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Wiley, Biotechnology Journal, 3(12), p. 1600583, 2017

DOI: 10.1002/biot.201600583

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Drying techniques for the visualisation of agarose-based chromatography media by scanning electron microscopy

Journal article published in 2016 by Mc Nweke, Mark Turmaine, Rg Graham McCartney, Dg Bracewell ORCID
This paper is made freely available by the publisher.
This paper is made freely available by the publisher.

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Abstract

The drying of chromatography resins prior to scanning electron microscopy is critical to image resolution and hence understanding of the bead structure at sub-micron level. Achieving suitable drying conditions is especially important with agarose-based chromatography resins, as over-drying may cause artefact formation, bead damage and alterations to ultrastructural properties; and under-drying does not provide sufficient resolution for visualisation under SEM. This paper compares and contrasts the effects of two drying techniques, critical point drying and freeze drying, on the morphology of two agarose based resins (MabSelect(TM) : dw ~ 85 µm and Capto(TM) Adhere: dw ~75 µm) and provides a complete method for both. The results show that critical point drying provides better drying and subsequently clearer ultrastructural visualisation of both resins under SEM. Under this protocol both the polymer fibres (thickness ~20 nm) and the pore sizes (diameter ~100 nm) are clearly visible. Freeze drying is shown to cause bead damage to both resins, but to different extents. MabSelect resin encounters extensive bead fragmentation, whilst Capto Adhere resin undergoes partial bead disintegration, corresponding with the greater extent of agarose crosslinking and strength of this resin. While freeze drying appears to be the less favourable option for ultrastructural visualisation of chromatography resin, it should be noted that the extent of fracturing caused by the freeze drying process may provide some insight into the mechanical properties of agarose-based chromatography media.