Polymer Displacement and Supermacroporous Cryogels - Two New Tools in Chromatographic Separation of Biomolecules and Bioparticles

University dissertation from Department of Biotechnology, Center for Chemistry and Chemical Engineering, P.O. Box 124, SE-221 00 Lund, Sweden

Abstract: Production of protein is often accompanied with difficulties due to the large amounts of different biological material resulting from fermentation. This makes purification necessary and affinity chromatography is usually involved as being biospecific, although suffering from limitations as low recovery due to ineffective elution and inability to handle particulate materials. Displacement of lactate dehydrogenase (LDH) from dye-affinity matrices with poly(ethyleneimine) (PEI) resulted in better recoveries and sharper elution profiles than traditional non-specific elution but with unchanged purification factors. Hence, displacement with PEI is a promising strategy for eluting proteins with reported low recoveries in dye-affinity chromatography. Displacement of (His)6-LDH, a hexahistidine-tagged thermostable LDH originating from a thermophilic bacterium, was carried out using a synthetic copolymer of vinyl imidazole and vinyl caprolactam as displacer from an immobilized metal affinity chromatography (IMAC) column. Complete elution of bound (His)6-LDH from Cu2+-imminodiacetic acid Sepharose was achieved at a co-polymer concentration of 5 mg/ml, corresponding to 3.7 mM imidazole units, while 160 mM free imidazole was needed to obtain the same result. Thus, efficiency could be significantly improved by a polymeric displacer having interacting groups of the same chemical nature as the corresponding low-molecular mass displacer. Continuous supermacroporous chromatographic columns with anion exchange ligands (2-(dimethylamino)ethyl group) and IMAC ligands (Cu2+-loaded iminodiacetic acid) have been developed allowing binding of E. coli cells and elution of bound cells with high recoveries and retained viability as well as direct capture of enzyme from non-clarified crude cell homogenate at high flow rates without mechanical obstruction. These poly(acrylamide)-based matrices were produced by radical co-polymerization of monomers in aqueous solution frozen inside a column (cryogelation). After thawing, the column contains a continuous cryogel matrix with interconnected 10 - 100 mm pores. These large pores make processing of particulate-containing material without blocking possible. Purification of (His)6-LDH from non-clarified crude extract without preliminary filtration or centrifugation was possible on the IMAC cryogel column. Totally, 210 ml crude homogenate was processed on a single five ml supermacroporous column at flow speeds up to 12.5 ml/min (625 cm/h) without impairment of column properties. E. coli cells bound to an IMAC-column were eluted with 80 % recovery using either 10 mM imidazole or 20 mM EDTA solutions and cells bound to an ion-exchange column at low ionic strength were eluted with 70 - 80% recovery at NaCl concentrations of 0.35 - 0.40 M, while it was possible for cells to pass unhindered through a column without ligands. Cells maintained viability after the chromatographic procedures. Mechanically the cryogel adsorbent is very stable. The matrix could easily be removed from the column, dried at 70 °C and kept in a dry state. After rehydration, (His)6-LDH was purified as efficiently as on a newly prepared column. Materials for manufacturing the cryogels are cheap, available and are simply mixed and frozen under specified conditions. Altogether, these qualities reveal that supermacroporous continuous cryogels is a very interesting alternative to existing methods of protein purification from particulate-containing crude extracts as well as an exciting new support for handling of microbial cells in a chromatographic mode.

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