SOP for Preparation of Sterically Stabilized Liposomes

Preparation of Sterically Stabilized Liposomes

1) Purpose

The purpose of this SOP is to describe the procedure for preparing sterically stabilized liposomes (also known as stealth liposomes). Sterically stabilized liposomes are coated with polymers such as polyethylene glycol (PEG) to extend their circulation time in the bloodstream, reduce recognition by the immune system, and enhance drug delivery to target tissues. This SOP outlines the formulation process, ensuring liposome stability and surface modification with PEG.

2) Scope

This SOP applies to personnel involved in the preparation of sterically stabilized liposomes for drug delivery applications. It includes instructions for lipid film formation, hydration, and PEGylation, as well as the quality control of the liposomes to confirm size, stability, and PEG coverage.

3) Responsibilities

Operators: Responsible for executing the procedure for preparing sterically stabilized liposomes and ensuring proper PEGylation.
QA Team: Responsible for reviewing the batch records and ensuring compliance with SOP guidelines and quality standards.
QC Team: Responsible for performing quality control tests to assess liposome size, PEGylation efficiency, and stability.

4) Procedure

4.1 Equipment Setup

The equipment required for preparing sterically stabilized liposomes must be cleaned, calibrated, and set up before use. The following equipment is essential for the process:

4.1.1 Required Equipment

Rotary evaporator
Sonicator or extruder
Vacuum pump
Magnetic stirrer
pH meter
Temperature-controlled water bath
Dynamic light scattering (DLS) instrument

4.1.2 Equipment Calibration

4.1.2.1 Ensure that the rotary evaporator is calibrated for temperature and vacuum pressure.
4.1.2.2 Verify the calibration of the sonicator or extruder for producing uniform liposomes.
4.1.2.3 Calibrate the pH meter using standard buffer solutions (pH 4.0, 7.0, and 10.0).

4.2 Lipid Film Formation

The first step in preparing sterically stabilized liposomes is the formation of a lipid film by dissolving lipids in an organic solvent and removing the solvent under reduced pressure.

4.2.1 Lipid Dissolution

4.2.1.1 Weigh the required amount of lipid components, including phospholipids, cholesterol, and PEGylated lipids (e.g., DSPE-PEG), based on 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 solution with a magnetic stirrer to ensure complete dissolution of the lipids.

4.2.2 Solvent Evaporation

4.2.2.1 Attach the round-bottom flask to the rotary evaporator and set the water bath to a temperature above the lipid phase transition temperature (typically 37°C).
4.2.2.2 Evaporate the solvent under reduced pressure to form a thin lipid film on the inner walls of the flask.
4.2.2.3 After solvent evaporation, dry the lipid film under vacuum for an additional 30 minutes to remove any residual solvent.

4.3 Hydration of Lipid Film

The lipid film is hydrated with an aqueous phase to form liposomes. This step involves the addition of a pre-warmed buffer or drug solution to the lipid film.

4.3.1 Preparation of the Aqueous Phase

4.3.1.1 Prepare the aqueous phase (e.g., phosphate-buffered saline or drug solution) according to the formulation protocol.
4.3.1.2 Adjust the pH of the aqueous phase to match the stability requirements of the liposomes.
4.3.1.3 Warm the aqueous phase to the required temperature (usually 37°C) in a temperature-controlled water bath.

4.3.2 Hydration Process

4.3.2.1 Add the warmed aqueous phase to the round-bottom flask containing the dried lipid film.
4.3.2.2 Vortex the mixture or stir gently for 30 minutes to ensure complete hydration of the lipid film and formation of multilamellar vesicles (MLVs).

4.4 Size Reduction to Form PEGylated Liposomes

The MLVs must undergo size reduction to form PEGylated liposomes of uniform size. This can be done using sonication, extrusion, or high-pressure homogenization.

4.4.1 Sonication Method

4.4.1.1 Transfer the MLV suspension to a sonicator and sonicate for 5 to 20 minutes, depending on the desired liposome size.
4.4.1.2 Monitor the temperature during sonication to ensure it does not exceed 40°C, which may destabilize the liposomes.
4.4.1.3 After sonication, allow the suspension to cool to room temperature.

4.4.2 Extrusion Method

4.4.2.1 Pass the liposome suspension through polycarbonate membrane filters using an extruder. Select the filter pore size (e.g., 100 nm or 200 nm) based on the desired liposome size.
4.4.2.2 Repeat the extrusion process 5 to 10 times to achieve uniform PEGylated liposomes.

4.5 Quality Control of Sterically Stabilized Liposomes

Once the PEGylated liposomes are prepared, they must undergo quality control testing to ensure proper size, stability, and PEG coverage. Perform the following tests:

4.5.1 Measure the particle size using dynamic light scattering (DLS) to confirm the size distribution.
4.5.2 Test the PEGylation efficiency by analyzing the surface coverage of PEG on the liposomes, using techniques such as zeta potential measurements or surface plasmon resonance.
4.5.3 Assess the stability of the liposomes by monitoring their size, morphology, and drug encapsulation efficiency over time under specified storage conditions.

4.6 Storage of PEGylated Liposomes

The sterically stabilized liposomes must be stored under appropriate conditions to ensure long-term stability and efficacy. Store the liposome suspension in sterilized, airtight containers at 4°C or as specified in the formulation protocol. Label all containers with the batch number, preparation date, and storage conditions. Periodically assess the liposomes for size, stability, and drug retention.

5) Abbreviations, if any

DLS: Dynamic Light Scattering
PEG: Polyethylene Glycol
QA: Quality Assurance
QC: Quality Control

6) Documents, if any

Batch Manufacturing Record (BMR)
Particle Size Analysis Report
PEGylation Efficiency Report

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
    PEGylation Method
    Size Reduction Method
    Sonication/Extrusion Time
    Operator Initials
    QA Signature
  
  
    Batch Number
    Lipid Name
    Weight in grams
    PEGylation/None
    Sonication/Extrusion
    Minutes
    Operator Name
    QA Name

Batch No.	Lipid Type	Weight	PEGylation Method	Size Reduction Method	Sonication/Extrusion Time	Operator Initials	QA Signature
Batch Number	Lipid Name	Weight in grams	PEGylation/None	Sonication/Extrusion	Minutes	Operator Name	QA Name