Preparation of Liposomes using High-Pressure Homogenization
1) Purpose
The purpose of this SOP is to describe the procedure for preparing liposomes using high-pressure homogenization. This technique applies high shear forces to lipid suspensions to reduce particle size and create uniform, small liposomes suitable for drug delivery and other applications. High-pressure homogenization is widely used for producing liposomes with consistent size distribution and stability.
2) Scope
This SOP applies to personnel involved in the preparation of liposomes using high-pressure homogenization. It includes steps for preparing lipid suspensions, setting up the high-pressure homogenizer, and performing size reduction through homogenization.
3) Responsibilities
- Operators: Responsible for setting up the high-pressure homogenizer, ensuring proper calibration, and conducting the homogenization process as per this SOP.
- QA Team: Responsible for reviewing the batch records and ensuring compliance with SOP guidelines and quality standards.
- QC Team: Responsible for conducting quality control tests to assess the size, stability, and encapsulation efficiency of the liposomes.
4) Procedure
4.1 Equipment Setup
Ensure that the high-pressure homogenizer and related equipment are properly cleaned, calibrated, and set up before starting the liposome preparation process.
4.1.1 Required Equipment
- High-pressure homogenizer
- Magnetic stirrer
- Rotary evaporator
- pH meter
- Temperature-controlled water bath
- Particle size analyzer (DLS or equivalent)
4.1.2 Equipment Calibration
- 4.1.2.1 Calibrate the high-pressure homogenizer, checking the pressure settings, nozzles, and other critical components.
- 4.1.2.2 Ensure the pH meter is calibrated with standard buffer solutions (pH 4.0, 7.0, and 10.0) before use.
- 4.1.2.3 Confirm that the water bath is capable of maintaining the required temperature for lipid hydration.
4.2 Lipid Film Preparation
The lipids must be dissolved in an organic solvent, and the solvent must be evaporated to form a lipid film. The lipid film will later be hydrated and subjected to high-pressure homogenization.
4.2.1 Lipid Dissolution
- 4.2.1.1 Weigh the required amount of lipids according to the formulation protocol and record the weights in the Batch Manufacturing Record (BMR).
- 4.2.1.2 Dissolve the lipids in an appropriate organic solvent (e.g., chloroform or ethanol) in a round-bottom flask.
- 4.2.1.3 Evaporate the solvent using a rotary evaporator to form a thin lipid film.
- 4.2.1.4 Dry the lipid film under vacuum for 30 minutes to remove residual solvent.
4.2.2 Hydration of Lipid Film
- 4.2.2.1 Hydrate the lipid film by adding the pre-warmed aqueous phase (e.g., phosphate-buffered saline or drug solution) to the flask.
- 4.2.2.2 Stir the mixture gently for 30 minutes to ensure complete hydration of the lipid film and formation of multilamellar vesicles (MLVs).
4.3 High-Pressure Homogenization
The liposome suspension is passed through the high-pressure homogenizer to reduce the size of the vesicles and achieve uniformity. The following steps outline the homogenization process:
- 4.3.1 Transfer the lipid suspension to the homogenizer inlet chamber.
- 4.3.2 Set the homogenizer to the desired pressure, typically between 500 and 1500 bar, depending on the target liposome size.
- 4.3.3 Pass the suspension through the homogenizer for multiple cycles (usually 5 to 10 passes) to achieve the desired size reduction.
- 4.3.4 After homogenization, collect the liposome suspension in a sterile container.
4.4 Quality Control
After the homogenization process, perform the following quality control tests to assess the size, encapsulation efficiency, and stability of the liposomes:
- 4.4.1 Measure the particle size of the liposomes using dynamic light scattering (DLS) or a similar particle size analysis method.
- 4.4.2 Evaluate the encapsulation efficiency by determining the concentration of the encapsulated drug or active ingredient.
- 4.4.3 Test the stability of the liposomes by storing them at specified conditions and assessing their size and morphology over time.
4.5 Storage of Liposomes
Once the liposomes have been homogenized and quality control tests have been completed, store them under appropriate conditions to maintain stability.
- 4.5.1 Transfer the liposome suspension to sterilized, airtight containers and store them at 4°C or as specified in the formulation protocol.
- 4.5.2 Ensure that all storage containers are labeled with the batch number, preparation date, and storage conditions.
- 4.5.3 Periodically assess the stored liposomes for size, stability, and drug retention to ensure they remain within specification during their shelf life.
5) Abbreviations, if any
- MLV: Multilamellar Vesicle
- DLS: Dynamic Light Scattering
- QA: Quality Assurance
- QC: Quality Control
6) Documents, if any
- Batch Manufacturing Record (BMR)
- Particle Size Analysis Report
- pH Calibration Log
7) References, if any
- FDA Guidelines for Liposomal Drug Products
- ICH Q7: Good Manufacturing Practice Guide
8) SOP Version
Version 1.0
Annexure
Annexure 1: Batch Manufacturing Record Template
| Batch No. | Lipid Type | Weight | Aqueous Phase | Homogenization Pressure | Operator Initials | QA Signature | |
|---|---|---|---|---|---|---|---|
| Batch Number | Lipid Name | Weight in grams | Buffer/Drug Solution | Pressure in Bar | Operator Name | QA Name | |