accordance with pharmacopeial guidelines.

2. Scope

This procedure applies to the Analytical Method Development (AMD) and Microbiology departments involved in the monitoring of pharmaceutical water systems used in formulation, cleaning, and analytical testing processes.

3. Responsibilities

• Microbiologist: Collects water samples, performs filtration, incubates media, and documents CFU counts.
• Analytical Scientist: Coordinates with water system personnel for timely sample scheduling.
• QA Officer: Reviews records for compliance and trends deviations or excursions.
• Head – AMD: Ensures adherence to approved procedures and interprets results for process control.

4. Accountability

The Head of Analytical Method Development is accountable for ensuring that all water bioburden estimation methods meet regulatory and internal quality requirements.

5. Procedure

5.1 Sample Collection

1. Collect samples in pre-sterilized, depyrogenated glass bottles (minimum 100 mL capacity).
2. Sample points should include user ends, storage tanks, and critical points in the PW/WFI loop.
3. Label with sample ID, location, date, time, and collector’s name.
4. Transport samples to the microbiology lab within 30 minutes of collection and analyze within 2 hours.
5. Record collection details in Annexure-1: Water Sampling Log.

5.2 Filtration and Media Preparation

1. Use 0.45 µm sterile membrane filters for sample filtration.
2. Filter 100 mL of sample aseptically using a membrane filtration assembly.
3. Rinse filter with 100 mL of sterile 0.1% peptone water to eliminate residual sanitizer (if applicable).
4. Place membrane onto:
   • Tryptic Soy Agar (TSA) for total aerobic microbial count
   • Sabouraud Dextrose Agar (SDA) for yeast and mold detection (optional)
5. Document filtration and media usage in Annexure-2: Filtration and Plate Log.

5.3 Incubation and Enumeration

1. Incubate TSA plates at 30–35°C for 3–5 days.
2. Incubate SDA plates at 20–25°C for 5–7 days (if included).
3. Observe plates daily and record colony counts.
4. Calculate bioburden as CFU/mL: CFU/mL = Total Colonies / 100.
5. Record results in Annexure-3: Bioburden Result Sheet.

5.4 Alert and Action Limits

1. Set limits based on type of water system:
   • Purified Water: Alert = 50 CFU/mL; Action = 100 CFU/mL
   • Water for Injection: Alert = 10 CFU/100 mL; Action = 20 CFU/100 mL
2. Any excursion must be investigated, and a deviation or out-of-specification (OOS) report generated.
3. Initiate CAPA and resampling where required.
4. Document in Annexure-4: Excursion Investigation Log.

5.5 Trends and Reporting

1. Compile monthly trend data for each sampling location.
2. Use line graphs or control charts to identify spikes, shifts, or system deterioration.
3. Review data in monthly quality meetings and recommend preventive actions.
4. Archive in Annexure-5: Monthly Water Bioburden Trend Chart.

6. Abbreviations

• PW: Purified Water
• WFI: Water for Injection
• CFU: Colony Forming Unit
• TSA: Tryptic Soy Agar
• SDA: Sabouraud Dextrose Agar
• SOP: Standard Operating Procedure

7. Documents

1. Water Sampling Log – Annexure-1
2. Filtration and Plate Log – Annexure-2
3. Bioburden Result Sheet – Annexure-3
4. Excursion Investigation Log – Annexure-4
5. Monthly Water Bioburden Trend Chart – Annexure-5

8. References

• USP <1231> – Water for Pharmaceutical Purposes
• WHO TRS 970 Annex 2 – Quality of Water in Pharmaceutical Use
• ICH Q10 – Pharmaceutical Quality System

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Water Sampling Log

Date	Sample ID	Location	Time	Collected By
19/05/2025	WB-127-A	User Point 3	09:00 AM	Rajesh Kumar

Annexure-2: Filtration and Plate Log

Sample ID	Volume Filtered	Media Used	Filter Lot	Operator
WB-127-A	100 mL	TSA	F045-25	Sunita Reddy

Annexure-3: Bioburden Result Sheet

Sample ID	Plate Count	CFU/mL	Limit	Status
WB-127-A	3	0.03	≤ 1.0	Pass

Annexure-4: Excursion Investigation Log

Date	Sample ID	CFU/mL	Deviation No.	CAPA
—	—	—	—	—

Annexure-5: Monthly Water Bioburden Trend Chart

[Graphical trend maintained offline. Attach to printed SOP for archival.]

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Included trend analysis and deviation tracking	Annual Review