Downstream processing of recombinant retroviral vectors

Liste des illustrations

• Figure 1 Retroviral particle structure
• Figure 2 Assays used for the quantitation of total retrovirus particles in vector stocks and transduction-competent particles.
• Figure 3 Flow chart for the downstream processing of retroviral gene therapy vectors.
• Figure 4 Analysis of the harvested supernatants during the retrovirus production phase.
• Figure 5 Effect of temperature on VSV-G pseudotyped retrovirus stability.
• Figure 6 Effect of NaCl concentration on VSV-G pseudotyped retrovirus infectivity.
• Figure 7 Effect of ionic strength on virus morphology by NSEM.
• Figure 8 Breakthrough curve for retroviral particles in Fractogel^® EMD Heparin (S) gel.
• Figure 9 Heparin affinity chromatography step gradient elution profile.
• Figure 10 Size exclusion chromatography elution profile.
• Figure 11 Western Blot analyses of heparin affinity chromatography fractions.
• Figure 12 Purification performance analysis by SDS-PAGE silver stained.
• Figure 13 Monitoring purity by NSEM in sequential purification steps.
• Figure 14 Scheme for the purification process of retroviral particles.
• Figure 15 Rate zonal sedimentation in continuous iodixanol gradients.
• Figure 16 Electrophoretic analyses of iodixanol gradient fractions.
• Figure 17 Size-exclusion chromatography elution profile.
• Figure 18 Electrophoretic analyses of size exclusion chromatography purified retrovirus particles.
• Figure 19 Electrophoretic analyses of subtilisin treated vs untreated retrovirus particles.
• Figure 20 Transmission electron micrographs of negatively stained virions using uranyl acetate staining.
• Figure 21 Distribution of infectious virus particles and total virus particles throughout the gradient.
• Figure 22 The effect of heparin on VSV-G retrovirus transduction to target cells.
• Figure 23 Binding of Env-protein deficient particles to heparin ligands.
• Figure 24 Binding affinity of RD114 (A) and VSV-G (B) pseudotyped retrovirus particles to heparin affinity ligands.
• Figure 25 Heparin affinity chromatography step gradient elution profile for RD114 pseudotyped vector.
• Figure 26 Potential attachment mechanisms for MoMLV-derived vectors.