Morgan, Peter Edward; (1997) Non-porous pseudoaffinity supports for the recovery of antibodies. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The major costs of production of monoclonal antibodies (MAb's) are incurred during downstream processing. Techniques which utilise the specificity of an affinity interaction have the potential to reduce the number of unit operations to a minimum and thus enable savings to be made in the overall costs of production. The preparation and detailed characterisation of two types of immobilised metal affinity adsorbent for the recovery of MAb's from Chinese hamster ovary (CHO) culture is described in this thesis. Non-porous metal chelating perfluorocarbon supports were prepared by coating particulate perfluorocarbon materials with polyvinyl alcohol which was subsequently activated and derivatised with iminodiacetic acid (IDA) functions. The preparation conditions were systematically optimised to produce stable coated and highly derivatised metal chelate adsorbents (2-5μmoles Zn2+/mL) Supports charged with zinc ions selectively bound MAb and non-specific adsorption to uncharged supports was not observed. The specific binding capacity of the best chelating perfluorocarbon adsorbent (chelating PTFEP; 9mg/mL) is close to that of a porous metal chelate adsorbent (chelating Streamline™; 13mg/mL). The chelating perfluorocarbons are very stable to treatment with a wide variety of chemicals, and this combined with their non-porous nature make them attractive alternatives to conventional affinity adsorbents. Non-porous magnetic chelators were produced from iron oxide particles made by the controlled alkali precipitation of iron salts. The iron oxide cores were silanised and subsequently coated with polyglutaraldehyde, epoxy activated and coupled with IDA. These supports are densely derivatised with IDA (30-60μmoles Me2+/mL) and exhibit high specific binding capacities for the test MAb's. Although Ni2+ charged supports were much more effective than Zn2+ charged supports for the recovery of MAb from PBS (~40mg/mL c.f ~21mg/mL), this advantage was lost when the adsorbents were employed in real process liquors. The presence of suspended cells appeared to have no effect on performance but some component(s) in the CHO medium selectively desorped (~50%) of nickel from charged supports. Nickel and zinc charged magnetic chelators were used to recover MAb directly from CHO fermentation's. MAb's bound to zinc and nickel charged adsorbents could be recovered at high yield and purity (~99%) and no detrimental effects of medium components or suspended material were observed.

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