Using Biological

Challenges in the
Validation of Steam
Sterilisation Processes:
How to Plan for Success

Mark Thompson

Sharon Smith
 Using Biological Challenges in the Validation of Steam Sterilisation Processes: How to Plan for Success

Although not a strict ‘requirement’ of the European Pharmacopoeia, the use of biological
challenges and Biological Indicators (BI’s) in the validation and requalification of steam
sterilisation processes is generally part of the overall validation strategy. This paper discusses
some of the problems that can arise and how to plan for success.

Introduction

Biological Indicators (BI’s) are ‘recommended’ for use in the qualification of steam sterilisation
processes in the European Pharmacopoeia (EP) whereas the US Pharmacopoeia (USP) ‘requires’
their use.

Regardless of the regulatory need or expectation, the use of a biological challenge or indicator in
addition to the thermal data provides an extra degree of assurance that the sterilisation process
is doing what is expected. After all, destroying micro organisms and spores is the objective of the
sterilisation process, the use of Biological Indicators is a direct challenge to this objective.

Specified and used properly, Biological Indicators will provide data which compliments the
thermal data. However, if specified and used incorrectly, the Biological Indicators will provide
data which ‘complicates’ the thermal data.

An example of this would be a challenge location that shows good thermal data, but where the BI
has survived. Failures of this nature will prompt some root cause analysis and the potential root
causes may include; the actual sterilisation process failure, human error or biological challenge
failures or errors. This paper addresses some of the most common biological challenge failures
and how to avoid them when considering the selection, use or analysis of biological challenges.

These include:

• Definition of the study objectives

• Ensuring the biological challenge is representative
• Biological challenge specification
• BI z value analysis.
• Ensuring the BI is within specification

Defining the Study Objectives

Many requalification and validation exercises fail because of badly defined objectives in the
protocols. Generally, the objective of a sterilisation process requalification is to demonstrate a
reliable and repeatable Sterility Assurance Level (SAL). For terminally sterilised products and
for equipment used in the later stages of aseptic manufacture, this will almost certainly be an SAL
of 10-6 or greater. For other processes, this will be based upon risk assessment and process
needs for bio burden control, but should however, still be defined as the primary objective in the
protocol.

A SAL of 10-6 is not demonstrated by just achieving a certain time / temperature relationship;
neither is it demonstrated by just killing Biological Indicators (BI’s). It requires knowledge or
worst case assumptions regarding the product or equipment bio burden, in relation to the
lethality the sterilisation process has demonstrated - this is where BI’s come in!

For example, with a terminally sterilised product, the pre sterilisation product bioburden will be
monitored on every fill. Based upon this historic data and evidence of in process control, a bio
burden assumption will be made, such as:-

Maximum bioburden population per filled vial < 103

Maximum D121 value of this bioburden < 0.5 minute

This would be a typical pre sterilisation bio burden limit for a terminally sterilised parenteral
product. To justify this there will be supporting population and heat shock data which
demonstrates control well within these limits.
 Using Biological Challenges in the Validation of Steam Sterilisation Processes: How to Plan for Success

Based upon such limits, a biological challenge will be selected. This could be Biological Indicators
with a spore population of 106 and a D121 value of 1 minute for example. If these biological
challenges are presented to the sterilisation process and complete kill is achieved then a 6 log
reduction of spores which have a D121 value of 1 minute has been demonstrated. This equates to a
12 log reduction of the defined bioburden worst case assumption (D121 value of 0.5 minutes).
Also, the defined bioburden worst case assumption of population per unit was 103 therefore the
12 log reduction starts at 103 and delivers a Sterility Assurance Level (SAL) of 10-9.

Known product
Bio burden 103
Population
6 logs of 1.0min D value
Limit. <103
102
will equate to 12 logs of
101
the 0.5 min bio burden value.

100

10-1

10-2 Sterilising Dose

10-3 (time)
10-4

10-5
Calculated SAL of 10-9
10-6 For micro organisms
10-7
With a D value of 0.5min
SAL of 10-9 attained
10-8 Based upon product
10-9
Bio burden
knowledge.

Figure 1: Spore Log Reduction Demonstrating Sterility Assurance Level

The logic shown in figure 1 is essential to demonstrate the defined Pharmacopoeia requirement
(EP and USP) of SAL of 10-6 or greater has been achieved. Simply killing Biological Indicators
does not demonstrate this. Indeed, it may not be necessary to completely kill the biological
challenge. Demonstrating a 3 or 4 log reduction in spore population may be sufficient for some
processes when the bioburden data is taken into account.

For porous load / equipment processes, the bioburden will not usually be monitored and it is
usual to make a worst case assumption of bioburden which may be:-

Maximum bioburden population < 106

Maximum D121 value of this bioburden < 1 minute

The objective is therefore not to kill all BI’s, but to achieve the required confidence level or SAL.
Killing BI’s is very likely to be a requirement to satisfy the objective, but it is not THE objective.
An SAL of 10-6 or greater may be achieved by demonstrating a log reduction and it may be
demonstrated by achieving a complete kill.

Ensuring the Biological Challenge is Representative

Cycle development or previous qualification studies should have identified and justified the
worst case locations within the product and within the load to be sterilised. These locations will
be challenged both thermally and biologically. The considerations for biological challenges
should include:-

• Which microbiological challenge?

o Geobacillus stearothermophilus
o Clostridiun sporogenese
o Bacillus coagulans
 Using Biological Challenges in the Validation of Steam Sterilisation Processes: How to Plan for Success

• Purchased Biological Indicator or direct inoculation onto equipment surfaces or into

product

• Self contained Biological Indicator (media and indicator dye included) or just the
Biological Indicator challenge (spores inoculated onto a paper strip, metal disk etc)

The decision should be based upon the method being as representative as possible and also on
access.

In terms of being representative, it is widely known that some surfaces or materials significantly
affect the D121 value of micro organisms and spores. An example of this would be rubber stoppers
and filter membranes; where sterility of these items is critical (e.g. aseptic fill), it is common to
directly inoculate spores in suspension onto the surfaces. In such a case the D121 value will need
to be re established as the D121 value of the spore suspension will no longer apply.

Figure 2: Inoculation of stoppers

To illustrate how the material or surface can affect the D121 value of
spores, the table below shows 17 different batches of stoppers that
have been directly inoculated and how the D121 value has increased.
The most extreme case being an increase in D value from 2.2
minutes (spore suspension) to 5.5 minutes on the inoculated
stopper.

Spore % Difference from

Stopper
Stopper Lot Suspension Spore Suspension
D121 value
D121 value D-value.
1 2.1 2.5 23.6
2 2.1 4.4 110.1
3 2.2 4.6 113.3
4 2.1 3.6 75.4
5 2.1 4.3 106.8
6 2.2 5.5 151.5
7 2.3 2.8 22.0
8 1.9 2.4 30.8
9 2.3 3.4 48.7
10 2.3 3.2 41.9
11 2.3 3.1 36.3
12 2.3 4.3 89.7
13 2.1 4.5 118.9
14 2.1 2.6 26.7
15 2.2 4.1 90.8
16 2.2 3.8 74.9
17 1.7 4.1 144.5

Table 1: Data Highlighting the Dramatic Variances between Manufacturer Spore Suspension D values
and Actual D values following Stopper Inoculation
 Using Biological Challenges in the Validation of Steam Sterilisation Processes: How to Plan for Success

Effect of Suspension D-value when Inoculated onto

Stopper
6

)5
C
14 Spore Suspension
2
1 D121 value
(3
e Stopper D121 value
u
la2
v
-
D1
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Stopper Lot No.

Figure 3: The Effects of Suspension D-value when Inoculated onto Stopper

With regard to BI access, A self contained Biological Indicator is not going to fit inside a filling
needle, or in the tip cap of a pre filled syringe. If these are critical areas (and they generally are)
they need to be challenged with a BI that can fit. This may be a thread BI, wire BI or again,
achieved via direct inoculation.

Figure 4: – Different types of surfaces and

materials will affect the D value of micro
organisms and spores

These issues need to be considered well in

advance so that the required BI’s can be
ordered and /or the D121 value determination
work (for direct inoculation) can be completed
before the study starts. The biological data
that is going to be generated should not be
considered in isolation. It must be reviewed
with the thermal data (time at temperature or
Fo value). Therefore, as far as is possible, the
biological challenge should be close to the
thermal data point.

Figure 5: Examples of a Fluids Load. Self

Contained BI and Thermocouple Located
Securely at the Slow to Heat Location within
the Bottle/Test Tube
 Using Biological Challenges in the Validation of Steam Sterilisation Processes: How to Plan for Success

Biological Challenge Specification

The D value of a spore population is defined as the time taken to achieve a 1 log reduction in the
spore population. Therefore the D value is an indication of how difficult spores are to destroy. It
follows that the higher the D value, the harder the spores are to destroy. The D value is usually
referenced to 121oC as this is the normal steam sterilisation minimum temperature. The D value
is written with this temperature as suffix:-

D121 value of x minutes

Where x is the time taken to achieve a 1 log reduction in population of spores.

The Pharmacopoeias specify a minimum D value of D121 > 1.5 minutes, and over 90% of
sterilisation validation and qualification exercises do apply this standard (D121 > 1.5 minutes).
However, this is taken as a recommendation and may be deviated from as described in the
terminal sterilisation example above.

The USP offers a calculation for determining the time required to achieve a guaranteed kill of a
population of spores. This calculation is:-

(Log population + 4) x D121 value = Guaranteed Kill Time.

Therefore the usual BI challenge of 1 million spores (population of 106) would require:-

(6 + 4) x D121 value = Guaranteed Kill Time.

This represents 10 times the D value for a Guaranteed Kill Time.

Example:

A sterilisation process running at 12oC for 15 minutes is validated with BI’s having a population
of 106 and a D121 value of 1.5 minutes. The USP calculation will calculate a guaranteed kill time of
15 minutes, therefore the validation study will show complete kill of all BI challenges.

The following year at requalification, additional BI’s are purchased with a population of 106 and
a D121 value of 2.1 minutes. The USP calculation will calculate a guaranteed kill time of 21
minutes. Therefore the validation study may NOT show complete kill of all BI challenges.
Nothing has changed with the lethality of the cycle, but the BI challenge is now more difficult.

This demonstrates that the specification of the biological challenge must be referenced and
justified by the validation approach being taken as discussed above. A population, D value and z
value (see below) specification must be established. The purpose of this is to ensure that the
challenge is good enough, but also to ensure problems are not caused during requalification
because the BI challenge has become harder.

BI z Value Analysis

The z value of a spore population is defined as the change in temperature that delivers a 1 log
change in D value. Generally, if a sterilisation process runs at 121oC and is controlled at a given
time at this temperature, it is not necessary to consider z value.

The z value does however need to be considered if sterilising at other temperatures, for example,
equipment at 134oC, or media at 118oC. The lower the z value, the more temperature sensitive
the spore population is. Therefore, a BI that has a low z value will be more easily killed at higher
temperatures than 121oC but will take longer to kill at temperatures below 121oC than a BI that
has a higher z value.

If a sterilisation process runs at 134oC, definition of a minimum z value as part of the BI

specification is required.
 Using Biological Challenges in the Validation of Steam Sterilisation Processes: How to Plan for Success

Example:

BI Lot A: D121 value of 2 minutes

z value of 13oC

These would have a D value at 134oC of 0.2 minutes.

BI Lot B: D121 value of 2 minutes

z value of 6.5oC

These would have a D value at 127.5oC of 0. 2 minutes and a D value at 134oC of 0.02 minutes.

Therefore two sets of BI’s each with a D121 value of 2 minutes, BUT the second set of BI’s with a z
value of 6.5oC would be killed in a tenth of the time. This is therefore not an acceptable challenge
to the sterilisation process. For this reason, any sterilisation cycle running at temperatures well
above the reference temperature of 121oC must have a minimum z value specification.

If a sterilisation process has some lethality delivered at temperatures below 12oC it is necessary
to define a maximum z value as part of the specification. This would apply to virtually all fluid
processes, particularly where Fo is used for control or cycle acceptance.

Fluids Sterilising Cycle Showing heating and cooling temperature spread

130

110
Fast to heat Slow to cool
location location
90

Slow to
De g C

70 heat
location Fast to cool
location
50

30

10
"1 4 6 :4 "
"1 5 0 :2 "
"1 5 4 :0 "
"1 5 7 :4 "
"1 1 :2 "
"1 0 5 :0 "
"1 0 8 :4 "
"1 1 2 :2 "
3 :1 0 "

"1 2 0 :4 "
"1 2 4 :2 "
"1 2 8 :0 "
"1 3 1 :4 "
"1 3 5 :2 "
"1 9 :0 "
"1 4 2 :4 "
3 :4 0 "

"1 5 0 :0 "
"1 5 3 :4 "
"1 5 7 :2 "
"1 0 1 :0 "
"1 0 4 :4 "
"1 0 8 :2 "
"1 1 2 :0 "
"1 1 5 :4 "
4 :1 0 "

"1 2 3 :0 "
"1 2 6 :4 "
4 :3 0 "
1"
2: 0
2: 0
2: 0
2: 1
3 :0 0
3: 0
3: 1
3: 1

3: 1
3: 0
3: 1
3: 1
3: 1
3 :3 1
3: 1

3: 0
3: 1
3: 1
4: 0
4: 0
4: 1
4: 0
4: 1

4: 0
4: 1
"1 4 3 :0

"1 6 :0

"1 6 :2

"1 9 :2

0 :2
2:
"1

Figure 6: Fluids Sterilising Cycle Showing Heating and Cooling Temperature Spread

Example:

BI Lot A: D121 value of 2 minutes

z value of 12oC

These would have a D value at 109oC of 20 minutes.

BI Lot B: D121 value of 2 minutes

z value of 6oC

These would have a D value at 115oC of 20 minutes and a D value at 109oC of 200 minutes.
 Using Biological Challenges in the Validation of Steam Sterilisation Processes: How to Plan for Success

Therefore, two sets of BI’s each with a D121 value of 2 minutes, BUT the second set of BI’s with a
z value of 6oC would take 10 times longer to kill at 109oC.

It may be the case that a fluids sterilisation cycle developed and successfully validated one year
(with BI lot A) fails requalification the second year, nothing has changed with the process, the
BI’s ‘appear’ to be the same, but BI lot B will be a lot harder to kill at temperatures below 121oC.
The temperature can be below 121oC for several hours of the cycle on a fluids load during heating
and cooling. In such an example the BI is a much more difficult challenge. This must be controlled
to ensure that the BI’s selected are a suitable challenge (therefore a maximum z value will be
specified) but also that validation and requalification exercises are repeatable with confidence.

Ensuring the BI is Within Specification

The BI or spore suspension will be supplied by the manufacturer with a certificate quoting
population, D value and possibly the z value data. Quality Control checks of the BI’s should
ensure that the BI’s are sourced from an approved and audited supplier and should be inspected
upon receipt of each delivered batch. This should include population verification and resistance
challenge. The Pharmacopoeias detail the requirements and acceptance criteria.

Over recent years there have been concerns with the shipping and storage of BI’s. It is likely that
the BI’s will have been subject to temperature, pressure and humidity variations during shipping
and that they could have been subject to x-ray several times as well. All of these factors could
potentially affect the heat resistance of the spores in use. Therefore, the BI’s may not provide the
challenge that the manufacturer claims. As an absolute minimum, a sub lethal cycle should be
run. This again is defined in the pharmacopoeia as a 121oC (+/-1oC) cycle for 6 minutes. The BI’s
should survive this cycle, demonstrating at least, a minimum level of resistance. However, a more
quantitative test that most companies build into their QC testing programme is to perform a
repeat D value determination, this requires a BIER vessel (Biological Indicator Evaluation
Resistometer). This will allow verification post shipping and storage that the BI’s meet the
manufacturers labelled claim (prior to shipping); the Pharmacopoeias put a +/-20% tolerance on
this.

There are many variables in this process, not least a compliant BIER vessel, but also the correct
method of presentation and media specification. The objective is to recreate the manufacturer’s
methods as closely as possible to demonstrate that the spore resistance has not changed
significantly since manufacture.

If the z value of the BI challenge is important then consideration should be given to verifying this
as well. This will require the D value determination to be run at three different temperatures.

To illustrate BI variability, the table below shows results obtained testing BI’s post shipping and
storage. This summarises testing across a one year period of BI batches from a variety of
manufacturers

Figure 7: BI Spore Strips

 Using Biological Challenges in the Validation of Steam Sterilisation Processes: How to Plan for Success

% Difference
Labelled Verified from Labelled Pass / Fail
Strip Lot
D121 value D121 value D-value.
(Spec. ±20%)
1 1.6 2.9975 87.3 Fail
2 2.7 1.6082 -40.4 Fail
3 1.7 2.7378 61.0 Fail
4 1.9 2.1943 15.5 Pass
5 1.9 1.9534 2.8 Pass
6 1.8 1.9935 10.8 Pass
7 1.8 2.0177 12.1 Pass
8 1.9 1.7878 -5.9 Pass
9 2.5 2.6480 5.9 Pass
10 1.8 1.8192 1.1 Pass
11 1.5 2.3764 58.4 Fail
12 1.7 1.4867 12.5 Pass
13 2.0 1.8087 9.6 Pass
14 2.0 1.6685 16.6 Pass
15 1.8 3.4442 91.3 Fail
16 1.6 1.2548 21.6 Fail
17 1.6 1.9265 20.4 Fail
18 1.5 2.8837 92.2 Fail

Table 2: Verification of Spore Strip D values

These spore strips results demonstrate a failure rate of over 40%. The actual D value differing
from the labelled D value by more than the +/-20% specified in the USP.

% Difference
from Pass / Fail
Labelled Verified
Ampoule Lot Labelled
D121 value D121 value
D-value.
(Spec. ±20%)
1 2.5 2.7052 8.2 Pass
2 1.9 1.8682 -1.7 Pass
3 1.7 1.9040 12.0 Pass
4 1.9 1.8924 -0.4 Pass
5 2.1 2.1349 1.7 Pass
6 2.1 2.0917 -0.4 Pass
7 2.0 2.3154 15.8 Pass
8 2.0 2.3027 15.1 Pass
9 1.8 1.3862 -23.0 Fail
10 1.7 1.2144 -28.6 Fail
11 1.6 1.1584 -27.6 Fail
12 1.9 1.9552 2.9 Pass

Table 3: Verification of Spore Ampoule D values

 Using Biological Challenges in the Validation of Steam Sterilisation Processes: How to Plan for Success

These BI spore ampoules results demonstrate a failure rate of 25%. The actual D value differing
from the labelled D value by more than the +/-20% specified in the USP.

If the BI challenge selected can differ high or low by these amounts, it needs to be verified so that
the study shows confidence in the biological results obtained. For this reason many sites build D
value determination into their QC testing of Biological Indicators.

Conclusion

A good steam sterilisation qualification study includes detailed analysis of the thermal data and
the biological lethality demonstrated. This study is only as good as the data. Getting reliable and
meaningful biological lethality data is not as easy as buying BI’s and demonstrating a kill. There
are many potential errors which will result in false positive or false negative results. The issues
listed and discussed above are the most commonly found errors and following the advice given
here will deliver more confidence in the whole qualification process. Many sites now appoint a
Site Microbiologist whose role includes taking responsibility for this subject and for reviewing
the whole process from qualification assumptions, the type of BI’s used, QC testing and data
analysis.

© Honeyman Group 2010