accordance with pharmacopeial standards.

2. Scope

This SOP applies to Analytical Method Development (AMD) activities for sterile formulations such as injectables, ophthalmics, and irrigation solutions in the formulation and microbiology laboratories.

3. Responsibilities

• Microbiologist: Develops, executes, and documents the sterility testing protocol.
• Analytical Scientist: Coordinates protocol integration with development batches and validation teams.
• QA Officer: Reviews the protocol, records, and validation data for GMP compliance.
• Head – AMD: Approves sterility protocol and ensures compliance with regulatory guidelines.

4. Accountability

The Head of Analytical Method Development is accountable for the scientific soundness and regulatory alignment of the sterility test protocol.

5. Procedure

5.1 Reference Guidelines

1. Refer to pharmacopeial guidelines:
   • USP <71> – Sterility Tests
   • EP 2.6.1 / IP 16 – Sterility Testing of Pharmaceutics
2. Assess product characteristics (formulation type, container closure, antimicrobial content) for method selection.

5.2 Selection of Test Method

1. Choose method based on product:
   • Membrane Filtration: For aqueous or alcohol-based solutions
   • Direct Inoculation: For oily, viscous, or non-filterable products
2. Document justification in Annexure-1: Method Selection Justification.

5.3 Preparation of Sterility Test Media

1. Prepare the following media per validated procedure:
   • Fluid Thioglycollate Medium (FTM) – for anaerobic and aerobic bacteria
   • Soybean Casein Digest Medium (SCDM) – for aerobic bacteria and fungi
2. Perform growth promotion test for each media batch.
3. Record in Annexure-2: Media Preparation and Suitability Log.

5.4 Preparation of Test Articles

1. Ensure aseptic sample handling under LAF unit.
2. Prepare the required volume of sample as per product monograph (e.g., 10 mL per unit).
3. Include negative controls (media only) and positive controls (inoculated with known microorganisms).
4. Log all preparations in Annexure-3: Sample Handling Record.

5.5 Execution of Membrane Filtration Method

1. Assemble sterile filtration unit and sterilize membrane using 0.1 N NaOH rinse and sterile water wash.
2. Pass sample through membrane filter (0.45 µm) using vacuum or peristaltic pump.
3. Rinse the membrane with sterile fluid to remove any inhibitory substances.
4. Transfer membrane aseptically into:
   • FTM: incubate at 30–35°C for 14 days
   • SCDM: incubate at 20–25°C for 14 days
5. Log execution details in Annexure-4: Filtration Execution Sheet.

5.6 Execution of Direct Inoculation Method

1. Directly inoculate sample volume (not more than 10% of the media volume) into:
   • FTM for anaerobes and aerobes
   • SCDM for fungi and aerobes
2. Incubate tubes under the same conditions as filtration method.
3. Monitor media for turbidity and color change.
4. Document in Annexure-5: Inoculation Log.

5.7 Observations and Reporting

1. Inspect test and control tubes every 2–3 days for microbial growth.
2. In case of growth, perform subculturing and identification of the contaminant.
3. Conclude the test after 14 days with documented results.
4. Summarize findings in Annexure-6: Sterility Test Summary Report.

6. Abbreviations

• FTM: Fluid Thioglycollate Medium
• SCDM: Soybean Casein Digest Medium
• SOP: Standard Operating Procedure
• LAF: Laminar Air Flow

7. Documents

1. Method Selection Justification – Annexure-1
2. Media Preparation and Suitability Log – Annexure-2
3. Sample Handling Record – Annexure-3
4. Filtration Execution Sheet – Annexure-4
5. Inoculation Log – Annexure-5
6. Sterility Test Summary Report – Annexure-6

8. References

• USP <71> – Sterility Tests
• EP 2.6.1 – Sterility
• ICH Q6A – Specifications

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Method Selection Justification

Product	Test Method	Justification
Injection A	Membrane Filtration	Aqueous solution, filterable

Annexure-2: Media Preparation and Suitability Log

Media	Batch No.	Growth Promotion Organisms	Result
FTM	FTM2025-01	B. subtilis, C. sporogenes	Pass

Annexure-3: Sample Handling Record

Sample ID	Volume	Control Type	Operator
STER-121	10 mL	Positive & Negative	Rajesh Kumar

Annexure-4: Filtration Execution Sheet

Sample	Filter Size	Rinse Volume	Transferred To
STER-121	0.45 µm	200 mL	FTM & SCDM

Annexure-5: Inoculation Log

Sample	Media	Volume Inoculated	Incubation
STER-121	FTM	1 mL	30–35°C / 14 Days

Annexure-6: Sterility Test Summary Report

All media remained clear for 14 days. No microbial growth was observed. Positive controls showed expected turbidity. The test passed for Injection A (Batch # INJ0525A).

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Included both direct inoculation and filtration protocols	Annual Review