4.1.1 Required Equipment

• pH meter (calibrated)
• Analytical balance
• Autoclave (for sterilization)
• Magnetic stirrer
• Glass beakers and volumetric flasks
• Filtration setup (for sterile filtration)
• Thermometer

4.1.2 Equipment Calibration

Before preparing the aqueous phase, all instruments must be checked for calibration. Calibration must be performed as follows:

• 4.1.2.1 Calibrate the pH meter using standard pH buffer solutions (e.g., pH 4.0, 7.0, and 10.0).
• 4.1.2.2 Ensure the analytical balance is calibrated to an accuracy of 0.001 g.
• 4.1.2.3 Verify that the autoclave is functioning correctly, and perform a spore test if necessary to confirm sterilization efficacy.
• 4.1.2.4 Record all calibration results in the equipment logbook.

4.2 Preparation of Buffers

Buffers are typically used as the aqueous phase in liposome formulations to maintain the pH and stability of the drug encapsulation environment. Commonly used buffers include phosphate-buffered saline (PBS), citrate buffer, and acetate buffer.

4.2.1 Buffer Selection

• 4.2.1.1 Select the buffer based on the compatibility of the drug and lipid components. PBS is commonly used for neutral liposomes, while acetate or citrate buffers may be preferred for pH-sensitive formulations.
• 4.2.1.2 Consult the formulation protocol or batch manufacturing record (BMR) for buffer concentration and composition.

4.2.2 Buffer Preparation

The following steps must be followed to prepare the buffer solution:

• 4.2.2.1 Weigh the required amount of buffer salts (e.g., sodium chloride, potassium phosphate) using an analytical balance.
• 4.2.2.2 Dissolve the salts in distilled or deionized water, stirring with a magnetic stirrer to ensure complete dissolution.
• 4.2.2.3 Measure the volume of the buffer solution using a volumetric flask, ensuring that the final volume is accurate to within ±0.1%.
• 4.2.2.4 Adjust the pH of the buffer solution to the desired level using a pH meter. Add small amounts of acid (e.g., hydrochloric acid) or base (e.g., sodium hydroxide) to achieve the correct pH.
• 4.2.2.5 Filter the buffer solution through a 0.22 μm filter to ensure sterility.

4.3 pH Adjustment and Osmolarity Testing

The pH and osmolarity of the aqueous phase must be carefully controlled to ensure compatibility with the lipid components and drug being encapsulated. Deviations in pH or osmolarity can lead to instability of the liposomes or degradation of the active ingredients.

• 4.3.1 After the buffer is prepared, measure the pH using a calibrated pH meter. If necessary, make small adjustments by adding acid or base, as described in Section 4.2.2.4.
• 4.3.2 Test the osmolarity of the aqueous phase using an osmometer. The osmolarity should be within the specified range (e.g., 270-320 mOsm/kg) to match physiological conditions and prevent osmotic stress on the liposomes.
• 4.3.3 Record the pH and osmolarity measurements in the Batch Manufacturing Record (BMR, see Annexure 1).

4.4 Sterilization of the Aqueous Phase

To maintain the sterility of the liposome formulation, the aqueous phase must be sterilized before use. The following methods can be used for sterilization:

4.4.1 Sterile Filtration

• 4.4.1.1 Pass the prepared buffer solution through a 0.22 μm filter to remove any microorganisms or particulate matter.
• 4.4.1.2 Ensure that the filtration equipment is sterile and that the filtration process is performed in a laminar flow hood to prevent contamination.
• 4.4.1.3 Store the filtered buffer in sterilized containers, and label them with the buffer name, concentration, pH, preparation date, and expiration date.

4.4.2 Autoclaving

If sterile filtration is not feasible, autoclaving can be used to sterilize the aqueous phase:

• 4.4.2.1 Transfer the buffer solution into autoclavable glass containers.
• 4.4.2.2 Autoclave the buffer solution at 121°C for 20 minutes.
• 4.4.2.3 After autoclaving, allow the solution to cool to room temperature under sterile conditions.
• 4.4.2.4 Inspect the containers for cracks or contamination after the autoclaving process. If contamination is detected, discard the buffer and repeat the process.

4.5 Storage of Aqueous Phase

The prepared and sterilized aqueous phase can be stored for future use in liposome formulations. The following storage guidelines must be adhered to:

• 4.5.1 Store the sterilized buffer solution in a cool, dry place, preferably between 2°C and 8°C, unless otherwise specified in the formulation protocol.
• 4.5.2 Ensure that all storage containers are labeled with the buffer name, concentration, pH, preparation date, and expiration date.
• 4.5.3 Record the storage conditions in the Raw Material Storage Log (see Annexure 2).
• 4.5.4 Conduct regular inspections of the buffer solutions to ensure that there are no signs of contamination or degradation (e.g., color change, precipitation).

5) Abbreviations, if any

• PBS: Phosphate-Buffered Saline
• BMR: Batch Manufacturing Record
• QC: Quality Control

6) Documents, if any

• Batch Manufacturing Record (BMR, see Annexure 1)
• Raw Material Storage Log (see Annexure 2)
• pH Meter Calibration Log
• Osmometer Calibration Log

7) References, if any

• FDA Guidelines for Sterile Drug Products
• ICH Q7: Good Manufacturing Practice Guide for Active Pharmaceutical Ingredients

8) SOP Version

Version 1.0

Annexure

Annexure 1: Batch Manufacturing Record Template

  

    
Batch No.
    
Buffer
    
pH
    
Osmolarity
    
Filtration Method
    
Operator Initials
    
QA Signature
  
  

    
Batch Number
    
Buffer Name
    
pH Value
    
mOsm/kg
    
0.22 μm Filter / Autoclave
    
Operator Name
    
QA Name
  
  

    
 
    
 
    
 
    
 
    
 
    
 
  

Annexure 2: Raw Material Storage Log Template

  

    
Date
    
Buffer Name
    
Batch No.
    
Storage Condition
    
Location
    
Operator Initials
  
  

    
DD/MM/YYYY
    
Buffer Name
    
Batch Number
    
2-8°C
    
Storage Area
    
Operator Initials