for size reduction)

Magnetic stirrer

Vacuum pump

pH meter

Temperature-controlled water bath

4.1.2 Equipment Calibration

• 4.1.2.1 Ensure the rotary evaporator is calibrated for temperature and vacuum pressure. Check the integrity of the seals.
• 4.1.2.2 Verify the calibration of the sonicator or extruder for producing uniform ULVs.
• 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 ULVs is to dissolve lipids in an organic solvent, followed by solvent evaporation to form a lipid film. The following steps outline this procedure:

4.2.1 Dissolving Lipids

• 4.2.1.1 Weigh the required amount of lipid components (e.g., phospholipids and cholesterol) 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 (e.g., chloroform or methanol) in a round-bottom flask.
• 4.2.1.3 Stir the solution using a magnetic stirrer to ensure complete dissolution.

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 slightly 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 walls of the flask.
• 4.2.2.3 After the solvent is fully evaporated, dry the lipid film under vacuum for an additional 30 minutes to remove residual solvent.

4.3 Hydration of the Lipid Film

The lipid film is hydrated with an aqueous phase to form liposomes. The hydration process can vary depending on whether small unilamellar vesicles (SUVs) or large unilamellar vesicles (LUVs) are desired.

4.3.1 Preparation of the Aqueous Phase

• 4.3.1.1 Prepare the aqueous phase, which may be a buffer solution (e.g., phosphate-buffered saline) or a drug solution as per the formulation protocol.
• 4.3.1.2 Adjust the pH of the aqueous phase using the pH meter to maintain liposome stability and drug compatibility.
• 4.3.1.3 Warm the aqueous phase to the desired temperature (typically 37°C) using 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 lipid film.
• 4.3.2.2 Vortex the mixture or stir gently for 30 minutes to allow complete hydration of the lipid film and formation of multilamellar vesicles (MLVs).

4.4 Size Reduction to Form ULVs

To convert multilamellar vesicles (MLVs) into unilamellar vesicles (ULVs), a size reduction technique must be employed. Common techniques include sonication, extrusion, or high-pressure homogenization.

4.4.1 Sonication Method

• 4.4.1.1 Transfer the MLV suspension to a sonicator.
• 4.4.1.2 Sonicate the suspension for 5 to 30 minutes, depending on the desired ULV size. Monitor the temperature during sonication to avoid overheating, which may destabilize the liposomes.
• 4.4.1.3 After sonication, allow the suspension to cool to room temperature before further processing.

4.4.2 Extrusion Method

• 4.4.2.1 Pass the MLV suspension through polycarbonate membrane filters using an extruder. The filter pore size will determine the final ULV size (e.g., 100 nm or 200 nm pores).
• 4.4.2.2 Repeat the extrusion process multiple times (usually 5 to 10 passes) to ensure a uniform ULV population.

4.5 Quality Control of ULVs

Once the ULVs are prepared, they must undergo quality control testing to ensure they meet the required size, stability, and encapsulation efficiency.

• 4.5.1 Measure the particle size of the ULVs using dynamic light scattering (DLS) or another particle sizing technique.
• 4.5.2 Test the stability of the ULVs by monitoring their size and morphology over time.
• 4.5.3 Evaluate the encapsulation efficiency by measuring the concentration of the encapsulated drug or active ingredient within the ULVs.

5) Abbreviations, if any

• MLV: Multilamellar Vesicles
• SUV: Small Unilamellar Vesicles
• LUV: Large Unilamellar Vesicles
• DLS: Dynamic Light Scattering
• QA: Quality Assurance
• QC: Quality Control

6) Documents, if any

• Batch Manufacturing Record (BMR)
• Particle Size Analysis Report
• pH 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	Aqueous Phase	Size Reduction Method	Sonication/Extrusion Time	Operator Initials	QA Signature
Batch Number	Lipid Name	Weight in grams	Buffer/Drug Solution	Sonication/Extrusion	Minutes	Operator Name	QA Name