| Summary: |
The AdTech-P2 Project is focused on some advanced technologies allowing the purification of target proteins. The Monolithic, Simulated Moving Bed(SMB) and Membrane technologies are included. A model system of bovine serum albumin(BSA) and myoglobin will be used. The main goal is the separation of four subclasses of glycoprotein immunoglobulin G (IgG), a major effector molecule of the humoral immune response in man.
With the approval of the cancer medication Rituxan for treatment of B-cell non-Hodgkins lymphoma (NHL) in 1997, many other monoclonal antibodies (mAbs) have been considered in cancer therapies. The majority of mAbs products in development are IgG's. As a consequence of the structural differences, the four IgG subclasses - IgG1, IgG2, IgG3 and IgG4 - show differences in some of their physicochemical characteristics and biological properties. Thus, selecting the appropriate subclass of antibody for specialized characteristics can yield antibodies with increased safety and efficacy.
Liquid chromatography is one of the dominant protein purification methods (ion exchange chromatography, hydrophobic interaction, reverse phase chromatography, etc.). Some singular packing materials for chromatographic separation of proteins have been developed considering the advantages linked to the perfusion chromatography. "Perfusion chromatography" allows better column efficiency and higher separation speed than with conventional packings. In the same direction, the monolithic stationary phases are characterized by having very large and interconnected pores-channels allowing high separation speed (forced convection plus diffusive transport).
The key concept behind the improved performance of large-pore, permeable packings is the "augmented" diffusivity by convection. This project treats of the separation and purification of proteins with emphasis on the adsorption kinetics of multi-component mixture of proteins: (i) on large pore particle with bi  |
Summary
The AdTech-P2 Project is focused on some advanced technologies allowing the purification of target proteins. The Monolithic, Simulated Moving Bed(SMB) and Membrane technologies are included. A model system of bovine serum albumin(BSA) and myoglobin will be used. The main goal is the separation of four subclasses of glycoprotein immunoglobulin G (IgG), a major effector molecule of the humoral immune response in man.
With the approval of the cancer medication Rituxan for treatment of B-cell non-Hodgkins lymphoma (NHL) in 1997, many other monoclonal antibodies (mAbs) have been considered in cancer therapies. The majority of mAbs products in development are IgG's. As a consequence of the structural differences, the four IgG subclasses - IgG1, IgG2, IgG3 and IgG4 - show differences in some of their physicochemical characteristics and biological properties. Thus, selecting the appropriate subclass of antibody for specialized characteristics can yield antibodies with increased safety and efficacy.
Liquid chromatography is one of the dominant protein purification methods (ion exchange chromatography, hydrophobic interaction, reverse phase chromatography, etc.). Some singular packing materials for chromatographic separation of proteins have been developed considering the advantages linked to the perfusion chromatography. "Perfusion chromatography" allows better column efficiency and higher separation speed than with conventional packings. In the same direction, the monolithic stationary phases are characterized by having very large and interconnected pores-channels allowing high separation speed (forced convection plus diffusive transport).
The key concept behind the improved performance of large-pore, permeable packings is the "augmented" diffusivity by convection. This project treats of the separation and purification of proteins with emphasis on the adsorption kinetics of multi-component mixture of proteins: (i) on large pore particle with bimodal pore size distribution and (ii) in monolithic columns, both of them for application in gradient SMB chromatography. Gradient SMB is a challenging manner to remove process inefficiencies in isocratic SMB systems and the application of proteins mixtures onto these two kinds of stationary phases will show the potential of the technique for recovery of proteins, in particular, of IgG MAbs.
The development of the separation process for protein purification will be carried out by means of screening the influence of concentration of salt and pH in mobile phase. The equilibrium and kinetics of proteins adsorption will be determined experimentally (for the model system and the multi-compound IgG mixture). These process characteristics will be used by the developed simulation software to evaluate the SMB behaviour (and performance) and to allow the design of the optimized operating unit (configuration, operating parameters, scale-up).
In order to concentrate and produce pure proteins free of salt from the collection streams of the gradient SMB system, the membrane separation will be included. The efficiency of this filtration step (UF/MF) extensively depends on molecular weight cut-offs of membranes and conditions of the feed-stock (salt concentration, pH, solvent strength), and, so, experimental analysis on this unit operation will be addressed.
A considerable amount of work has been done to find antibody purification methods; the protein purification technology is still in the growth phase. Also, there is an increased interest in the monolithic columns in preparative bio-separations. The LSRE, committed t |