Removal of envelope protein-free retroviral vectors by anion-exchange chromatography to improve product quality.
We have investigated the role of the retroviral lipid bilayer and envelope proteins in the adsorption of retroviral vectors (RVs) to a Fractogel DEAE matrix. Intact RVs and their degradation components (envelope protein-free vectors and solubilized vector components) were adsorbed to this matrix and eluted using a linear gradient. Envelope protein-free RVs (Env(-)) and soluble envelope proteins (gp70) eluted in a significantly lower range of conductivities than intact RVs (Env(+)) (13.7-30 mS/cm for Env(-) and gp70 proteins vs. 47-80 mS/cm for Env(+)). The zeta (zeta)-potential of Env(+) and Env(-) vectors was evaluated showing that envelope proteins define the pI of the viral particles (pI (Env(+)) < 2 versus 3 < pI (Env(-)) < 4) and that Env(+) and Env(-) vectors have similar zeta-potentials within pH 5 and 8. The results presented herein indicate that the adsorption of retroviral particles occurs through multi-point interaction of the envelope proteins with the cationic groups on the chromatographic matrix. The strength of this adsorption is thus dependent on the amount of envelope protein present in the viral lipid bilayer. In conclusion, AEXc enables the separation of gp70 proteins as well as envelope protein-free vectors constituting a significant improvement to the quality of retroviral preparations for gene therapy applications.
Rodrigues
Teresa
T
ITQB-UNL, Oeiras, Portugal.
Alves
Ana
A
Lopes
António
A
Carrondo
Manuel J T
MJ
Alves
Paula M
PM
Cruz
Pedro E
PE
D000327:Adsorption; D000818:Animals; D000838:Anions / Q000737:chemistry*; D002784:Cholesterol / Q000032:analysis; D002852:Chromatography, Ion Exchange / Q000295:instrumentation / Q000379:methods; D004279:DNA, Viral / Q000032:analysis; D003902:Detergents / Q000737:chemistry; D005822:Genetic Vectors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000648:ultrastructure; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D010316:Particle Size; D012313:RNA / Q000032:analysis; D015203:Reproducibility of Results; D012190:Retroviridae / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000648:ultrastructure; D014759:Viral Envelope Proteins / Q000737:chemistry*
D000838:Anions; D004279:DNA, Viral; D003902:Detergents; D014759:Viral Envelope Proteins; D012313:RNA; D002784:Cholesterol