What is a media fill test?
Aseptic process simulation (APS) is the ultimate step in verifying that an aseptic process is capable of producing a sterile product. Mandatory for aseptic processes, it can also verify the asepsis of axenic processes such as viral culture or fermentation.
Aseptic filling is the most critical step: the product is exposed to the environment. Replacing it with a culture medium – the media fill test – has been established practice for decades.
A topic still open to debate
The regulations require the test but say little about how to prepare, run and conclude it. Assessment therefore remains partly subjective, and a source of inspection observations. Relying on experienced experts lets you align your programme with current industry practice.
Troubleshooting your media fills
Our consultants support you with the difficulties you encounter in running your media fills.
Defining your media fill strategy
Meeting agency expectations while reducing the workload involved.
Keeping your APS register
Once the test is concluded, the register still has to be kept: who took part, which interventions each person performed, when their qualification expires. Our experts developed APSolute for this. The software links each APS batch to its filling line and produces the up-to-date qualification matrix on the day the inspector asks for it.
- APS batches: start and end dates, units filled, incubated and conforming, participants and interventions performed.
- Operator × activity qualification matrix, with each person's annual requalification due date.
- Planning: for a given date and line, the list of operators who need to take part in the next APS.
- Unalterable audit trail, signable PDF reports, numbered printouts in the name of their author.
Media fill test FAQ
References are to EU GMP Annex 1 in its revised version, in force since 25 August 2023.
Planning
How often should an APS be performed?
For initial validation, at least three consecutive successful simulations, covering all shifts likely to work on the process. After that, periodic revalidation normally takes place twice a year, i.e. roughly every six months, for each aseptic process, each filling line and each shift. For manual operations (aseptic filling or compounding), each operator performs three initial APS runs, then one APS roughly every six months.
Annex 1, § 9.38 and § 9.39
When must an APS be repeated outside the schedule?
After any significant change to practices, premises, utilities or equipment that may affect sterility assurance: HVAC system, process, number of shifts or operators, major site shutdown. Full initial validation must be repeated when the process has not been run for a long period, or when a new container or container-closure combination is introduced. It is also recommended to consider an APS after the last batch before a shutdown, before a long period of inactivity, and before decommissioning or relocating a line.
Annex 1, § 9.38 and § 9.49
Does an operator have to take part in every APS?
No, but each operator must take part in at least one successful APS per year. Unsupervised access to grade A and B areas is restricted to personnel who have passed their gowning qualification, reassessed at least once a year, and taken part in a successful APS. Their participation must cover the interventions they perform in production: having been present during the run is not enough.
Annex 1, § 9.38 and § 7.4
Design and execution
Which interventions should be included?
The inherent and corrective interventions representative of the routine process, performed in the same way and at a frequency comparable to production. Their selection and frequency follow from the assessment of risks to product sterility. Each intervention is recorded in the APS batch record, with its start and end time and the person who performed it. An APS must not be used to justify a practice that creates an unnecessary contamination risk.
Annex 1, § 9.34, § 9.35 and § 9.47
How many units should be filled, and for how long?
Enough units to simulate all the representative activities of the process, with a justification recorded in the CCS. In practice, the text cites a minimum of 5,000 to 10,000 units. For small batches, for example under 5,000 units, the number of APS units should be at least equal to the production batch size. The duration must be sufficient to challenge the process, the operators who intervene, shift changes and the ability of the environment to maintain suitable conditions.
Annex 1, § 9.40 and § 9.36 viii
And for campaign manufacturing?
The APS must reproduce the risks at the start and end of the campaign, and demonstrate that the campaign duration does not present a risk. An APS performed at the end of production or of a campaign can provide additional assurance, provided it is justified in the CCS. It does not replace routine APS runs.
Annex 1, § 9.36 xii and xiii
Are hold times part of the APS?
Yes. The APS plan must take into account the maximum permitted hold times for the sterile product and for equipment exposed during the aseptic process. For freeze-drying, it must reproduce the maximum interval between sterilisation of the freeze-dryer and its use. This is often where an inspector asks to see the link between the simulation and the sterilisation records.
Annex 1, § 9.36 iii, § 9.33 vii and § 8.122
Reading and concluding
Under what conditions should units be incubated?
The revised Annex 1 sets neither duration nor temperature: incubation conditions must be scientifically justified and validated to detect contamination with sufficient sensitivity. Units are swirled or inverted before incubation, then incubated without undue delay, in a transparent container. All integral units are incubated, including those with cosmetic defects. The record must reconcile units filled, incubated and not incubated, and justify every unit not incubated.
Annex 1, § 9.41, § 9.43, § 9.44 and § 9.47
Who reads the units?
Personnel trained and qualified to detect microbiological contamination, under conditions that make detection easier. Filled units are also inoculated with reference organisms and representative local isolates as a positive control. Organisms found in a contaminated unit are identified to species level where possible.
Annex 1, § 9.45 and § 9.43
What is the acceptance criterion?
The target is zero growth. A single contaminated unit is enough for the APS to fail. The most probable root cause(s) must then be investigated, corrective measures put in place and at least three consecutive successful APS runs performed. Batches manufactured since the last successful APS are reviewed and risk-assessed. Products manufactured on the line after the failure remain in quarantine, and production only resumes once revalidation has succeeded.
Annex 1, § 9.46
What happens to operators involved in a failed APS?
When the investigation links the failure to an operator's activity, that operator's activities are restricted until they have been retrained and requalified. An operator involved in a failed APS may also be disqualified. For access to grade B or interventions in grade A, their requalification should consider participation in a new successful APS.
Annex 1, § 9.46 vi and § 7.6
How do you track each operator's qualification?
Each participation has to be linked to the interventions performed and to an expiry date, and you need to be able to produce the status of personnel on a given date. That is what APSolute, the software developed by our experts, does. Discover APSolute →