Preparation of immuno-stimulating complexes (ISCOMs) by ether injection

preparation of liposomes, as a new, continuous and potentially scaleable method for the preparation of ISCOMs. Phosphatidylcholine (PC) and cholesterol (Chol) were dissolved in ether, which was injected into an aqueous solution, maintained at 55 degrees C, containing Quil A. The influences of the following variables on ISCOM formation were investigated: ratio of PC:Quil A:Chol used, pumping rate, total lipid mass and concentration of buffer salts and Quil A in the aqueous phase. All samples were characterized by negative stain transmission electron microscopy, photon correlation spectroscopy and sucrose ultracentrifugation gradient. It was demonstrated that ISCOMs could be produced by this method but the homogeneity of the preparation was influenced by the conditions used. Homogeneous ISCOM preparations were consistently produced only when the weight ratio of PC:Quil A:Chol was 5:3:2 with a total lipid mass of 20 mg, the Quil A dissolved in a 0.01 M phosphate buffer at a concentration of 6 mg in 4 ml, and the ether solution injected into the warmed buffer solution at a rate of 0.2 ml/min. Changing any of these variables resulted in more heterogeneous preparations in which ISCOMs typically co-existed with other colloidal structures such as worm-like and helical micelles, liposomes, lamellae and lipidic particles. (C) 2005 Elsevier B.V. All rights reserved.

Saponins from Quillaja saponaria Molina: Isolation, Characterization and Ability to Form Immuno Stimulatory Complexes (ISCOMs)

ISCOMs have received much attention as vaccine adjuvants due to their immunostimulatory effects. They are colloidal particles typically comprised of phospholipids, cholesterol and Quil A, a crude mixture of saponins extracted from the bark of Quillaja saponaria Molina. We have previously shown that ISCOMs can be prepared by ether injection wherein an ether solution of phospholipids and cholesterol in a mass ratio of 5:2 is injected into a solution of Quil A at a mass ratio of 7 lipids: 3 Quil A. The aim of this study was firstly to isolate and characterise discrete fractions of Quil A and secondly to investigate which of these fractions were able to form ISCOMs by the method of ether injection. Six fractions of Quil A were isolated by semi-preparative reverse phase high performance liquid chromatography (RP-HPLC) and characterised by analytical HPLC, liquid chromatography tandem mass spectrometry (LC-MS) and the qualitative Liebermann- Burchard and Molisch tests for triterpenoids and carbohydrates respectively. ISCOMs were subsequently prepared from the isolated fractions by the method of ether injection and the resulting preparations characterized by photon correlation spectroscopy (PCS) and negative stain transmission electron microscopy (TEM). The molecular weights of the major compounds in the fractions ranged from ∼1200 to ∼2300 Da; all fractions tested positive for triterpenoids and saccharides and four of the fractions were identified as QS-7, QS-17, QS-18 and QS-21 by analysis (LC-MS and analytical HPLC). Injection of ether solutions of lipids into aqueous solutions of QS-17, QS-18 or QS-21 all resulted in homogeneous ISCOM dispersions. The combination of lipids and QS-7 by ether injection produced lamellae and liposomes as the prominent structures and a minor amount of ISCOMs. The remaining two hydrophilic, low molecular weight fractions of Quil A did not produce ISCOMs, instead liposomes and helical structures predominated in the samples.