standards.

2. Scope

This SOP applies to non-sterile APIs and intermediates tested in the Analytical Method Development (AMD) department prior to formulation or downstream processing.

3. Responsibilities

• Microbiologist: Develops and executes bioburden testing protocols, including media selection, dilution schemes, and recovery validation.
• Analytical Scientist: Coordinates with microbiology to provide sample details and risk data.
• QA Executive: Verifies results, approves protocols and ensures traceability.

4. Accountability

The Head of Analytical Method Development is accountable for ensuring validated and compliant bioburden methods are available for all APIs under analysis.

5. Procedure

5.1 Sample Preparation

1. Weigh accurately 10 g (or prescribed quantity) of API under aseptic conditions.
2. Transfer into sterile container with 90 mL of sterile diluent (e.g., 0.1% peptone water).
3. Mix using vortex or shaker until the material is homogenized.
4. Prepare decimal dilutions as required (e.g., 10^-1, 10^-2, 10^-3).

5.2 Membrane Filtration or Plate Count

1. Choose method based on sample solubility and microbial load:
   • Membrane filtration for insoluble APIs or low CFU samples.
   • Pour-plate/spread-plate for soluble APIs or expected high counts.
2. Use TSA for Total Aerobic Microbial Count (TAMC) and SDA for Total Yeast and Mold Count (TYMC).
3. Incubate:
   • TAMC: 30–35°C for 3–5 days
   • TYMC: 20–25°C for 5–7 days

5.3 Recovery Validation

1. Spike API with known inoculum (~100 CFU) of challenge organisms (e.g., E. coli, S. aureus, C. albicans).
2. Perform test as per method and calculate recovery:
   
   % Recovery = (CFU recovered / CFU inoculated) × 100
3. Acceptable recovery: 70–130%

5.4 Method Suitability

1. Test for microbial inhibition or enhancement by:
   • Comparing recovery from API sample vs. control (buffer only)
   • Neutralizing antimicrobial properties if present using lecithin/polysorbate 80

5.5 Specified Organisms (Optional)

1. Test for presence/absence of:
   • Escherichia coli (Tryptic Soy Broth → MacConkey Agar)
   • Staphylococcus aureus (Soybean Casein Digest Broth → Mannitol Salt Agar)
   • Pseudomonas aeruginosa (Cetrimide Broth → Cetrimide Agar)
2. Incubate and record growth; report as “Absent” if no colonies observed.

6. Abbreviations

• SOP: Standard Operating Procedure
• TAMC: Total Aerobic Microbial Count
• TYMC: Total Yeast and Mold Count
• CFU: Colony Forming Units
• QA: Quality Assurance

7. Documents

1. Bioburden Method Validation Worksheet – Annexure-1
2. Recovery Validation Record – Annexure-2
3. Specified Organism Detection Log – Annexure-3

8. References

• USP <61> Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests
• USP <62> Tests for Specified Microorganisms
• Ph. Eur. 2.6.12 and 2.6.13
• ICH Q6A: Specifications

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name	Santosh Borkar	Neha Kapoor	Sunita Reddy
Designation	Microbiology Analyst	QA Reviewer	Head – AMD
Department	Analytical Method Development	QA	Analytical Method Development

11. Annexures

Annexure-1: Bioburden Method Validation Worksheet

API Name	Sample Weight	Diluent Used	Recovery Method	Media
Metformin HCl	10 g	0.1% Peptone Water	Membrane Filtration	TSA/SDA

Annexure-2: Recovery Validation Record

Organism	CFU Inoculated	CFU Recovered	Recovery (%)	Status
C. albicans	98	95	96.9%	Pass

Annexure-3: Specified Organism Detection Log

Organism	Media Used	Result	Status
E. coli	MacConkey Agar	No Growth	Absent

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
21/05/2025	2.0	Expanded to include recovery validation and specified organism detection	Annual Review and Audit Findings	Sunita Reddy
15/05/2022	1.0	Initial SOP Release	New SOP	QA Head