Preparation of Liposomes using Extrusion Method
1) Purpose
This SOP describes the preparation of liposomes using the extrusion method. The extrusion technique is used to reduce liposome size and achieve uniform size distribution. Liposomes are passed through membrane filters under pressure, resulting in small unilamellar vesicles (SUVs) with consistent size and characteristics.
2) Scope
This SOP applies to all personnel involved in the preparation of liposomes using the extrusion method. It covers the steps for setting up the extruder, preparation of the lipid and aqueous phases, and size reduction through extrusion.
3) Responsibilities
- Operators: Responsible for executing the extrusion procedure according to this SOP, ensuring proper calibration and cleaning of the extruder.
- QA Team: Responsible for ensuring that the extrusion process is conducted as per quality standards, verifying procedures, and reviewing batch records.
- QC Team: Responsible for conducting quality control tests to confirm the size, stability, and encapsulation efficiency of the prepared liposomes.
4) Procedure
4.1 Equipment Setup
Before beginning the extrusion process, ensure that all equipment is cleaned, calibrated, and set up correctly.
4.1.1 Required Equipment
- Extruder with polycarbonate membrane filters (100 nm or desired pore size)
- High-pressure nitrogen or air source
- Magnetic stirrer
- Rotary evaporator
- pH meter
- Temperature-controlled water bath
4.1.2 Equipment Calibration
- 4.1.2.1 Ensure the extruder pressure settings are calibrated correctly and that the membrane filters are intact.
- 4.1.2.2 Verify that 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 functioning correctly and can maintain the required temperature for the lipid hydration process.
4.2 Lipid Film Preparation
The lipids must be dissolved, and the organic solvent must be evaporated to form a thin lipid film. This will later be hydrated and extruded to form liposomes.
- 4.2.1 Weigh the required amount of lipid components according to the formulation protocol and record the weights in the Batch Manufacturing Record (BMR).
- 4.2.2 Dissolve the lipids in an appropriate organic solvent (e.g., chloroform, ethanol) in a round-bottom flask.
- 4.2.3 Evaporate the solvent using a rotary evaporator to form a thin lipid film.
- 4.2.4 Dry the lipid film under vacuum for 30 minutes to remove residual solvent.
4.3 Hydration of Lipid Film
The lipid film is hydrated using an aqueous phase, such as a buffer solution or drug solution, to form multilamellar vesicles (MLVs).
- 4.3.1 Add the pre-warmed aqueous phase to the round-bottom flask containing the lipid film.
- 4.3.2 Stir the mixture for 30 minutes to ensure complete hydration of the lipid film.
- 4.3.3 Adjust the pH of the aqueous phase using a calibrated pH meter to ensure stability of the liposomes and the encapsulated compound.
4.4 Extrusion Process
The multilamellar liposome suspension is passed through polycarbonate membrane filters under pressure to produce unilamellar liposomes of uniform size.
- 4.4.1 Transfer the hydrated lipid suspension to the extruder inlet.
- 4.4.2 Apply pressure using nitrogen or air, and pass the suspension through polycarbonate membranes with a pore size of 100 nm or the desired size.
- 4.4.3 Repeat the extrusion process 5 to 10 times to ensure uniform size reduction and consistency.
- 4.4.4 Collect the extruded liposome suspension in a clean, sterile container.
4.5 Quality Control
Once the extrusion process is complete, perform the following quality control tests:
- 4.5.1 Measure the particle size of the liposomes using dynamic light scattering (DLS).
- 4.5.2 Test the encapsulation efficiency of the liposomes by measuring the concentration of the encapsulated drug or compound.
- 4.5.3 Monitor the stability of the liposomes by storing them under the specified conditions and assessing size and morphology over time.
5) Abbreviations, if any
- MLV: Multilamellar Vesicle
- DLS: Dynamic Light Scattering
- SUV: Small Unilamellar Vesicles
- QA: Quality Assurance
- QC: Quality Control
6) Documents, if any
- Batch Manufacturing Record (BMR)
- Particle Size Analysis Report
- pH Meter 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 | Solvent | Aqueous Phase | Extrusion Passes | Operator Initials | QA Signature |
|---|---|---|---|---|---|---|---|
| Batch Number | Lipid Name | Weight in grams | Solvent Name | Buffer/Drug Solution | Number of Passes | Operator Name | QA Name |