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Fungal and Bacterial Spore Excursions in
Cleanrooms: Case Studies
Jim Polarine
Sr. Technical
Service Manager
Pharmig
November 29, 2018
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Copyright © 2017 STERIS Corporations. All Rights Reserved.
Outline
Fungal and Bacterial Spore Excursions: Case
Studies
• Bacillus cereus Contamination Case Study
• Fungal Spore Contamination Case Studies
• Contamination Sources
• Proactively prevent recontamination
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Review - Microflora in Cleanrooms (U.K.)
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Review - Microflora in Cleanrooms (U.K.)
Genus A/B
(6729) C/D
(2500)
Micrococci (and related) 38% 40%
Staphylococci 21% 11%
Bacillus (and related) 13% 10%
Pseudomonas (and related) <1% 8%
Corynebacterium (and related) 3% 5%
Rhodococci <1% N/A
Fungi N/A 3%
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Risk Assessments
• Pathogenic or Toxic Molds
• Does it affect product contact surfaces
• Where is it picked up onsite
• Create a risk map or diagram
• What are the possible assignable causes
• Proactively prevent the mold from reoccuring
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Where does the Investigation team begin?
Cause & Effect Diagram example:
Machine Method Materials
Aspergillus Contamination
Mother Nature
Man Measurement
HVAC Differential Pressures
Preventive Maintenance
Equipment Repair
Instrument Calibration
Change Controls
SOP Changes
Cleaning procedures
Raw materials
Cleaning products
Equipment Flow
Leak in Ceiling
Power Outage
Tornado
Frequency of Cleaning
Limits too high
Limits too low
Construction
Training
Personnel Monitoring
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Investigation Strategies:
Critical Zones
Trend history of organisms in the area versus the contaminant.
Sample Type Sample Frequency Minimum Data Review Period
Water
Daily ≥ 3 weeks prior to the excursion
Weekly ≥ 3 months prior to the excursion
Pure Steam Weekly
Monthly Quarterly
≥ 3 months prior to the excursion ≥ 3 months prior to the excursion ≥ 6 months prior to the excursion
Compressed Air/Gas Quarterly ≥ 1 year prior to the excursion
Environmental Monitoring Daily ≥ 2 months prior to the excursion
Environmental Monitoring Bi-Weekly or Monthly ≥ 6 months prior to the excursion
Personnel Applicable Facility SOP ≥ 3 months prior to the excursion
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Common Sources of Spores
• Items brought into the Cleanroom – Bags, Boxes, Intervention Equipment, Pallets, Pallet
Jacks, Scrubbers, Cart Wheels, Carts, Shoes, Shoe
Covers, Markers, Pens, Cellphones
– Raw Materials and components
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Bacillus cereus Contamination
Case Study
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Microorganism Resistance
Hierarchy
From McDonnell, “Antisepsis, Disinfection, and Sterilization: Types, Action, and Resistance” 2007, ASM Press
Bacillus cereus / sphaericus
Bacillus subtilis / G. stearothermophilus
Clostridium spp.
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Bacterial Spores in
Operations
•Bacillus subtilis
•Bacillus cereus
•Bacillus pumilus
•Bacillus licheniformis
•Bacillus sphaericus
•Bacillus thuringiensis
•Paenibacillus polymyxa
•Geobacillus spp.
•Clostridium difficile
B. cereus Group:
B. cereus, B. anthracis, B.
mycoides, B. thuringiensis,
B. pseudomycoides,
B. weihenstephanensis,
B. manliponensis
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Bacterial Endospore
Courtesy Dan Klein
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Bacterial Endospore Bacterial Spore Structure
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Bacillus cereus Case
Study
• ISO-7 and ISO-8 cleanrooms
• Spores tracked throughout the facility
• Process Vessels
–Source Locations
•Cleanroom Shoe Cover
•Fermenter
•Process Vessels
The Source was a Raw Material
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Exosporium – B. anthracis
Cote CK et al. 2011. Microbes and Infection 13(14-15):1146-55.
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Exosporium – B. anthracis
https://www.llnl.gov/str/Sep06/Velsko.html
Hydrophobicity helps adhere to fibers
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A schematic diagram illustrating a possible model for the exosporium of the B. cereus family.
Kailas L et al. PNAS 2011;108:16014-16019
©2011 by National Academy of Sciences
Model of B. cereus exosporium
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Bacillus Testing
Wyeth Spore Isolate T.K. Wyeth Spore Isolate B.cereus, 5.81, n=2
10-16-08
0.001.00
2.003.00
4.005.00
6.007.00
SK R
TU
1:4
Bleac
h
1:10
Ble
ach
CIP
100
CIP
200
Wes
cody
ne
Pro
klen
z 1
Sk Con
c. 1
:18
Formulation
Av
era
ge
Lo
g R
ed
uc
tio
n
20 min.
40 min
60 min
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Fungal Spore Contamination
Case Study
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Molds common to cleanrooms
and cold rooms •Aspergillus spp.
•Penicillium spp.
•Stachybotrys spp.
•Cladosporium spp.
•Mucor spp.
•Scopulariopsis spp.
•Trychophyton spp.
•Chaetomium spp.
•Acremonium
•Candida albicans (yeast)
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Aspergillus
• ISO-5 Cleanroom
– Source
• High Impingement Spraying Device
• Broken Pipes
• HVAC Shut Down
• Exceeding Limits in ISO-7 areas
– Dock Doors proximal to ISO-7
cleanroom
– Storage room with limited control
– No limits for mold spores (Establish
Limits)
– Limited control for incoming and
outgoing items
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High Pressure
Impingement Sprayers
Holes
in Walls
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Sources of Aspergillus
Behind Cleanroom Door Kickplate
Black Sharpie
Marker Tip
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Courtesy Dan Klein
Aspergillus brasiliensis
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Aspergillus Investigation
• Sporicidal usage in pass-through
• Clean and dirty area on the dock
• Better control of gowning area
• Cart Wheel control methods
• Better gowning control
Photo: Terra Universal
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NECC Fungal
Contamination
There was no investigation by the firm when levels exceeded the action
limits and no identification of isolates. No documented corrective
actions were taken to remove microbial contamination (bacterial and
fungal) from the facility. (FDA WL October 2, 2012)
83 out of 321 vials of methylprednisolone acetate contaminated (fungal
contamination found)
64 deaths and 750 Illnesses
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Aspergillus brasiliensis
Courtesy Bruce Ritts
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Courtesy Bruce Ritts
Aspergillus spores Aspergillus brasiliensis
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Spiny Spores
Courtesy Dave Shields
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Courtesy Dan Klein
Cleanroom Fungi
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Penicillium
• Two ISO-7 Cleanrooms
• Action Levels of 10 and picking up >100 Fungal Spores
– Engineering Investigating
– HVAC
– Duct Work
– HEPA Filters
– Cooling Coils (two hits)
– Wall Coverings
– Airflow Vents
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Penicillium Investigation
• Entry and Exit Procedures
• Gowning Procedures (Triple Gowning)
• Cart Wheels
• Construction (Current Maintenance Log)
– Further Investigation
– Use of Sporicides and Frequency
– Plastic Containers in the Cleanroom
– Coldroom Cleaning Procedures (0-
5°Celsius)
– Documentation of Cleaning Process
– Designate Assignable Causes
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Penicillium Investigation
Cleanroom Cooling Coils
Plywood from a
Wooden Pallet from
Construction
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Penicillium
Courtesy Ann Larson
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Penicillium Spores
Courtesy Bruce Ritts
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Glass Evaluation
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Stainless Steel Evaluation
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Flooring Evaluation
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Cladosporium Spores
Courtesy Bruce Ritts
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Paecilomyces Spores
Courtesy Bruce Ritts
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Leptosphaerulina Spores
Courtesy Bruce Ritts
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Testing Bacterial Spore
Formers
• Bacterial spores are significantly more
challenging to inactivate than
vegetative cells (worst case for a
strain)
• Bacterial spores generally require
sporicidal agent to inactivate
• Spore form is most likely form of
Bacillus, Paenibacillus, etc. to be
encountered in classified area
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Testing Bacterial Spore
Formers
Can you consistently test
against “vegetative” Bacillus?
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Testing Bacterial Spore
Formers in DET
• Bacterial spore formation is a stochastic event
• Sporulation occurs within sub-populations (i.e. the entire suspension does not sporulate at the same time)
• Sporulation kinetics is complicated, depending on a multitude of environmental factors-NOT just nutrient scarcity
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Paenibacillus 24 Hour Culture
Courtesy of Jamie Knutson, STERIS Laboratories
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Paenibacillus 18 Hour Culture
Courtesy of Jamie Knutson, STERIS Laboratories
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18 Hour Paenibacillus Culture
Courtesy of Jamie Knutson, STERIS Laboratories
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Contamination Sources
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Contamination Sources
Facility • Poor design
• Aging facility
• Maintenance
• Cleaning and
disinfection
People • Gowning rooms
• Attire (clothing, shoe covers,
hoods, face masks, goggles, etc)
• Conduct
• Standard Operating Procedures
Materials • Pass through sterilizers
(autoclaves, dry heat ovens,
depyrogenation tunnels, etc.)
• Decontamination chambers (EO,
VHP, UV, etc.)
• Material handling airlocks
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Industry References
• USP 41 <1072> Disinfectants and Antiseptics
• Annex 1 (2008) and MHRA Orange Guide (2015)
• FDA Aseptic Processing Guide (2004)
• FDA, MHRA, HPRA, CFDA, ANSM, ANVISA, & EMA Expectations
• Industry Articles (Ex. Scott Sutton, Jose Martinez, Tim Sandle, Richard Prince,
Rebecca Smith, Jeanne Moldenhauer), Crsytal Booth
• PDA Cleaning and Disinfection TR No. 70 (October, 2015)
• PDA TR No. 69 on Biofilms (2015)
• The CDC Handbook - A Guide to Cleaning & Disinfecting Cleanrooms (Tim Sandle
2016)
• A Guide to Disinfectants and their use in the Pharmaceutical Industry (Pharmig 2006)
• USP 41 <1116> Microbiological Control and Monitoring of Aseptic Processing
Environments
• USP 41 <1115> Bioburden Control of Non-Sterile Drug Substances and Products
• PIC/S Guide to Good Practices for the Preparation of Medicinal Products in
Healthcare Establishments (2014)
• WHO Annex 6
• PHSS Technical Monograph #20 “Bio-contamination characterization, control,
monitoring and deviation management in controlled/GMP classified areas
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Acknowledgements
Thank you audience!
STERIS Life Sciences
• Dan Klein – Sr. Manager, Research and Development
Microbiology and Clinical Affairs
• Bruce Ritts and Stacey Gish– STERIS R&D, Scanning
Electron Microscopy
• Dave Shields – Manager, STERIS Laboratories
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Introduction
Jim Polarine
• Senior Technical Service Manager
• STERIS Corporation | Life Sciences
• Mobile: 314-616-9931
• E-Mail: [email protected]
• Web: www.sterislifesciences.com
STERIS Technical Services
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Thank you for your attention!
Fungal and Bacterial Spore Excursions:
Case Studies