Media Fill Test — Sterile API Manufacturing (Aseptic Process Simulation)

1. Objective

To demonstrate that the aseptic API manufacturing process (including personnel, equipment, materials, and environment) can reliably produce a sterile product by simulating routine filling/processing using microbiological growth medium instead of the API.

2. Scope

Applies to all aseptic fill/finish operations and associated supporting operations used to manufacture sterile API batches (e.g., compounding, filling, stoppering, transfer, isolation, lyophilization if applicable). Includes all operators, lines, isolators, and shifts that form part of normal operations or represent worst-case.

3. References

• Company SOP for Aseptic Process Simulation / Media Fill

• Sterility assurance and validation SOPs

• Environmental monitoring and gowning SOPs

• Change control, deviation and CAPA procedures
  (Note: adapt references to your site/regulator)

4. Responsibilities

• QA: Approve protocol, witness runs, review results, release/decline validation report.

• Production/Tech Ops: Execute media fill steps, perform line set-up, complete batch records.

• Microbiology: Prepare/validate media, incubate, read and report results.

• Engineering: Ensure equipment qualification and set parameters.

• Validation Lead: Overall execution, data consolidation, final report.

5. Test material & media

• Growth medium: Tryptic Soy Broth (TSB) or equivalent validated growth medium for bacteria and fungi. Use sterile, validated lots.

• Volume: Use normal fill volumes (actual product volumes). If multiple volumes used in production, include each volume or worst-case (highest/lowest as applicable).

• Containers: Use representative containers (vials, ampoules, syringes, stoppers, seals) identical to production.

• Simulants: Where appropriate, include worst-case excipients (e.g., surfactants) if they affect viability.

6. Acceptance / Success criteria (recommended)

• Primary: No microbial growth in the filled units and process samples at the end of incubation (i.e., zero positive units).

• Environmental & personnel: Action/alert levels per your EM program must be within limits during the run. Any excursion requires investigation.

• Runs: Minimum of 3 consecutive successful media fill runs for initial qualification. For routine requalification, frequency per SOP (e.g., annually or after major changes).

• If contamination occurs: treat as a failed run and follow investigation/CAPA; if isolated and explainable, QA may allow repeat per SOP.

Note: Some companies/regulators accept limited positive units in certain non-critical components — check local regulatory expectations. Best practice: aim for zero.

7. Worst-case considerations (must be simulated)

• Longest open processing time

• Most operators interacting with the process (maximum human involvement)

• Highest number of aseptic manipulations/transfers

• Smallest container/needle size (harder to handle) or highest container exposure

• Most exposed container orientation (e.g., inverted vs upright)

• Shifts or times when environmental monitoring tends to be poorest

8. Run design & sample size (practical recommendations)

• Number of runs: Minimum 3 consecutive successful runs for initial validation.

• Unit count per run: Simulate a typical batch — ideally use the same number of units as a smallest routine commercial batch or a statistically significant sample that includes high-risk manipulations. If full batch simulation is impractical, follow SOP-defined representative sampling but include worst-case steps.

• In-process samples: Include environmental settle plates, surface contact plates, operator glove prints (finger dab plates), and air samples at key times (start, mid, end).

• Positive controls: In each run, include intentionally inoculated control unit(s) or media blanks to confirm medium viability (e.g., one media unit inoculated with low CFU) — these must show growth to confirm the test is valid.

• Negative controls: Sterile media sealed and incubated to confirm no contamination introduced during handling.

9. Execution steps (protocol flow)

1. Pre-Run

   • Approve protocol and personnel qualification.

   • Verify equipment IQ/OQ/PQ status.

   • Environmental monitoring: baseline checks pre-run.

   • Prepare media aseptically in cleanroom or buffer area per microbiology SOP.

   • Label media units to look like product (but clearly indicate “MEDIA FILL — DO NOT RELEASE”).

2. Gowning

   • Full aseptic gowning per SOP; document operator identity and gowning checks.

3. Set-up

   • Assemble line/isolation as for normal production; use standard supplies and procedures.

4. Simulated production

   • Perform all aseptic manipulations exactly as in routine production (transfers, fills, stoppering, capping, crimping, labeling).

   • Record timing of critical steps (open vial time, transfers, interruptions).

5. In-process monitoring

   • Place active air samplers and settle plates at critical locations.

   • Collect glove prints and surface contact plates at predefined times and operators.

6. Post-run handling

   • Seal and transport media units to microbiology lab per chain-of-custody; avoid shaking/overheating.

7. Incubation

   • Incubate media units per microbiology SOP (see recommended incubation below).

8. Reading and recording

   • After incubation, inspect visually and perform subculture/identification on positives.

   • Document lot numbers, operator IDs, environmental data, and all deviations.

9. Report

   • Validation report summarizing run conditions, environmental data, results, investigation for any positives, and conclusions.

10. Recommended incubation (typical)

• Primary: Incubate at 20–25°C for 7 days (to favor fungi and some bacteria) and 30–35°C for 7 days (to favor mesophilic bacteria). Many labs use a two-stage incubation (7 days at each temp) — total 14 days.

• Controls: Positive and negative controls incubated the same way.

• Reading: Inspect daily if desired; final read at end of full incubation. Any turbidity/cloudiness = positive.

(If your microbiology SOP uses different temperatures/durations, follow that SOP.)

11. Environmental monitoring & data to collect during runs

• Active air counts at key locations (CFU/m³)

• Passive settle plates (CFU/plate)

• Surface contact plates (CFU/25 cm²)

• Operator glove prints (CFU)

• Temperature/Humidity logs (if relevant)

• Differential pressure and airflow status

• Any excursions or alarms

12. Documentation & batch record

• Media fill protocol with signature approvals

• Run log with time-stamped actions

• Environmental monitoring results

• Incubation logs and microbial results

• Positive control/negative control results

• Deviations and investigation records

• Final validation report and conclusion

13. Handling positives / investigation

If any filled unit or associated environmental/operator sample shows growth:

1. Quarantine all related materials and data.

2. Immediate review of incubation and chain-of-custody to rule out lab contamination.

3. Identify organism and compare with EM isolates (fingerprinting if available).

4. Assess location, operator, step where positive likely occurred (time-stamp correlation).

5. Review gowning, technique, equipment, HEPA performance, and air/surface data.

6. Classify as critical/non-critical contamination; decide if run fails.

7. Corrective actions: retrain personnel, equipment maintenance, repeat media fill runs (as required), revise SOPs.

8. CAPA: implement corrective/preventive actions and monitor effectiveness.

14. Trending & Ongoing Program

• Maintain a media fill logbook with all runs, dates, operators, and results.

• Trend EM results and media fill performance; investigate upward trends or recurrent anomalies.

• Requalification frequency: per company SOP (commonly annually, after major changes, or after confirmed contamination).

15. Report content (validation report)

• Protocol summary and objectives

• Run details and dates

• Media used and validation of medium

• Incubation conditions and controls

• Environmental monitoring data

• Results (tabulated)

• Deviations and investigations

• Conclusion and disposition (pass/fail)

• Signatures from QA, Microbiology, Validation Lead

16. Practical tips & common pitfalls

• Use representative operators (routine staff, not only highly trained specialists).

• Simulate interruptions and typical handling disturbances (e.g., break/change of operator) only if they are part of routine — but document deliberately.

• Don’t over-manipulate just to pass; test true worst-case conditions.

• Ensure positive control demonstrates medium viability — otherwise entire run is invalid.

• Keep media at appropriate temperatures during transport and avoid excessive agitation.

• If isolators/closed systems are used, still perform worst-case personnel interventions (glove breaches, transfers through ports) as relevant.

17. Example results table (format to include in report)

(Populate with your actual numbers.)

18. Post-failure remediation examples

• Repeat media fill after corrective actions (e.g., retrain personnel, replace HEPA filter)

• Deep clean and requalification of equipment

• Review and tighten gowning controls

• Enhanced monitoring for subsequent runs

19. Variations for special processes

• Lyophilization: include stoppering/open vial exposure simulation before lyophilization; post-stoppering incubation strategy may differ.

• Isolator/closed systems: simulate worst-case through glove port manipulations and transfer via rapid transfer ports (RTPs).

• Terminal sterilization steps: if product is terminally sterilized, APS may focus more on pre-sterilization aseptic steps and sterile transfers.

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