Mold Monitoring and Control in Pharmaceutical Manufacturing Areas

• Microbiology Consultants, LLC

Introduction

Recent high profile product recalls associated with mold contamination has resulted in more attention from the FDA to fungal isolation in environmental monitoring and product testing in the pharmaceutical industry. Companies need to anticipate these FDA concerns especially with respect to the upcoming regulatory inspections and institute remediation when mold is found in their products and manufacturing facilities to protect patient safety.

High profile recalls for fungal contamination include:

• The 2009 - 2011 pharmaceutical tablet recalls due to moldgenerated Tribromoanisole taints from wooden pallets.^1,2
• The July, 2012 FDA warning letter received by Sanofi Pasteur’s sterile product manufacturing facility in Toronto, Canada for mold contamination.³
• The October, 2012 New England Compounding Center of Framingham, Massachusetts recall of three lots consisting of a total of 17,676 single-dose vials of the steroid, preservative-free methylprednisolone acetate after a multistate outbreak of fungal meningitis caused by the mold Exserohilium rostratum. ^4-6

Tribromoanisole Taints

Tribromophenol (TBP)-treated lumber from South America was used for the construction of wooden pallets for commerce in the Caribbean. The unintended consequences was that high humidity in Puerto Rico promoted mold growth on the pallets resulting in the fungal methylation of TBP to the volatile, odorous taint tribromoanisole (TBA) detectable at very low concentrations (ppt). TBA was absorbed into High Density Polyethylene (HDPE) bottles used for pharmaceuticals, which were transported on the pallets and used to package the tablets. When the user opened the sealed bottles they were sickened by the highly unpleasant odor of the TBA taint from the container headspace. Follow-up toxicological investigations determined that, although the odor may deter the patient taking the drug, it had no toxicological impact.⁷ As taints were not as well understood in the pharmaceutical as in the food industry, the PDA established a task force, which published the 2014 PDA Technical Report No. 55 Detection and Mitigation of 2, 4, 6 Tribromoanisole and 2, 4, 6 Trichloroanisole Taints and Odors in the Pharmaceutical and Consumer Healthcare Industries.

Mold Contamination in a Manufacturing Facility

In October 2011 the Sanofi Pasteur’s sterile product manufacturing facility in Toronto, Canada experienced flooding that lead to water damage. The consequences were fungal colonization in water damaged building materials, adverse trends in fungal isolation during environmental monitoring, questions from the Australian health authorities as to the state of validation of their sterility test for BCG tuberculosis vaccine, multiple 483 observations from an April, 2012 FDA inspection and a subsequent July 12, 2012 FDA warning letter.³ The FDA warning letter highlighted for a period from August 2010 through April 2012, fifty-eight non-conforming mold isolations occurred without adequate investigation and corrective action, insufficient frequency of monitoring in relation to the duration of media fills, poor aseptic technique in the aseptic processing areas, inadequacy of the firm’s disinfectant/sporicidal agent effectiveness studies with respect to fungal spores and poor facility maintenance. The firm decided in July 2012 to halt production and repair the building and recall four vaccine lots precipitating a global shortage of BCG vaccine and significant financial losses for the company.

Multi-State Meningitis Outbreak Due to Mold Contaminated Compounded Sterile Preparations

In October 2012, the FDA inspected the sterile compounder New England Compounding Corporation (NECC), Framingham, MA for cause in response to a multi-state outbreak of fungal meningitis. The inspection was against the GMP regulations as applied to sterile product manufacturing facilities.⁸ The 483 observations revealed issues with bacterial and fungal contamination in the clean rooms used for sterile compounding. The environmental monitoring records show the clean rooms and ancillary rooms and areas had counts of bacteria and molds that frequently exceeded the action level.

Furthermore, the 483 observations cited dozens of samples of methyl prednisolone acetate contained either greenish black foreign matter or white filamentous material. Sterility testing by the FDA Northeastern Regional Laboratory confirmed the presence of the environmental fungus Exserohilium rostratum as well as other bacteria and molds. Despite extensive cleaning in anticipation of regulatory inspections, discolorations were observed on several pieces of equipment at the facility like autoclaves used in the manufacture of sterile product, including the injectable steroid. In addition, dark particulates and white filamentous substances covered the louvers of the Heating Ventilation and Air Conditioning (HVAC) return behind the autoclaves. The inspectors also noted that large equipment used for excavation in a waste recovery area was producing airborne particulates outside the facility, approximately 100 feet from the intake of the NECC’s HVAC system.

As of the final CDC update in October 2015, this multi-state outbreak resulted in 753 fungal meningitis infections in at least 20 states and 64 deaths due to Exserohilium rostratum from contaminated preservative free MPA steroid injections.⁹ In December, 2015, NECC President Barry Cadden and Supervising Pharmacist Glenn Chin were indicted on racketing charges and 25 counts of second-degree murder for knowingly distributing contaminated compounded sterile products.

Comprehensive reviews of fungal-associated drug product and medical device outbreaks and recalls beyond the three discussed above are published in the technical literature.^10,11 These outbreaks included products are diverse as wooden tongue depressors, skin-moisturizing lotion, contact lens solutions, and compressed tablets.

Current Situation

As a result of these high profile events, pharmaceutical microbiologists, quality organizations, and manufacturing management have to review their company’s position as to mitigating fungal contamination risk and may have found the following:

• Insufficient attention may be given to fungal isolation and trending during environmental monitoring,
• disinfectant effectiveness studies may not adequately address disinfectant activity against fungal spores,
• a lack of appreciation as to water damage promotion of fungal growth within pharmaceutical facilities, and
• most pharmaceutical microbiology laboratories lack the capability to reliably identify fungi to genus, and especially species.

To successfully monitor and control fungal contamination trending/ tracking rules for fungal isolation especially in Class C and D (ISO 7 and 8) areas need to be defined, fungal identification capabilities must be available to support environmental monitoring programs, the potential sources of fungi in a manufacturing environment should be understood, aggressive corrective actions to fungal excursions must be in place, and the environmental monitoring and remediation program must be adequately documented and available during regulatory inspections.

Choice of Fungal Isolation Media

What is the preferred fungal isolation media? Sabouraud Dextrose Agar (SDA), Malt Extract Agar (MEA), Potato Dextrose Agar (PDA), Rose Bengal Agar, or V8 Agar? The pioneering French mycologist Sabouraud, for the cultivation of dermatophytes, developed SDA. The low pH (5.6) and high carbohydrate content is favorable for the growth of fungi, especially dermatophytes, and inhibitory to bacteria in clinical samples. The addition of antibiotics, an option unavailable to Sabouraud in 1892, such as chloramphenicol may further increase the selectively of the medium by suppressing the growth of bacterial colonies that can overgrow the plates. SDA is the required medium for the Total Combined Yeast and Mold Count described in USPand is widely used in environmental monitoring in the pharmaceutical industry. However, the low pH may inhibit the recovery of some fungi. In 1992, the Emmons modification to the original formulation adjusted the pH from 5.6 to 6.9 and reduced the dextrose from 40 to 20 g/L. The authoritative Difco Manual reports a good cultural response with 8 fungal and 1 filamentous bacterial species when incubated at 25 ± 2°C with the plates inverted for 5 days.¹²

To summarize, the media of choice (Table 1) for indoor air monitoring moldcontaminated buildings are MEA for general fungal isolation, DG18 for xerophilic fungal isolation, and V8 agar for Stachybotrys and Chaetomium species.¹³ However, in comprehensive comparison of different media for environment monitoring in pharmaceutical cleanrooms using settling and contact plates, supported the use of SDA for the widest range of isolation of different fungi and MEA for the greatest number of isolates.¹⁴

In a related study, Weissfeld et al (2013) evaluated the trade organization, Controlled Environmental Testing Association (CETA) recommendation that the use of a single medium, Typtic Soy Agar (TSA) is acceptable for environmental monitoring in sterile compounding pharmacies.¹⁵ This is a position generally supported by recommendations in the USP general informational chapter. What the study found by analyzing more than 5 years of environmental monitoring data using a volumetric air sampler with two media was that MEA yielded more than 2.5 times more fungal isolates than TSA from samples collected at the same locations. However, superior fungal recovery on MEA reported may be the result of the higher incubation temperature and shorter incubation time for the TSA in this study (72 hours at 35 ± 2°C) that would favor bacterial over fungal isolation compared to that of the MEA (7 days at 28± 2°C).

More recently Gordon et al (2014) compared the different incubation conditions used for microbiological environmental monitoring and found that the highest recovery for total microbial counts from areas with personnel transit was with the general microbiological growth medium TSA incubated at 30-35°C and for molds with mycological medium SDA incubated at 20-25°C.¹⁶ Based on their experience, single-plate strategies using either a two-temperature incubation or an intermediate incubation temperature of 25-30°C yielded reasonable recoveries of total aerobic counts and mold counts. Notably, the 30-35°C incubation followed by a 20-25°C incubation had the lowest mold recovery. However, a laboratorybased study performed in parallel was inconclusive. These overall findings were generally supported by additional studies.¹⁷

In the absence of a consensus, the author recommends two possible approaches to fungal monitoring. TSA may be used with an incubation schema of 2-3 days incubation at 30-35°C to encourage bacterial isolation especially those derived from human skin followed an additional 2-5 days at 20-25°C to allow for fungal growth or to simultaneously collect air and surface samples on TSA and MEA and incubate the plates for 48-72 hours at 30-35°C and 5-7 days at 20-25°C respectively.

Fungal Isolation and Enumeration Methods

Are active air samplers, settling plates, contact plates, or spore traps the best indicators of fungal contamination of a cleanroom? Active air samplers give a quantitative measurement of the colony-forming units per volume of air during a short sampling time but may be invasive, distorting the laminar airflow whereas settling plates are passive sampling devices that may monitor the air cleanliness over a 4 hour period.

What is the preferred incubation temperature? 20-25, 25-30 or 30- 35°C? Although an incubation temperature of 20-25°C is widely used in the pharmaceutical industry to recover molds and 30-35°C to recover bacteria, the technical literature suggests that a 25-30°C incubation temperature may be preferable based on the optimal growth temperature of common environmental molds.²⁰

Are there seasonal differences in the number and types of fungi in cleanrooms? Although the seasonal numbers of outside fungal counts is highest in the summer and early fall, the environmental controls, i.e. temperature and humidity, space pressurization, HEPA filtered air and number of air changes should make the fungal counts within a pharmaceutical manufacturing facility largely independent of the season of the year (Table 2).

What species are indicative of fungal growth in the building materials of a pharmaceutical facility? Studies on water-damaged buildings compare the airborne counts and dominant fungal species in the outside and inside air and fungal species associated with water damaged construction materials from the walls and ceilings. In general, the ratio of indoor/outdoor fungal counts is Cladosporium herbarium, C. cladosporioides, non-sporulating isolates and Alternaria alternata (50%), Pencillium viridicatum (26%), P. aurantiogriseum (16%), and 7% were comprised of Asperigillus versicolor, A. sydowii, P. variable, P. brevicompactum, P. crustosum and P. chrysogenum. The dominant fungi in outdoor air were similar with Cladosporium herbarium, C. cladosporioides, non-sporulating isolates, Alternaria alternata and Eppicoccom nigrum and multiple species of the genera Penicillium and Aspergillus. Fungal isolates from visibly damaged wallboard were Penicillium aurantiogriseum, P. viriicatum, Paecilomyces variotti, Chaetomium globosum, Memnonilla echinataand Stachybotrys chartarum. The toxicogenic fungus Stachybotrys chartarum was more frequently detected in lactophenol cotton blue mounts than isolated on solid media, as it is not a prolific sporulating mold.²²

What is the experience in pharmaceutical manufacturing facilities? Comprehensive reports are not common. Microorganisms isolated over an 8-year period in a sterile product manufacturing plant summarized in Table 3 are informative reinforcing the view that the majority of the bacteria isolated are Gram-positive cocci from human skin and fungi are most prevalent in support areas to the aseptic processing areas.²³

Skin-borne Gram-positive cocci predominant in all grades of cleanroom. However, in the less controlled Grade C and D areas, the proportion of Gram-positive cocci decreased, and Gram-positive non-spore-forming rods and Gram-negative rods increased while in the Grade D areas there was a marked increase in fungi. This can be attributed to more personnel traffic; the entry of non-sterile equipment and components, the presence of sinks, and reduced number of air changes and space pressurization. The percentage of occurrence of different fungi in descending order23 was Cladosporium spp. (28%), Penicillin spp. (19%), Curvularia spp. (10%), Aspergillus fl avus (7%), Alternaria spp. (6%), A niger (6%), Fusarium spp. (6%), A. fumigatus (5%), Exserohilium spp. (3%), A. terreus (2%), Rhizopus spp. (2%), Bipoloris spp. (2%), Aspergillus spp. (1%) and Mucor spp. (1%).

The Role of Compendial Testing in Fungal Monitoring and Control

Is the sterility test media and incubation condition conducive for fungal isolation? The USPsterility test uses two culture media incubated at two different incubation temperatures. The use of Fluid Thioglycollate Medium incubated at 30-35°C for at least 14 days and Soybean-Casein Digest Medium incubated at 20-25°C for at least 14 days is a compromise solution to the isolation of a wide range of microorganisms.²⁴ Analysis of sterility test failure shows that typically the microorganisms associated with the failure is less likely to be isolated in both media and greater than a third of the failure occur between the 7 and 14 day of incubation.^25,26 The author expects that fungal contamination would most likely to detected using the Soybean-Casein Digest Medium incubated at 20-25°C.

Disinfection Programs

Within a disinfection program, fungal control must be considered. Table 4 highlights the relative resistance of microorganisms to commonly used disinfectants. Note the relative resistance of fungal spores. The choice of disinfectants widely used in the pharmaceutical industry in the U.S. that are most suitable for different microorganisms is found in Table 5.

Sample Size

Should active air sampling use two media for bacteria and molds and is the sample size 500 or 1000 L per medium? It may be prudent to move from a single general microbiological growth medium like soybean-casein digest agar to two media such as soybean-casein digest agar and malt extract agar or Sabouraud dextrose agar, if mold begins to be isolated. Logically in terms of evaluating the results against industry action levels (Table 6) a sample size of 1000 L or 1 M³ per media makes sense.

Product Recalls

What fungi have been implicated in pharmaceutical product recalls, infectious contamination outbreaks, and nosocomial infections? A recent US survey (27) of 144 recalls, for the seven year period from 2004 through 2011 of non-sterile branded pharmaceutical drug products (5%), over-the-counter drug products (42%), cosmetics (31%), medical devices (14%) and dietary supplements (eight per cent of the total recalls) for microbiologically-related issues highlighted that the majority of these recalls (72%) were associated with objectionable microorganisms4. The most frequently cited microorganisms in the recalls were the Burkholderia cepacia (34 occurrences), unspecified fungal contamination (19 occurrences), Bacillus cereus (nine occurrences), Pseudomonas aeruginosa (six occurrences), Elizabethkingia meningoseptica (five occurrences), Enterobacter gergovia (five occurrences), Pseudomonas putida (three occurrences), Pseudomonas spp. (two occurrences) and Salmonella spp. (two occurrences). Seventeen other occurrences were associated with a single species. As noted above, fungal contamination is the second most frequent cause of product recall behind B. cepacia. The unspecified fungal identification may imply multiple fungal contaminants or the inability of the manufacturers to identify the implicated fungi. This author believes that the pharmaceutical industry is doing a poor job in area of mycology.

Clinical Experience

Superficial infections of the skin and nails are the most common fungal infections in humans and affect around 25% of the population worldwide. Mucosal yeast infections of the oral and genital tracts are common especially vulvo-vaginal candidiasis in women of childbearing years. Invasive fungal infections that have a much lower incidence rate are more serious due to their high mortality rates. According to the literature, the top five opportunistic invasive fungal infections in descending order are caused by Cryptococcus neoformans, Candida albicans, Pneumocystis jirovecii, Asperigillus fumigatus and Rhizopus oryzae. ²⁸

Facility Inspection

What are the best methods for facility inspection? The options are visual inspection, blue light, water activity measurements and optical scopes. Visual inspection for signs of water damage and mold growth should be routine and damaged areas remediated. The detection of mold that may not be sporulating may be assisted by the use of blue light, as mold will fluoresce. Water activity measure devices are widely used by the building industry to detect poorly dried lumber and other water damaged building materials. These inexpensive devices can be purchased at building supply chains. Optical scopes may be useful for detecting mold growth in ductwork, and behind walls and ceilings.

Is environmental monitoring fl awed as prolific spore-forming molds are over represented? An argument can be made that fungal species that grow on building materials without sporulation will not contribute greatly to airborne and surface fungal contamination so their underrepresentation in environmental monitoring will reflect their potential for drug product contamination.

What is the origin of most fungi found in cleanrooms? Plants, soil, people or facilities? There is a strong seasonal pattern of airborne fungal spores related to seasonal plant growth especially in the Eastern states peaking in the summer and early fall. Whether these phyllophane fungi enter a building will depend on the construction of the building, its ventilation and environmental controls.

Risk Mitigation

As required by 21 CFR 211.42 facilities must be constructed of nonporous material that do not absorb water and promote fungal growth. If floods occur the maintenance staff needs to react to water damage within a facility aggressively and fully dry or replace damaged walls and ceilings within 72 hours to prevent mold growth. Adequate temperature and humidity controls must be in place to discourage fungal growth. In addition, cellulosic materials such as cardboard and wooden pallets must be excluded from GMP areas as they can become wet and support fungal growth. Cleaning and disinfection procedures must be in place to prevent people and mobile equipment tracking fungi into our facilities.

Studies to qualify site disinfection programs must adequately address fungal mycelia and spores as well as bacteria. Routine environmental monitoring data trending and annual reviews must fl ag new fungal isolates and ability of the disinfectants and sporicidal agents to achieve an adequate log reduction within a specified contact time confirmed. These activities must be adequately documented.

Fungal Identification

Experienced mycological expertise must be available in-house or in supporting contract-testing laboratories for classical fungal identification that is based largely on colony appearance and cellularmorphology. Larger companies with greater resources should build fungal identification capabilities by the implementation of proteomic and genotypic identification methods.

Most mycological laboratories have relied on phenotypic identification using colony morphology, color and sporulation, cellular diagnostic features like conidiophores, and carbohydrate utilization pattern.²⁹ However, these methods are time-consuming, subjective, and depend largely on the skill and experience of the mycologist in the laboratory.

Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI TOF) mass spectrometric methods are improving the quality and timeliness of microbial identification and reducing identification costs.^30-32 They are being implemented in both clinical and pharmaceutical microbiology laboratories as their first-line identification methods.

These proteomic methods are supplemented with genotypic methods especially when the fungal isolate is associated with product failures and the isolate is not in the MALDI TOF mass spectrometry library. The commercial suppliers of these technologies recognize these database limitations and are updating their libraries every 3 to 6 months and allow for in-house reference entries to be continuously added to the database,

The application of rapid rRNA base sequencing methods results in more timely and accurate species-level identification that has improved clinical outcomes were there may be a high mortality rate with systemic fungal infection and is applicable to the pharmaceutical industry.^33,34 There are two major sequencing targets for fungal identification the D1/D2 region of the large ribosome subunit (LSU) and the internal transcribed spacer regions (ITS1/ITS2). To identify a fungal isolate the steps are extract the nucleic acid from the fungal mycelium, amplify ITS gene and sequence compared to valid sequences using a recognized database with identification performed using relevant matches within database.

This may be a challenge in that mycologists estimate there are 1.5 million fungal species and less than 1% of this number are sequenced for the ITS region. Public ITS databases are available include International Nucleotide Sequence Database Collaboration (INSDC): GenBank, European Molecular Biology Laboratory (EMBL) and DNA Database of Japan (DDBJ). Of the 165,000 fungal ITS sequences available in INSDC less than half have full species names and an estimated 10% have incorrect names. Because of these challenges, there is a need for software to search validated databases for correct identifications.

Conclusion

Molds that outcompete bacteria at lower water activities have a high potential for contaminating pharmaceutical products, so need our attention. The high mortality rates with fungal infection amongst immune-compromised patients highlights the overall patient risk. The diversity of recent product recalls for mold contamination and infection outbreaks is a reminder to regulators, clinicians and pharmaceutical manufacturers alike of the dangers associated with product contamination. Pharmaceutical manufacturers need to give more attention to mold monitoring, identification and risk mitigation.

References

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3. FDA Warning Letter dated July 12, 2012 to Olivier Charmell, Sanofi, Paris, France http:// www.fda.gov/iceci/enforcementactions/warningletters/2012/ucm312929.htm
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8. New England Compounding Corporation FDA Form 483 Media Call, October 26, 2012 http://www.fda.gov/downloads/newsevents/newsroom/mediatranscripts/ucm327225.pdf
9. Centers for Disease Control and Prevention. 2015 Multistate outbreak of fungal meningitis and other infections www.cdc.gov/hai/outbreaks/meningitis.html
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Author Biography

Dr. Tony Cundell consults with a number of pharmaceutical, con-sumer health and beverage companies, microbiology instrument manufacturers, contract testing laboratories and sterile compounding pharmacies in the areas of microbial risk assessment, regulatory affairs, and microbiological testing.

He is a member of the 2015-2020 U.S. P Microbiology Committee of Experts. Recently he received the 2016 PDA Martin Van Trieste Pharmaceutical Science Award for outstanding contributions to the advancement of pharmaceutical science.

Tony Cundell has a Ph.D. in Microbiology from the Lincoln University, New Zealand.