• 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 pH meter using standard buffer solutions (pH 4.0, 7.0, and 10.0).
• 4.1.2.3 Confirm that the sonicator or high-pressure homogenizer is functioning properly and calibrated for size reduction.

4.2 Lipid Film Formation

The first step in preparing pH-sensitive liposomes is dissolving the lipid components in an organic solvent, followed by solvent evaporation to form a lipid film. The following steps outline this process:

4.2.1 Lipid Dissolution

• 4.2.1.1 Weigh the required amount of lipid components, including pH-sensitive lipids such as phosphatidylethanolamine (PE) or other anionic lipids, based on the formulation protocol.
• 4.2.1.2 Dissolve the lipids in an appropriate organic solvent (e.g., chloroform or ethanol) in a round-bottom flask.
• 4.2.1.3 Use a magnetic stirrer to ensure complete dissolution of the lipid components.

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 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 phase may contain buffers, drugs, or other active ingredients.

4.3.1 Preparation of the Aqueous Phase

• 4.3.1.1 Prepare the aqueous phase according to the formulation protocol, which may include a buffer solution or drug solution.
• 4.3.1.2 Adjust the pH of the aqueous phase using a calibrated pH meter to match the pH sensitivity of the liposomes (typically pH 5.5 to 7.4).
• 4.3.1.3 Warm the aqueous phase to the desired 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 full hydration of the lipid film and formation of multilamellar vesicles (MLVs).

4.4 Size Reduction to Form pH-Sensitive Liposomes

To convert MLVs into unilamellar vesicles, size reduction techniques such as sonication or extrusion are employed.

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 size of the liposomes.
• 4.4.1.3 Monitor the temperature during sonication to prevent overheating, which could destabilize the liposomes.

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 liposome size (e.g., 100 nm).
• 4.4.2.2 Repeat the extrusion process 5 to 10 times to ensure size uniformity.

4.5 Testing pH Sensitivity

Once the liposomes are prepared, they must be tested for pH sensitivity. This can be done by incubating the liposomes at different pH levels and monitoring their size or release of encapsulated material.

• 4.5.1 Incubate liposomes in buffers of varying pH (e.g., pH 5.5, 6.5, and 7.4) at 37°C for 1 to 2 hours.
• 4.5.2 Measure the particle size of the liposomes using dynamic light scattering (DLS) to determine changes in size or aggregation.
• 4.5.3 Assess the release of encapsulated material by measuring the concentration of the drug or active ingredient released into the solution at different pH levels.

4.6 Quality Control of pH-Sensitive Liposomes

Perform quality control tests on the pH-sensitive liposomes to ensure they meet size, encapsulation efficiency, and pH-responsiveness specifications.

• 4.6.1 Measure the particle size using dynamic light scattering (DLS) or another particle sizing technique.
• 4.6.2 Test the encapsulation efficiency by analyzing the concentration of the encapsulated drug or active ingredient in the liposomes.
• 4.6.3 Assess the stability of the liposomes by storing them at specified conditions and monitoring their size and morphology over time.

5) Abbreviations, if any

• DLS: Dynamic Light Scattering
• MLV: Multilamellar Vesicle
• QA: Quality Assurance
• QC: Quality Control

6) Documents, if any

• Batch Manufacturing Record (BMR)
• Particle Size Analysis Report
• pH Sensitivity Test Results

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
    
pH Sensitivity Test Conditions
    
Sonication/Extrusion Time
    
Operator Initials
    
QA Signature
  
  

    
Batch Number
    
Lipid Name
    
Weight in grams
    
Buffer/Drug Solution
    
pH 5.5, 6.5, 7.4
    
Minutes
    
Operator Name
    
QA Name