Using Biological Challenges in the Validation of Steam Sterilisation Processes

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.


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.

103

102

101

100

10-1

10-2

10-3

10-4

10-5

10-6

10-7

10-8

10-9

SAL of 10-9 attained

Based upon product

Bio burden

knowledge.

Sterilising Dose

(time)

Calculated SAL of 10-9

For micro organisms

With a D value of 0.5min

6 logs of 1.0min D value

will equate to 12 logs of

the 0.5 min bio burden value.

Known product

Bio burden

Population

Limit. <103

103

102

101

100

10-1

10-2

10-3

10-4

10-5

10-6

10-7

10-8

10-9

SAL of 10-9 attained

Based upon product

Bio burden

knowledge.

Sterilising Dose

(time)

Calculated SAL of 10-9

For micro organisms

With a D value of 0.5min

6 logs of 1.0min D value

will equate to 12 logs of

the 0.5 min bio burden value.

Known product

Bio burden

Population

Limit. <103

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


• 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.

Stopper Lot

Spore

Suspension

D121 value

Stopper

D121 value

% Difference from

Spore Suspension

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

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.


0

1

2

3

4

5

6

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

D-value (121C)

Stopper Lot No.

Effect of Suspension D-value when Inoculated onto Stopper

Spore Suspension

D121 value

Stopper D121 value

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


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.


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.

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.

Fluids Sterilising Cycle Showing heating and cooling temperature spread

10

30

50

70

90

110

130

"12:43:00"

"12:46:40"

"12:50:20"

"12:54:01"

"12:57:40"

"13:01:20"

"13:05:01"

"13:08:41"

"13:12:20"

"13:16:01"

"13:20:40"

"13:24:21"

"13:28:01"

"13:31:41"

"13:35:21"

"13:39:01"

"13:42:40"

"13:46:20"

"13:50:01"

"13:53:41"

"13:57:20"

"14:01:00"

"14:04:41"

"14:08:20"

"14:12:01"

"14:15:40"

"14:19:20"

"14:23:01"

"14:26:40"

"14:30:21"

Deg C

Slow to cool

location

Fast to cool

location

Fast to heat

location

Slow to

heat

location


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


Strip Lot Labelled

D121 value

Verified

D121 value

% Difference

from Labelled

D-value.

(Spec. ±20%)

Pass / Fail

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.

Ampoule Lot Labelled

D121 value

Verified

D121 value

% Difference

from

Labelled

D-value.

(Spec. ±20%)

Pass / Fail

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


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

Using Biological Challenges in the Validation of Steam Sterilisation Processes

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