Fungal Contamination in Pharmaceutical Production Areas 6_Cundell_pres.pdf · Understanding, Investigating, and Remediating Mold Contamination in Pharmaceutical Production Areas Tony

Understanding, Investigating, and Remediating Mold Contamination in

Pharmaceutical Production Areas

Tony Cundell, Ph. D.

Consulting Microbiologist,

Scarsdale, NY

June 7, 2016 IVT Microbiology Workshop 1

FDA : More Attention Drawn to Mold Contamination

• High profile product recalls associated with fungal contamination has resulted in more attention from the FDA to mold isolation in environmental monitoring and product testing

• Pharmaceutical companies need to anticipate these concerns especially with respect to the upcoming FDA inspections and patient safety


Recent Fungal Contamination Recalls

High profile recalls for fungal contamination include:

• The 2009-2011 pharmaceutical tablet recalls due to fungal-generated TBA taints

• The July, 2012 FDA warning letter received by Sanofi Pasteur’s sterile product manufacturing facility in Toronto, Canada for fungal contamination.

• The New England Compounding Center of Framingham, Massachusetts, October, 2012 recall of three lots consisting of a total of 17,676 single-dose vials of the steroid, preservative-free methylprednisolone acetate after a multi-state outbreak of fungal meningitis caused by Exserohilium rostratum


Implicated Products


TBA-Tainted Tablet Recalls

• Tribromophenol(TBP)-treated lumber from South America was used for the construction of wooden pallets.

• Consequences– 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

– TBA was absorbed into HDPE bottles, which were transported on the pallets and used to package the tablets.

• When the user opened the bottles they were sickened by the highly unpleasant odor of the TBA taint


Sanofi BCG Vaccine Recall• In October, 2011 Sanofi Pasteur’s sterile product

manufacturing facility in Toronto, Canada experienced flooding that lead to water damage.

• Consequences– Adverse trends in fungal isolation during environmental monitoring

in Building 86,

– 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

– July 12, 2012 FDA warning letter

• The firm decided in July, 2012 to halt production and repair the building and recall four vaccine lots.


Sanofi Recall

• The July 12, 2012 FDA warning letter highlighted:

– For a period from August 2010 through April 2012 58 non-

conforming mold isolations occurred without adequate investigation and corrective action.

– Insufficient frequency of monitoring in relation to the duration of the fills.

– Poor aseptic technique in the aseptic processing areas,

– Inadequacy of the firm’s disinfectant/sporicidal agent effectiveness studies with respect to fungal spores

– Poor facility maintenance.

• This contributed to a worldwide shortage of BCG vaccine.


New England Compounding Center Recall

• In October, 2012 the FDA inspected the sterile compounder NECC for cause

– The inspection was against the GMP regulations as applied to sterile product manufacturing facilities.

– The 483 observations revealed issues with bacterial and fungal contamination in the clean rooms.

– 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.


NECC Recall483 observations:• Dozens of samples of methylprednisolone acetate contained

either greenish black foreign matter or white filamentous material. Sterility testing confirmed the presence of fungi.

• 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.

• 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 NECC’s HVAC system.


NECC Recall

• 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.

• In December 2015, NECC President Barry Caddenand Supervising Pharmacist Glenn Chin were indicted on racketing charges and 25 counts of second-degree murder for knowingly distributing contaminated compounded sterile products.


Common Environmental Fungi

Considerations with fungal isolates include:

• Their potential pathogenicity

• Ability to grow on solid microbiological media

• Diagnostic features for specification:• Colony morphology, color and sporulation

• Cellular morphology especially conidiophores, and conidia size, shape and color

• Carbohydrate utilization patterns


Cladosporium spp.

• Pathogenicity: Common saprophytic molds

• Growth: Moderately rapid with rich conidia formation within 7 days at 25°C. Most strains do not grow at 37°C.

• Colony Morphology: Surface greenish brown to black with grayish velvety nap, becoming heaped and folded. Reverse side is black.

• Cellular Morphology: Hyphae are septate and dark. Conidiophores are branched, vary in length, and usually produce two or more conidial chains. Conidia are brown, round to oval (3-6 x 4-12 micron) and are usually smooth.


Cladosporium cladosporides


Alternaria spp.

• Pathogenicity: Common saprophyte

• Growth: Rapid; mature within 5 days at 30°C

• Colony Morphology: Surface at first greyish white and woolly and later greenish black or brown with a light border. Reverse is black.

• Cellular Morphology: Hyphae are septate and dark. Conidiophores are septate, variable length, and has a zigzag appearance. The conidia are large (8-16 x 23-50 micron) and brown with both transverse and longitudinal septations.


Alternaria alterna


Aspergillus fumigatus

• Pathogenicity: Ubiquitous in nature. Causes invasive disseminated aspergillosis, allergic aspergillosis and fungal sinusitis.

• Growth: Rapid growth in 3 days. Has the ability to grow at 45°C.

• Colony Morphology: Surface is velvety or powdery, various shades of green with a narrow white border turning dark gray with age. Reverse white or tan.

• Cellular morphology: Septate hyphae. Conidiophore smooth and relatively short. The phialides are uniseriate, compact, with a columnar formation. Conidia smooth, round an small (2-3.5 micron in diameter)


Asperigillus fumigatis


Stachybotrys chartarum• Pathogenicity: Produces several mycotoxins that affect

humans after ingestion, inhalation or percutaneous absorption. Implicated in sick building syndrome.

• Growth: Moderately rapid, usually mature within 7 days but prefers high cellulose media than standard laboratory media.

• Colony morphology: Surface is white at first, becoming dark gray to black with age. Reverse is at first white then dark.

• Cellular morphology: Hyphae are septate. Conidiophore are simple or branched bearing clusters of 3-10 phialides. Conidia are dark, oral (4.4 x 9 micron), single cells, forming clusters on the phialides.

18June 7, 2016 IVT Microbiology Workshop

Stachybotrys chartarum


Media for Environmental Monitoring

• The media of choice for indoor air monitoring mold-contaminated buildings are MEA for general fungal isolation, DG18 for xerophilic fungal isolation, and V8 agar for Stachybotrys and Chaetomium species (Anderson and Nissen, 2000).

• However, in comprehensive comparison of different media for environment monitoring in pharmaceutical cleanrooms using settling and contact plates, (Gebalaand Sandle, 2013) supported the use of SDA for the widest range of isolation of different fungi and MEA for the greatest number of isolates.


Fungal Media



• 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.

• This is a position supported by recommendations in the USP general informational chapter <1116>.



• What the study found by analyzing more than 5-years of environmental monitoring data using a volumetric air sampler (SAS 180, BioScience International, Rockville, MD) 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).


Active Air Samplers


Media for Environmental Monitoring• 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 TSA incubated at 30-35°C and for molds with SDA incubated at 20-25°C. 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 count and mold counts.

• Notably, the 30-35°C incubation followed by a 20-25°C incubation had the lowest mold recovery. However, a laboratory-based study performed in parallel was inconclusive.


Recommended Approaches

• The presenter recommends two possible approaches to fungal monitoring:

• Tryptic soy agar (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.

• 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.


Airborne Monitoring Options


Surface Monitoring Options


Industry Response to FDA Concerns

Pharmaceutical microbiologists have review their company’s position as to mitigating fungal contamination risk and found the following:

• Insufficient attention may be given to fungal isolation and trending during environmental monitoring

• Disinfectant effectiveness studies may not address disinfectant activity against fungal spores

• Most pharmaceutical microbiology labs lack the capacity to reliably identify fungi to genus and especially species necessary for manufacturing investigations


Environmental Monitoring

• Trending/tracking rules for fungal isolation especially in Class D (ISO 8) areas need to be defined.

• Fungal identification capabilities must be available to support EM programs

• Potential sources of fungi should be understood

• Aggressive corrective actions to fungal excursions must be in place

• The EM program must be adequately documented


EM Requirements


FDA Aseptic Processing Guidance EU GMPs Annex 1

Active Air

sampling

Passive air

sampling

(optional)

Active Air

sampling

Passive air

sampling

Surface

Class 100: 1

cfu/m3

Class 100: 1

cfu/plate

Grade A: <1

cfu/m3

Grade A: <1

cfu/plate

Grade A: <1

cfu/ 25-30

cm2

Class 10,000: 5

cfu/m3

Class 10,000: 5

cfu/plate

Grade B: 10

cfu/m3

Grade B: 5

cfu/plate

Grade B: 5

cfu/25-30 cm2

Class 100,000:

100 cfu/m3

Class 100,000: 50

cfu/plate

Grade C: 100

cfu/m3

Grade C: 5

cfu/plate

Grade C: 5

cfu/25-30 cm2

Grade D: 200

cfu/m3

Grade D: 100

cfu/plate

Grade D: 100

cfu/ 25-30

cm2

Risk Mitigation

• Facilities must be constructed of non-porous material that do not absorb water and promote fungal growth

• Maintenance staff need to react to water damage within a facility aggressively and replace damaged walls and ceilings within 72 hours

• Adequate temperature and humidity controls must be in place

• Cellulosic materials such as cardboard and wooden pallets must be excluded from GMP areas

• Procedures must be in place to prevent people and mobile equipment tracking fungi into our facilities


Disinfectant Effectiveness Studies

• Studies to qualify site disinfection programs must adequately address fungal mycelia and spores

• EM annual reviews must flag new fungal isolates and ability of the disinfectants and sporicidal agents to achieve an adequate log reduction with a specified contact time confirmed

• These activities must be adequately documented


Fungal Identification in the Pharmaceutical Industry

• Experienced mycological expertise must be available in-house or in supporting contract testing labs for classical fungal identification.

• Companies must build fungal identification capabilities by the implementation of proteomic and genotypic identification methods


Fungal Identification: Phenotypic

• Most mycological laboratories have relied on phenotypic identification using:

– colony morphology, color and sporulation,

– cellular diagnostic features like conidiophores

– carbohydrate utilization patterns

– These methods are time-consuming, subjective and depend largely on the skill and experience of the mycologist in the laboratory

Larone, D.H. 2002 Medically-important Fungi: A Guide to Identification.

Fourth Edition ASM Press, Washington, D.C.


Fungal Identification: Proteomic

• MALDI TOF mass spectrometric methods are improving the quality and timeliness of microbial identification and reducing costs.

• They are being implemented in both clinical and pharmaceutical microbiology labs as first line identification methods as the databases expand.

• The proteomic methods are supplemented with genotypic methods.

• Phenotypic microarrays can be used to demonstrate metabolic diversity of our microbial isolates


Fungal Identification: Genotypic

• The application of rapid rRNA base sequencing methods results in more timely and accurate species-level identification that has improved clinical outcomes and is applicable to the pharmaceutical industry.

• 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).

Dong J., M.J. Loeffelholz and M. R. McGinnis 2012. Sequence-based fungal identification and classification. In Molecular Microbiology: Diagnostic Principles and Practice Second Edition ASM Press pp669-676


Fungal Target rRNA Sequences


Fungal Identification: Genotypic• To identify a fungal isolate :

– amplify ITS gene and sequence– compared to valid sequences using database– Identification can be performed using relevant matches with

database

• Mycologists estimate :– there are 1.5 million fungal species– Less than 1% of this number are sequenced for the ITS region.

• Public ITS Databases are available:– International Nucleotide Sequence Databases (ISND: GenBank,

European Molecular Biology Laboratory (EMBL) and DNA Database of Japan (DDBJ)

• 165,000 fungal ITS sequences available in ISDN– less than half have full species names – an estimated 10% have incorrect names

• There is a need for software to search validated databases for correct identifications.


Microbial Identification and Strain Typing

• State-of-the-industry phenotypic identification methods are being replaced by state-of-the-art proteomic and genotypic methods

• Strain typing is improving the quality and timeliness of environmental monitoring tracking/trending and product contamination investigations and corrective actions.


Fungal Identification Systems


Identification System Mechanism Data Size

Biolog FF System Substrate Utilization

plus photograph

library

400 species from 120

genera

Bruker Biotyper MALDI TOF mass

spectrometry

110 species from

around 40

different genera

BioMerieux Vitek MS MALDI TOF mass

spectrometry

X species from around

Y different genera



Identification

System

Mechanism Data Size

MicroSeq Fungal

Identification

system

Target amplification

and D2 LSU base

sequencing

V1.0 900 species

V2.0 1113 species

CR/Accugenix Target amplification

and ITS base

sequencing

1703 unique species

comprising 471 genera (2013)

SmartGene Target amplification

and ITS base

sequencing

1574 full-length ITS

sequences representing 516

species of 103 genera of mold

and 184 sequences of 8

genera of yeast (2008)



Identification

System

Mechanism Data Size

International

Nucleotide

Sequence

Databases

(GenBank/EMBL/D

DBJ)

Target amplification

and D2 and IST base

sequencing. Blast

searches for

homologous

sequences in public

databases

165,000 ITS sequences in INSD,

around 50% lack a species

name and around 10% with

incorrect names (2010)

Recommended Fungal SurveysAsk your customers the following questions:

• How many fungal IDs do you conduct each week?

• Are these fungal IDs conducted in-house or at a contract lab?

• What ID methods do you employ? – i.e. classical mycological methods,

– Biolog FF,

– MicroSeq D1 rRNA sequencing,

– MALDI TOF mass spectrometric methods,

– MicroSeq ITS rRNA sequencing.

• What is your required turnaround time for an ID?

• If you had the services of fungal ID center would the number of fungal IDs increase to how many per week?


Economic Mycology

The benefits of fungi deserves highlighting and include:

• Yeast for wine, beer, vinegar, and bread making.

• Fungi in cheese making

• Fungal fruiting bodies as food and garnishing

• Industrial fermentation for the production of chemicals, vitamins and antibiotics

• Rhizozymal fungi for plant and tree growth


Conclusions

• The speaker believes that pharmaceutical microbiologist must work to do a better job with the isolation, identification and control of mold in pharmaceutical manufacturing areas.

• Thanks for your attention.


Documents

Fungal Contamination in Pharmaceutical Production Areas 6_Cundell_pres.pdf · Understanding, Investigating, and Remediating Mold Contamination in Pharmaceutical Production Areas Tony