The Steam Sterilization Cycle - A3P

Sterilization & Decontamination
in Research and Pharmaceutical
Presented by
Daniel Caron
Rencontres A3P Canada
September 13 & 20, 2007

Principles of Steam Sterilization
Steam is the ideal sterilant for items that can
withstand moisture and high temperatures
because it is:
• nontoxic
• readily available
• fairly easy to control

Principles of Steam Sterilization
Steam is Water in the Vapor State
Steam Heats by Condensing and Giving Up Energy
• 6.8X More Energy Required
– Boiling from liquid to Vapor at 212F than heating from 70F-
212F
• By-product is Condensation
– Remains on product
– Rolls off

Principles of Steam Sterilization
Three factors are critical to assure successful
steam sterilization:
• time
• temperature
• moisture

Principles of Steam Sterilization
TIME
All organisms do not die at the same time. The number of
survivors is usually plotted on a semi-logarithmic scale. A
straight line survivor curve usually results.

Microbial Death Rate Curve that
Illustrates Concept of D-ValueD
Number of Survivors
10,000
Log Number of Survivors
4
1,000
3
100
2
10
1
1
0.1
D-Value
D-Value is the time to reduce
the microbial population by
90% or 1 log
0
-1
0.01
0 1 2 3 4 5 6 7 8 9 10 11 12
Exposure Time (Minutes)
-2

Principles of Steam Sterilization
Temperature
•The second critical factor in steam sterilization is the
temperature of the saturated steam.
•Increasing temperature dramatically reduces the time
needed to achieve sterilization.

Time to Achieve Equivalent Microbial Lethality at
Different Exposure Temperatures
Population 10E6, D value 2 minutes
285° F (140° C)
( .13 Minutes )
270° F (132° C)
( 0.9 Minutes )
250° F (121° C)
12 Minutes
212° F (100° C)
80 Hours
200° F (93° C)
321 Hours
176° F (80° C)
643 Hours
10 20 30 40 50 60 100 300 500 700
MINUTES
HOURS

Principles of Steam Sterilization
Moisture
•Steam denatures or coagulates proteins.
•Dry heat is an oxidation process with different kinetics - it
requires much higher temperatures and longer exposure times.
•B. stearothermophilus spores - killed in 12-15 minutes at 121°C
(250 °F) when saturated steam is used
•more than 6 hours are required to dry heat at 121°C.

The following illustration shows the basic
components of a typical prevacuum steam sterilizer:
Chamber
Safety
Valve
Air
Filter
Jacket
Safety
Valve
Jacket / Chamber
Steam Supply
Pressure
Regulator
PC
Jacket
Steam
Supply
Chamber
Temperature
Sensor
Steam Supply Jacket
Bleed
Valve
Jacket
Trap
Chamber
Trap
Vacuum
System
To Waste

Principles of Steam Sterilization
The Steam Sterilization Cycle
A steam sterilization cycle consists of three
phases:
– Heating (pre-conditioning) phase
– Sterilization (exposure) phase
– Cool-down (post conditioning) phase

The Steam Sterilization Cycle
Heating (pre-conditioning) phase -
Steam enters the sterilizer chamber and air
is removed by either:
gravity displacement
mechanically (prevacuum)

The Steam Sterilization Cycle
Sterilization (exposure) phase -
Load is exposed to steam at a set temperature
(measured and controlled by a temperature
sensor in the drain line or product) for a set
time.

The Steam Sterilization Cycle
Cool-down (post-conditioning) phase -
Sterilizer chamber is exhausted to atmospheric
pressure followed by circulating air through the
chamber or by drawing vacuum. Jacket heat
may or may not be maintained during the
drying phase.

Basic Gravity and Prevac Cycles
40
30
20
10
0
Pre-Vac
Gravity
-10
-20
-30
9

Basic Gravity Cycle
Applications:
• Empty glassware
• Instruments
Basic Gravity Cycle
• Open, empty pans, vats, carboys

Basic Liquid Cycle
(Modified gravity cycle)
Generally used to process
• Media/Liquids in open containers
– Water
– TSB
– TSA
• Filled bottles / tubes with loose caps
• Generally at 121 o C

Basic Gravity Cycle
Pre-conditioning and Exposure
Two Methods of Controlling the Cycle
• Control by Temperature Probe in Drain
– Timed
• Control using Probe in Product
– F o

Basic Gravity Cycle
Pre-conditioning and Exposure
May use F o
load probe in liquid loads if it is
available in the sterilizer
• Advantages
– produces desired lethality
– minimal effect on product
– optimizes the exposure period
Can use time at temperature if probes are not
available (overkill method)

What is F 0
F0 as a Function of Temperature
Lethality Curve
1.545
123 °C
1.227
122 °C
1.00
121.1 °C
F 0 Value
.50
118.1 °C
.245 115 °C
.008
110 °C
Fo Calculation
*Reference Temperature 121.1°C
Z - Value = 10 °C

Gravity Cycle
Post- conditioning
Liquid Cycles are returned to atmospheric pressure
slowly to
• Prevent boil-over
• Prevent liquid loss
Rate of slow exhaust depends on
• Volume of liquid per flask / tube
• Total load capacity
• If there is a jacket cooling process built into the sterilizer

Prevac or Pulsing Vac Cycles
Generally used to process dry goods
• articles in open trays
• porous goods such as textiles
• open, unfilled glassware/plastic bottles
• rubber stoppers
• filter cartridges
• Disc filters

Prevac Cycle Loads
Metal articles
Porous goods (textiles)
Glassware (open)
Glassware in pouches
Plastics (open)
Plastics in pouches
Rubber stoppers
Filter cartridges
Disk filters
2.1.3

Prevac Cycle
Pre-conditioning
Number and depth of vacuums during pre-conditioning
can be user selectable
• based on degree of difficulty to penetrate product with steam
– Tygon tubing may take several pulses/vacs to penetrate to
center of tube
– Long lengths may require deeper vacuum
– Large metal items may require several pulses to heat

Prevac Cycles
Product Constraints
Ability of the product to withstand ;
• vacuums
• pressure / vacuum rates
– Peel pouches may burst
– Covers may be blown off of covered pipe ends
May need to control rates if available on sterilizer

Prevac Cycle
Exposure Phase
• Timed
• Based on drain line temperatures
• Generally at 121 o C but could be at 132 o C
• Overkill method employed

Post-conditioning phase
Single deep vacuum, hold
Used to dry the load
Basic cycle draws a single deep vacuum and holds
Hold time varies
• may be 20-45 minutes or longer
• Time depends on
– material that comprises load
– packaging
– level of dryness required

Post-conditioning
Pulsing Vac Cycle
Pulls vacuum with alternating pressurization with
air
Used for peel pouched items, items with low
specific heat (rubber goods)
• May be unheated or optional heated

Gravity Cycles
30
25
20
15
10
5
0
-5
-10
-15
-20
-25
GRAVITY DISPLACEMENT
>Provided on both gravity and prevacuum sterilizers
>Operates on the downward air displacement air-removal principle.
>Used for sterilizing nonporous heat and moisture stable goods at 250 or 270°F
(121 or 132°C).
Gravity

Isothermal Cycles
4
3.5
3
2.5
2
1.5
1
0.5
0
Isothermal
ISOTHERMAL CYCLE- Optional cycle
>Designed for low temperature sterilization of heat sensitive and heat coagulable materials.
>Used for pasteurization, melting agar, inspissation (increase in viscosity by dehydration of highprotein
containing media), or fractional sterilization (Tyndallization)
>Temperatures are controlled at 78C(172F), 88C (190F), and 104C(219F)

Liquid Cycles
16
14
12
10
8
6
4
2
0
Liquids
LIQUID CYCLE- Modified Gravity Cycle
>Provided on both gravity and prevacuum sterilizers
>Used for sterilizing liquids, in flasks or test tubes with vented closures,
at 250°F (121°C).
>Optimal Solution Cooling is on Century Units

Prevac Cycles
40
30
20
10
0
Pre-Vac
-10
-20
-30
PREVACUUM
>Available only on prevacuum sterilizers
>Employs a mechanical air-removal system
>For efficient, high-volume processing of porous heat and
moisture stable materials, such as fabrics, wrapped hard goods,
and container systems at 250 or 270°F (121°C or 132°C).

Decontamination Cycles
Optional cycle designed to decontaminate all effluents from the sterilizer
chamber prior to discharge to drain
During purge, pre-steam pulses, and heat-up phases, steam is
admitted through the drain port (located on the sterilizer chamber floor).
Helps to heat any condensate on the chamber floor to sterilizing
temperatures.
Potentially contaminated air is pushed upward and discharged
through the vent in the top of the chamber.
Gaseous exhaust filtered through a 0.2 micron (0.2 micrometer) bacterial
retentive filter before exiting to the sterilizer vacuum system, building drain
system, and surrounding environment.

Decontamination Cycle
The temperature is monitored and controlled using a
temperature probe located after the bacterial retentive filter
housing.
The bacterial retentive filter is also sterilized during the
exposure phase.
After the exposure phase the decontaminated condensate
that has accumulated in the drain line and chamber floor is
discharged via the drain port located on the chamber floor.
All liquid effluents are decontaminated prior to discharge to
drain.

Standard Steam Flow

Decontamination Cycles
DECONTAMINATION (EFFLUENT DECONTAMINATION) CYCLE

Decontamination Cycles

LABORATORY CYCLES
Preconditioning Exposure Postconditioning
Gravity Purge, Heat –up Timed Vacuum Dry
Prevac
Purge, Prevac
Pulses, Heat-up
Timed
Vacuum Dry
Liquids Purge, Heat-up Timed
F0 Optional
Slow Exhaust
Isothermal Purge, Heat-up Timed Slow Exhaust
Decontamination
Purge, Prevac
Pulses, Heat-up
Timed
Vacuum Dry

B Process Cycles
>General purpose steam sterilization cycle applicable for all dry goods and porous loads.
>Suitable products include filters, equipment parts, instruments, textiles, and rubber goods.
>Primarily used for production, clean room supplies and utensils, and production support.
>Features a preconditioning air removal phase using vacuum and steam pulses.
>Drying can be accomplished by deep vacuum, vacuum pulsing, or fast exhaust.

B Process Cycles
Continued
>General purpose steam sterilization cycle applicable for liquids in open / vented containers
e.g., culture media with vented closures.
>May be used for microbial decontamination of liquids in vented/open containers or vented
red/orange bags.
>Utilizes a forced air preconditioning and a user programmable rated slow exhaust.
>Steam-to-jacket is turned off during slow exhaust.

CA Process Cycles
>Designed for liquid products in vented or sealed containers.
>Primarily used for production, media preparation, and R&D purposes when liquids are in open
containers or cooling time is less critical.
>Employs forced air removal during preconditioning.
>No circulating water in the sterilizer jacket is used.
>Maintains sterile air overpressure during the cooling phase to cool the load and prevent the
product from boiling.

C Process Cycles
>Designed for liquid products in vented or sealed containers.
>Primarily used for production, media preparation, and R&D purposes when liquids are in
open containers or cooling time is less critical.
>Employs forced air removal during preconditioning and indirect cooling by circulating water
in the sterilizer jacket.
>Maintains sterile air overpressure during the cooling phase to cool the load and prevent the
product from boiling.

CF Process Cycles
>Designed for liquid products (LVPs or SVPs) in vented or sealed containers.
>Primarily used for production, media preparation, and R&D purposes when liquids are in open
containers or cooling time is less critical.
>Employs forced air removal during preconditioning and indirect cooling by circulating
water in the sterilizer jacket.
>Maintains sterile air overpressure during the cooling phase to cool the load and prevent the
product from boiling.
>A fan is present in the sterilizer chamber to assist in cooling the product during post
conditioning.
>The circulated air with fan and jacket cooling process significantly reduces the cycle time by
approximately 50% over a jacket cooling process, depending on the application.

Steam-Air-Mixture (A/C)
>Cycle designed for terminal sterilization of liquids in plastic or glass containers (e.g. pouches,
bottles, LVPs, SVPs and blister packs) that must be dry at the end of the cycle.
>The cycle employs an over pressurized air/steam mixture to sterilize.
>Counter pressure is maintained based on the product temperature through the entire cycle.
>Cooling is performed by fan-circulated air, cooled by indirect jacket cooling.
>Allows immediate post-cycle product processing (e.g. labeling and packaging).

Steam-Air-Mixture (A/C)

Superheated Water (RP)
>Designed for terminal sterilization of liquids in plastic or glass containers (e.g. pouches, bottles,
and blister packs).
>Employs an over pressurized water spray to sterilize; No pure steam is required.
>Air counter pressure is employed throughout the entire cycle based on the product temperature.
>Extremely efficient cooling time.

RP Process Cycles

G Process Cycles
>Designed for terminal sterilization of liquids in glass containers (vials, bottles).
>For non-vented containers only.
>Employs a purified water spray during cooling only.
>Air counter pressure is also employed during the cooling based on the product temperature.

GΔT Process Cycles
>Designed for terminal sterilization of liquids in glass containers (vials, bottles).
>For non-vented containers only.
>Employs a temperature controlled purified water spray during cooling only.
>Air counter pressure is also employed during the cooling based on the product temperature.
>The cooling time, delta and final load temperature are configurable to protect the load from
thermal shock.

Process Cycles
Products to Sterilize Cycle End Support Air
Media Precond. Exposure Postcond Cooling Media
Open containers,
vented flasks, textiles,
components, utensils
Open / vented containers,
sealed bottles
Open / vented containers,
sealed bottles
Open / vented containers,
sealed bottles
B DRY Steam Steam-to-jacket off;
Heat loss by conduction
CA DRY Steam X
Steam-to-jacket off;
Air overpressure
C DRY Steam X
Air overpressure/
Jacket water
CF DRY Steam X
Air circ./ Jacket water/
Fan on, during cooling
only
Sealed glass bottles G WET Steam X
Water/jacket water/ Air
overpressure
Sealed glass fragile bottles GΔT WET Steam X
Water temp, controlled /
jacket water/ Air circ
Sealed glass or Flexible containers
E.g. plastic bags, blisters, syringes,
containing liquids
Sealed glass or Flexible containers;
E.g. plastic bags, blisters, etc.
A/C DRY Steam X X X
Air / Jacket water/ Fan
on during entire cycle
RP WET Water X X X
Water/ Air circ./ Jacket
water

Cycles for Solutions
CYCLE TYPE
STERILIZING
MEDIA
PRE-
CONDITIONING
LOAD STATUS AT END OF
CYCLE
SPEED OF COOLING
B PROCESS
WITH SLOW
EXHAUST WITH
RATES
STEAM
FORCED AIR;
WITH OR
WITHOUT RATES
DRY EXTERNALLY AND
APPROXIMATELY AT BOILING
TEMPERATURE
SLOW
CA Process
STEAM
FORCED AIR;
WITH OR
WITHOUT RATES
RELATIVELY DRY EXTERNALLY
AND AT COOLING TEMPERATURE
SET POINT (VACUUM DRYING
MAY BE ADDED AFTER COOLING)
SLOWER THAN C PROCESS, BUT
FASTER THAN B PROCESS
C Process
STEAM
FORCED AIR;
WITH OR
WITHOUT RATES
RELATIVELY DRY EXTERNALLY
AND AT COOLING TEMPERATURE
SET POINT (VACUUM DRYING
MAY BE ADDED AFTER COOLING)
MODERATE
CF Process
STEAM
FORCED AIR;
WITH OR
WITHOUT RATES
DRY EXTERNALLY AND AT
COOLING TEMPERATURE SET
POINT (VACUUM DRYING MAY BE
ADDED AFTER COOLING)
MODERATELY FAST
(FASTER THAN C PROCESS,
BECAUSE OF FAN)

Cycles for Solutions
Continued
CYCLE TYPE
STERILIZING
MEDIA
PRE-CONDITIONING LOAD STATUS AT END OF CYCLE SPEED OF COOLING
G
STEAM
FORCED AIR; WITH
OR WITHOUT RATES
WET, AND AT COOLING
TEMPERATURE SET POINT
(VACUUM DRYING MAY BE ADDED
AFTER COOLING)
MODERATE
GΔT
STEAM
FORCED AIR; WITH
OR WITHOUT RATES
WET, AND AT COOLING
TEMPERATURE SET POINT
(VACUUM DRYING MAY BE ADDED
AFTER COOLING)
MODERATE; MORE
CONTROLABLE
THAN
G PROCESS
A/C
STEAM (MIXED BY
FAN WITH
STERILE AIR
COUNTER-
PRESSURE)
AC HEATING (STEAM
AND STERILE AIR
COUNTER-
PRESSURE IN
CHAMBER w/ FAN)
DRY EXTERNALLY AND AT
COOLING TEMPERATURE SET
POINT; READY FOR LABELING OR
PACKAGING
MODERATELY FAST
RP
STERILE
SUPERHEATED
WATER (with
STERILE AIR
COUNTER-
PRESSURE)
STERILE
SUPERHEATED
WATER w/ STERILE
AIR COUNTER-
PRESSURE
WET EXTERNALLY AND AT
COOLING TEMPERATURE SET
POINT
MODERATELY FAST

Common Problems and
Misconceptions About Steam
Sterilization

Common Problems
Possible sources of
air in chamber:
Air is generally a deterrent to sterilization
PACK
STERILIZER CHAMBER
AIR POCKET
AIR
‣Leak (during vacuum)
in piping or door gasket
‣Insufficient prevacuum
‣Air entrained in steam
STEAM
+ AIR
STEAM
+ AIR
SPORES
STERILIZER DRAIN

Common Problems
Steam Quality Issues
Wet Steam
Wet steam is undesirable - it has
less energy than dry steam and
it can cause wet loads
STERILIZER CHAMBER
PACK
Water Droplets
•The packaging used for
sterile products prevents recontamination
when dry, but
its bacterial retentive
properties will be adversely
affected by moisture.
•Wet loads can be
considered to be nonsterile.
•Causes - Wet steam may
be caused by excessive
pressure drops on the boiler
due to high demands.
STEAM
+ Water
STERILIZER DRAIN
STEAM
+ Water

Misconception
Assumption: Everything Can Be Sterilized
Facts
• Closed valves on containers do not permit
steam penetration. Open all valves
• O-rings and seals retard steam penetration. May
need to remove o-rings or extend processing
times
• Upright, empty containers are difficult to remove
air from with a gravity cycle

Test Tubes in Rigid Container
Fact: Solid bottom
containers retard steam
penetration
• Bottom set of tubes in
container
• Top set of tubes above
container

Effects of Rigid Bottom Container
Temperature
° F
260
240
220
200
180
160
140
120
100
80
60
40
20
Top Test Tube Rack
Bottom Test Tube Rack
0 2 4 6 8 10 12 14 16 18 20
Time (Minutes)

Misconception
Assumption: Exposure Time for a Liquid Cycle
Means That the Product is at Temperature for that
Period
Fact: Exposure time must account for product comeup
and desired time at temperature

Come-up Rates for Two Different Flask
Sizes
140
120
100
80
60
40
Chamber Drain
Media Load 1
Media Load 2
20
0

Misconception
Assumption: The attainment of 121.1 0 C is a
significant requirement to sterilize.
Fact: The objective of steam sterilization is to deliver
lethality. F 0
accumulates at all spore killing
temperatures although it is slower at lower
temperatures.

Misconception
Assumption: A steady state temperature distribution
range of +/- .5 0 C is necessary for all loads.
Fact: Desirable for terminal sterilization in final
containers. Not required for sterilization of production
equipment. Distributions of 1 0 C to 2 0 C are acceptable

Misconception
Assumption: Fixed load patterns are required for
proper sterilization
Fact: It is not as much the location of the items, but
the material makeup, wrapping, mass, loading
procedures and orientation of the load that define
“difficult-to-sterilize locations”

QUESTIONS ?

MERCI !