Aseptic Process Simulation: Dual Incubation of Vials and Routine Sterility Test Is Not Mimick

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Aseptic Process Simulation (APS), also known as a Media Fill Test, is one of the most critical validation exercises performed in sterile pharmaceutical manufacturing. Its purpose is to simulate the aseptic filling process using a sterile nutrient medium instead of the actual drug product to demonstrate that the process, equipment, environment, and personnel can maintain sterility throughout production.

In this article, we’ll explore the principle of aseptic process simulation, the dual incubation approach used for filled vials, and why routine sterility testing is not simulated as part of the media fill process. This topic is particularly relevant to microbiologists, quality assurance teams, and regulatory professionals ensuring compliance with USP <71>, EU GMP Annex 1, and WHO TRS 961.

1. What Is Aseptic Process Simulation (APS)?

The aseptic process simulation is a risk-based validation exercise that assesses the capability of an aseptic manufacturing process to consistently produce sterile products without microbial contamination. Instead of using the actual formulation, a sterile growth medium such as Soybean–Casein Digest Medium (SCDM or Tryptic Soy Broth) is filled into the same type of containers (vials, ampoules, syringes) under identical environmental and operational conditions.

The APS is designed to simulate the worst-case manufacturing scenario — including longest filling duration, maximum operator interventions, and equipment challenges — to demonstrate sterility assurance of the process.

2. Principle of Dual Incubation in Aseptic Process Simulation

After the filling of media vials is completed, the containers are incubated for a total of 14 days under two distinct temperature conditions to promote the growth of both bacterial and fungal contaminants, if any were introduced during filling.

The incubation scheme is generally as follows:

  • First Phase (7 Days at 20–25°C): Supports the growth of fungi, molds, and psychrophilic microorganisms that thrive at lower temperatures.
  • Second Phase (7 Days at 30–35°C): Encourages the growth of mesophilic bacteria, including potential contaminants from the environment or operators.

The vials are visually examined daily or at defined intervals for turbidity, clumping, or microbial growth. If even one vial shows growth, the APS is considered a failure until a complete investigation proves otherwise.

3. Stepwise Flow of Aseptic Process Simulation

  1. Preparation: Sterilization of all components, media, and equipment used for simulation.
  2. Setup: Media filling setup under the same conditions as actual product filling (Grade A environment with Grade B background).
  3. Filling Operation: Aseptically fill the sterile medium into containers following routine production parameters.
  4. Interventions: Perform all routine and non-routine interventions (e.g., change parts, stopper addition, line stoppages) that might occur during actual filling.
  5. Sealing and Incubation: After filling, vials are sealed and transferred to incubation conditions — first at 20–25°C, then at 30–35°C.
  6. Observation and Result Interpretation: Any evidence of microbial growth indicates a loss of aseptic control and requires a complete root cause investigation.

4. Purpose of Dual Incubation

The dual incubation approach ensures the recovery of a wide range of microorganisms by mimicking conditions suitable for both fungi and bacteria. A single incubation temperature cannot support the growth of all possible contaminants. Therefore:

  • 20–25°C: Allows detection of fungi and environmental molds.
  • 30–35°C: Promotes growth of common bacterial contaminants.

This two-phase incubation process provides comprehensive microbial coverage and ensures regulatory compliance as per USP <71>, Ph. Eur. 2.6.1, and Annex 1.

5. Why Routine Sterility Test Is Not Simulated in APS

It is important to note that routine sterility testing is not included in the aseptic process simulation. Although both tests deal with sterility assurance, they serve distinct purposes and are not interchangeable.

Key Reasons:

  • Different Objectives:
    • Media Fill (APS): Evaluates the aseptic filling process, operator technique, and environmental controls.
    • Sterility Test (USP <71>): Confirms the sterility of finished product units from the batch.
  • Test Environment:

    APS is performed in the production filling line under actual aseptic conditions. The sterility test, however, is performed in a microbiology lab isolator or cleanroom, which already has validated aseptic conditions.

  • Different Risk Profiles:

    The sterility test involves aseptic sampling and incubation of final product units to verify the sterility of a batch, whereas APS is a process validation study designed to challenge the system, not to test a product batch.

  • Regulatory Guidance:

    According to EU GMP Annex 1 (2022 Revision), “Media fills are designed to simulate aseptic process conditions and are not intended to replicate sterility testing.” Therefore, sterility testing simulation is not a regulatory expectation.

  • Different Frequency and Documentation:

    APS is conducted semi-annually or when major changes occur, while sterility testing is routine and batch-specific. Their validation and control requirements differ accordingly.

6. Acceptance Criteria for APS

The acceptance criteria are straightforward:

  • No growth should be observed in any of the incubated vials after 14 days of dual incubation.
  • If growth is detected in one or more vials, the APS is deemed unsuccessful, and a root cause investigation (RCA) must be initiated.
  • Revalidation is required following any process modification or failure.

7. Regulatory References

  • United States Pharmacopeia (USP <71> Sterility Tests)
  • European Pharmacopoeia (Ph. Eur. 2.6.1 Sterility)
  • EU GMP Annex 1 (Manufacture of Sterile Medicinal Products)
  • WHO TRS 961, Annex 6 – Aseptic Processing Guidelines
  • ISO 13408-1: Aseptic Processing of Health Care Products

8. Conclusion

The Aseptic Process Simulation (APS) serves as the cornerstone of sterility assurance in aseptic manufacturing. The dual incubation of vials at 20–25°C and 30–35°C ensures detection of a wide range of microorganisms, while the exclusion of routine sterility test simulation emphasizes that APS is a process validation activity — not a product sterility verification test.

In simple terms, media fill validates the process, and sterility test verifies the product. Both complement each other to uphold the highest standards of aseptic quality and patient safety.

💬 About the Author

Siva Sankar is a Pharmaceutical Microbiology Consultant and Auditor with extensive experience in sterility testing, validation, and GMP compliance. He provides consultancy, training, and documentation services for pharmaceutical microbiology and cleanroom practices.

📧 Contact: siva17092@gmail.com
Mobile: 09505626106

📱 Disclaimer: This article is for educational purposes and does not replace your laboratory’s SOPs or regulatory guidance. Always follow validated methods and manufacturer instructions.

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