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