TY - JOUR

T1 - Recombinant protein purification using gradient-assisted simulated moving bed hydrophobic interaction chromatography. Part I

T2 - Selection of chromatographic system and estimation of adsorption isotherms

AU - Palani, Sivakumar

AU - Gueorguieva, Ludmila

AU - Rinas, Ursula

AU - Seidel-Morgenstern, Andreas

AU - Jayaraman, Guhan

N1 - Funding Information: We acknowledge the support given by the Bioprocess Engineering Group at MPI and the Leibniz Institute for Neurobiology in Magdeburg. The financial support of Deutscher Akademischer Austausch Dienst (DAAD), Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 578) and the travel grant for S.P. from GDCh allowing to attend the HPLC 2009 conference are gratefully acknowledged.

PY - 2011/9/16

Y1 - 2011/9/16

N2 - The design of gradient simulated moving bed (SMB) chromatographic processes requires an appropriate selection of the chromatographic system followed by the determination of adsorption isotherm parameters in the relevant range of mobile phase conditions. The determination of these parameters can be quite difficult for recombinant target proteins present in complex protein mixtures. The first part of this work includes the estimation of adsorption isotherm parameters for streptokinase and a lumped impurity fraction present in an Escherichia coli cell lysate for a hydrophobic interaction chromatography (HIC) matrix. Perturbation experiments were carried out using a Butyl Sepharose matrix with purified recombinant protein on buffer equilibrated columns as well as with crude cell lysate saturated columns. The Henry constants estimated for streptokinase were found to exhibit in a wide range a linear dependence on the salt concentration in the mobile phase. These parameters were applied in subsequent investigations to design a simulated moving bed (SMB) process capable to purify in a continuous manner recombinant streptokinase from the E. coli cell lysate.

AB - The design of gradient simulated moving bed (SMB) chromatographic processes requires an appropriate selection of the chromatographic system followed by the determination of adsorption isotherm parameters in the relevant range of mobile phase conditions. The determination of these parameters can be quite difficult for recombinant target proteins present in complex protein mixtures. The first part of this work includes the estimation of adsorption isotherm parameters for streptokinase and a lumped impurity fraction present in an Escherichia coli cell lysate for a hydrophobic interaction chromatography (HIC) matrix. Perturbation experiments were carried out using a Butyl Sepharose matrix with purified recombinant protein on buffer equilibrated columns as well as with crude cell lysate saturated columns. The Henry constants estimated for streptokinase were found to exhibit in a wide range a linear dependence on the salt concentration in the mobile phase. These parameters were applied in subsequent investigations to design a simulated moving bed (SMB) process capable to purify in a continuous manner recombinant streptokinase from the E. coli cell lysate.

KW - Adsorption isotherm parameters

KW - Hydrophobic interaction chromatography

KW - Perturbation method

KW - Recombinant streptokinase

KW - Simulated moving bed

UR - http://www.scopus.com/inward/record.url?scp=80051866110&partnerID=8YFLogxK

U2 - 10.1016/j.chroma.2011.06.111

DO - 10.1016/j.chroma.2011.06.111

M3 - Article

C2 - 21816402

AN - SCOPUS:80051866110

VL - 1218

SP - 6396

EP - 6401

JO - Journal of Chromatography A

JF - Journal of Chromatography A

SN - 0021-9673

IS - 37

ER -