Isolation of influenza viral proteins by size-exclusion and ion-exchange high-performance liquid chromatography: the influence of conditions on separation

Improved particle counting and size distribution determination of aggregated virus populations by asymmetric flow field‐flow fractionation and multiangle light scattering techniques

“…The particle volume (V), equal to (4/3)p( ffiffiffiffiffiffiffi ffi 5=3 p R g ) 3 , was determined for spheres, once the RMS radius was measured experimentally as described in the Materials and Methods section. The formulae for obtaining radius (R m ) from R g were based on the full integration of the peak area that includes the mass distribution for the particle of interest.…”

“…Ultracentrifugation is often used for virus purification and concentration, 1,2 but relatively low-virus concentrations in the samples make this application difficult to use for quantitative analysis. Size exclusion chromatography (SEC) could also be applied for the study of viral samples 3 ; however, in SEC separation, the interaction of virus with the stationary phase may bias the original monomer-aggregate profile of the sample. A more practical technique may be asymmetric flow field-flow fractionation (AFFFF), which has been gaining widespread use.…”

Reversed phase monolithic analytical columns for the determination of HA1 subunit of influenza virus haemagglutinin

“…For this reason various chromatographic methods for determining the concentration of the influenza virus and profiling of its proteins started to emerge. Phelan and Cohen managed to separate the major proteins of the detergent-disrupted influenza virus by RP-HPLC on conventional porous media, however, individual protein recoveries were low [20], as was the case with the ion-exchange chromatography [21]. Size exclusion chromatography showed higher potential to separate viral proteins with recoveries >90% [21].…”

“…Phelan and Cohen managed to separate the major proteins of the detergent-disrupted influenza virus by RP-HPLC on conventional porous media, however, individual protein recoveries were low [20], as was the case with the ion-exchange chromatography [21]. Size exclusion chromatography showed higher potential to separate viral proteins with recoveries >90% [21]. A 2D HPLC method was reported for quantitative detection of virus proteins in vaccines, utilizing SEC and a non-porous silica RP column [22].…”

Protein purification

“…Recent widespread use of both ionic and nonionic detergents has, in many cases, allowed proteins with unusual solubility properties to be isolated. Particularly illustrative of this is the purification of integral plasma membrane proteins[130][131][132]. Ionic detergents (e.g.…”