SEPARATION OF PROTEINS BY ION EXCHANGE AND MEMBRANE CHROMATOGRAPHY: BUFFER COMPOSITION, INTERFERING IMPURITIES AND FOULING CONSIDERATIONS
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Efficient separation of target protein from impurities is crucial in bioseparation for large scale production and purity of the target protein. Two separation process approaches were considered in this study. The first approach focused on identifying major impurity and optimization of solution properties for target protein purification. The second approach consisted of designing an adsorbent that interacted specifically with the target molecule. The first study included modification of protein solution properties (pH, ionic strength, buffer ions) in order to maximize lysozyme purification by a strong cation exchange resin. The interaction of phytic acid, a major impurity, present in transgenic rice extracts, that contributes to decreased lysozyme adsorption capacity on SP Sepharose was evaluated. The target protein was lysozyme, which is used in a purified form as a baby formula additive to reduce gastrointestinal tract infections. At constant ionic strength, lysozyme in pH 4.5 acetate buffer had a higher binding capacity and stronger binding strength than at pH 6.0. Lysozyme in sodium phosphate buffer of pH 6.0 exhibited lower adsorption capacity than in pH 6 Tris buffer. Binding capacity and strength were significantly affected by phytic acid in all studies buffers. The second study consisted of surface modification of microfiltration membranes for protein purification and separation and reduces fouling. This study describes adsorption and fouling of chemically modified microfiltration membranes with bovine serum albumin (BSA) and immunoglobulin G (IgG). Least fouling resulted with polyethylene glycol (PEG) membranes when BSA protein was used. Amine-functionalized membranes showed specific interaction with BSA. There was multi-layer deposition of IgG on amine-functionalized membrane. G3 membrane synthesized to selectively bind IgG seemed a noble option to separate IgG from a protein mixture.