4) Procedure

4.1 Equipment Setup

Before beginning the preparation of lipid components, all equipment must be inspected, cleaned, and calibrated. Proper equipment setup ensures the accuracy and consistency of the lipid preparation process. The following equipment is required for the preparation of lipid components:

4.1.1 Required Equipment

• Analytical balance (with a sensitivity of 0.0001g)
• Clean glass beakers (various sizes depending on the volume of lipid solutions)
• Magnetic stirrer with a hot plate (capable of maintaining precise temperature control)
• Temperature probe (for monitoring the temperature during lipid dissolution)
• Volumetric flasks (for measuring solvents accurately)
• Filtration setup (for filtering lipid solutions if required)
• Vacuum pump (optional, for solvent evaporation)
• Desiccator (for storage of lipid films)

4.1.2 Equipment Inspection and Calibration

All equipment must be inspected and calibrated before use to ensure accuracy. The following steps should be taken for equipment preparation:

• 4.1.2.1 Verify that the analytical balance is calibrated within the acceptable range. The calibration should be performed using certified calibration weights, and the results should be recorded in the equipment logbook.
• 4.1.2.2 Ensure that all glass beakers, flasks, and stir bars are clean, dry, and free of any contaminants. Glassware should be rinsed with the appropriate solvent and dried before use.
• 4.1.2.3 Check that the magnetic stirrer and hot plate are functioning correctly. The temperature control feature must be tested to ensure that it can maintain the specified temperature during the lipid dissolution process.
• 4.1.2.4 If a filtration setup is required, ensure that the filters are correctly assembled and tested for integrity. Use membrane filters with the appropriate pore size, depending on the lipid solution’s viscosity and particle size.
• 4.1.2.5 Record all calibration and inspection activities in the equipment logbook. Any discrepancies or issues with the equipment should be reported to the maintenance team before proceeding.

4.2 Weighing and Handling Lipid Components

The accurate weighing and handling of lipid components are essential to ensure the quality of the liposome formulation. Lipid components such as phospholipids, cholesterol, and other excipients must be weighed precisely according to the formulation requirements. The following steps outline the procedure for weighing and handling lipids:

4.2.1 Weighing Lipids

• 4.2.1.1 Tare the analytical balance before weighing each lipid component to ensure that the weight is accurate.
• 4.2.1.2 Weigh each lipid component (e.g., phospholipids, cholesterol) individually according to the batch manufacturing record (BMR) specifications. Ensure that the lipid components are free from any impurities or contaminants.
• 4.2.1.3 Record the weight of each lipid component in the BMR (see Annexure 1). Any deviations from the specified weight must be documented and reported to the QA team for review.
• 4.2.1.4 Store the weighed lipids in clean, labeled containers until they are ready to be dissolved. Ensure that the lipids are protected from moisture and light during storage to prevent degradation.

4.3 Dissolving Lipid Components

Lipids must be dissolved in an appropriate solvent to form a uniform lipid solution. The solvent used for dissolving lipids must be carefully selected based on the solubility properties of the lipid components. The most commonly used solvents include chloroform, methanol, and ethanol. The following steps outline the procedure for dissolving lipids:

4.3.1 Solvent Selection

The choice of solvent depends on the lipid being dissolved and its role in the liposome formulation. The solvent must be of analytical grade, and its purity must be verified by the QC team before use:

• 4.3.1.1 Chloroform is commonly used for dissolving phospholipids due to its excellent lipid solubility and ability to form stable lipid films upon evaporation.
• 4.3.1.2 Methanol and ethanol may be used for dissolving cholesterol and other lipid components. Ensure that the solvents are anhydrous to prevent moisture from affecting lipid stability.
• 4.3.1.3 For lipids sensitive to oxidation, use solvents that have been purged with an inert gas (e.g., nitrogen or argon) to reduce oxygen exposure.

4.3.2 Dissolution Process

• 4.3.2.1 Transfer the weighed lipid components into a clean glass beaker or flask.
• 4.3.2.2 Add the solvent in a volume that is sufficient to fully dissolve the lipid components. Stir the mixture using a magnetic stirrer to facilitate dissolution.
• 4.3.2.3 Maintain the temperature of the solution between 20°C-40°C using a hot plate. Excessive heat should be avoided as it can degrade lipid components.
• 4.3.2.4 Continue stirring the solution until all lipid components are fully dissolved. This may take anywhere from 10 to 60 minutes, depending on the lipid type and solvent used.
• 4.3.2.5 If necessary, filter the lipid solution through a membrane filter to remove any undissolved particles. Record any filtration steps in the BMR.

4.4 Lipid Film Formation

Once the lipids are fully dissolved, the lipid solution must be dried to form a thin lipid film. This is a critical step in the liposome preparation process, as the lipid film will later be hydrated to form liposomes. The following steps outline the lipid film formation process:

• 4.4.1 Transfer the lipid solution into a round-bottom flask suitable for rotary evaporation.
• 4.4.2 Set up the rotary evaporator and apply vacuum to remove the solvent. Rotate the flask slowly to ensure that the lipid film forms evenly across the inner surface of the flask.
• 4.4.3 Continue the evaporation process until the solvent is fully removed, and a thin, dry lipid film forms on the wall of the flask. This process may take between 30 minutes and 2 hours, depending on the type of lipid and solvent used.
• 4.4.4 If necessary, apply gentle heating (<40°C) during the evaporation process to speed up solvent removal, but ensure the temperature does not exceed the lipid degradation threshold.
• 4.4.5 Once the solvent has been completely removed, stop the rotation and vacuum. Allow the lipid film to cool to room temperature before proceeding to the next step.
• 4.4.6 If the lipid film is not to be used immediately, it can be stored for future use. Transfer the lipid film into an appropriate container (e.g., a glass vial) and seal it tightly to prevent exposure to air or moisture.
• 4.4.7 Store the lipid film under an inert atmosphere (e.g., nitrogen or argon) in a desiccator or freezer at -20°C or lower, depending on the stability requirements of the lipid components.
• 4.4.8 Label the storage container with the lipid film name, batch number, preparation date, and expiration date. Record the storage details in the storage log (see Annexure 2).

4.5 Handling and Storage of Lipid Components

Proper handling and storage of lipid components are crucial for maintaining their integrity and preventing degradation. Lipids are highly sensitive to oxidation and moisture, and incorrect storage conditions can result in lipid degradation, affecting the quality of the final liposome formulation. The following handling and storage precautions must be followed:

• 4.5.1 Minimize the exposure of lipid components to air, light, and moisture during both the preparation and storage processes. Use containers that are air-tight and opaque to protect the lipids from environmental factors.
• 4.5.2 For storage, ensure that lipid components are kept in temperature-controlled environments. Sensitive lipids should be stored at -20°C or lower, and containers should be kept in a desiccator to prevent exposure to humidity.
• 4.5.3 If handling lipid powders, wear appropriate personal protective equipment (PPE), including gloves and lab coats, to avoid direct skin contact and contamination of the lipid material.
• 4.5.4 Lipid components should be handled in a fume hood or glove box, especially if organic solvents are involved, to protect operators from inhalation hazards and ensure product safety.
• 4.5.5 Always use solvents that are free from water and other contaminants to prevent unwanted reactions or degradation of the lipid components. Solvents should be stored in sealed containers under an inert atmosphere.
• 4.5.6 Any unused lipid components must be stored in their original containers, properly labeled with the component name, batch number, and expiration date. If the lipid is stored in a new container, transfer all necessary labeling information.
• 4.5.7 Ensure that storage areas are regularly inspected and monitored for temperature and humidity. Any deviations from the specified storage conditions must be documented, and corrective action should be taken immediately.
• 4.5.8 When using lipid components in liposome formulations, ensure that the components are within their expiration dates and meet all QC requirements before use.

5) Abbreviations, if any

• PPE: Personal Protective Equipment
• BMR: Batch Manufacturing Record
• QA: Quality Assurance
• QC: Quality Control

6) Documents, if any

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

7) References, if any

• FDA Guidelines for Lipid-Based Drug Formulations
• International Conference on Harmonisation (ICH) Q7: Good Manufacturing Practice Guide for Active Pharmaceutical Ingredients
• USP <1163> Good Distribution Practices for Bulk Pharmaceutical Excipients

8) SOP Version

Version 1.0

Annexure

Annexure 1: Batch Manufacturing Record Template

Batch No.
Lipid Component
Weight
Solvent
Dissolution Time
Operator Initials
QA Signature


Batch Number
Lipid Name
Weight in grams
Solvent Name
Minutes
Operator Name
QA Name


 
 
 
 
 
 
 

Annexure 2: Raw Material Storage Log Template

Date
Material Name
Batch No.
Storage Condition
Location
Operator Initials


DD/MM/YYYY
Material Name
Batch Number
Temperature/Humidity
Storage Area
Operator Initials