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Recombinant protein purification, Hydrophobic Interaction Chromatography (HIC), Simulated Moving Bed Chromatography (SMBC), Adsorption isotherms, Equilibrium theory
Abstract:
Liquid chromatography plays an important role in the recombinant protein purification processes, right from product capture to product polishing. High throughput techniques like simulated moving bed (SMB) chromatography, which operate in simulated counter-current mode, offer advantages like product enrichment, increase in productivity and decrease in eluent consumption compared to the batch chromatography. Investigation of SMB chromatography for downstream processing of recombinant proteins will be useful to demonstrate the potential of SMBC as an industrial tool to cope with higher production titers.
In this work, streptokinase (SK) was over expressed as soluble intracellular protein in E.coli. The complex protein mixture containing recombinant streptokinase was chosen as a case study for protein purification using SMBC. HIC matrices were chosen for the SMBC process, based on a screening strategy which included evaluation of selectivity and column regeneration.
Model-based design saves time and material during the multivariable process development. Model based design and optimisation of the simulated moving bed (SMB) chromatographic process requires accurate estimation of adsorption isotherm parameters in the range of elution conditions. Adsorption isotherm parameters for streptokinase (SK) were determined based on isocratic elution techniques with purified recombinant streptokinase on a buffer equilibrated column. The linearity of the isotherms were validated by perturbation techniques on a column saturated with crude cell lysate. The Henry's constants for streptokinase were found to exhibit a linear dependence on the salt concentration range of 100-200 mM in the mobile phase.
Adsorption isotherm parameters determined were used to design a three-zone open-loop simulated moving bed-hydrophobic interaction chromatography (SMB-HIC) process. The fourth zone was used for independent regeneration and pre-equilibration of the columns. Theoretical studies were carried out based on the equilibrium model and the equilibrium stage model, to find the suitable operating parameters. Two-step salt gradient conditions were simulated by choosing two different salt concentrations in the feed and desorbent streams.
Experiments were conducted and the theoretical model predicting the separation zone was validated. Continuous separation was demonstrated for streptokinase from contaminants present in a clarified solution of E. coli cell lysate. Although simulation results predicting more ideal separation behavior were at variance with experimental results, the trends from the experiments for product purity were in agreement with the theoretical trends. The effect of various parameters such as desorbent flow rate and switch times was investigated on the target product concentration. This work demonstrates the potential of the SMB process for the recombinant protein purification. It also demonstrates the efficiency and limitations of a design-based approach based on knowledge of isotherm parameters for the product and impurities. Future studies would need to incorporate more complex models, such as those incorporating adsorption-desorption kinetics and non-linear isotherms.
