Affinity chromatography is a crucial method of protein purification, used during the manufacture of many therapeutic antibodies. The protein A ligand is the most common choice for selective purification of IgG molecules. Aggregated product is often found in protein A elution pools; this is generally attributed to the effect of low pH (elution) on protein structure. However, there is evidence that other parameters of the chromatography process influence aggregation phenomena. Physical and chemical transitions, like concentration of protein on the adsorbent surface and change in buffer composition, may challenge the structural integrity of proteins, increasing their propensity for aggregation.<br/>To advance understanding of how adsorption processes might impact antibody stability, neutron reflectivity was used to characterize the structure of adsorbed IgG on model surfaces. In the first model system IgG was adsorbed directly to silica and demonstrated a flat-on-surface orientation. In the second model system, protein A was attached to a silica surface to produce a configuration representative of a silica chromatography resin. Adsorbed IgG structures extended up to 230 Å away from the surface and are suggestive of two IgG molecules bound to protein A with a skewed orientation.<br/>To complement our simplified planar NR model systems, SANS studies on model packed columns where the concentrations, interactions, and flow fields experienced are much more comparable to typical chromatographic separations, were carried out. To that end, we have worked closely with ISIS and NIST staff to construct a SANS compatible flow cell to closely mimic an affinity column and provide the ability to both change buffer conditions in situ on the same sample and study the effect of flowing buffers through. The flow through cell was packed with Prosep-Ultra plus resin (Protein A coated silica beads) and loaded with increasing levels of IgG representative of bioprocessing. This experiment suggests that the resin has a complex structure composed of a range of pore sizes and within this structure the IgG does not adsorb uniformly across these pore surfaces – a process largely dependent on its concentration. Upon increasing concentration, IgG molecules migrate from the external pores (i.e. closest to the bead surface) onto the internal, smaller ones. This hypothesis can be supported by the fact that an additional peak is visible during the SANS experiment at higher IgG concentrations.

Neutron scattering to characterise protein interactions with solid-liquid interfaces in bioprocessing

255th National Meeting (2018)

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