Validation and Management of Heat Sterilization

(Autoclave and Dry Heat Oven)

CBE Pty Ltd

This training program is copyright to CBE Pty Ltd and may not be
modified, reproduced, sold, loaned, hired or traded in any form without
its express written permission.

CBE 106 V1

Introduction

Module Outcomes
On completion of this module the participant should be able to:
List the essential cGMP requirements for sterilisation validation
specifically autoclaves and hot air sterilisers/dry hear ovens
List the IQ, OQ and PQ requirements for heat sterilisation processes
Differentiate between two sterilisation approaches (overkill and
bioburden)
Calculate and use an Fo for autoclave sterilisation validation
Interpret a basic print-off for a sterilisation process.

CBE 106 V1

Introduction

Module Topics
How does heat sterilization work

Critical process parameters and metrics

Developing a validation process / cycle

Bioburden reduction vs. overkill cycles

Content of protocols and reports

CBE 106 V1

Introduction

Useful References
PIC/S Guide to Good Manufacturing Practices - PE 009 2014 Annex
1
FDA Recommendations for Submitting Documentation for Sterilisation
Process Validation, November 1994

ANSI/AAMI/ISO 11134 Sterilisation of HealthCare products

requirements for validation and routine control Industrial moist heat
sterilisation (1993)
ISPE Good Automated Manufacturing Practices (GAMP)

BP Appendix XVIII Methods of Sterilisation - Monograph for Biological

Indicators
ANSI/AAMI ST79:2006 Comprehensive guide to steam sterilisation
and sterility assurance in health care facilities

AAMI TIR 13:1997 Principles of industrial moist heat sterilization

CBE 106 V1

Regulatory Agencies

Useful References
PDA Technical Monograph 1 Validation of Steam Sterilisation
Cycles 2007
PDA Technical Report 3, (TR3) Validation of Dry Heat Processes
Used for Sterilization and Depyrogenation (under revision)

USP <1035 > Biological Indicators

USP <1211> Sterilisation and Sterility Assurance of Compendial
Articles

CBE 106 V1

Regulatory Agencies

Define Sterile (IJ Pflug)

Sterile
Free from viable microorganisms.
Sterilisation
Any physical or chemical process which destroys all life forms, with special
regard to microorganisms (including bacteria and sporogenous forms), and
inactivates viruses. Therefore the terms "sterile" and "sterilization", in a strictly
biological sense, describe the absence or destruction of all viable
microorganisms. In other words, they are absolute terms: an object or system is
either "sterile" or "not sterile".
The destruction of a microbial population subjected to a sterilization process
follows a geometrical progression to be 100% certain the article is sterile it
would require infinite sterilisation.
Sterility Assurance Level (SAL)
For practical purposes the probability of finding a non-sterile unit (PNSU =
Probability of Non Sterile Unit) must therefore be lower than 10-6.
CBE 106 V1

BP/ EP Monograph - XVIII

Sterility is the absence of viable micro-organisms.
The sterility of a product cannot be guaranteed by
testing; it has to be assured by the application of a suitably
validated production process.
It is essential that the effect of the chosen sterilisation
procedure on the product (including its final container or
package) is investigated to ensure effectiveness and the
integrity of the product and that the procedure is validated
before being applied in practice
Revalidation is carried out whenever major changes in the
sterilisation procedure, including changes in the load, take
place.
CBE 106 V1

Industry Rules -Terminal Sterilisation

(BP/EP)
Wherever possible, a process in which the product is
sterilised in its final container (terminal sterilisation) is chosen.
If terminal sterilisation is not possible, filtration through a
bacteria-retentive filter or aseptic processing is used;
Wherever possible, appropriate additional treatment of the
product (for example, heating of the product) in its final
container is applied.

In all cases, the container and closure are required to

maintain the sterility of the product throughout its shelf-life.

CBE 106 V1

Why Are Autoclaves Essential?

Easiest way to sterilise large volumes of heat tolerant
materials.
More effective than dry heat (lower temperature /shorter
time
Not as messy as chemicals and more reliable
No need for radiation shielding etc.
Once validated, simple indicators used to tell autoclaved and
non autoclaved material apart the temp/time/pressure trace
is used to confirm sterilization occurred.
Can deliver > 1012 sterility assurance

CBE 106 V1

Heat Sterilisation Methods

Moist Heat (Steam)
Air in autoclave chamber is
displaced by saturated steam
Condensing water vapour
acts as a conductor of heat

Dry Heat Oven or Tunnel

Heated dry air is distributed
throughout an oven or tunnel
by convection or radiation

CBE 106 V1

10

Common Types of Autoclaves

Production autoclave.
Usually large
Loads one side (Grade C), unloads the other (Grade B)
Used to sterilize production equipment
May be used to terminally sterilize filled product (can have one
opening)
If faulty, potential critical impact on sterile core or batch disposition
Microbiology Laboratory Autoclave
May be large or small
Usually loads and unloads from same side - Sterilized items do not
unload directly into production environment
Used to sterilize equipment as well as media. Also used to
decontaminate materials before disposal

CBE 106 V1

Definitions: D-Value, Z-Value and Fo

What is the D value?
refers to decimal reduction time - The time required at a certain
temperature to kill 90% (eg reduce population by log 1) of the organisms
being studied. Thus after an organism is reduced by 1 D, only 10% of the
original organisms remain. Dependant on microbe and initial numbers.
Eg D value of 1.5 means it takes 1.5minutes to reduce 1 log (to 10%)
@121oC. A Dvalue of 2.0 means more resistant while a Dvalue of 1min
means less resistant.

What is a Z value?
Refers to the temperature change required to produce a 1 log reduction
in D value.

CBE 106 V1

Definitions: D-Value, Z-Value and Fo

What is F0?
The number of minutes to kill a specified number of microbes with a Z
value of 10oC at a temp of 121.1oC.
Often confused with the time the chamber is held at elevated
temperature and pressure and in practice is the same thing.
Fos accumulate as the sterilisation cycle progresses very little
accumulation below 112oC.

Overkill
Use many more microbes than would find on items typically autoclaved.
Negates the need to test sample for bioload before running the cycle.
Use a sterilisation time exceeding what is necessary to kill a large
number of microbes. Negates the need to determine D value of microbe.
Overkill is generally defined as a 12 log reduction in bioload

CBE 106 V1

Autoclave Operating Mechanism

Steam enters the chamber
jacket, passes through an
operating valve and enters the
rear of the chamber behind a
baffle plate. It flows forward
and down through the
chamber and the load, exiting
at the front bottom.
A pressure regulator maintains
jacket and chamber pressure
at a minimum of 15 psi, the
pressure required for steam to
reach 121C (250F).
Overpressure protection is
provided by a safety valve.
CBE 106 V1

14

Monitoring of Sterilisation Processes

Biological measurements
Required to demonstrate that
sterilisation process was
effective

Physical measurements
Time, temperature, pressure,
vacuum.
Required to calculate sterility
assurance levels (SAL)

Chemical measurements
Autoclave tape or other
indicators such as Bowie Dick

CBE 106 V1

15

Hows Does An Autoclave Sterilize?

Steam held at elevated
temperature and pressure for
time is used to transfer moist
heat.
The steam condenses on a
surface and releases energy
The energy splits open the cell
wall.
Heat acts to denature proteins,
effectively killing all cells
present.
Effectiveness is reliant on
saturated steam condensing
CBE 106 V1

Thermal Monitors - Thermocouples

(HSA Guidance)
The number of thermal monitors used (10) and their location
in the chamber should be described. A diagram is helpful.
Accuracy of thermocouples should be 0.50C.
Thermocouples should be calibrated before and after a
validation experiment at two temperatures: 00C and 1250C.
Any thermocouple that senses temperature more than 0.50C
away from the calibration temperature bath should be
discarded. Stricter limits i.e., <0.50C, may be imposed
according to the users experience and expectations.
Temperature recorders should be capable of printing
temperature data in 0.10C increments.
CBE 106 V1

17

Biological Indicators (BIs)

A characterized preparation of a specific microorganism that
provides a defined and stable resistance to a specific
sterilization process.
Typically spore-forming bacteria
Used to:
Assist in the PQ of the sterilization equipment and
Assist in the development and establishment of a validated
sterilization process for a particular article.
Monitor established sterilization cycles
Periodically revalidate sterilization processes
Evaluate the capability of processes used to decontaminate
isolators or aseptic clean-room environments.

CBE 106 V1

18

Examples of Biological Indicators

Sterilisation
Method

Organism
(Spore type)

Identification

No. Viable
Organisms

D value

Steam

Bacillus stearothermophilus
Clostridium sporogenes
Bacillus subtilis spp

NCTC 10007
NCIB 8157
ATCC 7953
NCTC 8594
NCIB 8053
ATCC 7955

1.0105 to
5.0106 per
unit

Typically 1.5 min to

2.5 min @ 121C

Dry Heat

Bacillus subtilis

NCIB 8058
ATCC 9372

1.0106 to
5.0106 per
unit

1min to 3 min @
160C Typically 1.9
min @ 160C

Radiation

Bacillus pumilus (min. dose of

25kGy)
Bacillus cereus (for higher
dose levels)

NCTC 824
NCIB 8982
ATCC 14884
SSI C 1/1

>107 - 108 per

indicator unit

~3 kGy (0.3 MRad)

Ethylene
Oxide

Bacillus subtilis, variety Niger

NCTC 10073
ATCC 9372

1.0106 to
5.0107 per
unit

2.5 min to 5.8 min

@ ETO 600mg/l
60% RH and 54C
Typically 3.5

Filtration

Pseudomonas diminuta

ATCC 19146

recommend
107

NA

CBE 106 V1

19

Example Dvalues of Organisms

AVERAGE VALUES OF D AND Z FOR SOME REPRESENTATIVE
MICROORGANISMS Wallhauser 1980

Microorganism

D121

Clostridium botulinum

0.2

10

Bacillus stearothermophilus

2.0

Bacillus subtilis

0.5

10

Bacillus megaterium

0.04

Bacillus cereus

0.007

10

Clostridium sporogenes

0.8 - 1.4

13

Clostridium histolyticum

0.01

10

CBE 106 V1

20

Calculation of Fo
In mathematical terms, F0 is expressed as follows:

CBE 106 V1

21

Fo Calculations BP/EP
Fo = D121(Log No- Log N) = D121Log IF
D121 = D-value of the reference spores (5.1.2) at 121 C,
N0 = initial number of viable micro-organisms,
N = final number of viable micro-organisms,
IF = inactivation factor.

IF = No/N = 10 t/D
t = exposure time
D = D-value of micro-organism in the exposure conditions.

CBE 106 V1

22

Fo Tables
Points to Note
1. 121.1 = Fo of 1min
2. Below around 112 very little
accumulated Fos
3. Increase/decrease is
exponential slight changes
have a big impact.
4. The F0 value of a saturated
steam sterilisation process is
the lethality expressed in
terms of the equivalent time
in minutes at a temperature of
121 C delivered by the
process
CBE 106 V1

23

PNSU, SAL and Overkill

Sterility assurance level (SAL) is the reciprocal of Probability
of a Non-Sterile Unit (PNSU).
The purpose of a BI challenge is to establish that the
biological lethality is equivalent to the physically determined
F0, generally measured by thermocouples.
SAL = Fo / Dvalue
With a Dvalue of 1.5min and a Fo of 18min = we have an 12 log reduction.
If we started with 106 we would end up with 10-6 which is the PNSU so
we have an SAL of 1012

Overkill generally means that you develop a cycle that gives

a complete kill of BIs with a No of 106 and then you double
that cycle otherwise can use a reduced cycle approach
Overkill is really over overkill and only sutiable for equipment.
CBE 106 V1

24

Example Calculation of SAL

Generally in sterilisation we are required to achieve an SAL of 106
(minimum) and often an additional 6 log reduction (overkill situation).
For example if a material has a bioburden of 400cfu then to reduce
the bioburden to 1 = log (400) = (2.60). This shows that only a 2.6
log reduction is needed to bring the population to 1 and therefore the
total log reduction required for sterilisation with SAL of 106 = 2.6+6 =
8.60 to achieve this we need a total sterilisation time at 121oC with
a Dvalue of 2.0 = 2.0 x 8.6 = 17.2 min.
For BI challenge, with a starting population of 106 and a Dvalue of
2.0, to reduce the population to 10 -6 we need 2.0 x 12 logs = 24
minutes at 121oC to achieve overkill conditions.
CBE 106 V1

25

Critical parameters needed for

successful sterilization

Article wrapping
Chamber load pattern
Air removal (steam displacement or vacuum)
Moisture (saturated steam)
Pressure / vacuum conditions
Temperature
Cycle Time and Dwell Time
Contact with surfaces:
Packaging permeable to moist heat
Items designed to allow contact
Items designed to allow air removal

CBE 106 V1

26

What Can Go Wrong ?

Effective sterilization is dependant on:
initial bioload of incoming materials
Microbe resistance to heat (Dvalue) of that bioburden
Time the autoclave is held at a sterilizing temperature
Ability of steam to penetrate items being sterilized
Steam Penetration:
As steam is used to transfer heat, tightly wrapped items, or long tubing
may not be properly penetrated. Would represent worse case for
validation.
Air Pockets:
Trapped air creates localised dry heat conditions reducing lethality rates
CBE 106 V1

The Problem of Air

Pockets of trapped air result in localized dry heat conditions which
reduces the SAL.
Autoclaves without vacuum are considered non-GMP
Air removal relies on
Vacuum pre-pulsing the chamber before introduction of steam
generally 3 - 4 times
Careful consideration of the load pattern and contents

Known issues with air removal:

Extended length of transfer tubing
Filters mis-orientated to trap air
Tank valves closed off to prevent removal

Air inlet at end of the cycle must be sterilized via an air filter filter
must be periodically integrity tested.

CBE 106 V1

28

Steam Supply Quality

Expected to test steam quality regularly = WFI minus bioload.
HTM-2010 (UK Standard) sets our requirements for steam
quality wrt validation and monitoring
HSA Guidance states Steam quality must be tested
periodically to ensure that:

moist heat (rather than dry-heat) sterilising conditions are achieved;

superheating does not occur;
wet loads are avoided;
non-condensable gases is below 3.5%; and
mineral and organic impurities (including bacteria and pyrogens) are below
specified maximum levels.

The three basic steam quality tests are the superheat test,
dryness value and non-condensable gas tests.

CBE 106 V1

29

Saturation Temperatures and

Pressures for Steam

CBE 106 V1

30

Operating Characteristics of Steam

Sterilisers
Air Removal Options
Gravity displacement:
Steam enters and displaces the
residual air through an open
vent

Vacuum air removal:

Air is removed with a
mechanical pump prior to dwell
time.

Pressure is needed to achieve

high temperatures (steam)
Must release pressure slowly
for liquids (slow exhaust)

Items must be allowed to dry

before removal from chamber

CBE 106 V1

31

Example time/temperature/pressure
Print-off.

CBE 106 V1

32

Sterilisation Cycle Development

Two basic approaches are employed to develop
sterilisation cycles for moist heat processes:
Overkill, used for equipment and for heat stable products, and,
Probability of Survival (Bioburden Approach), used for heat
sensitive products.

Need to specify cycle conditions

Heat lability, or not, of the artlcles being sterilised

Pre-vac. conditions
Time/temperature and Fo requirements
Load patterns and orientations
Wrapping
Slow or fast exhaust

CBE 106 V1

Cycle Development Overkill Method

Assumes all bioburden to be biological indicator species - worst
case assumption. Requires a 12 log reduction of a resistant
biological indicator with a known D-value of > 1 min.
End point is SAL > 106 (In reality much higher)
Consider a safety margin where the product demonstrates
susceptibility for microbial growth and can handle extended heat
exposure.
Bioburden and resistance data are not required to determine the
required F0 values.
Cycle parameters are chosen to ensure that the coldest point within
the load receives an F0 that will provide, at a minimum, the SAL level
chosen for the cycle - typically F012
Overkill is always run with equipment loads
CBE 106 V1

34

Cycle Development Probability of Survival Method

Used for semi heat labile product,
The sterilisation process is validated to achieve the
destruction of a pre-sterilisation bioburden to a level of at
least 100, with a minimum safety factor of an additional
six-log reduction (1 x 106) or
SAL of 106,
Requires D-value of bioburden to be measured and
monitored.

CBE 106 V1

35

Cycle Development Demonstration of Sterility Assurance

For both approaches, must establish the cycle needed to
provide the minimum F0 values.
Must do heat distribution and heat penetration studies
to determine the amount of heat delivered to the slowest
heating unit in each load.
Validation studies must show that each unit receives the
minimum F0 value to achieve the SAL.
Must evaluate each load pattern:
Thermometrics
Lethality

CBE 106 V1

36

Cycle Development Demonstration of Sterility Assurance

For lethality studies, use a defined resistant challenge
organism such as Geobacillus stearothermophilus
exposed to the product being validated.
On establishment of the BIs resistance in a given
product, provided the D-values of any potential
bioburden or environmental isolates exhibits a lower Dvalue than the reference BI, it is safe to assume that the
cycle will exhibit sufficient lethality overall.
Problem is that it is very difficult to experimentally
establish Dvalues so in practice this is not done.

CBE 106 V1

37

Wrapping Articles and Load Descriptions

(Must develop equipment wrapping program)

Must completely seal the wrap

Generally 2 - 3 sealed layers
Overwrapped articles retain
moisture
Must include BI and T/C when
validating artlcle
Must specify load in autoclave
Number and type of artlcles
Specific location (diagram / photo)
Load pattern must appear in
operating procedure
CBE 106 V1

38

Steam Sterilizers and Validation

Kill microbes with a very high
degree of assurance even
under worst case conditions
Protect the contents of the load
from deterioration or instability
Can deliver more Fos for
Equipment loads than for
Product

CBE 106 V1

Its all about the bugs!

39

Validation Principles
The basic principles for validation of a heat sterilizing
process are:
Must use BIs to demonstrate lethality
Must use thermometrics/ thermocouples
Cycle development and description of load patterns
are pre-requisites
Can do time/temperature or Fo approach for control
Calibrate thermocouples both pre and again post
Must include worst case conditions
Maximum and minimum loads/ patterns
One run of reduced cycle time / temperature
Cold start for at least one of three runs per load pattern
CBE 106 V1

40

Validation Approach and Sequences

DQ: Has the item been specified

correctly ?
IQ: does equipment meet the URS
requirements? Is everything that
was on the box, in the box? Is the
unit installed properly. Are support
programs in place for ongoing
operation of A/C?

User
Specification

Functional
Specification

Design
Specification

OQ: does the A/C operate

properly? Does the unit hold temp
and pressure correctly?

PQ: validation of autoclave cycles

and loading patterns need to
show sterilization.

CBE 106 V1

Is based on

PQ

Is based on

OQ

Is based on

IQ

Commissioning

Implementation

Overview of Sterilisation Validation

(Scope of Works)
URS

+ Functional Specn

Load Patterns

Cycle
Development

Controllers

Validate and
Calibrate

Document Cycles
& Controls

CBE 106 V1

Design Specn

DQ

Build

FAT, SAT and IQ

Protocols

Steam
Fluids/Air

OQ - Empty &
Full Chamber

Thermo. +
Lethality

PQ - Penetration
Validate Lethality

Thermo. +
Lethality

42

Validating Load Patterns

(Why are load patterns important?)
Sterilization relies on steam penetration. Need to validate each
set load patterns
Very important to show what you put in an autoclave comes out
sterile consistently
Bis: When to use spore strips and when to use solutions
How to validate?
3x successful runs each loading pattern
Place BI with each item in worse case spot. Place
thermocouple next to BI, but not touching item.
How often to re qualify? annually expected
Loading patterns should be documented and adhered to.
Worse case validated can use less but not more equipment
CBE 106 V1

Pre-Qualification Activities GMP DQ Considerations

Materials of construction proposed and the quality of finish

Clean-ability of the design;
Air breaks on drain lines;
Location of drains;
Method by which the chamber maintains leak tight conditions to prevent
back flow of non-sterilised air into the chamber;
Interlocking of doors;
The door type (swing or lift);
A microbial retentive vent filter with provision for in-situ sterilization and
integrity testing,
Able to insert validation sensors through entry port
Controller / HMI features security and configuring / prints/downloads
Alarm features
Nominated cycles

CBE 106 V1

44

Installation Qualification (IQ)

Confirm item has been built according to design specifications
Materials of construction are suitable for GMP standards.
The vendor must provide evidence of a satisfactory
completion Factory Acceptance Test (FAT) showing that the
item meets fabrication, functional and preliminary
performance standards prior to shipment.
The item is installed in a safe manner and hooked up to the
appropriately qualified services (water, steam, air) and
drainage.
The statutory documentation for the pressure vessel design,
plumbing and electrical connections have been provided.
Should do an empty chamber map.
A typical acceptable range of temperature in the empty chamber is
1oC when the chamber temperature is not less than 121oC
CBE 106 V1

45

Autoclave - Installation Qualification

(Critical Services)
Steam supply to the autoclave chamber is qualified as WFI
grade or clean steam.
Clean steam is produced using Water for Injection (WFI) and
is tested to the relevant WFI pharmacopoeial requirements
except for bioburden.
Need sampling ports to collect the steam
The clean steam generator must be validated and have
sufficient capacity to meet the peak loads.
The autoclave has a sterilisable vent filter in place that is
capable of being integrity tested.

CBE 106 V1

46

Autoclave - Operational Qualification

Empty Chamber Thermal Mapping
Verify the heat distribution pattern in an empty chamber
Repeat annually to re-confirm operation of autoclave
Conduct cold start and hot start

Controller Reliability
Ensure each step in the PLC is in the correct sequence and is
repeatable. Failure modes should include failure and restart of
the critical services and include:
Electrical power loss,
Loss of equipment or instrument compressed air loss,
Service loss: jacket or pure steam, cooling water, vacuum,
Other critical service.
CBE 106 V1

47

Operational Qualification Control

Systems
Control System Verification:

Sterile Door Security,

Program Change/Alteration Security,
Cycle program Back Up and Recovery,
Calculation of F0 Accuracy,
Independence of Controlling and Monitoring Thermocouples,
Accuracy of Printout Record.

Alarm and display indicators,

Ensure these indicate the correct status of the autoclave for each cycle,

Door Interlock
must work correctly not allowing access during the cycle,

Gasket Integrity/ Leak testing

Verify positive/negative pressure seal of all door gaskets.
Bowie Dick Test to demonstrate air removal from chamber
CBE 106 V1

48

Operational Qualification
The operation of the autoclave shall be evaluated
according to a written OQ protocol.
Empty chamber temperature distribution studies,
Full and minimum load chamber heat distribution studies**.
A minimum of three replicate cycles should be carried out for
chamber heat distribution studies. An analysis of the data should
identify:
The lowest temperature in the chamber (i.e. cold spot(s)) where a
measurable temperature distribution exists,
Any movement of the cold spot between the repeats of the same
cycle or between cycle types (i.e. empty, minimum and full loads).
**Could be done instead as part of PQ.
CBE 106 V1

49

PQ of Autoclave
PQ: validation of autoclave cycles and loading patterns.
What SAL do you need?
Need to show a 106 or 1012 reduction of microbes.
What is your starting bioload?
Spore strips have >106 CFU.
What is the microbes D value?
For Geobacillus stearothermophilus, this is around 1.5 2.0
Must use physical, chemical and biological indicators (Bis).

CBE 106 V1

PQ of Autoclave
Heat Distribution Study how does steam circulate around
the contents ? Is it consistent ? Can be done with
thermocouples only.
Heat Penetration Study how quickly does the heat penetrate
the item or liquid.
Maximum Loads
Minimum Loads what does this mean ?

Worst Case Conditions

Reduced time and temperature
If overkill needed 50% of cycle to show >10-6 production cycle is doubled
to achieve 12 log reduction.
CBE 106 V1

Performance Qualification
(Heat Penetration Studies)
Heat penetration studies carried out for each load configuration
for each nominated cycle with the aim to:
Identify any cold spots within the load;
Measure the accumulated Fo for each challenge location within
the nominated load.
Microbiological challenge (lethality) studies carried out as part of
heat penetration studies (reduced exposure).
Product degradation (maximum exposure)
Load lag time or come up determination look for slowest to heat
location
BI is Geobacillus stearothermophilus with a certified D-value
between 1.5 and 2.0 and a verified spore count of between 5x105
and 5x106,
CBE 106 V1

52

Load Equilibration Time

Equilibration time, that is, the time for the penetration thermocouples to show the
same temperature as the chamber.
Ideally equilibration time should be less than 15 seconds for chambers less than
800Litres and 30 seconds for larger chambers.
If the equilibration time is exceeded it diagnoses:

140
120
100
80
60
40
20
0

CBE 106 V1

Inadequate air removal

OR
Inadequate steam penetration OR
Excessive non-condensable gases

Chamber
Load

53

Acceptance Criteria PQ
The steriliser must meet current GMP Standards for Installation
and Operation,
The differential between the hottest and coldest thermocouple
at any time during the dwell phase should not exceed 2oC,
Minimum of three acceptable consecutive sterilisation runs per
load pattern for a full (maximum) load and a minimum load
pattern,
The sterilisation hold time for the reference thermocouple(s)
must not be less than the nominated cycle sterilisation hold
time,
The reference probe must be within - 0.5oC to +0.5oC of the
nominated cycle conditions.
CBE 106 V1

54

Acceptance Criteria PQ

The general thermal profiles of the vacuum, heat-up and sterilisation hold
phases for all thermocouples must be defined for each of the studies to
provide a basis for the review of the autoclaves physical performance.
Meets all minimum F0 requirements for the nominated load conditions,
All thermocouples should achieve a SAL value nominated for the cycle with a
D-value of 1.0 of the BI in water. (If alternatives are used justification should
be provided),
All biological indicators (BIs) subjected to heat are:
rendered non-viable when incubated (i.e. there must be no growth from
the recovered spore inoculum),
For a Reduced Cycle provide the cycle minimum SAL when calculated
back to the full cycle time.
F0 for a cycle with complete lethality and for a cycle with survivors.

CBE 106 V1

55

Example Acceptance Criteria

(Equipment Load)

Four pulses of vacuum down to 25 kPa

3 positive pulses of steam to 160 kPa
Sterilisation set-point temperature 124oC for lowest T/C
All T/Cs within range 124oC -126oC during dwell
T/C does not fluctuate by > 1oC during dwell
Sterilisation dwell time 15 minutes
Accumulate > 30 Fo
All Bis show no growth
Post sterilisation drying time 20 minutes
Leak rate tests remain within specification
At least 9 of 10 T/Cs remain within calibration

CBE 106 V1

56

Final Validation Report

CBE 106 V1

57

Final Validation Report

Ensure documentation has been completed and approved in
line with the site quality procedures,
Ensure an adequate training program has been performed to
ensure operators manage the process consistently.
Summarise all validation activities in a Validation Summary
Report.

Report against the Validation Protocol.

Close out of all Deviations
Validation Certification.
Ensure system is under Change Control.

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58

Annual Re-validation Example

(Include the following tests)
1 Chamber leak rate test
2. Air removal and steam penetration test (Bowie Dick Test)
3. Heat distribution studies for empty chamber (1x)
4. Heat penetration studies for standard production loads:
Load #1 Filling Components
Load #2 Filling Machine Cap Components
Load #3 Filling Machine Stopper Components

5. Biological challenge testing for standard loads

6. Steam condensate quality test
7. Planned preventative maintenance schedule, including instrument calibration

Three consecutive cycles shall be tested for each load configuration to

demonstrate consistency of autoclave performance.
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Routine Monitoring of Autoclaves

Sequential number runs and a running log

Must double sign prints to verify cycle conditions met
Record conditions met and any alarms activated
Chamber Leak Rate Test (weekly)
Physical indicator on each item in each load
Bowie Dick Test (Optional)
BIs are not routinely included in the cycle
Reliance on controlling probe (directly correlated to the
worst case (coldest) location for the validation probe
For product loads usual ot probe a number of dummy
vials in the load for added assurance.
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Auditor Considerations
What do GMP auditors look for in an audit

Was re-validation conducted in time frame?

Focus on PQ primarily but interest in IQ/OQ for newer autoclaves
Coolest and warmest positions clearly stated in validation report?
Preventative maintenance program, SOPS, leak rate test data ?
Cycle time / Fo is it sufficient for tested D values?
Was validation equipment within calibration (pre and post use))
Traces for validation and most recent cycles consistency ?
Are vacuum cycles used appropriately?
Is anything thing not listed on the loading pattern present in the
autoclave? Enough room for steam to circulate through chamber?
Deviations from protocols. Are conclusions valid and justified?
Can site demonstrate terminally sterilised product is stable?
CBE 106 V1

Validation of Dry Heat Sterilisation

Processes

CBE 106 V1

BP/EP XVIII Monograph

Minimum conditions of 160 C for at least 2 hours for sterilisation.
(Other combinations of time and temperature may be used provided that it has been
satisfactorily demonstrated that the process chosen delivers an adequate and
reproducible level of lethality when operated routinely within the established
tolerances.)

Dry heat sterilisation is carried out in an oven equipped with forced

air circulation or other equipment specially designed for the purpose.
The steriliser is loaded in such a way that a uniform temperature is
achieved throughout the load. Knowledge of the temperature within
the steriliser during the sterilisation procedure is usually obtained by
means of temperature-sensing elements inserted into representative
containers together with additional elements at the previously
established coolest part of the loaded steriliser.
The temperature throughout each cycle is suitably recorded.
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63

Depyrogenation of Glassware
Dry heat is used for depyrogenation purposes and results in
complete destruction of micro-organisms
It is accepted that validation of depyrogenation means also
SALs much greater than 10-6.
Dry heat at temperatures greater than 220C is frequently
used for sterilisation and depyrogenation of glassware. In this
case demonstration of a 3-log reduction in heat resistant
endotoxin can be used as a replacement for biological
indicators. (BP/EP)
Spores of Bacillus subtilis (for example, var. niger ATCC 9372,
NCIMB 8058 or CIP 77.18) are recommended as biological
indicators.
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Example of Depyrogenation Cycle

Description

Also need
Load Pattern Description
Location of T/Cs whroughout the chamber
Cycle ranges for parameters
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65

Installation Qualification
Calibration of monitoring devices
Preventative Maintenance program developed
All filters are listed with the following information

identification
type
size
change frequency
air capacity
flow rate
integrity testing requirements
the air downstream from the filter should be tested for total and
viable particulates to ensure the filters do not shed or leak
particles

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66

Operational Qualification Considerations

PLC Reliability
Blower Rotation - verify RPM and correct direction
Heater Elements integral
Air flow rate throughout the chamber
HEPA filter installation integrity (inlet and exhaust) in clod
condition
Chamber non-viable particle monitoring - Grade A in cold
condition
Room Balance chamber positive ot room at all times

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Operational Qualification Considerations

For Ovens:
Door interlocks
Gasket integrity

For Tunnels:
Belt velocity and chart recorder speed calibrated

Empty chamber heat distribution profile

Temperature profile wall and chamber
Minimum of 3 studies
Record all critical process parameters
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HAO Performance Qualification for

Depyrogenation
Expected to apply endotoxin the inside of glass vials
Techniques and methods for recovering and testing endotoxin must
be validated.
should recover a minimum of 50% of applied endotoxin from glass
surfaces.
Recovery studies should be performed at the level of expected
endotoxin.

Need to challenge with >10,000 Endotoxin Units (EUs)

Acceptance criteria is > 3 log reduction demonstrated on 3
consecutive runs for each load pattern.

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69

Performance Qualification
Loaded chamber heat distribution
Loaded chamber heat penetration (min and max load
patterns)
Biovalidation
Sterilisation cycles only

Depyrogenation verification
Endotoxin challenge studies must indicate at least a 3-log
reduction for all locations for all runs

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Example HAO Control Probe Print

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USP <1211>
Dry-Heat Sterilization/Depyrogenation
A dry-heat sterilization/depyrogenation system is
supplied with heated, HEPA filtered air, distributed
uniformly throughout the unit by convection or radiation
and employing a blower system with devices for sensing,
monitoring, and controlling all critical parameters.
A typical acceptable range in temperature in the empty
chamber is 15oC when the unit is operating at not less
than 250 .

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Example Acceptance Criteria for HAO

Cycle Conditions
Must meet the nominated ranges of the cycle conditions
Thermometrics
All thermocouple locations shall indicate temperatures continuously
in excess of 220oC for a period of at least 2 hours 15 minutes,
during the exposure phase of the cycle.
The timing of the exposure phase of the cycle starts from the
slowest to heat thermocouple reaching 220 oC and finishes with the
fastest to cool thermocouple falling below 220 oC.

Pyrometrics - > 3 log reduction

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Maintaining the Validated State

(Annual Routine Re-validation)
Routine requalification program containing at least:
Annual requalification of the sterilisation process (for example,
heat distribution on representative load(s), determination of min.
F0 values), at least annually.
Preventive maintenance program giving the scheduled
maintenance measures required, SOPs for their performance,
responsibilities, requirements for documentation.
Change control procedure specifying under what circumstances
a re-validation is needed e.g repairs.

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Sterile Filtration Basics

CBE Pty Ltd
This training program is copyright to CBE Pty Ltd and may not be
modified, reproduced, sold, loaned, hired or traded in any form without
its express written permission.

CBE 106 V1

Introduction

75

Useful References
PIC/S Guide to Good Manufacturing Practices - PE 009
2014 Annex 1
PDA Technical Report No.26: Sterilizing Filtration of
Liquids
PDA Technical Report No.40: Sterilising Filtration of Gases
FDA Guidance for Industry Sterile Drug Products
Produced by Aseptic Processing Current Good
Manufacturing Practice 2004)

CBE 106 V1

Regulatory Agencies

Filter Types

Depth Filter

CBE 106 V1

Membrane Filter

77

Membrane Filters
Thin polymer films that have many microscopic pores
which can be of different pore sizes (0.1, 0.22, 0.45 etc)
Retain microorganisms by sieving, entrapment or
adsorption (or a combination thereof) e.g.
Size exclusion (combination of sieving and entrapment); is very
reliable
Size of filter pores required to screen out:
Yeast 0.45 -1.2 m
Bacteria 0.2 m
Viruses and mycoplasmas 0.01-0.1m

Membrane filtration is usually employed for heatsensitive products;

Most are hydrophobic in nature
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Examples of Membrane Filters

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79

Filter Selection and System Design

Criteria

Retention Capability
Integrity Testing
Filtration Rate and throughput
Materials of construction

Hydrophobicity
Durability
Toxicity
Leachables / Extractables
Particle Shedding
Gas/Filter Compatibility

Water Blockage
Design Consideration for
Condensation Control
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Applications
Sterilising (Membrane/Cartridge/Disc) filters are used in
pharmaceutical manufacture for:

Bulk Product Filtration

Steam Sterilisation in place (SIP)
Gas Filters
Vent Filters

Other (Depth) filters are used for:

Clarifying bulk product
Reducing bioburden and filtering viruses (nano-filtration)
Reducing endotoxin (positively charged filters)

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General Principles
from PIC/S GMP- Annex 1
Filtration alone is not considered sufficient when
sterilisation in the final container is possible.
If product cannot be sterilised in the final container,
solutions or liquids can be filtered:
Through a filter of nominal pore size of 0.22 micron or less
Into a previously sterilised container

Such filters can remove most bacteria and moulds but

NOT all viruses or mycoplasmas
Consideration should be given to complementing the
filtration process with some degree of heat treatment

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General Principles
PIC/S GMP- Annex 1
For products which do not undergo terminal sterilisation,
a second further filtration (double filtration) is
recommended:**
immediately prior to filling
as close as possible to the filling point

Fibre shedding characteristics should be minimal

** This is also an FDA recommendation

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General Principles
PIC/S GMP- Annex 1
Filter integrity should be verified before and immediately after use
by:
Bubble point,
Diffusive flow
Pressure hold test

or
or

The time taken to filter a known volume of bulk solution and the
pressure difference to be used across the filter should be
determined during validation and any significant differences from
this during routine manufacturing should be noted and investigated.
Integrity of critical gas and air vent filters should be confirmed after
use.
Integrity of other filters at appropriate intervals.
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General Principles
PIC/S GMP- Annex 1
The same liquid filter should not be used for more than
one working day unless such use has been validated.
The filter should not affect the product by removal of
ingredients from it or by release of substances into it**

** this often requires leachables and exractables studies to verify the

suitability of a filter under conditions of use.

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85

Filtration Time Limits

Time limits should include, for example, the period
between the start of bulk product compounding
and its sterilization, filtration processes etc.
The time limits established for the various
production phases should be supported by data.
Bioburden and endotoxin load should be assessed
when establishing time limits for stages such as
the formulation processing stage.

The total time for product filtration should be

limited to an established maximum to prevent
microorganisms from penetrating the filter.
Such a time limit should also prevent a significant
increase in upstream bioburden and endotoxin
load.
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86

Filtration Efficacy
A sterilizing grade filter should be validated to
reproducibly remove viable microorganisms
from the process stream, producing a sterile
effluent.
Currently, such filters usually have a rated pore
size of 0.2 m or smaller.
Use of redundant sterilizing filters should be
considered in many cases.
Validation should include microbiological
challenges. The microorganism
Brevundimonas diminuta (ATCC 19146) is
generally used
A challenge concentration of at least 107
organisms per cm2 of effective filtration area
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87

Pre- Filtration Bioburden Requirements

"Since the effectiveness of the filtration process is also
influenced by the microbial burden of the solution to be
filtered, the determination of the microbiological quality of
solutions prior to filtration is an important aspect of the
validation of the filtration process in addition to the
establishment of the other parameters of filtration procedure,
such as pressures, flow rates, and filter unit characteristics.
USP
Universally accepted that pre-sterilisation bioburdens are
monitored consensus limit is < 10cfu/100mL

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88

What we know about filtration

Products can alter the size of micro-organisms
Osmotic pressure, pH can change organism size
Large incident bioburden can cause grow-through

Filters have limited number of retentive pores once

pores are saturated can get breakthrough
Industry evidence of very small micro-organisms
Experiences of penetration of very small bacteria
through 2 in series 0.2micron filters

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89

GMP Records
Must record the integrity testing of filters in the batch
record filtration is generally a critical step generally a
printout verified by dated signature.
The limits should be included in the record
The limits should reflect the validation reports
If there are initial failures this must be recorded as a
deviation even if resolved.
Integrity testing devices must be qualified

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90

Filter Validation Studies

Supplier Responsibility

Show correlation between

integrity test result and P.
diminuta reduction
Provide instructions,
specifications and limits for test
Determine bubble point of
product compared to water

User responsibility

Prove sensitivity of test in situ

Perform test in accordance with
test specifications
Record integrity test results
Provide product samples for
bubble point ratio determination

Require a protocol supplied by vendor and approved by client

Methodology: ASTM F838-83 standards or comparable
Must use product to do the microbial challenge
Once conditions established in the laboratory same conditions used in use
Integrity limits established and verified after each use.

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The following table (adapted from Carleton & Agalloco[1]) lists the elements
that comprise a sterile filtration validation study.
[1] Validation of Pharmaceutical Processes Sterile Products 2nd Ed Carleton & Agalloco

Validation Element

Filter Manufacturer

Filter User

Filter Reproducibility

Validation

Qualify Manufacturer

Sterilisation

Provide Recommendation

Operate within recommendation

Validation
Provide procedures and limits

Follow manufacturers procedures

Validation

Integrity Test
Correlate test with bacterial retention

Operation

Provide Limits for operating, temperature, pressure

Bacterial Retention

Validation

Perform integrity ratio work if wetting with

product
Establish operating parameters within the limits
provided
Provide product/process details
Review and Authorise Report
Provide product/process details

Extractables

Validation

Establish and document acceptance criteria

Review and Authorise report
Document compatibility

Compatibility

Provide information on materials

Perform Studies

Adsorption / binding

Provide information regarding known issues

Perform Studies

Particulates

Provide data for removal

Verify limits are achieved

Fibers

Meet non-fibre releasing claim (21CFR 210.3 b(6))

Preflush filters according to recommendations

Endotoxins

Perform analysis

Verify low endotoxins from filters

Perform testing (Class VI plastics, cytotoxicity) and

provide results

Obtain results and reports.

Toxicity

CBE 106 V1

Review and document conclusions

92

In Conclusion
These guys are you best friends

CBE 106 V1

93

Steve Williams,
Director, CBE Pty Ltd
www.cbe-ap.com.au
+61(0)417116476
[email protected]

CBE 106 V1