equipment is necessary for the process:

4.1.1 Required Equipment

• Rotary evaporator
• Round-bottom flask
• Vacuum pump
• Magnetic stirrer
• Sonicator or extruder
• pH meter
• Temperature-controlled water bath

4.1.2 Equipment Calibration

• 4.1.2.1 Calibrate the rotary evaporator and vacuum pump, checking for leaks and ensuring proper vacuum pressure.
• 4.1.2.2 Ensure the pH meter is calibrated with standard buffer solutions (pH 4.0, 7.0, and 10.0).
• 4.1.2.3 Verify the sonicator or extruder is functioning correctly and capable of reducing the size of liposomes to the desired level.

4.2 Lipid Dissolution

The lipids used in the formulation must be dissolved in an appropriate organic solvent to facilitate film formation. Follow the steps below:

• 4.2.1 Weigh the required amount of lipids according to the formulation protocol and record the weights in the Batch Manufacturing Record (BMR).
• 4.2.2 Transfer the lipids into a clean round-bottom flask.
• 4.2.3 Add the selected organic solvent (e.g., chloroform, ethanol, or methanol) to the flask, ensuring the lipids are completely dissolved.
• 4.2.4 Stir the solution using a magnetic stirrer to ensure uniform dissolution of the lipids.

4.3 Solvent Evaporation

Once the lipids are dissolved, the organic solvent must be evaporated to form a thin lipid film. The following steps outline the solvent evaporation process:

• 4.3.1 Place the round-bottom flask containing the lipid solution on the rotary evaporator.
• 4.3.2 Set the water bath temperature to a value slightly above the lipid phase transition temperature (usually around 35°C-40°C).
• 4.3.3 Apply vacuum pressure to evaporate the solvent while rotating the flask to ensure an even distribution of the lipid film.
• 4.3.4 Continue rotating and evaporating until all the solvent has been removed, typically within 1-2 hours.
• 4.3.5 After solvent evaporation, allow the lipid film to dry further under reduced pressure for 30 minutes to remove any residual solvent traces.

4.4 Hydration of the Lipid Film

After the solvent has been evaporated, the lipid film must be hydrated with an aqueous solution to form liposomes.

4.4.1 Preparation of the Aqueous Phase

• 4.4.1.1 Prepare the aqueous phase (e.g., phosphate-buffered saline or a drug solution) as per the formulation protocol.
• 4.4.1.2 Adjust the pH of the aqueous solution using a calibrated pH meter, ensuring the pH is within the specified range.
• 4.4.1.3 Warm the aqueous solution in a water bath to a temperature above the lipid phase transition temperature (usually 37°C).

4.4.2 Lipid Film Hydration

• 4.4.2.1 Add the pre-warmed aqueous solution to the flask containing the lipid film.
• 4.4.2.2 Vortex or stir gently for 15 to 30 minutes to allow complete hydration of the lipid film, resulting in the formation of multilamellar liposomes.

4.5 Liposome Size Reduction

The liposomes formed after hydration are typically multilamellar and heterogeneous in size. Size reduction techniques such as sonication or extrusion must be employed to produce small unilamellar vesicles (SUVs).

4.5.1 Sonication

• 4.5.1.1 Place the liposome suspension in a sonicator bath.
• 4.5.1.2 Sonicate the suspension for 5 to 30 minutes, depending on the desired size. Ensure that the temperature is monitored and maintained to avoid overheating.
• 4.5.1.3 After sonication, cool the liposome suspension to room temperature.

4.5.2 Extrusion

• 4.5.2.1 Pass the liposome suspension through polycarbonate membrane filters using an extruder to achieve the desired size and uniformity.
• 4.5.2.2 Repeat the extrusion process several times (usually 5 to 10 passes) to ensure size consistency.

4.6 Quality Control

The quality of the liposomes must be assessed to ensure they meet the required size, encapsulation efficiency, and stability. Perform the following tests:

• 4.6.1 Measure the particle size of the liposomes using dynamic light scattering (DLS) or a similar particle sizing technique.
• 4.6.2 Assess the encapsulation efficiency by determining the concentration of the encapsulated drug in the liposome suspension.
• 4.6.3 Evaluate the stability of the liposomes by storing them at the specified conditions and monitoring their size and morphology over time.

5) Abbreviations, if any

• 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	Sonication Time	Operator Initials	QA Signature
Batch Number	Lipid Name	Weight in grams	Solvent Name	Buffer/Drug Solution	Minutes	Operator Name	QA Name