Standard Operating Procedure for Conducting Freeze-Thaw Stability Studies in BA/BE Analysis
| Department | BA-BE Studies |
|---|---|
| SOP No. | SOP/BA-BE/174/2025 |
| Supersedes | SOP/BA-BE/174/2022 |
| Page No. | Page 1 of 9 |
| Issue Date | 17/04/2025 |
| Effective Date | 20/04/2025 |
| Review Date | 17/04/2026 |
1. Purpose
To define the process for conducting freeze-thaw stability studies for plasma samples to assess the effect of multiple freezing and thawing cycles on analyte concentration in bioavailability and bioequivalence (BA/BE) studies.
2. Scope
This SOP applies to all plasma samples used in validated bioanalytical methods for BA/BE studies that undergo multiple freeze-thaw cycles prior to or during analysis.
3. Responsibilities
- Bioanalytical Analyst: Prepares, stores, and
processes stability samples as per protocol.
Supervisor/Principal Investigator: Reviews results for accuracy and regulatory compliance.
QA Officer: Verifies documentation and data integrity, and ensures SOP adherence.
4. Accountability
The Head of Bioanalytical Operations is accountable for ensuring that all freeze-thaw stability studies are performed in compliance with regulatory guidelines and documented appropriately.
5. Procedure
5.1 Sample Preparation and Labeling
- Prepare six replicates each of Low QC (LQC) and High QC (HQC) samples in plasma.
- Aliquot into labeled tubes with:
- Study ID
- Analyte name
- QC level
- Freeze-Thaw Cycle Number
- Preparation Date
- Record in Annexure-1: Freeze-Thaw Sample Log.
5.2 Freeze-Thaw Cycle Execution
- Store samples at -20°C or -70°C for a minimum of 12 hours to ensure complete freezing.
- Thaw samples at room temperature (~25°C) until completely liquified.
- Repeat freezing and thawing for a total of three cycles.
- Ensure uniform timing across replicates within a batch.
5.3 Analysis of Stability Samples
- Analyze freeze-thaw samples together with freshly prepared calibration curve and QC samples.
- Use validated LC-MS/MS method as per method validation protocol.
- Record measured concentrations and compare with nominal values.
5.4 Acceptance Criteria
- Mean concentration of LQC and HQC samples must be within ±15% of nominal values.
- %CV should be ≤15% for replicate values at each QC level.
- Results must be documented in Annexure-2: Freeze-Thaw Stability Summary.
5.5 Documentation and Reporting
- Prepare a summary report including:
- Measured concentrations
- QC sample charts
- Acceptance status
- Submit to QA for review and approval before including in validation or study report.
6. Abbreviations
- BA/BE: Bioavailability/Bioequivalence
- LQC: Low Quality Control
- HQC: High Quality Control
- %CV: Percent Coefficient of Variation
- QA: Quality Assurance
7. Documents
- Freeze-Thaw Sample Log – Annexure-1
- Freeze-Thaw Stability Summary – Annexure-2
8. References
- ICH M10: Bioanalytical Method Validation
- US FDA Guidance for Bioanalytical Method Validation
- Internal Method Validation Protocol
9. SOP Version
Version: 2.0
10. Approval Section
| Prepared By | Checked By | Approved By | |
|---|---|---|---|
| Signature | |||
| Date | |||
| Name | |||
| Designation | |||
| Department |
11. Annexures
Annexure-1: Freeze-Thaw Sample Log
| Sample ID | QC Level | Cycle No. | Freeze Date | Thaw Date | Operator |
|---|---|---|---|---|---|
| BE174-FT-01 | LQC | 3 | 14/04/2025 | 17/04/2025 | Sunita Reddy |
Annexure-2: Freeze-Thaw Stability Summary
| QC Level | Nominal (ng/mL) | Mean Measured | %CV | % Difference | Status |
|---|---|---|---|---|---|
| LQC | 20.0 | 19.1 | 4.2 | -4.5% | Accepted |
| HQC | 1500.0 | 1498.2 | 2.8 | -0.1% | Accepted |
Revision History:
| Revision Date | Revision No. | Details | Reason | Approved By |
|---|---|---|---|---|
| 01/01/2022 | 1.0 | Initial SOP release | New inclusion | QA Head |
| 17/04/2025 | 2.0 | Updated freeze-thaw validation parameters and documentation format | ICH M10 alignment | QA Head |