Validation of aseptic processes relies upon prospective, concurrent, and retrospective validation as well as re-validation.

Prospective studies include installation and operational qualification for a new or renovated facility as well as product simulation studies and a prospective process validation with the original product according to PIC/S
Document PI 006.

Concurrent validation includes a process validation with the same requirements as for prospective studies but performed during routine production on qualified equipment.

Retrospective validation uses the data of earlier manufacturers but is not a recommended technique for aseptic processes.

Re-validation includes:

  • Regular performance of process simulation studies.
  • Monitoring of environment, disinfection procedures, equipment cleaning, and sterilization (including containers and closures).
  • Routine maintenance and re-qualification of equipment, e.g. autoclaves, ovens, HVAC (heating, ventilation, and air conditioning) systems, water systems, etc.
  • Regular integrity testing of product filters, containers, closures, and vent filters
  • Re-validation after changes

Process simulation studies (media fills) are simulating the whole process in order to evaluate the sterility confidence of the process.

Process simulation studies include formulation (compounding), filtration, and filling with suitable media.

Simulations are made to ensure that the regular process for commercial batches repeatedly and reliably produces the finished product of the required quality. 

The methods for simulating an aseptic process vary according to the process used for the various types of products, i.e. liquid, semi-liquid, and solid dosage forms.


  • The media fill should emulate the regular product fill situation regarding equipment, processes, personnel involved, and time taken for filling as well as for holding.
  • Where filling takes place over extended periods, i.e. longer than 24 hours, the process simulation test should extend over the whole of the standard filling period. In order to prevent excessively high numbers of units being filled, it is usually acceptable to just run the machine for a reasonable time, if the validity of the simulation is not diminished by this procedure.
  • Sterile filtered air should be used instead of inert gases in Process simulations and breaking a vacuum because inert gases will prevent the growth of aerobic microorganisms.
  • Where anaerobes are detected in the environmental monitoring or sterility testing, the use of inert gas should be considered for a process simulation because the inert gas is supporting the growth of anaerobes.
  • Before enumerating the different process simulation test procedures some preliminary explanations are necessary for the preparation of liquid media as it is used for the majority of the process simulation tests.
  • Where a liquid nutrient medium is used it should be prepared in a similar manner to the product. The medium should be dissolved in Water for Injection in a standard manufacturing vessel. If heat is required to dissolve it then only minimal heat should be used.
  • The pH of the medium should be measured and, if necessary, adjusted to bring it into the required range. The medium should be aseptically filtered into an aseptic holding vessel using the normal production filter and processing procedure. It may be also acceptable to sterilize the media.
  • All aseptic holding vessels should be covered by a process simulation test on a regular basis unless a validated, pressure hold or vacuum hold test is routinely performed.


Test Performance

  • The process simulation test should follow as closely as possible the routine aseptic manufacturing process and include all critical subsequent manufacturing steps.
  • All equipment should remain the same for the routine process (wherever practicable). Appropriate combinations of container size and opening, as well as speed of the processing line, should be used (preferably at the extremes).
  • The process simulation test should represent a “worst-case” situation and include all manipulations and interventions likely to be represented during a shift.
  • Worst-case conditions – the largest container with the widest mouth as it is exposed longer to the environment. However, the exception for this is small ampoules run at the highest speed as the ampoules may be unstable and cause frequent jams thus necessitating frequent operator intervention.
  • The fill volume of the containers should be sufficient to enable contact of all the container-closure seal surfaces when the container is inverted and also sufficient to allow the detection of microbial growth.
  • If batches are produced smaller than 3000 units, the minimum number of containers used for the process simulation should be equal to the commercial batch size.
  • Simulation tests should be performed on different days and hours during the week and not only at the beginning of a work day.
  • If the same process is conducted in a separate clean room, this should also be validated.
  • In order to find the possible source of contamination it may be good advice to video tape the aseptic fill and also number the individual vials or segregate vials in chronological order during incubation.

Selection of Growth Medium

The criteria for the selection of growth medium include:

  • Low selectivity,
  • Clarity,
  • Medium concentration
  • Filterability

Low Selectivity:

  • The medium selected should be capable of supporting a wide range of microorganisms, which might reasonably be encountered and be based on the in-house flora (e.g. isolates from monitoring etc.).
  • Media used in the evaluation must pass a growth promotion test. The control organisms used should include those relevant strains of test micro-organisms identified by relevant Pharmacopoeias as being suitable for use in the growth promotion test.
  • Growth promotion tests should demonstrate that the medium supports recovery and growth of low numbers of microorganisms, i.e. 10-100 CFU/unit or less.
  • Growth promotion testing of the media used in simulation studies should be carried out after completion of the incubation period of media fill to demonstrate the ability of the media to sustain growth if contamination is present.
  • Growth promotion testing should be demonstrated within 5 days at the same incubation temperature as used during the simulation test performance.


  • The medium should be clear to allow for ease in observing turbidity.

Medium Concentration:

  • The recommendations of the supplier should be followed unless alternative concentrations are validated to deliver equal results.


The medium should be capable of being filtered through the same grade as used in production( In case a filter is used in the aseptic manufacturing process)

Incubation Conditions:

  • Incubate at 20-25°C for a minimum of 7 days after first reading and followed immediately by incubation at 30-35°C   for a total minimum incubation time of 14 days.
  • Prior to incubation the containers with the microbiological growth medium should be inverted or otherwise manipulated to ensure that all surfaces, including the internal surface of the closure, are thoroughly vetted by the medium.
  • The containers should not be completely filled with the medium in order to provide sufficient oxygen for the growth of obligate aerobes.
  • Similarly, containers should not be overlaid with inert gases even though the product.
  • The microorganisms present in the containers of the simulation test should be identified to genus but preferably species level to aid determination of the possible sources of the contamination.

Reading of the Test

During the inspection, the containers after incubation should be compared to a known sterile container for comparison as some microbial growth shows up as a faint haze which is difficult to detect unless there is a control container to compare against. Visual Inspectors should be trained for this task.

Test Frequency

  • Simulation tests should be distinguished between “start-up” and “on-going” simulation tests.
  • A “start-up” simulation test consists of three consecutive satisfactory simulation tests per shift and should be carried out before routine manufacturing can start.

Examples of “Start–up” simulation tests are performed

  • New processes
  • New equipment
  • After critical changes in processes, Equipment, or environment and significant personnel changes (a new shift)
  • Modifications in equipment directly in contact with the product
  • Modifications in the HVAC system.
  •  An “on-going” simulation test consists of one satisfactory simulation test per shift and is mainly performed for the periodic monitoring of aseptic conditions during routine manufacturing but also after less critical changes of processes, equipment, or environment or if processing lines stand idle for more than 6 months.
  • “On-going” simulation tests should be performed with each shift of each process line at least twice per year under the condition that there were no changes in the normal production procedures and no action limits were
  • Exceeding an action level demands a re-validation.
  • Depending on the result of the follow-up investigation this re-validation may require the inclusion of one to three satisfactory process simulation tests.


  • After the incubation period of the media-filled containers, visually examined the containers for microbial growth.
  • Contaminated containers should be examined for evidence of container/closure damage which might compromise the integrity of the packaging system.
  • Damaged containers should not be included as failures (positives) when evaluating results.
  • The number of containers used for media fills should be sufficient to enable a valid evaluation.
  • For small batches, the number of containers for media fills should at least equal the size of the product batch. The target should be zero growth and the following should apply.

When filling fewer than 5000 units, no contaminated units should be detected.

When filling 5,000 to 10,000 units:

a) One (1) contaminated unit should result in an investigation, including consideration of a repeat media fill.
b) Two (2) contaminated units are considered cause for revalidation, following the investigation.

When filling more than 10,000 units:
a) One (1) contaminated unit should result in an investigation.
b) Two (2) contaminated units are considered cause for revalidation, following the investigation.

  • For any run size, intermittent incidents of microbial contamination may be indicative of low-level contamination that should be investigated.
  • Investigation of gross failures should include the potential impact on the sterility assurance of batches manufactured since the last successful media fill.
  • All contaminating microorganisms whether or not an alert or action limit has been exceeded should be identified to at least genus and preferably species where practicable to determine the possible source of contamination.
  • If a process simulation test fails then due account should be taken of products filled between the last successful test and the test failure.
  • Recording any deviations during the simulation test is important to trace later on the exact cause and evaluate the consequences.
  • The investigation should identify batches that could be affected during this time period and the disposition of the affected batches should be re-assessed.

Non-viable monitoring

  • The location chosen for monitoring should be checked to ensure that the positions reflect the worst case.
  • For room monitoring, the counts should be performed in locations where there is most operator activity.
  • For the filling environment, the counts should be performed adjacent to the filling zone and where components are exposed in such a way as to detect operator activity within these areas.
  • Monitoring with sampling probes located in such a way that they monitor the air from the HEPA filter rather than the air immediately surrounding the critical zones should be avoided.
  • However, the location of the sample device should not compromise the laminarity of the airflow in the critical zone.
  • Initial validation should be checked to confirm that worst-case positions have been adequately identified. These may be reconfirmed during process simulation tests.

Air Borne Microbial/Microbial Monitoring

  • A combination of the methods identified in Annex 1 of the EU/PIC/S GMP guide for monitoring microbial levels is used in environmental monitoring programmes.
  • Microbial monitoring should be performed in and around areas of high operator activity. It is not unusual to see settle plates and air sample locations well away from such areas.
  • A typical example is where settle plates are located well to the rear of the filling machine where there is little or no operator activity. The same may be true for air sampling.
  • It is important, to observe operator activity over a period of time and ensure that the monitoring sites are so located as to monitor operator activity.
  • The process simulation test provides an ideal opportunity to confirm that the worst-case locations have been identified by the use of additional monitoring during the test.
  • A useful monitoring technique is to monitor the filling needles at the end of the filling session.
  • Additional monitoring around the affected area prior to disinfection may provide useful information as to the cause.




  • The basic principles of process validation are described in Annex 15 to the EU/PIC/S Guide to GMP
  • PIC/S Document PI 006 (Recommendations on Validation Master Plan, Installation and Operational Qualification, Non-Sterile Process Validation, Cleaning Validation) and apply also to aseptic processing.
  • Annex I to the EU/ PIC/S Guide to GMP provides the basic requirements for the manufacture of sterile products including those aseptically processed.

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