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Full Steam Ahead

Biological indicators (BIs) 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 microorganisms and spores is the objective of the sterilisation process and the use of BIs is a direct challenge to this objective.

When specified and used properly, BIs will provide information which compliments the thermal data.However, if used incorrectly, this accrued information will simply complicate the thermal data.

An example of this would be a challenge location that shows good thermal data, but the BI has survived. Failures of this nature will prompt some root cause analysis, and the potential causes will include the actual sterilisation process failure, human error and biological challenge failures or errors.This article addresses five of the most common failures and how to avoid them when considering the selection, use or analysis of biological challenges, as well as their solutions. These involve: 
  • Defining the study objectives properly 
  • Ensuring the biological challenge is representative 
  • Biological challenge specification 
  • BI z value analysis 
  • Ensuring the BI is within specification
Defining 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 bioburden control, but should still be defined as the primary objective in the protocol. An SAL of 10-6 is not demonstrated by just achieving a certain time/temperature relationship; neither is it demonstrated by just killing BIs. It requires knowledge or worst case assumptions regarding the product or equipment bioburden, linked to what lethality the sterilisation process has demonstrated – this is where BIs 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 bioburden assumption will be made, such as: 

  • Maximum bioburden population per filled vial is less than 103
  • Maximum D121 value of this bioburden is less than 0.5 minutes 
This would be a typical pre-sterilisation bioburden 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 BIs with a spore population of 106 and a D121value of one minute, for example. If these biological challenges are presented to the sterilisation process and complete kill is achieved, then a six log reduction of spores, which have a D121 value of one 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 an SAL of 10-9.

The logic shown in Figure 1 is essential to demonstrate that the defined pharmacopoeia requirement (EP and USP) of SAL of 10-6or greater has been achieved. Simply killing BIs does not demonstrate this. Indeed, it may not be necessary to completely kill the biological challenge. Demonstrating a three or four 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 is less than 106 
  • Maximum D121 value of this bioburden is less than one minute
The objective is therefore not to kill all BIs, but to achieve the required confidence level, SAL. Killing BIs is very likely to be a requirement to satisfy the objective, but it in itself 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; for example, Geobacillus stearothermophilus, Clostridium sporogenese or Bacillus coagulans 
  • Purchased BI or direct inoculation onto equipment surfaces or into product 
  • Self-contained BI (media and indicator dye included) or just the BI challenge (spores inoculated onto a paper strip or metal disk and so on)
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/materials significantly affect the D121value of micro organisms and spores. An example of this would be rubber stoppers and filter membranes.Where sterility of these items is critical (for example 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 reestablished as the D121 value of the spore suspension will no longer apply.

To illustrate how the material or surface can affect the D121 value of spores, Table 1 shows 17 different batches of stoppers that have been directly inoculated and how the D121value has increased.The most extreme being an increase in D value from 2.2 minutes (spore suspension) to 5.5 minutes on the inoculated stopper.With regard to BI access, a self-contained BI 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 could be either a thread,wire or via direct inoculation.

These issues need to be considered well in advance so that the required BIs can be ordered and/or the D121value 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 F0 value).Therefore, as far as is possible, the biological challenge should be close to the thermal data point.

Biological Challenge Specification

The D value of a spore population is defined as the time taken to achieve a one 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 one log reduction in population of spores. The pharmacopeias specify a minimum D value of D121 is not less than 1.5 minutes, and over 90 per cent of sterilisation validation and qualification exercises do apply this standard. 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.

For example, a sterilisation process running at 121ºC for 15 minutes is validated with BIs having a population of 106 and a D121value of 1.5 minutes. The USP calculation will calculate a guaranteed kill time of 15 minutes. The validation work will show complete kill of all BI challenges. The following year at requalification, additional BIs 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 re-qualification because the BI challenge has become harder.

z Value Analysis

The z value of a spore population is defined as the change in temperature that delivers a one 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 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 temperatures higher 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, a definition of a minimum z value as part of the BI specification is required.

For 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 BIs each with a D121 value of 2 minutes, but the second set of BIs with a z value of 6.5ºC would be killed in a one 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 121ºC it is necessary to define a maximum z value as part of the specification. This would apply to virtually all fluid processes, particularly where F0 is used for control or cycle acceptance.

For example:

BI Lot A: D121 value of 2 minutes z value of 12ºC
These would have a D value at 109 ºC of 20 minutes

BI Lot B: D121 value of 2 minutes z value of 6ºC
These would have a D value at 115 ºC of 20 minutes and a D value at 109 ºC of 200 minutes

Two sets of BIs each with a D121 value of two minutes, but the second set of BIs with a z value of 6ºC would take 10 times longer to kill at 109ºC.

It may be that a fluids sterilisation cycle developed and successfully validated in one year (with BI Lot A) fails requalification in the second year.Nothing has changed with the process – the BIs ‘appear’ to be the same – but BI Lot B will be much 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 BIs 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 for the BI. Quality control (QC) of the BIs should include ensuring the BIs are sourced from an approved and audited supplier and upon receipt each delivered batch of BIs should be QC checked, which 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 BIs. It is likely that the BIs will have been subject to temperature, pressure and humidity variation during shipping and could have been x-rayed several times as well. All of these factors could potentially affect the heat resistance of the spores in use. Therefore, the BIs 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 six minutes.The BIs 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 is to perform a repeat D value determination – this requires a BI evaluation resistometer (BIER) vessel. This will allow verification post shipping and storage that the BIs meet the manufacturer’s labelled claim (prior to shipping); the pharmacopoeias put a +/-20 per cent tolerance on this.

There are many variables in this process, not least a compliant BIER vessel, but also the correct method of presentation, media specification and so on.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, Table 2 shows results obtained testing BIs postshipping and storage.This summarises a typical year’s testing of BI batches from a variety of manufacturers. 

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

The BI spore ampoules results in Table 3 demonstrate a failure rate of 25 per cent. The actual D value differs from the labelled D value by more than the +/-20 per cent specified in the USP. If the BI challenge selected can differ high or low by these amounts, it needs to be checked so that the study can have confidence in the biological results obtained. For this reason many sites build D value determination into their QC testing of BIs.


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 biological indicators 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 biological indicators used, quality control testing and data analysis.

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Mark Thompson is Managing Director of the Honeyman Group Ltd. Mark’s prolific background in the pharmaceutical and medical device industry spans more than 20 years. Various engineering management roles with the world’s largest household names including Eli Lilly, Rhone Poulenc Rorer, and Smith & Nephew Medical have seen Mark leading teams through an array of projects driven by capacity relief, new product introduction, profit improvement and regulatory compliance. He is a Chartered Engineer and member of Institute of Electrical Engineers. As Managing Director of the Honeyman Group for the last 10 years, Mark primarily specialises in sterile product manufacture, bioburden control and sterilisation processes.

Sharon Smith is the Microbiology Team Leader at the Honeyman Group Ltd. Sharon is at the forefront of the growing number of pioneering analytical projects that the company is being asked to undertake. Having developed an extensive and impressive knowledge of microbiological analysis, data interpretation and troubleshooting, and with a particular interest in D and z value determinations, Sharon leads the microbiology team in the development of new methods and techniques.
Mark Thompson
Sharon Smith
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