Aqueous Phase Preparation for Liposomal Formulations
1) Purpose
This SOP provides guidelines for the preparation of the aqueous phase used in liposomal formulations. The aqueous phase is a critical component in liposome production, as it serves as the medium for the encapsulation of water-soluble drugs and other active pharmaceutical ingredients (APIs). The aqueous phase must be prepared with accuracy to ensure the efficacy and stability of the final liposome product.
2) Scope
This SOP applies to all personnel involved in the preparation of the aqueous phase for liposome formulations in research, development, and commercial production settings. It includes steps for selecting and preparing buffers, adjusting pH, and maintaining sterility during the process.
3) Responsibilities
- Operators: Responsible for the accurate preparation of the aqueous phase according to this SOP. Operators must ensure that all equipment is properly cleaned, sterilized, and calibrated before use.
- QA Team: Responsible for overseeing the preparation process, ensuring that all steps are carried out in compliance with SOP requirements, and verifying the accuracy of documentation.
- QC Team: Responsible for testing the quality of the aqueous phase, including pH, osmolarity, and sterility, before it is used in the liposome formulation process.
4) Procedure
4.1 Equipment Setup
All equipment used in the preparation of the aqueous phase must be thoroughly cleaned, sterilized, and calibrated before starting the process. The following equipment is required:
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 |