solutions for controlling fungal and bacterial spores in ... · solutions for controlling fungal...

1/

Solutions for Controlling Fungal

and Bacterial Spores in Cleanrooms

Jim Polarine

Technical Service

Manager

PDA Midwest

Chapter

May 11, 2017

2/Copyright © 2014 STERIS Corporations. All Rights Reserved. CONFIDENTIAL and PROPRIETARY to STERIS Corporation

Common sources of Bioburden

•Disinfectant Qualification Studies

•Bringing the Cleanroom Online

•FDA WLs and 483s

Agenda


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



Cleanrooms (U.K.)



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%


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.


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)


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


Aspergillus niger

http://genome.jgi-psf.org/Aspni5/Aspni5.home.html

http://nigergallery.blogspot.com/2012/10/aspergillus-niger.html


Penicillium

Courtesy Ann Larson


Cleanroom Fungi

Courtesy Dan Klein


Exosporium – B. anthracis

Cote CK et al. 2011. Microbes and Infection

13(14-15):1146-55.


Inoculum Preparation

Fungal Spores

Cultures need to be

incubated for a

sufficient length of

time before

harvesting spores



Disinfectant Qualification Studies

•Bringing the Cleanroom Online

•FDA WLs and 483s

Agenda


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.


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


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


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


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


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


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


Efficacy Testing


Fungal Testing

Comparison

H2O2/PAA Conc. Quat Conc. Quat


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


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


Surface Preparation

Autoclaving may not be acceptable for some

surfaces (Saniflex)


Surface Tension Issue


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


Surface Conditions Effect Performance

Courtesy Bruce Ritts




Bringing the Cleanroom Online

•FDA WLs and 483s

Agenda


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


Cleaning and Disinfection

Efficacy - In situ study


Time 0

Red = Spore formers Green = Other


After 1X Cleaning - No

Sporicide


After 2X Cleaning – No

Sporicide


After 3X Cleaning - No

Sporicide


After Sporicide




Bringing the Cleanroom Online

FDA WLs and 483s

Agenda


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.


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


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


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


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

[email protected]

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