SOP for Microfluidization Technique for Liposome Preparation

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 correct and the system is free of any leaks. Confirm the functionality of the homogenization chamber.
4.1.2.2 Ensure the pH meter is calibrated using standard buffer solutions (pH 4.0, 7.0, and 10.0) before use.
4.1.2.3 Confirm that the sonicator is functioning correctly and capable of reducing the initial lipid suspension size.

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

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