Removal of envelope protein‐free retroviral vectors by anion‐exchange chromatography to improve product quality

Abstract

Original PaperRemoval of envelope protein-free retroviral vectors by anion-exchange chromatography to improve product qualityWe have investigated the role of the retroviral lipid bilayer and envelope proteins in the adsorption of retroviral vectors (RVs) to a Fractogel TM 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 (f)-potential of Env + and Env -vectors was evaluated showing that envelope proteins define the pI of the viral particles (pI (Env + ) a 2 versus 3 a pI (Env -) a 4) and that Env + and Env -vectors have similar f-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.

Lentiviral Vector Purification Using Nanofiber Ion-Exchange Chromatography

“…The absence of RT activity and lack of RNA genome and/or envelope proteins cause the formation of deficient/non-infectious vectors and contribute to heterogenicity of LV harvests in their production. Although minimizing the formation of these product-related impurities is part of the upstream optimization, ion-exchange chromatography has the potential to remove soluble retroviral envelope proteins and particles lacking the envelope protein from the biologically active retroviral particles with the envelope proteins 36 . With the expansion of ligand types and chromatography modes, additional opportunities for the application of nanofibers in other viral vector DSP steps (e.g., polishing) are possible.…”

Impact of physicochemical parameters on in vitro assembly and disassembly kinetics of recombinant triple‐layered rotavirus‐like particles

“…Isoelectric point determination using titration curves based on zeta potential measured with DLS, at different pH, is simpler and more accurate than electrophoresis. Recent works used DLS for pI determination of adenovirus type 5 (Trilisky and Lenhoff, 2007) and retrovirus (Rodrigues et al, 2008); however, this is the first report where the pI of a VLP was determined using this technique. Also, Schaldach et al (2006) determined experimentally the pI of three viruses (Norwalk, MS2, and Qb) in order to study their interaction with charged surfaces.…”

Lentiviral Vector Bioprocessing

“…LVs have a net negative charge at neutral pH on their surface [228] due to the composition of the external envelope and their overall isoelectric point, therefore an AEX chromatography with quaternary amines (QA) or diethylaminoethyl (DEAE) ligands are viable options for their capture and elution. With retro or lentiviral vectors, elution tends to be stepped, with the sequential increases in salt concentrations firstly eluting loosely bound components to improve purity before a final high salt buffer elutes the vector.…”

“…For a weak ligand such as DEAE, this profile can resemble pH 8 and NaCl concentrations of 0.1 and 0.65 M [190]. Stepped elution is particularly effective with vectors, whereby their large size and variable surface composition allow for interaction at multiple sites on the stationary phase and are thus retained to a greater extent compared to individual feed components [228]. This does, however, require greater salt concentrations or pH shift to elute, which necessitates immediate dilution to maintain stability due to osmotic effects [223] or envelope degradation [8,9].…”