1/
Solutions for Controlling Fungal
and Bacterial Spores in Cleanrooms
Jim Polarine
Technical Service
Manager
PDA Midwest
Chapter
May 11, 2017
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Common sources of Bioburden
•Disinfectant Qualification Studies
•Bringing the Cleanroom Online
•FDA WLs and 483s
Agenda
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Review - Microflora in
Cleanrooms (U.K.)
• Tim Sandle
• PDA J Pharm Sci and Tech 2011, 65:392-403
• A Review of Cleanroom Microflora: Types,
Trends, and Patterns
• Examined isolates from 2000-2009 in U.K.
• Grade A/B and C/D
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Review - Microflora in
Cleanrooms (U.K.)
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Review - Microflora in
Cleanrooms (U.K.)
GenusA/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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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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Molds common to cleanrooms
and cold rooms
•Aspergillus spp.
•Penicillium spp.
•Stachybotrys spp.
•Cladosporium spp.
•Mucor spp.
•Scopulariopsis spp.
•Trychophyton spp.
•Chaetomium spp.
•Candida albicans (yeast)
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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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Aspergillus niger
http://genome.jgi-psf.org/Aspni5/Aspni5.home.html
http://nigergallery.blogspot.com/2012/10/aspergillus-niger.html
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Penicillium
Courtesy Ann Larson
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Cleanroom Fungi
Courtesy Dan Klein
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Exosporium – B. anthracis
Cote CK et al. 2011. Microbes and Infection
13(14-15):1146-55.
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Inoculum Preparation
Fungal Spores
Cultures need to be
incubated for a
sufficient length of
time before
harvesting spores
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Common sources of Bioburden
Disinfectant Qualification Studies
•Bringing the Cleanroom Online
•FDA WLs and 483s
Agenda
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Testing Against Fungal
Spores
• Trichophyton mentagrophytes is US EPA standard (easily killed)
• Cleanroom users test Aspergillus brasiliensis (typically the most difficult to kill mold)
Disinfectant Time KillPhenolic Disinfectant
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
A. niger T. mentag. C. albicans
Microorganism
Ave.
Lo
g R
ed
ucti
on
3 min.
5 min.
10 min.
15 min.
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Case Study on Substrates
Efficacy (log reduction) of Low pH phenolic: (1:256 ) against test microorganisms on representative surfaces
Surface
Staphylococc
us
epidermidis
Pseudomonas
aeruginosa
Corynebacteri
um
glutamicum
Candida
albicans
Aspergillus
brasiliensis
Penicillium
chrysogenum
Stainless Steel 6.62 >6.10 b 4.18 >4.31 b <3.00c 4.95
Glass 6.85 6.42 5.26 >5.80 b 2.98 5.11
Aluminum 6.35 5.69 5.14 >3.93 b <3.00c 3.48
Epoxy 4.36 4.45 4.48 3.19 <3.00c <3.00c
Enamel >6.05b >5.72 b 5.45 >3.92 b <3.00c 2.83
Acrylic 4.53 6.06 4.49 2.92 <3.00c <3.0 c
Mipolam 4.36 3.87 4.29 4.37 <3.00c 3.25
Vinyl 4.08 3.68 3.93 2.61 <3.00c 2.1
Hardwood 5.18 >4.54 b 5.26 3.2 <3.00c 2.59
Melamine Covered
Wood >5.38 b >5.64 b >5.09 b >5.12 b 3.65 3.95
Plastic >5.73 b >5.32 b >5.05 b >4.04 b <3.00c 2.44
Plexiglas >5.90 b 5.62 4.83 >4.40 b <3.00c 3.85
Chromium 6.55 5.95 6.63 4.08 <3.00c 2.61
a Disinfectant Efficacy = (Log MSP(positive control)
- Log MSP(test coupons)
), where MSP(Positive Control)
= Mean surviving population on
positive control coupons; MSP(test coupon)
= Mean surviving population on test coupons after disinfectant treatment; b Each of
triplicate coupons showed no growth after disinfectant treatment; c Each of triplicate coupons showed TNTC growth
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Hard Surface Test Results
-1.00
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
Glass Stainless
Steel
Enamel Epoxy Miplan Vinyl Acrylic
Avera
ge L
og
Red
ucti
on
Surface
Hard Surface ComparisonH2O2/PAA RTU
Bacillus subtilis ATCC 19659
1 min
5 min
Mipolam
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Environmental Isolate Testing
2 Log Reduction
Target
3 Log Reduction
Target
Spore Forming Microorganisms
0
1
2
3
4
5
6
7
8
9
10
70%
IPA
Klercide A
LpHse
Ves
phen
e IIse
Klercide B
Hyd
rogen
Per
oxide
Spo
rKlenz
RTU
Hyp
oChlor
ite(1:1
0)
Lo
g R
ed
ucti
on
± S
D
A. niger
B. pumilus
B. subtilis
0
1
2
3
4
5
6
7
8
9
10
Lo
g R
ed
ucti
on
±S
D
Vegetative Microorganisms
C. albicans
M. luteus
P. aeruginosa
S. aureus
S. maltophilia
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Spore Testing
19
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
6% Hydrogen Peroxide SK RTU
Ave
. Lo
g R
edu
ctio
n6% H2O2 vs. Spor-Klenz RTU
Standard Time Kill Study 13 Jun 2007B. subtilis spores 19659 Baseline = 6.60 log10
1 min
3 min
5 min
10 min
3, 5, 10 minute contact 1, 3, 5 minute contact
STERIS Corporation
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70% IPA Efficacy against Molds
Fungicidal Activity of 70% Isopropyl Alcohol using Time Kill Method
0
1
2
3
4
5
6
7
A. niger 16404 A. fumigatus 28282 A. fumigatus 96918 T. mentagagrophtes
9533
S. chartarum 16275 P. notatum 10108
Organism
Lo
g R
ed
ucti
on
30 sec 60 sec 120 sec
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H2O2/PAA RTU against Molds
Fungicidal Activity of H2O2/PAA RTU using Time Kill Method
0
1
2
3
4
5
6
7
A. niger 16404 A. fumigatus 28282 A. fumigatus 96918 T. mentagagrophtes
9533
S. chartarum 16275 P. notatum 10108
Organism
Lo
g R
ed
ucti
on
30 sec 60 sec 120 sec
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Efficacy Testing
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Fungal Testing
Comparison
H2O2/PAA Conc. Quat Conc. Quat
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Temperature = 0°C
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
Ave
rag
e L
og
Re
du
cti
on
Formulation
Time Kill - Affect of Temperature (0°C) on Formulations against B. subtilis ATCC 19659
Baseline = 6.83
30 sec contact
1 min contact
3 min contact
5 min contact
10 min contact
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Temperature = 60°C
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
Ave
rag
e L
og
Re
du
cti
on
Formulation
Time Kill - Affect of Temperature (60°C) on Formulations against B. subtilis ATCC 19659
Baseline = 6.83
30 sec contact
1 min contact
3 min contact
5 min contact
10 min contact
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Surface Preparation
Autoclaving may not be acceptable for some
surfaces (Saniflex)
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Surface Tension Issue
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Surface Type and Condition
• Visually smooth
surfaces can be
irregular
• Older or damaged
surfaces can be
more challenging
• Glass and stainless
steel typically the
least challenging
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Surface Conditions Effect Performance
Courtesy Bruce Ritts
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Common sources of Bioburden
Disinfectant Qualification Studies
Bringing the Cleanroom Online
•FDA WLs and 483s
Agenda
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Case Study:
Construction Event
• Worst Case Events
• 9X Clean [1X Sporicide + 2X Phenolic
repeated on days 1,2,3]
• Fogging
• VHP®
• Triple Clean– Defined 3X Disinfectants and Sporicide (Different
Definitions)
– EM frequency (Static and Dynamic)
– Release of the room
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Cleaning and Disinfection
Efficacy - In situ study
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Time 0
Red = Spore formers Green = Other
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After 1X Cleaning - No
Sporicide
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After 2X Cleaning – No
Sporicide
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After 3X Cleaning - No
Sporicide
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After Sporicide
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Common sources of Bioburden
Disinfectant Qualification Studies
Bringing the Cleanroom Online
FDA WLs and 483s
Agenda
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Recent FDA WL
“no use of sporicidal disinfectant on surfaces
inside aseptic filling room (b)(4), although your
environmental monitoring detected spore-
forming organisms there; and”
FDA Warning Letter 1/17/17.
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FDA WL
“Our inspection found multiple deficient practices at
your facility that pose a significant microbiological
contamination risk. For example, your cleaning and
disinfection program lacked use of a sporicidal
agent. Significantly, the microbe identified in the
sterility failures is a spore-former. In addition, our
inspection identified poor facility maintenance. This
included leaking pipes in the cleanroom ceiling,
chipped and cracked floors in the batch tank room, and
blue and black particulates as well as dust on tanks
next to the ingredient charging ports.”
FDA WL 12/23/16
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Recent FDA 483
Specifically,
a. Despite your firm’s use of sporicidal disinfectants, a
variety of spore forming bacteria are routinely recovered from your
environmental and personnel monitoring within the aseptic
processing areas.
b. Non-sterile disinfectants are routinely used by your
employees during cleaning of aseptic processing area, including
the critical work area and buffer room. For example, the technician
was observed disinfecting the interior walls, followed by the aseptic
processing work surfaces using the same mop head.
c. Your firm has not conducted disinfectant efficacy studies
to demonstrate that the disinfectants and application methods (e.g.
spray, wipe, mop, aerosol, etc.) used to clean the walls, ceilings,
work surfaces and other items in the work areas can sufficiently
reduce bioburden.
GMP Trends July 15, 2016
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FDA Warning Letter -
Investigations
“There was no investigation by the firm when levels exceeded their action limits and there was no identification of the isolates. No documented corrective actions were taken to remove microbial contamination (bacteria and mold) from the facility.”
FDA Warning Letter 10-26-12
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Summary Slide
•Common sources of bioburden when starting
up the cleanroom
•Disinfectant Qualification Studies
•Bringing the cleanroom online
•FDA WLs and 483s
44/
Control of Fungal and Bacterial
Spores in Cleanrooms
Thank you for
your attention