “wild type”. This term was used by geneticists as an artificial definition to simplify laboratory experimentation. For pharmaceutical manufacturing the “wild type” term should be limited to those microorganisms recovered, isolated and identified from the pharmaceutical EM program and/or bioburden tests.

The bacterial stress response enables bacteria to survive adverse and fluctuating conditions in their immediate surroundings. The stress response in bacteria involves a complex network of elements that acts by external stimulus, which aim to temporarily increase tolerance limits. These stress responses are often very specific, each specialized for a kind of stress (i.e. chemical, water and nutrients deprive). Those stress responses facilitate bacteria a transition from one environment to a sub-optimal environment.¹⁴ The nutrients enable the bacteria to essentially re-wire its metabolism when it encounters a new environment.^15,19 In theory, the organism “adapted” to low nutrient or high stress environment will rewire their metabolism by ON and OFF preexisting alleles to deal with the high nutrient, moisture, and mesophilic temperature conditions of the traditional microbial recovery method. The relocation of natural microorganisms to the laboratory can result in their adaptation to these favorable conditions. Wild-type natural isolates of Bacillus subtilis are difficult to work with compared to laboratory ATCC strains that have undergone domestication processes of mutagenesis and selection. B subtilis ATCC strain have improved capabilities of transformation (uptake and integration of environmental DNA), growth, and loss of abilities needed “in the wild”. Bacterial adaptive responses in pharmaceutical manufacturing include development of spores and competence, activation of motility, synthesis of proteases, and changes in energy production systems. Most of these adaptions have been encountered in Bacillus subtilis.^20,21 Staphylococcus aureus can be sorted into at least 10 dominant human lineages. Genomes of bacteria within the same lineage are mostly conserved, except for mobile genetic elements. Mobile genetic elements that are common in S. aureus include bacteriophages, pathogenicity islands, plasmids, transposons, and staphylococcal cassette chromosomes. These elements have enabled S. aureus to continually evolve and gain new traits. There is a great deal of genetic variation within the S. aureus species. Fitzgerald et al. (2001) revealed that approximately 22% of the S. aureus genome is non-coding and thus can differ from bacterium to bacterium. An estimated 20% to 30% of the human population are long-term carriers of S. aureus.²²

The adaption is accompanied by complex changes that include the repression of some protective features that are essential in nature or for virulence. Escherichia coli is an undesirable finding in the manufacturing area. The presence of E. coli or any other fecal microorganism means a possible control breach including bad hygiene practices by operators. E. coli culture strains (e.g. ATCC) are well-adapted to the laboratory environment, and, unlike wildtype strains, have lost their ability to thrive in the intestine. E. coli laboratory strains have lost their ability to form biofilms which is considered a virulence characteristic.²³ Therefore, any E.coli EI is better that any ATCC strain and should be used for GPT.

Candida albicans is a common inhabitant of the human gut flora. It is detected in the gastrointestinal tract and mouth in 40–60% of healthy adults. C. albicans thrives and used genome-wide experimental approaches to uncover the genes required to proliferate in their environment. The laboratory adapted C. albicans is not considered a human pathogen. In addition, C. albicans is a rare EI as it does not proliferate so well outside the human body.^24,25

Conclusion

The utilization of EI 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 EI is present. There is not sustainable data that support regulatory agencies continuing the enforcement of of EI for GPT. The fact is EI usually recovered from EM programs are not representative of the pharmaceutical manufacturing environment bioburden due to the limitation of the EM methods.⁹ Well-controlled manufacturing suites are such a hostile environment for organisms to be established. EI within pharmaceutical suites are influenced by many factors besides the manufacturing process, much of them antimicrobial in nature. The presence of organism within process areas is incidental and accidental in nature and their residence is transient. In fact, most of EI will be recently dispersed or recovered prior to any stress that might come. However, there are exceptions such as Bacillus spp, capable to survive 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.^26,27 Table 1 summarized that the author considered the pros and cons of the utilization of EI for GPT. The list is not all inclusive and many exceptions may apply.

Growth promotion testing do not challenge organisms under such stress conditions encountered during the sampling process or their recovery from pharmaceutical articles. Despite the facts discussed, the analysis of EI in GPT is still the best information available to evaluate the effectiveness of microbial contamination controls.

Implementing standard operating procedures and grappling with the practicalities of EI selection and culture maintenance that will sustain the cultural characteristics of such wild-type isolates around some degree of regulatory uncertainty is what is required. Unfortunately, there is no guidance on the level of characterization as described by compendia.

“The views and opinions of the author expressed herein do not necessarily state or reflect those of Janssen Biotech Inc. and Johnson and Johnson Family of companies.”

References

1. Food and Drug Administration (FDA) (2004) “Guidance for Industry - Sterile Drug Products Produced by Aseptic Processing - Current Good Manufacturing Practice”, Rockville, Maryland, USA
2. FDA (2009) “Guidance for Industry – Q8(R2) Pharmaceutical Development, ICH, revision 2”, Rockville, Maryland, USA
3. World Health Organization Points to Consider, Environmental Monitoring of Clean Rooms in Vaccine Manufacturing Facilities (November 2012) World Health Organization (WHO), Geneva, Switzerland.
4. World Health Organization— Presentation transcript: Manufacture of sterile medicines – Advanced workshop for SFDA GMP inspectors, Nanjing, November 2009.
5. Japanese Pharmacopoeia 17th Edition (2016) (XV, General Information section 11.4.1 concerned with Media Fill Tests.
6. Canada / Health Products and Food Branch Inspectorate; Good Manufacturing Practices Questions and Answers; http://www.hc-sc.gc.ca/dhp-mps/compli-conform/gmp-bpf/question/gmp-bpf-eng.php (access Feb 2017)
7. USP General Chapters <61> “Microbial Examination of Non-Sterile Products: Microbial Enumeration Tests”;
8. Sandle, T. (2011). A Review of Cleanroom Microflora: Types, Trends, and Patterns, J. PDA J Pharm Sci Technol, 65(4): pp 392-403.
9. Salaman-Byron Angel L (2018) Limitations of Microbial Environmental Monitoring Methods in Cleanrooms Am Pharma Rev 21(3): pp 12 - 19
10. Anderson SW et al (2007) Characterization of small colony variant Stenothrophomas maltophilia isolated from the sputum specimens from five patience with cystic fibrosis Clinical Microbiology 45(2):pp 529-35
11. Grosso-Becerra et al. (2014) Pseudomonas aeruginosa clinical and environmental isolates constitute a single population with high phenotypic diversity BMC Genomics, 15:318
12. Cundell T et al (2010) Equivalence of Quality Control Strains of Microorganisms Used in the Compendial Microbiological Tests: Are National Culture Collection Strains Identical? PDA J Pharm Sci Technol. 64(2): pp137-55
13. Salazar-Pena M (2010) System Biology of Glucose sensing and repression in Aspergillus niger: Lesson for genomics and transcriptomics 2010 ISBN: 978-91-7385-439-9.
14. Ron, Eliora Z. (2012). “Bacterial Stress Response”. In Rosenberg, Eugene; Prokaryotes: a handbook on the biology of bacteria (4th ed.). Berlin: Springer.
15. Filloux, AAM (editor) (2012). Bacterial Regulatory Networks Caister Academic Press London UK
16. Requena, JM (editor) (2012). Stress Response in Microbiology. Caister Academic Press
17. Jacob F (1977) Evolution and tinkering. Science 196: pp 1161–1166.
18. Hoekstra HE, Coyne JA (2007) The locus of evolution: evo devo and the genetics of adaptation. Evolution 61: 995–1016.
19. Hottes AK et al (2013) Bacterial Adaptation through Loss of Function PLoS Genetics Vol 9(7) e1003617
20. Hecker M and Völker U. (2001) General stress response of Bacillus subtilis and other bacteria Adv Microb Physiol. 44: pp 35-91.
21. Sandle, T (2014) The Risk of Bacillus cereus to Pharmaceutical Manufacturing American Pharmaceutical Review.
22. Fitzgerald, J. R., Sturdevant, D. E., Mackie, S. M., Gill, S. R., & Musser, J. M. (2001). Evolutionary genomics of Staphylococcus aureus: insights into the origin of methicillin resistant strains and the toxic shock syndrome epidemic. Proceedings of the National Academy of Sciences, 98(15), 8821-8826
23. Vidal O, et al (1998). Isolation of an Escherichia coli K-12 mutant strain able to form biofilms on inert surfaces: involvement of a new ompR allele that increases curli expression”. Journal of Bacteriology. 180 (9): pp 2442–9.
24. Kerawala C and Newlands C, eds. (2010). Oral and maxillofacial surgery. Oxford: Oxford University Press. pp. 446-447
25. Erdogan A and Rao SS (2015). “Small intestinal fungal overgrowth”. Curr Gastroenterol Rep. 17 (4): pp 16
26. Fux CA, Shirtliff M, Stoodley P, Costerton JW (February 2005). “Can laboratory reference strains mirror “real-world” pathogenesis?”. Trends in Microbiology. 13 (2): pp 58–63
27. Palková, Z (2004) Multicellular microorganisms: laboratory versus nature, EMBO J Vol 5(5): pp 470-475.

Author Biography

Angel L. Salaman-Byron MS, PhD is Principal Process Scientist at Janssen Biotech Malvern, PA, USA. Dr. Salaman-Byron received his PhD degree in Medical Microbiology from the University of Puerto Rico, Rio Piedras Campus. Besides his scientific contribution during his dissertation research Dr. Salaman-Byron has contributed to the pharmaceutical literature with publications appearing in American Pharmaceutical Review and Pharmaceutical Technology as well as White papers contributor. Dr. Salaman-Byron has worked with oral dosage small molecules as well as large molecules manufacturer and aseptic filling processes, such as at Wyeth, Pfizer, Amgen and AbbVie.

Key Words

pharmaceutical, cleanroom, environmental Isolate, growth promotion