Purification of a functional gene therapy vector derived from Moloney murine leukaemia virus using membrane filtration and ceramic hydroxyapatite chromatography

Abstract

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.

Strategy for selection of methods for separation of bioparticles from particle mixtures

“…Intracellular particulate products produced with microorganisms Table II Extracellular particulate products produced with microorganisms (Beards et al, 1993;Buque-Taboada et al, 2004;Kometani et al, 1997;Kumagai, 1999;Leuenberger, 1985;Matsumae et al, 1995;Michielsen et al, 2000a,b;Miller, 1985;Nakayama, 1985;Spassov et al, 1996;Takahashi, 2003) VLPs produced produced with microorganisms (Andrews et al, 1995;Cruz et al, 2000;Kitano et al, 1987;Kuiper et al, 2002;Meijer et al, 1996;Moran, 1999;Tsoka et al, 2000) Solid products produced with immobilized catalysts (Takahashi, 2003;Davison et al, 1997;Kasche and Galunsky, 1995;Kuhl et al, 1990;Lee et al, 1999a;Zmijewski et al, 1991) Production of solid products and solid by-products with enzymes (Blacker and Holt, 1997;Youshko et al, 2002) (Bowden et al, 1991;Fischer et al, 1992;Fischer et al, 1995;Gram et al, 1994;Hellebust et al, 1989;Hoess et al, 1988;Honda et al, 2000;…”

“…Again it is clear that the production of these bioparticles gives rise to a particle-particle separation problem. Purification of VLPs is currently performed with a wide variety of separation techniques, such as centrifugation (Cruz et al, 2000;Tsoka et al, 2000), filtration (Cruz et al, 2000;Kuiper et al, 2002;Tsoka et al, 2000), extraction without dissolution (Andrews et al, 1995;Kitano et al, 1987), and chromatography (Cruz et al, 2000;Kuiper et al, 2002;Tsoka et al, 2000) that are used in a large train of separation operations. Direct VLP isolation by particleparticle separation would reduce the number of process steps and might thus lower the downstream processing costs.…”

Surface Modification of Retroviral Vectors for Gene Therapy

“…Alternatively viruses may be concentrated and purified by ultra-filtration or dialysis protocols, separating virus and contaminants according to size using semipermeable membranes. Conventional column based methods lead to problems regarding large-scale preparations, but use of tangential flow devices can circumvent this aspect (Geraerts et al 2005;Kuiper et al 2002).…”

Performance and characterization of a nanophased porous hydroxyapatite for protein chromatography

“…Besides purification of nucleic acids (Bachvarov and Ivanov, 1983;Colman et al, 1978;Freitag, 2000, 2002;Johnson and Ilan, 1983;Kuiper et al, 2002;Pakroppa et al, 1975;Shoyab and Sen, 1979;Udvardy et al, 1979), a prime application of hydroxyapatite adsorbents is the purification of antibodies at small scale as well as industrial scale (Aoyama and Chiba, 1993;Bowles et al, 1988;Giovannini and Freitag, 2001;Jungbauer et al, 1989;Luellau et al, 1998;Pavlu et al, 1986;Poiesi et al, 1989;Zola and Neoh, 1989). Another standard adsorbent for large-scale separation of antibodies in the biopharmaceutical industry is staphylococcal Protein A affinity chromatography (Boschetti and Jungbauer, 2000;Jungbauer and Wenisch, 1989).…”