Characterization of lysozyme adsorption in cellulosic chromatographic materials using small-angle neutron scattering

Abstract

Measurements of the nanoscale structure of chromatographic adsorbents and the associated distribution of sorbed protein within the media can facilitate improvements in such media. We demonstrate a new technique for this purpose using small-angle neutron scattering (SANS) to characterize the nano- to microscale structure of the chromatographic media and sorbed protein under conditions relevant for preparative chromatographic separations. The adsorption of lysozyme on cellulosic S HyperCel™ (Pall Corporation), a strong cation exchanger, was investigated by SANS. The scattering spectrum is reduced to three contributions arising from (1) the chromatographic medium, (2) discrete protein molecules, and (3) the distribution of sorbed protein within the medium. These contributions are quantified for a range of protein loadings. The total concentration of protein in the chromatographic media can be quantified from the SANS spectrum and the protein is observed to retain its tertiary structure upon adsorption, within the resolution of the method. Further analysis of the SANS spectra shows that protein adsorption is uniform in the media. These measurement techniques provide new and valuable nanoscale information about protein sorption in chromatographic media.

Characterization of cross-linked cellulosic ion-exchange adsorbents: 2. Protein sorption and transport

“…Small-angle neutron scattering on these materials suggests that protein adsorbs fairly uniformly within the particle (Koshari et al, 2015), including the possibility of protein partitioning throughout most of the particle volume. The globular formations that can be seen in the electron micrographs makes the determination of other structural characteristics somewhat difficult.…”

“…Further evidence that HyperCel displays polymer-modified-like characteristics is apparent from small-angle neutron scattering (Koshari et al, 2015) and from SEM imaging of adsorbed phases, in which the pore architecture could be more clearly seen in STAR AX HyperCel when protein was adsorbed, a system where definite structural characteristics could not be resolved via TEM in a phase devoid of protein. The work presented provides a good complement to the investigation of the structural characteristics for the same materials, as adsorption of protein has a definite impact on the native architecture of the beads, and by extension, the transport mechanisms that govern the movement of solutes through these stationary phases.…”

Ionic strength-dependent changes in tentacular ion exchangers with variable ligand density. I. Structural properties

“…We are not aware of previous use of SAXS to characterize such sorbents, although neutron scattering has recently been used to characterize lysozyme adsorption on cellulosic adsorbents [32]. …”

Double probe approach to protein adsorption on porous carbon surfaces

“…[22,23,24] These techniques are unique in that they can detect the spatial structure and organisation of molecules adsorbed inside the porous medium. [25] For a full understanding of how proteins are adsorbed in porous carbons, the use of…”

“…[8] Upon adsorption, proteins can denature or change their conformation, and hence their activity, and thus may present a health hazard. [10,11] For lysozyme, an enzyme of molecular weight M W =14.6 kDa, the maximum adsorption on untreated porous carbon surfaces occurs at its isoelectric point, around pH 11.…”