Microfluidization Technique for Liposome Preparation
1) Purpose
The purpose of this SOP is to provide step-by-step instructions for the preparation of liposomes using the microfluidization technique. Microfluidization is a high-shear method used to reduce the size of liposomes and achieve uniformity in size. It is an efficient technique to generate small unilamellar vesicles (SUVs) or large unilamellar vesicles (LUVs) with high encapsulation efficiency and improved stability.
2) Scope
This SOP applies to personnel involved in liposome preparation using microfluidization. The document covers the process of setting up the microfluidizer, preparing lipid solutions, and using the microfluidizer to form uniform liposomes.
3) Responsibilities
- Operators: Responsible for executing the microfluidization technique as per this SOP and ensuring proper calibration and maintenance of the equipment.
- QA Team: Responsible for ensuring that the liposome production process complies with quality standards, verifying the procedures, and reviewing batch records.
- QC Team: Responsible for conducting quality control tests to assess the size, stability, and encapsulation efficiency of the prepared liposomes.
4) Procedure
4.1 Equipment Setup
The microfluidizer and other equipment must be prepared and calibrated before the liposome preparation process begins.
4.1.1 Required Equipment
- Microfluidizer
- High-pressure pump
- Magnetic stirrer
- Sonicator (for pre-processing)
- pH meter
- Temperature-controlled water bath
4.1.2 Equipment Calibration
- 4.1.2.1 Calibrate the microfluidizer by ensuring that the pressure settings are
4.2 Lipid Preparation
Lipids must be dissolved and prepared for microfluidization. This includes the preparation of the lipid-solvent solution and the aqueous phase (buffer or drug solution).
4.2.1 Lipid Dissolution
- 4.2.1.1 Weigh the required amount of lipid components (e.g., phospholipids and cholesterol) according to the formulation protocol. Record the weights in the Batch Manufacturing Record (BMR).
- 4.2.1.2 Dissolve the lipids in an appropriate organic solvent, such as chloroform or ethanol, in a round-bottom flask.
- 4.2.1.3 Stir the lipid-solvent mixture with a magnetic stirrer until fully dissolved.
4.2.2 Solvent Evaporation
- 4.2.2.1 Evaporate the solvent under reduced pressure using a rotary evaporator to form a thin lipid film inside the round-bottom flask.
- 4.2.2.2 Dry the lipid film under vacuum for 30 minutes to remove any residual solvent.
4.2.3 Hydration of the Lipid Film
- 4.2.3.1 Hydrate the lipid film by adding the pre-warmed aqueous phase (e.g., phosphate-buffered saline or drug solution) to the flask.
- 4.2.3.2 Stir the mixture gently for 30 minutes to ensure complete hydration of the lipid film and formation of multilamellar vesicles (MLVs).
4.3 Microfluidization
The liposome suspension (multilamellar vesicles) is passed through the microfluidizer to achieve size reduction and uniformity. The following steps outline the microfluidization process:
- 4.3.1 Transfer the hydrated lipid suspension to the microfluidizer inlet chamber.
- 4.3.2 Set the microfluidizer to the required pressure, typically between 10,000 and 30,000 psi, depending on the desired liposome size.
- 4.3.3 Pass the suspension through the microfluidizer at least 5 to 10 times to achieve the desired liposome size and uniformity.
- 4.3.4 Collect the processed liposome suspension from the microfluidizer outlet into a clean, sterile container.
4.4 Size and Quality Control
After microfluidization, quality control tests must be performed to assess the size, stability, and encapsulation efficiency of the liposomes. Follow these steps:
- 4.4.1 Measure the particle size of the liposomes using dynamic light scattering (DLS) or a similar particle sizing method.
- 4.4.2 Test the encapsulation efficiency by measuring the concentration of the encapsulated drug or active compound in the liposome suspension.
- 4.4.3 Assess the stability of the liposomes by monitoring their size, morphology, and drug retention over time.
4.5 Storage of Liposomes
Once the liposomes are prepared and quality control tests are complete, they must be stored under appropriate conditions to ensure stability.
- 4.5.1 Store the liposome suspension in sterilized, sealed containers at 4°C or as specified in the formulation protocol.
- 4.5.2 Ensure that the storage containers are properly labeled with the liposome batch number, preparation date, and storage conditions.
- 4.5.3 Periodically test the stored liposomes for size, stability, and drug content to ensure that they meet the required specifications during their shelf life.
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 | Microfluidization Passes | Operator Initials | QA Signature |
|---|---|---|---|---|---|---|---|
| Batch Number | Lipid Name | Weight in grams | Solvent Name | Buffer/Drug Solution | Number of Passes | Operator Name | QA Name |
