Purification of a functional gene therapy vector derived from Moloney murine leukaemia virus using membrane filtration and ceramic hydroxyapatite chromatography.
The ability of membrane ultra- and diafiltration and two chromatography media, Matrex Cellufine Sulfate (Millipore) and Macro-Prep ceramic hydroxyapatite (Bio-Rad), to adsorb, elute, and purify gene therapy vectors based on Moloney murine leukaemia virus (MoMuLV) carrying the 4070A amphotropic envelope protein was studied. Membrane ultra- and diafiltration provided virus concentration up to 160-fold with an average recovery of infectious viruses of 77 +/- 14%. In batch experiments, Macro-Prep ceramic hydroxyapatite (type 2, particle size 40 microm) proved superior to Matrex Cellufine Sulfate for MoMuLV vector particle adsorption. Furthermore, functional vector particles could be eluted using phosphate buffer pH 6.8 (highest titres from >or=300 mM phosphate) from the Macro-Prep adsorbent, with higher specific titres (cfu/mg protein) than the starting material. Similar results were obtained when this ceramic hydroxyapatite was packed into a column and used in a liquid chromatography system. Recovery of transduction-competent virus was between 18 and 31% for column experiments and 32 and 46% for batch experiments.
D002516:Ceramics / Q000737:chemistry*; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D017886:Durapatite / Q000737:chemistry*; D015870:Gene Expression; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000302:isolation & purification*; D007475:Ion Exchange Resins / Q000737:chemistry*; D008567:Membranes, Artificial; D008979:Moloney murine leukemia virus / Q000235:genetics* / Q000302:isolation & purification; D011108:Polymers; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014462:Ultrafiltration / Q000295:instrumentation / Q000379:methods
D007475:Ion Exchange Resins; D008567:Membranes, Artificial; D011108:Polymers; C080965:sulfate cellufine; D017886:Durapatite