A novel purification strategy for retrovirus gene therapy vectors using heparin affinity chromatography

Abstract

Membrane separation and chromatographic technologies are regarded as an attractive alternative to conventional academic small-scale ultracentrifugation procedures used for retrovirus purification. However, despite the increasing demands for purified retroviral vector preparations, new chromatography adsorbents with high specificity for the virus have not been reported. Heparin affinity chromatography is presented here as a novel convenient tool for retrovirus purification. The ability of bioactive retroviral particles to specifically bind to heparin ligands immobilized on a chromatographic gel is shown. A purification factor of 63 with a recovery of 61% of functional retroparticles was achieved using this single step. Tentacle heparin affinity supports captured retroviral particles more efficiently than conventional heparin affinity chromatography supports with which a lower recovery was obtained (18%). Intact, infective retroviral particles were recovered by elution with low salt concentrations (350 mM NaCl). Mild conditions for retrovirus elution from chromatographic columns are required to preserve virus infectivity. VSV-G pseudotyped retroviruses have shown to be very sensitive to high ionic strength, losing 50% of their activity and showing membrane damage after a short exposure to 1M NaCl. We also report a complete scaleable downstream processing scheme for the purification of MoMLV-derived vectors that involves sequential microfiltration and ultra/diafiltration steps for virus clarification and concentration respectively, followed by fractionation by heparin affinity chromatography and final polishing by size-exclusion chromatography. Overall, by using this strategy, a 38% yield of infective particles can be achieved with a final purification factor of 2,000.

Highly efficient concentration of lenti- and retroviral vector preparations by membrane adsorbers and ultrafiltration

“…But many chromatographic media are inapplicable because of the relatively labile nature of lenti- and retroviral vectors [11]. Affinity chromatography strategies are based on streptavidin-biotin interaction [31], heparin binding [32] and immobilized metal affinity [33]. In anionic exchange chromatography the negatively charged viral vectors bind to the positively charged chromatographic material.…”

“…We found that only one hour of exposure to high salt (1 M NaCl) on ice is enough to inactivate 16.0 ± 6.4% of the LVs (Additional file 1, Figure S1). Others observed a reduction by 50% of infectious retroviral particles when exposed to high salt for one hour at room temperature [32]. Therefore, further purification steps are necessary [32].…”

“…Others observed a reduction by 50% of infectious retroviral particles when exposed to high salt for one hour at room temperature [32]. Therefore, further purification steps are necessary [32]. However, anionic exchange requires no modification of the viral vectors such as biotinylation or his-tagging and is fast, versatile and easy to scale up [11,35,36].…”

“…Elution of the viral particles was achieved by application of high salt buffer. However, due to toxic effects of salt buffers with high molarity [32,38] subsequent rebuffering is necessary. We used UF units for desalting and concentration, leading to high titer LV preparations with a biological titer in the range of 2 to 5 × 10 9 IP/ml.…”

Scale-up and manufacturing of clinical-grade self-inactivating γ-retroviral vectors by transient transfection

“…Although significant advances in downstream processing of gRV vectors have been made in the past several years using chromatography- or centrifugation-based strategies, 27,28 the susceptibility of gRV vector particles to temperature, pH, ionic strength, shear stress and freeze–thaw cycles 29 have limited the purification and concentration strategies for large-scale clinical manufacturing. To support production of high-titer SIN gRV vectors by transfection without the need for further concentration, several modifications have been made to the viral backbone to improve RNA processing and increase titer of non-concentrated vector stocks as described previously.…”