Cross-flow filtration—an improved detergent removal technique for the preparation of liposomes

Abstract

Using a cross-flow (tangential) filtration device, a lipid-detergent mixed micellar solution is filtered under pressure through a suitable membrane with a molecular weight exclusion limit of 10–50 kDa. In contrast to commonly used dead-end ultrafiltration filters, the solution flows tangential to the membrane surface. A wave-like flow of the solution over the membrane is effected due to the shape of the membrane, preventing clogging during filtration. Detergent monomers pass through the membrane and are collected as waste filtrate, whereas the mixed micelle solution is continuously pumped as retentate through the device until liposome formation is completed. Buffer solution is introduced into the system at the same rate as the filtrate is removed therefrom to keep the retentate volume constant. Variations of the following parameters were studied in detail: (1) membrane cutoff; (2) filtration area; (3) flow rate; (4) starting lipid/detergent ratio; (5) choice of detergent; and (6) cholesterol content. We determined the kinetics of detergent removal and compared filtration time, amount of filtrate, vesicle size and stability of the resulting liposome dispersions. Cross-flow filtration under optimum conditions results in liposome formation within minutes and in principle makes unlimited upscaling possible.