To investigate the complex process dynamics of liposome preparation, we’ve developed the analytical tools based on well known methods in the field of liposome characterisation, centrifugation and ultracentrifugation of liposome dispersions.
The lipid powder prepared by spray-drying technology was wetted and hydrated to produce the initial liposome dispersion. The presence of optically dense particles in the dispersions, in addition to the liposomes, was detected using an optical microscope. These particles were separated from liposomes in the dispersions using centrifugal force. The resulting sediment dispersed in the buffer (pH65 °C = 5,5) had a significantly lower drug substance : phospholipid mass ratio compared to the (final) lyophilisate after the membrane filtration process and thus represented an unwanted fraction in the dispersion. By optimising the wetting and hydration processes of the lipid powder, we sought to reduce the amount of sediment. The individual impacts of pH buffer solution, mixing time during hydration of the lipid powder, type of stirrer and type of lipid powder were systematically evaluated, and the most significant impacts were determined by multivariate data analysis. We have also shown the applicability of centrifugation for the investigation of wetting and hydration processes of the lipid powder when scaling up to production. Preparation of the initial liposome dispersions was followed by high-pressure homogenisation of the dispersions to reduce size of the liposomes and membrane filtration to ensure sterility of the final product. The liposome dispersions from the different stages of the high pressure homogenisation process were ultracentrifuged and the sediment was dispersed in the buffer (pH65 °C = 5,5). Chromatographic and AF4-MALS methods were used to prove that the particles in the sediment were not liposomes. The UV VIS spectrophotometry based on the change in absorbance of the drug substance was identified as a potential method for quantification of the amount of drug substance in the dispersed sediment and also as a method for quantification of the amount of (unwanted) sediment in the dispersions during the high-pressure homogenisation process, as the mass ratio of drug substance : phospholipids in the sediment did not change during the high pressure homogenisation process. Furthermore, we demonstrated the usefulness of quantification of the sediment in dispersions after the high-pressure homogenisation process to ensure the feasibility of membrane filtration of the liposome dispersions. To further understand the membrane filtration process of dispersions, we have applied AF4 MALS method based on the radius of gyration of the particles in the dispersed sediment.
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