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Effects of bed compression on protein separation on gel filtration chromatography at bench and pilot scale
Author(s) -
Kong Darryl YC,
Gerontas Spyridon,
McCluckie Ross A,
Mewies Martin,
Gruber David,
TitchenerHooker Nigel J
Publication year - 2018
Publication title -
journal of chemical technology and biotechnology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.64
H-Index - 117
eISSN - 1097-4660
pISSN - 0268-2575
DOI - 10.1002/jctb.5411
Subject(s) - chromatography , theoretical plate , chemistry , size exclusion chromatography , sepharose , porosity , agarose , compression (physics) , protein purification , elution , bovine serum albumin , materials science , analytical chemistry (journal) , composite material , organic chemistry , enzyme
BACKGROUND Poorly packed chromatography columns are known to reduce drastically the column efficiency and produce broader peaks. Controlled bed compression has been suggested to be a useful approach for solving this problem. Here the relationship between column efficiency and resolution of protein separation are examined when preparative chromatography media were compressed using mechanical and hydrodynamic methods. Sepharose CL‐6B, an agarose based size exclusion media was examined at bench and pilot scale. The asymmetry and height equivalent of a theoretical plate (HETP) was determined by using 2% v/v acetone, whereas the void volume and intraparticle porosity ( ϵ p ) were estimated by using blue dextran. A protein mixture of ovalbumin (chicken), bovine serum albumin (BSA) and γ'‐ globulin (bovine) with molecular weights of 44, 67 and 158 kDa, respectively, were used as a ‘model’ separation challenge. RESULTS Mechanical compression achieved a reduction in plate height for the column with a concomitant improvement in asymmetry. Furthermore, the theoretical plate height decreased significantly with mechanical compression resulting in a 40% improvement in purity compared with uncompressed columns at the most extreme conditions of compression used. CONCLUSION The results suggest that the mechanical bed compression of Sepharose CL‐6B can be used to improve the resolution of protein separation. © 2017 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.