Biological Indicators for Heat and Gas Sterilisation Validation

In short: A biological indicator (BI) is a known population of resistant bacterial spores used to prove that a sterilisation cycle really kills microorganisms. This article explains the key terms (D-value, z-value, log reduction), the test organisms normally used for steam, dry heat and gas processes, and what EU GMP Annex 1 and FDA guidance say about using them. It is written for QA, microbiology and validation staff and pharmacy students.

What is a biological indicator?

EU GMP Annex 1 (2022) defines a biological indicator as a population of microorganisms inoculated onto a suitable medium (for example a solution, container or closure) and placed within a steriliser, load or room location to determine the efficacy of a sterilisation or disinfection cycle. The challenge organism is chosen and validated for its resistance to the given process, and the incoming lot’s D-value, count and purity define the quality of the BI.

Physical measurements (temperature, pressure, time) show what the equipment did. A BI shows what the cycle did to living organisms. The two together give confidence that the process delivers the intended sterility assurance.

Key terms

• D-value: the time (at a fixed temperature, or the dose) needed to reduce the population of a given organism by 90 per cent, that is by one log10.
• z-value: the temperature change that changes the D-value ten-fold (Annex 1 glossary). To find it, determine the D-value at three or more temperatures, plot log10 of D against temperature and take the negative reciprocal of the slope. The intended process temperature should lie within the tested range.
• Log reduction: the number of tenfold drops in population. A cycle that takes a BI from 10⁶ spores to fewer than 1 has achieved more than a 6-log reduction.
• Initial population (N₀): the number of spores on each indicator unit, stated on the supplier’s certificate.
• F₀: equivalent minutes at 121 °C delivered to the load, calculated from temperature data (used for steam).

A simple worked example (illustrative numbers)

Time to reduce a population from N₀ to a target N is: time = D × (log10 N₀ − log10 N).

If a BI has N₀ = 10⁶ spores per unit and D = 2 minutes at the test temperature, then bringing it down to 10⁻³ (one unit in a thousand with a survivor) needs 2 × (6 + 3) = 18 minutes of exposure. A cycle designed for a large safety margin beyond this is often called an overkill cycle. These figures are only to show the arithmetic; real values come from the BI certificate and your validation protocol.

Choosing the test organism

Pick an organism that is more resistant to the process than the bioburden of your product, so that if the BI is killed, the natural bioburden is killed with a margin.

The table follows the FDA guidance for biological indicator premarket submissions (October 2007), which lists these spores as the ones generally used. If an organism not listed is used, the user must show it is the most resistant to the process, for example from the manufacturer’s data or published studies. The pharmacopoeias (Ph. Eur. 5.1.2, USP <55> and <1229.5>) give detailed organism, strain and resistance requirements; their full text is subscription-based, so check the current edition used by your site.

For steam processes, spores of G. stearothermophilus typically show D-values at 121 °C of a few minutes, varying with the supplier, the carrier material and the primary packaging of the BI.

Using BIs in validation

Moist heat (autoclave)

• Place BIs at the locations shown to be the coolest or the hardest to reach (cold spots, inside the most difficult load items), based on the temperature distribution and heat penetration studies. FDA’s aseptic processing guidance states that biological indicators in numerous positions in the load, including the most difficult places, are a direct means of confirming sterilisation efficacy.
• Run the BIs through the validated cycle. After exposure, incubate them under the supplier’s conditions together with an unexposed positive control. All exposed BIs must show no growth.
• A half cycle (reduced exposure) is often used to show that the full cycle gives a large margin. If the load, cycle or equipment changes, re-evaluate whether new BI studies are needed.

Dry heat

Heat transfer is less efficient and temperature distribution is usually less uniform than in a steam steriliser, so the distribution study and the placement of BIs at cold spots are especially important. Use the validated cycle for the product or component, for example sterilising and depyrogenating glassware.

Gas processes (ethylene oxide, hydrogen peroxide and similar)

Gas sterilisation depends on several interacting variables: gas concentration, humidity, temperature, time, load configuration and packaging. BIs are therefore needed in development, validation and monitoring. Place them in the most difficult-to-reach positions in the load.

What GMP expects

EU GMP Annex 1 (2022) says:

• All sterilisation processes must be validated, by physical measurements and where appropriate by biological indicators (§8.36).
• Suitable BIs placed at appropriate locations should be considered as an additional method to support validation. They should be stored and used according to the supplier’s instructions, and positive controls should be tested for each sterilisation cycle where BIs are used to monitor a process (for example with ethylene oxide). Precautions must be taken to avoid transferring microbial contamination from the BIs to the manufacturing or other testing processes. BI results alone must not override other critical parameters and process design (§8.42).
• The reliability of BIs is important: suppliers should be qualified, and transport and storage controlled. Before using a new lot, verify the population, purity and identity of the indicator organism. Other certificate values, such as D-value and z-value, can normally be taken from the qualified supplier’s batch certificate (§8.43).
• Each ethylene oxide cycle should be monitored with suitable BIs distributed at defined worst-case locations that were shown during validation (§8.76).

In the United States, 21 CFR 211.113(b) requires written procedures to prevent microbial contamination of drug products purporting to be sterile, including validation of any sterilisation process.

Practical tips

1. Keep BIs within expiry and stored as per the label, away from sterilants, heat and light.
2. Check the supplier certificate for organism, population, D-value, z-value, expiry and lot number before use.
3. Always include an unexposed positive control from the same lot.
4. Decontaminate and discard used BIs safely; positive BIs are a microbial hazard.
5. Treat any positive BI after a validated cycle as an investigation trigger (check the load, placement, cycle data and BI integrity).
6. Keep validation and monitoring records (cycle printouts, BI results, certificates) for at least 1 year after the expiry date of the respective product batch (EU GMP Chapter 4 §4.11, or 5 years after certification by the Qualified Person if longer; 21 CFR 211.180(a)), longer if local law or the marketing authorisation requires.

Frequently asked questions

Why is Geobacillus stearothermophilus used for steam sterilisation? Its spores are highly resistant to moist heat, so if they are killed the product bioburden is expected to be killed with a margin.

Is a BI better than a chemical indicator? They do different jobs. A chemical indicator shows exposure to some conditions but does not prove microbial kill. A BI challenges the process with living spores. Neither replaces the temperature and pressure records.

What does a positive BI mean? Growth after exposure means the spores survived. Check whether the BI was faulty or mishandled, and review the cycle and load before deciding on the impact.

Can BI results override physical parameters? No. Annex 1 states that BI results alone should not be used to override other critical parameters and process design.

What is the difference between D-value and z-value? D-value is how long (at one temperature) to reduce the spore count by 90 per cent. z-value is how much the temperature must change to change D tenfold.

See also

• Production of sterile pharmaceutical products
• Process validation
• What is the sterility test?
• Sterile product building, facility and clean area classification

References

1. European Commission, EudraLex Volume 4, Annex 1: Manufacture of Sterile Medicinal Products, C(2022) 5938 final, 22 August 2022 (fully applicable since 25 August 2024): glossary (biological indicator, z-value), §8.36, §8.42, §8.43, §8.76.
2. US FDA, Guidance for Industry and FDA Staff: Biological Indicator (BI) Premarket Notification [510(k)] Submissions (October 2007), Table 2, recommended spores.
3. US FDA, Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing, Current Good Manufacturing Practice (September 2004).
4. 21 CFR 211.113(b) and 211.180(a), US Code of Federal Regulations.
5. European Commission, EudraLex Volume 4, Part I, Chapter 4: Documentation, §4.11.
6. European Pharmacopoeia general chapter 5.1.2 Biological indicators of sterilisation, and USP general chapters <55> and <1229.5> (subscription-based; consult the current edition).