Scope

This procedure applies to injectable drug products, APIs, water for injection (WFI), and other raw materials tested for endotoxins in the Analytical Method Development (AMD) department using LAL-based BET methods.

3. Responsibilities

• Microbiologist: Prepares dilutions, selects suitable LAL reagent lot, and conducts validation runs including inhibition/enhancement studies.
• Instrument Operator: Operates the kinetic BET system and analyzes data.
• QA Executive: Reviews raw data, validation summary, and compliance with pharmacopeial acceptance criteria.

4. Accountability

The Head of Analytical Method Development is accountable for ensuring that validated BET methods are available and suitable for routine testing of applicable products.

5. Procedure

5.1 Preparation

1. Use depyrogenated glassware and certified endotoxin-free consumables.
2. Select an LAL reagent compatible with the detection method (gel clot, chromogenic, turbidimetric).
3. Prepare dilutions of Control Standard Endotoxin (CSE) as per CoA instructions.

5.2 Validation Plan

1. Perform three key validation studies:
   • Linearity and sensitivity of the method
   • Inhibition/Enhancement testing
   • Precision and repeatability
2. Define Maximum Valid Dilution (MVD) using:
   
   MVD = (Endotoxin Limit × Sample Concentration) / λ

5.3 Inhibition/Enhancement Test

1. Spike test samples with known CSE concentration (~2 λ).
2. Compare recovery in test matrix to recovery in LAL reagent water (control).
3. Acceptable range: 50%–200% recovery.
4. If inhibition is observed, dilute sample or add neutralizers such as polysorbate 80 or lecithin.

5.4 Linearity and Sensitivity

1. Run CSE standards in duplicate at concentrations: 0.25 λ, 0.5 λ, 1.0 λ, 2.0 λ.
2. Plot standard curve and calculate r value (must be ≥ 0.980).

5.5 Precision and Repeatability

1. Test at least six replicates at the target λ concentration.
2. Calculate %RSD (Relative Standard Deviation). Acceptance: RSD ≤ 25%.

5.6 Documentation

1. Record all calculations and raw data in Annexure-1 and Annexure-2.
2. Summarize validation results in BET Method Validation Report (Annexure-3).
3. Retain chromatograms, gel clot tubes, or instrument logs as per GMP record retention policy.

6. Abbreviations

• BET: Bacterial Endotoxin Test
• LAL: Limulus Amebocyte Lysate
• CSE: Control Standard Endotoxin
• RSD: Relative Standard Deviation
• λ: Labelled sensitivity of LAL reagent

7. Documents

1. Inhibition/Enhancement Raw Data Sheet – Annexure-1
2. Linearity and Precision Calculation Log – Annexure-2
3. BET Method Validation Summary Report – Annexure-3

8. References

• USP <85> Bacterial Endotoxins Test
• Ph. Eur. 2.6.14 Bacterial Endotoxins
• FDA Guidance for Industry: Pyrogen and Endotoxins Testing

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name	Ravindra Jadhav	Neelam Tiwari	Sunita Reddy
Designation	Microbiology Analyst	QA Reviewer	Head – AMD
Department	Analytical Method Development	QA	Analytical Method Development

11. Annexures

Annexure-1: Inhibition/Enhancement Test Data

Sample	Spiked (EU/mL)	Recovered (EU/mL)	Recovery (%)	Status
API-235	0.5	0.46	92%	Pass

Annexure-2: Linearity and Precision Data

CSE Level	Mean OD	Time to Threshold	Replicates
0.5 EU/mL	0.812	28.5 min	6

Annexure-3: BET Method Validation Report Summary

BET method validated for injectable product INJ-2025-011 using kinetic-chromogenic detection. Linearity r = 0.992. Inhibition/Enhancement: 89–102% recovery. Precision RSD = 6.4%. Method deemed valid.

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
21/05/2025	2.0	Updated MVD formula and added precision requirement section	Annual Review	Sunita Reddy
10/06/2022	1.0	Initial SOP Release	New SOP	QA Head