The mechanical stirring method uses mixing and shear to promote vesicle formation by phospholipids in an aqueous phase. Agitation also helps disperse the formulation and influence the liposome size distribution. The process typically includes raw material preparation, initial dispersion, high shear mixing, solvent removal, drug loading, and downstream processing and storage. The operating conditions depend on the lipid composition, drug properties, and equipment configuration.
Raw material preparation
Lipid selection and composition
Common membrane forming materials include egg yolk lecithin and soybean lecithin. Cholesterol may be added to adjust membrane fluidity and stability. The initial process specifies a phospholipid to cholesterol molar ratio of 4:1 to 6:1. The actual ratio should be determined according to the formulation and its intended properties.
Aqueous phase and drug preparation
Hydrophilic drugs can be dissolved in water for injection or phosphate buffer at pH 7.0 to 7.4. For hydrophobic drugs, a suitable organic solvent may be selected according to their solubility. Examples include anhydrous ethanol and ether. Solvent selection should account for formulation compatibility, residual solvent limits, and handling safety. Solvents with unacceptable toxicological risks should be avoided.
Preparation of the initial dispersion
Dissolve the lipid materials in anhydrous ethanol to obtain a lipid solution at 10 to 20 mg/mL. Transfer the solution to a jacketed stirred reactor and adjust the temperature to 30 to 40°C. Slowly add the aqueous phase containing the dissolved drug or the selected buffer solution.
Maintain the stirring speed at 100 to 300 r/min for 5 to 10 minutes to obtain a relatively uniform initial dispersion. Provide suitable ventilation during operation to prevent organic solvent vapors from accumulating.
High shear mixing and particle size control
After the initial dispersion has formed, increase the stirring speed to 800 to 1500 r/min. Continue mixing at 30 to 40°C for 15 to 30 minutes. Mechanical shear helps improve dispersion and can affect the resulting liposome size distribution.
Stirring speed and mixing time alone do not determine the final particle size. Lipid composition, formulation concentration, reactor geometry, and agitator design also influence the result. If a defined particle size or size distribution is required, verify the process using particle size measurements. Mixing conditions or additional size control steps may need to be adjusted based on the results.
Organic solvent removal
After initial dispersion and high shear mixing, remove the residual organic solvent by reduced pressure distillation where appropriate. The original process specifies a reactor pressure of negative 0.08 to negative 0.09 MPa and a temperature of 35 to 40°C, with agitation maintained during solvent removal.
The endpoint should be determined according to the solvent used, formulation characteristics, and residual solvent requirements. The absence of a noticeable solvent odor is not sufficient to confirm that the residual solvent level meets specification.
For pharmaceutical formulations, residual ethanol should be assessed against the applicable pharmacopoeial requirements and residual solvent guidelines. A limit of 5000 ppm is used in relevant guidance under specified conditions, but it should not be treated as a universal limit for every formulation.
Drug loading and pH adjustment
If the drug was not added during initial dispersion, it may be introduced at a later stage according to the drug properties and formulation design. The original process specifies an additional 10 minutes of stirring after adding the free drug to promote its association with the lipid system.
The loading method depends on drug solubility, lipid affinity, and the intended location of the drug within the liposome. Additional stirring alone does not ensure adequate encapsulation. Encapsulation efficiency and free drug content should be evaluated for the specific formulation.
After the relevant processing steps, adjust the formulation to pH 7.0 to 7.4 using phosphate buffer. If further improvement in particle size distribution is required, use the measurement results to guide changes in stirring conditions or the selection of suitable shear mixing equipment.
Downstream processing and storage
Sterilizing filtration
For liposome formulations suitable for filtration, a 0.22 μm membrane filter may be evaluated for sterilization. Before implementation, confirm that the liposome size and formulation properties are compatible with the selected filter. The process should be validated for sterilizing performance, product recovery, and post-filtration quality.
Separation of free drug
Centrifugation or ultrafiltration may be selected to separate unencapsulated drug, depending on the drug and liposome properties. The original process specifies centrifugation at 10000 to 15000 r/min for 20 to 30 minutes. Actual centrifugation conditions should be established based on the rotor radius, relative centrifugal force, and formulation properties. Rotational speed alone is insufficient to define the separation conditions.
Storage
The finished liposome formulation may be stored at 4°C or sealed after lyophilization, provided the selected approach is suitable for the formulation. Storage conditions, shelf life, and quality requirements should be established through stability evaluation.
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