Critical Assessment of Purification and Analytical Technologies for Enveloped Viral Vector and Vaccine Processing and Their Current Limitations in Resolving Co-Expressed Extracellular Vesicles

Abstract

Viral vectors and viral vaccines are invaluable tools in prevention and treatment of diseases. Many infectious diseases are controlled using vaccines designed from subunits or whole viral structures, whereas other genetic diseases and cancers are being treated by viruses used as vehicles for delivering genetic material in gene therapy or as therapeutic agents in virotherapy protocols. Viral vectors and vaccines are produced in different platforms, from traditional embryonated chicken eggs to more advanced cell cultures. All these expression systems, like most cells and cellular tissues, are known to spontaneously release extracellular vesicles (EVs). EVs share similar sizes, biophysical characteristics and even biogenesis pathways with enveloped viruses, which are currently used as key ingredients in a number of viral vectors and licensed vaccine products. Herein, we review distinctive features and similarities between EVs and enveloped viruses as we revisit the downstream processing steps and analytical technologies currently implemented to produce and document viral vector and vaccine products. Within a context of well-established viral vector and vaccine safety profiles, this review provides insights on the likely presence of EVs in the final formulation of enveloped virus products and discusses the potential to further resolve and document these components.

Investigating Environmental Matrices for Use in Avian Influenza Virus Surveillance—Surface Water, Sediments, and Avian Fecal Samples

“…Another method to concentrate AIV from smaller water volumes took advantage of formaldehyde-stabilized chicken erythrocytes for AIV binding; erythrocytes express sialic acid receptors utilized by influenza viruses to attach to permissive host cells ( 23 ). In addition, other methods such as ultracentrifugation, chromatography, and PEG precipitation were described for influenza A virus purification from cell culture supernatants but not from surface water ( 24 – 26 ). No general best-practice enrichment technology has been identified so far.…”

Rapid isolation and quantification of extracellular vesicles from suspension‐adapted human embryonic kidney cells using capillary‐channeled polymer fiber spin‐down tips

“…Alternatively, the initial increase in EV yield may reflect an accelerated expression rate during the cell number growth phase, slowing as that process reaches a steady state. Though none of the identified works have monitored EV release during the production of therapeutic vectors/products, it would be interesting to assess potential relationships between the productivity of mAb‐ or viral vector‐producing cell lines and EV release characteristics [ 63 , 64 , 65 , 66 ].…”

Efficient clinical-grade γ-retroviral vector purification by high-speed centrifugation for CAR T cell manufacturing

“… 28 , 38 Continuous flow centrifugation is currently being used for large-scale vaccine manufacture. 39 However, shear effects exerted on viral particles through high centrifugal forces cause structural damage, leading to decreased overall recovery. 40 Given the volume constraint of ultracentrifugation-based methods and the complexity of continuous-flow centrifugation, we hypothesized that high-speed centrifugation as a single step would allow the purification and concentration of γ-RV volumes sufficient for early-phase clinical studies.…”