Zulqarnain, Kamran; (2001) Scale-up of affinity separation based on magnetic support particles. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

There is an incentive to introduce affinity separation techniques earlier in downstream processing due to their superior selectivity and recovery capabilities. However, as most conventional affinity separations tend to use porous adsorbents they are susceptible to fouling from colloidal matter present in early stage feedstocks. There is evidence to suggest that non-porous matrices are more resistant to fouling and therefore potentially more suitable for particulate and foulant feedstreams. To obtain a comparable surface area to typical porous supports for ligand immobilisation, the dimensions of a non-porous particle have to be much smaller, and their separation from cell debris again presents a problem. Magnetic separation allows the recovery of such small particles in the presence of biological matter with relative ease. This thesis describes the preparation and characterisation of sub-micron magnetic particles using simple chemical precipitation techniques amenable to scale up, to produce suitable iron oxide matrices for the manufacture of affinity supports. These adsorbents were subsequently used in laboratory scale experiments for the selective recovery of proteins from crude liquors. Preparative scale recovery of magnetic adsorbents was then carried out using high gradient magnetic separation (HGMS) technology. An optimal ratio of Fe2+/Fe3+, 1:2, was identified to produce non-porous superparamagnetic crystals (~ 12 nm in diameter) with a specific surface area of 110 m2 g-1, a saturation magnetism of ~40 emu g-1 combined with low residual magnetism. These particles were silanised and subsequently coated with polyglutaraldehyde, epoxy activated, capped with ammonium groups and coupled with Cibacron Blue. The selectivity and interaction of these supports were assessed by using enzymes (lactate, malate and alcohol dehydrogenases) that are known to have an affinity for Cibacron Blue. High binding capacities (> 110 mg g-1) and affinities (kd < 1.2 μM) were obtained. Furthermore, the presence of suspended solids in the homogenate appeared to have no negative affect on the performance of the adsorbents in the recovery of the target molecule. In a separate study lysozyme magnetic particles (LMP) were prepared from the polyglutaraldehyde coated supports and successfully used to recover Fv fragments from clarified E. coli lysate. On a preparative scale using HGMS, Fv fragments of high purity (98 %) were eluted in a single step with a purification factor of 54.1. A parallel use of the LMP was to release periplasmic α-amylase from E. coli cells. This technique compared favourably to the standard release method of using free lysozyme.

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