Large-scale production of lipoplexes with long shelf-life

Large-scale production of lipoplexes with long shelf-life
Jule Clementa, 1, , , Karin Kiefera, 1, Andrea Kimpflera, Patrick Garidelb and Regine Peschka-S?ss
January 2005
European Journal of Pharmaceutics and Biopharmaceutics
Abstract
The instability of lipoplex formulations is a major obstacle to overcome before their commercial application in gene therapy. In this study, a continuous mixing technique for the large-scale preparation of lipoplexes followed by lyophilisation for increased stability and shelf-life has been developed. Lipoplexes were analysed for transfection efficiency and cytotoxicity in human aorta smooth muscle cells (HASMC) and a rat smooth muscle cell line (A-10 SMC). Homogeneity of lipid/DNA-products was investigated by photon correlation spectroscopy (PCS) and cryotransmission electron microscopy (cryo-TEM). Studies have been undertaken with DAC-30?, a composition of 3?-[N-(N,N'-dimethylaminoethane)-carbamoyl]-cholesterol (DAC-Chol) and dioleylphosphatidylethanolamine (DOPE) and a green fluorescent protein (GFP) expressing marker plasmid. A continuous mixing technique was compared to the small-scale preparation of lipoplexes by pipetting. Individual steps of the continuous mixing process were evaluated in order to optimise the manufacturing technique: lipid/plasmid ratio, composition of transfection medium, pre-treatment of the lipid, size of the mixing device, mixing procedure and the influence of the lyophilisation process.
It could be shown that the method developed for production of lipoplexes on a large scale under sterile conditions led to lipoplexes with good transfection efficiencies combined with low cytotoxicity, improved characteristics and long shelf-life.