microorganisms as per pharmacopeial requirements.

2. Scope

This SOP applies to Analytical Method Development (AMD) and Quality Control (QC) departments involved in testing ophthalmic formulations including eye drops, ointments, and gel-based sterile products.

3. Responsibilities

• Microbiology Analyst: Performs sterility test procedures, records observations, and maintains aseptic conditions.
• QC Analyst: Assists in sample preparation and documentation.
• QA Executive: Reviews and approves sterility test results, deviations, and trend reports.

4. Accountability

The Head of Analytical Method Development is accountable for ensuring that sterility testing is performed in compliance with USP <71>, EP 2.6.1, and WHO TRS guidelines.

5. Procedure

5.1 Selection of Method

1. Use membrane filtration method for aqueous ophthalmic solutions.
2. Use direct inoculation method for viscous/semi-solid ophthalmic products (e.g., gels or ointments).

5.2 Environmental and Equipment Conditions

1. Perform testing under aseptic conditions in a certified Grade A laminar airflow hood located in Grade B cleanroom.
2. Use sterilized equipment including filtration units, forceps, scissors, and media bottles.

5.3 Media Preparation

1. Use Fluid Thioglycollate Medium (FTM) for anaerobic and aerobic bacteria.
2. Use Soybean Casein Digest Medium (SCDM or TSB) for fungi and aerobic bacteria.
3. Perform growth promotion testing (GPT) using standardized organisms before use.

5.4 Membrane Filtration Method

1. Filter prescribed quantity (usually 10 mL) of ophthalmic product through 0.45 µm membrane filter using sterile filtration assembly.
2. Rinse filter with sterile fluid (e.g., Fluid A or Fluid D) to remove preservatives or antimicrobial agents.
3. Transfer one membrane into FTM and another into SCDM using sterile forceps.
4. Incubate FTM at 30–35°C and SCDM at 20–25°C for 14 days.

5.5 Direct Inoculation Method

1. Inoculate required volume (typically 0.2–1 mL or 200 mg) of ophthalmic ointment or gel into each medium.
2. Swirl gently to disperse; incubate as described in 5.4.

5.6 Observation and Interpretation

1. Check test media daily for turbidity or visible microbial growth.
2. If no growth observed after 14 days, report as “Sterile.”
3. If growth is observed, perform microbial identification and root cause analysis.

5.7 Validation and Controls

1. Validate neutralization of antimicrobial agents before routine testing.
2. Include positive controls (challenge microorganisms) and negative controls (uninoculated media).

6. Abbreviations

• FTM: Fluid Thioglycollate Medium
• SCDM: Soybean Casein Digest Medium
• GPT: Growth Promotion Test
• USP: United States Pharmacopeia
• EP: European Pharmacopeia

7. Documents

1. Sterility Test Log – Annexure-1
2. Environmental Monitoring Report – Annexure-2
3. Growth Promotion Certificate – Annexure-3

8. References

• USP <71>: Sterility Tests
• EP 2.6.1: Sterility
• WHO TRS 961 Annex 6: Good Manufacturing Practices for Sterile Products

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name	Dr. Ruchi Sharma	Amol Khedekar	Sunita Reddy
Designation	Microbiologist	QA Reviewer	Head – AMD
Department	Analytical Method Development	QA	Analytical Method Development

11. Annexures

Annexure-1: Sterility Test Log

Sample ID	Method	Media Used	Volume Tested	Incubation Period	Result
OPH-2025-001	Membrane Filtration	FTM, SCDM	10 mL	14 Days	Sterile

Annexure-2: Environmental Monitoring Report

Date	Area	Airborne Particulates	Surface CFU	Personnel Monitoring	Status
20/05/2025	Grade A LAF	Nil	1 CFU	Nil	Pass

Annexure-3: Growth Promotion Certificate

Media lots used for this test were challenged with ATCC strains of Staphylococcus aureus, Bacillus subtilis, Aspergillus brasiliensis, Clostridium sporogenes, and Candida albicans. All media supported visible growth within 5 days.

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
21/05/2025	2.0	Added environmental monitoring and validation criteria for neutralization	Annual Review and GMP Compliance	Sunita Reddy
01/03/2022	1.0	Initial SOP Release	New SOP	QA Head