BOSCH Bio DecontaminationH2O2

Bio-decontamination with H2O2 - Vapor
Bio-Decontamination with H2O2 Vapor

in Pharmaceutical Industry
Dr. Johannes Rauschnabel,
Robert Bosch GmbH
SBM Experts Congress 13th-14th of September, 2007

Packaging Technology
PA-PH/EPP | 10/09/2007 | Robert Bosch GmbH 2007. All rights reserved, also regarding any disposal, exploitation, reproduction, editing,
distribution, as well as in the event of applications for industrial property rights.
Structure
Introduction
VHP for Isolators
Results
More Applications
Summary
Structure
Introduction
z
Chemistry /
Biocidal Effect
Challenges
Regulatory
VHP for Isolators
Results
More Applications
Summary
Introduction
Hydrogen Peroxide is an old sterilant well established in food industry,

where liquid H2O2 is applied for disinfection of packaging material and
plastic bottles.
H2O2 has a broad antimicrobial activity spectrum and shows low toxicity.
Residues of hydrogen peroxide decompose into neutral species, which

underlines the environmental friendliness of this agent:
2 H2O2 ' 2 H2O + O2
But: hydrogen peroxide can decompose with fast kinetics under some
circumstances it can explode at high concentrations.
For complex geometries disinfection with liquid peroxide has limits. For
this case H2O2 in the vapor state is widely used.
Chemistry / Biocidal Effect
The hydrogen peroxide molecule has a

unique geometry with a high energy angle,
which makes splitting of O-O-bond easy:
H2O2 ' 2 HO
2 HO ' H-O-H + O2
The decomposition can be catalyzed by

metallic surfaces (Cu, Pb, Mg) which
can be used to decompose after use.
On the other side this makes the need for
noble surfaces in the equipment to be
disinfected.
Chemistry / Biocidal Effect
The biocidal effect of hydrogen peroxide

comes from the oxygen atom/radical,
which is released under influence of the
enzyme catalase at the microbiotic surface.
The oxidative power of oxygen radicals
cause irreversible damage to enzymic
systems and DNA.
A minimum of humditiy at the surface

to be treated helps to promote this metabolismism especially bacterial spores
need a basic humidity to show any biochemical activity.
Structure
Introduction
z
Chemistry /
Biocidal Effect
Challenges
Regulatory
VHP for Isolators
Results
More Applications
Summary
Challenges
Vaporized Hydrogen Peroxide is used

to disinfect bottles and pharmaceutical
equipment interior, such as isolators
(mini-cleanrooms on top of machinery).
Pioneer in that technique was AMSCO,

later bought by Steris Co., with their
VHP technology back in the 80 ies.
T [C]
Disadvantage of vaporizing H2O2 are the

two physical states present in the system,
when using diluted H2O2 for evaporation.
This results in different concentrations and
thereby in different inactivation efficacy.
Gas + liquid
Gas state
25
liquid state
35
79
Conc. H2O2 [%]
Challenges
Slow evaporation of e.g. 35%

H2O2 separates the both components
(destillation) during transition into
vapor. Condensation on the surfaces
makes the next separation
(concentration change).
Sterilisation means absence of viable

microbiotics. In complex geometries
this is a challenge, because homogeneous inactivation is required. This can be
questioned with vapor/liquid mixtures.
H202 vapor is known as bio-decontaminating, not sterilizing surfaces

though 6 log inactivation can be achieved/proven.
Structure
Introduction
z
Chemistry /
Biocidal Effect
Challenges
Regulatory
VHP for Isolators
Results
More Applications
Summary
10
Regulatory
In Guidance for Industry: Sterile Drug

Products Produced by Aseptic
Processing Current Good Manufacturing Practice published by FDA
in September 2004, within Appendix 1:
Aseptic Processing Isolators the need
for decontaminated surfaces is explained:
1. Surface Exposure
Decontamination procedures should ensure full exposure of all isolator
surfaces to the chemical agent. The capability of a decontaminant to
penetrate obstructed or covered surfaces is limited.
11
Regulatory
Continuation Aseptic Guidance:

2. Efficacy
The decontamination method should render the inner surfaces of the isolator free
of viable microorganisms. Multiple available vaporized agents are suitable for
achieving decontamination. Cycles should be developed with an appropriate
margin of extra kill to provide confidence in robustness of the decontamination
processes. Normally, a four- to six-log reduction can be justified depending on the
application.
E. Filling Line Sterilization
Where decontamination methods are used to render certain product contact
surfaces free of viable organisms, a minimum of a six-log reduction should be
demonstrated using a suitable biological indicator.
12
Regulatory
Continuation Aseptic Guidance:

E. Filling Line Sterilization
Where decontamination methods are used to render certain product contact
surfaces free of viable organisms, a minimum of a six-log reduction should be
demonstrated using a suitable biological indicator.
In EC GUIDE to Good Manufacturing Practice, Annex 1 a list of

sterilization techniques (heat, moist heat, radiation, ethylene oxide)
does not include hydrogen peroxide. The description of
sterilization/decontamination of isolators does not specifically discuss
issues coming from bio-decontamination instead sterilization.
13
Regulatory
In EC GUIDE to Good Manufacturing

Practice, Annex 1 a list of sterilization
techniques (heat, moist heat, radiation,
ethylene oxide) does not include
hydrogen peroxide. The description of
sterilization/decontamination of isolators
does not specifically discuss issues
coming from bio-decontamination
instead sterilization.
14
Regulatory
PIC/S Recommendation Isolators used for Aseptic Processing and

Sterility Testing; Chapter An expansion of the detailed points to be
considered for the implementation of the principles to isolators subjected
to a sporicidal process collects 14 points for the sporicidal process. Point
9.4.1 mentions H2O2 bio-decontamination:
The agent selected for gas generation
should be sporicidal. The agent used for
gas generation or other means of application
should be capable of rapidly killing bacterial
endospores, fungal spores and vegetative
microorganisms.
Peracetic acid, hydrogen peroxide and
formaldehyde are used.
15
Structure
Introduction
VHP for Isolators
Results
More Applications
Summary
16
VHP for Isolators
Vaporized hydrogen peroxide is

widely used to decontaminate the
inside of pharmaceutical isolators.
The good old pioneering days
applied a mixture of steam and
H2O2 to be on the safe side,
but validation was a nightmare.
Nowadays several H2O2 gas generators are on the market

different philosophy, but more or
less reliable technology.
Cycle development and validation

has become a routine job.
17
VHP for Isolators
Isolator design principle

Air Management
System (HVAC)
Unidirectional Air
Flow Unit
Aseptic Processing
Chamber
18
VHP for Isolators

Example (isolated vial/bottle fill line in Austria)
19
VHP for Isolators
Liquid H2O2 is dropped on a heated

plate inside the filter level of an isolator. Recirculation and continous
injection of peroxide goes to saturation
of atmosphere including strong visible
condensation
H2O2
Closed loop system:
Heat plate
Filter
Open loop system:

Liquid hydrogen peroxide is evaporated
outside of the isolator in a gas generator;
which carries the vapor with air into the
Isolator
Dry air
H2O2
isolator, where it is recirculated.

Some o-l-systems perform flash evaporation.
20
10
VHP for Isolators
H2O2
Closed loop system:
Simple air management
Vapor passes filters first
Incomplete evaporation
Limited injection rate
Saturation/condensation soon
Open loop system:
Complete (flash) evaporation
Controlled atmosphere
Capability for high gas conc.
Air management more complex
More sophisticated generator
H2O2
21
VHP for Isolators

Preparation (Phase I)
- Expanding gloves, load monitoring goods
Conditioning (Phase II)

- Injection of H2O2 with a high rate to
achieve a high gas concentration
H2O2-conc.
- Preparation of isolator atmosphere

- Preheating of peroxide duct work
I+ II
Biodecontamination (Phase III)

- Injection with a lower rate to replace
III
IV
Process time
inactivated peroxide and keep level
Aeration (Phase IV)

- Removal of H2O2 from injection system
Idealized process cycle
- Removal of peroxide from all isolator surfaces including filters.

22
11
VHP for Isolators
Bosch SafeVAP (open loop system)

Features:
z
Venturi transport
3-step evaporation
Inj.rates up to 80g / min
Controlled atmosphere
SafeVAP = Safe Vacuum Assisted Peroxide Evaporation

23
VHP for Isolators
Description
Compressed air
HVAC
Particle filter
Blower
Evaporator
Filter
Venturi
Peroxide
source
(35%)
Scale
Air to
isolator
Isolator
Exhaust air
Peroxide duct
HEPA H14
Aseptic Processing Chamber
24
12
VHP for Isolators

Isolator vol. 8 m
Description
Cycle time 22 min

Aeration 1,5
hours
Peroxide: 310 g
Inj. rate I: 25 g/min
Inj. rate II: 9 g/min
800
H2O2 concentration [ppm]
700
600
500
25000
20000
15000
400
10000
300
200
5000
100
0
H2O concentration [ppm]
0
-100
5:
:0
15
06
-5000
1:
:1
15
04
:1
15
7:
02
:
15
1
:0
23
8:
:2
15
59
4:
:3
15
58
:
15
6
:5
40
:
15
4
:5
46
3
:5
52
5:
Time
25
VHP for Isolators
Cycle Development for H2O2 biodecontamination include

z
OQ-studies
tests with chemical indicators (CIs)
Tests with bio indicators (BIs),

which are stainless steel coupons
inoculated with 6 log of the most
resistant viable (spores of
Geobacillus stearothermophilus).
To ease handling (and to put an extra

challenge on the biological test
system), BIs wrapped in Tyvek became
industrial standard.
26
13
VHP for Isolators

Air
flow
study
Worst location CIs

for H2O2
concentration
Air
velocity
mapping
D-value
determination
Evaluate
aeration
time
Worst location
BIs 3 times
Safety
Evaluation
Worst location with

triplicate BIs
Critical
design
area
Testing
Complete
Biological
Verification
Temperature
mapping
Physical
System
Evaluation
Chemical
System
Evaluation
Biological
System
Characterization
and Optimization
27
Structure
Introduction
VHP for Isolators
Results
z
Recent Studies
Results
More Applications
Summary
28
14
Recent Studies
Many studies were performed at

Bosch during the last 3 years
(PhD thesis). Basis for these
studies was a reference isolator
equipped with the SafeVAP system
and placed in the Bosch PharmaLab.
29
Recent Studies
st
an
da
r
co
nt
ro
(E ls
S
(E -1)
SF
( E G)
SE
(E P )
(E S-G
S)
(E 443
S 5
( A - 43 )
lu 01
m
in )
iu
m
(P )
O
(E M)
PD
(P M)
VD
F
(F )
P
( H M)
( P YP
A- A )
12
(P G F
VC )
(C -T
G R
( P - TI )
VC SS
-S )
C
H
)
(P ( P
EE C
K- )
N
(P at)
ET
(
P
( P PVC )
EE -U
K
(P - P )
E- VX
U
H )
M
W
(V )
M
(T Q )
R
FX
)
D-Value in min
5
4,5
4
3,5
3
2,5
2
1,5
1
0,5
0
Material
av
er
ag
e
Goal of this study was to learn

the influence of different surfaces
(material, finish) on inactivation
efficacy of bioburden.
Prestudy
Mainstudy
Poststudy
Humidity Study
Influence of relative humidity
at different H2O2 gas concentration levels was studied
and compared with dew point
analysis.
2100
20000
1900
1700
15000
1500
1300
10000
1100
900
5000
700
Water concentration [ppm]
D-Values of tested Materials in Pre-, Main- and Poststudy
Material of Construction Study:
Hydrogen peroxide concentration [ppm]
500
300
00:00
00:02
00:05
00:08
00:11
00:14
0
00:17
Duration of bio-decontamination phase [hh:mm]

W ater concentration (humidity level
Hydrogen peroxide concentration
4)
3)
2)
1)
30
15
Structure
Introduction
VHP for Isolators
Results
z
Recent Studies
Results
More Applications
Summary
31
Results
Objective of material study:
z
Examine influence of different

construction material surfaces
on inactivation of spores
Determine variable kill kinetics
(D-Value)
Test organism: spores of Geobacillus stearothermophilus
ATCC 12980 with population
of > 1*106 spores per BI.
Exposure
time [min]
10
15
20
25
Results
Survival
Fractio
nal
Killing
32
16
Results
Results on some materials (50 different surfaces tested)
5
4,5
4
3,5
3
2,5
2
1,5
1
0,5
0
st
a
nd
ar
d
co
nt
ro
(E ls
S
(E -1)
SF
(E G)
SE
(E P )
(E S-G
S)
(E 443
S- 5 )
4
(A
l u 301
m
in )
iu
m
(P )
O
M
(E )
PD
(P M)
VD
F
(F )
P
(H M)
(P YP
A- A )
1
(P 2G F
VC )
(C -T
G R
( P - TI )
VC SS
-S )
C
H
)
(P (P
EE C
K- )
N
(P at)
ET
(
P
(P PVC )
EE -U
K
(P -P )
E- VX
U
H )
M
W
(V )
M
(T Q)
R
FX
)
D-Value in min
D-Values of tested Materials in Pre-, Main- and Poststudy
av
er
ag
e
Material
Prestudy
Mainstudy
Poststudy
No reliable kill
33
Results
Results on standard control (commercial Bio indicator made from SSL)
1,2
1100
1,0
1000
0,8
900
0,6
800
0,4
700
0,2
600
0,0
Average
Concentration
of H2O2 in ppm
D-Value of
Standard
Contorls in min
D-Values of Standard Controls and average Hydrogen Peroxide

Concentration during the Cycles
500
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
Number of Cycle for Material Study

D-Value Standard Controls
Concentration of Hydrogen Peroxide
34
17
Results
Summary of the study:
z
Most materials tested show good inactivation properties.

Plastic/elastomeric materials have generally higher D-values
compared with stainless steel and glass.
Standard commercial available BIs show a certain spread of D-values
(0,2 min to 0,9 min late positives included in the calculation), which
represents microbiobolgical accuracy.
A correlation between wetting properties, surface roughness and the
entire D-values can be recognized.
Critical materials of construction should either be replaced or need
prolonging of the cycle time.
35
Results
The results were recently published:
PDA journal, Vol. 61, No. 4, July-August 2007

36
18
Results
Procedure for Humidity study:
z
Separate evaporation of water

and hydrogen peroxide (35%)
Control of humidity level with
NIR probe, dew point sensor
and standard humidity sensor
D - value determination of
commercial BIs (SSL)
Investigation on inactivation
in hindered surfaces with
high aspect ratio
35% liq. H2O2

H2O
37
Results
2100
20000
1900
1700
15000
1500
1300
10000
1100
900
5000
700
Hydrogen peroxide concentration [ppm]
Details:
500
300
00:00
00:02
00:05
00:08
00:11
00:14
0
00:17
Duration of bio-decontamination phase [hh:mm]

Water concentration (humidity level 4)
Hydrogen peroxide concentration
38
19
Results
400 ppm:
25000
20000
15000
10000
5000
0
0
10
12
14
16
18
20
14
16
18
20
D-value [min]
39
Results
600 ppm
25000
20000
15000
10000
5000
0
0
10
12
D-value [min]
40
20
Results
800 ppm:
25000
20000
15000
10000
5000
0
0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
D-value [min]
41
Results
Comparison
Wrapped vs.
Nude BIs :
3,0
D-value [min]
2,5
2,0
1,5
1,0
0,5
0,0
Humidity
level 1
Humidity
level 2
Humidity
level 3
Humidity
level 4
Humidity level
BIs wrapped in Tyvek
unpacked BIs
42
21
Results
Correlation of injected H2O2-amount during Cond.phase with D
values (mean average)
Mittelwert 800ppm 1. FL
Mittelwert 800ppm 4.FL
14,00
D-Wert [min]
12,00
10,00
8,00
6,00
4,00
2,00
0,00
0
100
200
300
400
500
600
700
Injected H2O2 amount [g]
43
Results
Summary:
z
The higher the cgas phase concentration, the more independent is the
inactivation effect from the humidity level.
At lower concentrations the same kill as at higher concentration can be
achieved with higher humidity (condensation)
The faster the concentration rises during conditioning phase (flash
evaporation), the less peroxide is needed to achieve a good kill.
(Nano/micro) condensation occurs at all humidity levels. Visible
concentration is not needed to get short inactivation times.
44
22
Structure
Introduction
VHP for Isolators
Results
More Applications
Summary
45
More Applications
Vaporized H2O2 is applied with all kinds

of isolators: fill/finish isolators, sterility
testing isolators, hospital isolators
A new trend is fumigation of cleanrooms

and animal testing facilities with hydrogen
peroxide vapor.
H2O2 in vapor state could potentially replace

alcoholic spray&pray procedures in material
transfer chambers and material locks for
thermolabile products.
Bio-decontamination of tubs with presterilized,

nested syringes can also be performed with H2O2.
46
23
Structure
Introduction
VHP for Isolators
Results
More Applications
Summary
47
Summary
Hydrogen peroxide vapor is widely used in pharmaceutical industry. One

major application is disinfection of isolator interior.
Closed and open loop systems are established in the market, which are
technically robust and validatable.
Inactivation effect on bioburden differs depending on the material of

construction (material and finish).
Gas concentration and humidity effect dominate the kill efficacy: high gas
concentrations through flash evaporation help to achieve efficient
processes.
New applications of H2O2 vapor include cleanroom fumigation and

decontamination of thermolabile materials in transfer chambers,
48
24
Thank you for your attention !
johannes.rauschnabel@de.bosch.com
49
25

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Bio-decontamination with H2O2 - Vapor

Bio-Decontamination with H2O2 Vapor

SBM Experts Congress 13th-14th of September, 2007

Bio-decontamination with H2O2 - Vapor

VHP for Isolators

Bio-decontamination with H2O2 - Vapor

VHP for Isolators

Bio-decontamination with H2O2 - Vapor

Hydrogen Peroxide is an old sterilant well established in food industry,

Residues of hydrogen peroxide decompose into neutral species, which

Bio-decontamination with H2O2 - Vapor

Chemistry / Biocidal Effect

The hydrogen peroxide molecule has a

The decomposition can be catalyzed by

Bio-decontamination with H2O2 - Vapor

Chemistry / Biocidal Effect

The biocidal effect of hydrogen peroxide

A minimum of humditiy at the surface

Bio-decontamination with H2O2 - Vapor

VHP for Isolators

Bio-decontamination with H2O2 - Vapor

Vaporized Hydrogen Peroxide is used

Pioneer in that technique was AMSCO,

Disadvantage of vaporizing H2O2 are the

Bio-decontamination with H2O2 - Vapor

Slow evaporation of e.g. 35%

Sterilisation means absence of viable

H202 vapor is known as bio-decontaminating, not sterilizing surfaces

Bio-decontamination with H2O2 - Vapor

VHP for Isolators

Bio-decontamination with H2O2 - Vapor

In Guidance for Industry: Sterile Drug

Bio-decontamination with H2O2 - Vapor

Continuation Aseptic Guidance:

Bio-decontamination with H2O2 - Vapor

Continuation Aseptic Guidance:

In EC GUIDE to Good Manufacturing Practice, Annex 1 a list of

Bio-decontamination with H2O2 - Vapor

In EC GUIDE to Good Manufacturing

Bio-decontamination with H2O2 - Vapor

PIC/S Recommendation Isolators used for Aseptic Processing and

Bio-decontamination with H2O2 - Vapor

VHP for Isolators

Bio-decontamination with H2O2 - Vapor

VHP for Isolators

Vaporized hydrogen peroxide is

Nowadays several H2O2 gas generators are on the market

Cycle development and validation

Bio-decontamination with H2O2 - Vapor

VHP for Isolators

Isolator design principle

Bio-decontamination with H2O2 - Vapor

VHP for Isolators

Bio-decontamination with H2O2 - Vapor

VHP for Isolators

Liquid H2O2 is dropped on a heated

Closed loop system:

Open loop system:

isolator, where it is recirculated.

Bio-decontamination with H2O2 - Vapor

VHP for Isolators

Closed loop system:

Simple air management

Vapor passes filters first