Preparation of chemically modified bead cellulose resins and their application to protein purification : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University
A bead cellulose matrix, Perloza TM, was chemically modified to prepare inexpensive resins for chromatography. Conventional and novel resins were produced. Adsorption and elution methods suitable for industrial chromatography were developed. An agarose matrix, Sepharose TM, was used for comparison. Matrix activation with carbonyldiimidazole (CDI) was optimised for Sepharose and Perloza. Improved, reliable performance was obtained using column solvent exchange, with an imidazole tracer. Substitution efficiency of 75-98% was obtained for aminoacyl ligands/spacer arms by minimising water content. The aqueous carboxymethylation level obtained for Perloza was 0.3-0.4 mMoles/g dry. This was increased to 1.3-2 mMoles/g dry, using 75-80% DMSO solvated Perloza, Epichlorohydrin and bisepoxide activation levels (+/- organic solvents) were low. Etherification of Perloza with allyl bromide or allyl glycidyl ether resulted in high allylation levels (> 1.50 mMoles/g), even in aqueous media. Matrix allyl groups were reacted with bromine water or aqueous N-bromosuccinimide, to produce (predominantly) bromohydroxypropyl groups. Subsequent attachment of amine and thiol ligands, by nucleophilic substitution, was simple and efficient. Allyl matrices were also used for free radical addition of sulphite and various thiols (mercaptoethanol, mercaptoacids, glutathione). Efficient addition was found without thermal or chemical catalysis. Addition of mercaptoacetic acid followed by carboxylate titration was the preferred measure of (allyl) activation level. Addition of several other thiols occurred at 60°C. The usefulness of allyl chemistries was exemplified by preparation of ion exchange resins. Their physical and chromatographic properties compared favourably with commercial resins. They combined good laboratory performance with high flow rates and simple, cheap preparation suited to large scale use. Mixed mode resins were prepared from CDI and allyl matrices. These contained charged (secondary amine or carboxylate) and hydrophobic (alkyl spacer arm and/or ligand) groups. The milk clotting enzyme chymosin, was adsorbed to these resins at high and low ionic strength. Near homogeneous chymosin was eluted by a pH change, which induced electrostatic repulsion. Alkyl carboxylate resins were preferred. They were simple to prepare, use and regenerate, despite the use of crude broths. The presence of charged groups could cause non-specific adsorption, interference with target protein adsorption and greater fouling. Weak acid and base hydrophobic groups (e.g. pyridyl) were attached to matrices and titration confirmed that uncharged and charged forms were obtained in a pH range (5-9) suitable for protein chromatography. At low ligand density, the salt promoted hydrophobic adsorption properties of these resins (uncharged from) were similar to those of Phenyl Sepharose. At higher ligand density, retention was longer, eventually leading to adsorption independent of ionic strength. Complete elution was obtained by pH adjustment (to the partially ionised resin form). Chymosin was strongly adsorbed to uncharged pyridyl (hydrophobic ionisable) resins and rapidly eluted by a small pH change. High ligand density (strong adsorption) is favourable for large scale use because the ionic strength of feedstreams does not need to be adjusted prior to loading. Strong adsorption to mixed mode and weakly ionisable resins was also found for amylase. Rapid elution (and significant purification) was again obtained by a small pH change. Subtilisin was adsorbed likewise by most hydrophobic ionisable resins and recovered efficiently at pH 5.2. However, subtilisin adsorption to mixed mode resins was comparatively weak, possibly reflecting the weaker hydrophobicity of subtilisin compared to amylase. The adsorption of catalase on Phenyl Sepharose and (low ligand density) pyridyl Perloza was equivalent, at pH 7.5. Catalase was eluted by a pH change from the Perloza resin, whereas elution from Phenyl Sepharose required addition of ethylene glycol. This indicated that pyridyl Perloza resins would be useful for chromatography of very hydrophobic proteins.