Affinity chromatography is an indispensable method of protein purification used during the manufacture of many therapeutic antibodies. The protein A ligand is a popular choice for selective purification of immunoglobulin G (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 facets of the chromatography process influence aggregation phenomena. Physical and chemical transitions, such as concentration of protein on the adsorbent surface and change in buffer composition, may challenge the structural integrity of proteins, increasing their propensity for aggregation. The influence of elution from protein A on the aggregation rate of an IgG4 during a subsequent low pH hold was investigated. IgG4 was incubated in elution buffer after protein A chromatography and the monomer concentration in neutralised samples was quantified by size exclusion chromatography. Rate constants for monomer decay over time were determined by fitting exponential decay functions to the data. Similar low pH experiments were implemented in the absence of a chromatography step. The IgG4 demonstrated highly pH-dependent and apparently concentration-independent aggregation behaviour. The findings suggested that aggregation was governed predominantly by a pH-dependent unfolding/ re-folding equilibrium. Elution from protein A was found to increase aggregation rates by half an order of magnitude, while other aspects of the aggregation kinetics appeared un-affected. In order to advance understanding of how adsorption processes might impact antibody stability, neutron reflectivity was used to characterise the structure of adsorbed IgG on model surfaces. In the first model system IgG was adsorbed directly to silica and demonstrated a side-on orientation with high surface contact. A maximum dimension of 60Å in the surface normal direction and high density surface coverage were observed under acidic pH conditions. In the second model system protein A was attached to a silica surface to produce a configuration representative of a glass chromatography resin. Interfacial structure was probed during sequential stages from ligand attachment, through IgG binding and elution. Adsorbed IgG structures extended up to 230Å away from the surface and showed dependence on surface blocking strategies. The data was suggestive of two IgG molecules bound to protein A with a somewhat skewed orientation and close proximity to the silica surface. The findings provide insight into the orientation of adsorbed antibody structures under conditions encountered during chromatographic separations. The outcomes of this work provide a broad scope for future investigations. Based on preliminary work, using neutron measurement techniques to monitor aggregate formation inside glass chromatography resins is suggested as an interesting direction.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:763092 |
| Date | January 2016 |
| Creators | Mazzer, A. |
| Contributors | Bracewell, D. G. ; Dalby, P. A. ; O'Hara, J. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1478395/ |
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