206 research outputs found
A validated model for the simulation of protein purification through affinity membrane chromatography
No full textA mathematical model is proposed for the description of protein purification through membrane affinity chromatography. The model describes all the three stages of the chromatographic cycle and takes into account convection, axial dispersion and binding reaction kinetics in the porous membrane matrix, while boundary layer mass transfer resistance is shown to be negligible. All the model parameters have a precise physical meaning which enables their evaluation through separate experimental measurements, independent of the chromatographic cycle. Model testing and validation has been performed with experimental chromatographic cycles carried out with pure IgG solutions as well as with complex mixtures containing IgG1, using new affinity membranes. The comparison between model calculations and experimental data showed good agreement for all stages of the affinity cycle. In particular, for loading and washing steps binding kinetics was found so fast that adsorption equilibrium was sufficient to describe the observed behavior; as a result, the model simulations are entirely predictive for the adsorption and washing phases. On the contrary, in the elution step the reaction rate is comparable to that of the other simultaneous transport phenomena.
The model is able to predict the performance of chromatographic purification of IgG from complex mixtures simply on the basis of the parameter values obtained from pure IgG solutions
Influence of protein adsorption kinetics on breakthrough broadening in membrane affinity chromatography
No full textExisting mathematical models developed to describe membrane affinity chromatography are unable to match the complete breakthrough curve when a single Langmuir adsorption isotherm is used, because important deviations from the observed behaviour are systematically encountered in the simulation of breakthrough broadening near saturation. The relevant information required to overcome that limitation has been obtained by considering simultaneously both loading and washing curves, thus evaluating the adsorption data at equilibrium and recognizing what are the appropriate adsorption mechanisms affecting the observed behavior. The analysis indicates that a bi-Langmuir binding kinetics is essential for a correct process description up to the saturation of the stationary phase, together with the use of the relevant transport phenomena already identified for the experimental system investigated. The input parameters used to generate the resulting simulations are evaluated from separate experiments, independent from the chromatographic process. Model calibration and validation is accomplished comparing model simulations with experimental data measured by feeding pure human immunoglubilin G (IgG) solutions as well as a cell culture supernatant containing human monoclonal IgG1 to B14-TRZ-Epoxy2 bio-mimetic affinity membranes. The simulations obtained are in good agreement with the experimental data over the entire adsorption and washing stages, and breakthrough tailing appears to be associated to the reversible binding sites of the bi-Langmuir mechanism. Remarkably, the model proposed is able to predict with good accuracy the purification of IgG from a complex mixture simply on the basis of the results obtained from pure IgG solutions
Advances in membrane affinity chromatography for the recovery of antibodies
No full textRecovery of antibodies with Protein A affinity chromatography columns has become the standard for the biotech industry. Membrane affinity chromatography has never taken off the ground due to the lower capacity of membrane supports compared to chromatographic beads.
In this work new affinity membranes endowed with high capacity for IgG will be presented. These membranes have been experimentally characterized using a novel approach that integrates experiments with a mathematical modelling. All the relevant parameters like static and dynamic binding capacity, selectivity and purity of the recovered antibody have been evaluated and will be discussed. Indications towards scale-up and comparison with the state of the art of affinity chromatography will be addressed
A simulation model for membrane affinity chromatography processes
No full textChromatography represents one of the most important and widely used unit operation in the biotechnology industry. However this technique suffers from several limitations such as high pressure drop, slow mass transfer through the diffusive pores and strong dependence of the binding capacity on flow rate. One of the alternatives that are receiving increasing attention is represented by membrane chromatography, for which it is imperative to develop a reliable simulation model able to describe the process performance in a predictive way.
In the present work a novel model is proposed that can describe all the chromatographic steps involved in the membrane affinity chromatography process for protein purification. The mathematical description is based on the species continuity equation coupled with a proper binding kinetic equation, and suitable to describe adequately the dispersion phenomena occurring both in the micro-porous membranes as well as in the extra-column devices used in the system. The model considers specifically all the different chromatographic steps, namely adsorption, washing and elution.
Model validation is achieved by comparing simulation results with an extensive set of experimental data which have been obtained for the purification of immunoglobulin G from a cell culture supernatant, using several different innovative affinity membranes and using a broad spectrum of operating conditions.
The few relevant fitting parameters of the model were derived from a calibration with experimental affinity cycles performed with pure IgG solutions, then the model is used to describe experimental data obtained in chromatographic cycles carried out with complex feeds as the cell culture supernatant.
Simulations reveal a good agreement with experimental data in all the chromatography steps, both in the case of pure IgG solutions and for the cell culture supernatant considered
Experimental and theoretical study of membrane adsorbers for the primary capture of protein manufacturing
No full textMembrane technology plays an important role in bioprocessing, however the use of membrane adsorbers for the primary capture is still not implemented due to the limited binding capacity of membranes with respect to chromatography beads. In this work, affinity membranes endowed with high capacity have been applied to the purification of monoclonal antibodies.
To this aim novel membrane matrices have been functionalized with affinity ligands using Protein A as well as synthetic ligands that exhibit affinity for the Fc portion of antibodies. These new affinity membranes have been characterized both in batch and in chromatographic experiments using pure protein solutions and a cell culture supernatant. The effect of operating conditions on process performance have been studied in detail in order to find the optimal conditions for binding and elution steps.
A fundamental study of transport and kinetic phenomena involved in the process has been performed. As a result, a rather complete mathematical model for the affinity membrane process has been developed and validated with the data acquired with different promising membrane materials. The model is predictive for all the different stages, adsorption, washing and elution, and it is a useful tool to assess the use of this technology in large scale process
Novel affinity membranes for IgG purification
No full textMembrane chromatography is a purification technique that is receiving increasing attention by the biotechnology industry. However, the industrial application of this process has been limited by the relative low binding capacity of membranes with respect to chromatography beads.
In this work the purification of IgG using novel affinity membranes endowed with improved performances will be discussed. The membranes have been prepared using a new family of pre-activated epoxy membranes functionalized with natural and synthetic affinity ligands that show high specificity towards the Fc portion of antibodies.
The resulting affinity membranes have been fully characterized in batch and dynamic experiments with both pure IgG solutions and a cell culture supernatant containing monoclonal IgG1. The influence of membrane structure parameters and ligand density on separation performance has been investigated to draw indications for material improvements. The relevant process parameters like the maximum binding capacity, the affinity equilibrium constant and selectivity have been obtained in complete adsorption, washing and elution chromatographic cycles; the results for the different affinity membranes tested were critically analyzed and compared with data of commercially available membrane adsorbers.
Considerations about the application of the improved affinity materials in industrial scale will be addressed
Experimental evaluation and theoretical analysis of affinity membrane adsorbers
No full textMembrane affinity chromatography is an innovative technique that is receiving increasing attention by the biotechnology industry. Membrane chromatography is not limited by diffusion as the process based on resin beads, since the main transport mechanism is due to convection through the porous structure; therefore it is particularly suitable for purifying large bio-molecules such as IgG.
In the present work the purification of immunoglobulin G using novel affinity membranes endowed with improved performance is discussed. The membrane adsorbers studied derive from the functionalization of epoxy membranes with natural and synthetic affinity ligands that show high specificity towards IgG.
The resulting affinity membranes are fully characterized in complete adsorption, washing and elution affinity cycles. The separation performance of each affinity support has been determined by feeding both pure IgG solutions and a cell culture supernatant. The relevant process parameters, like maximum adsorption capacity, affinity equilibrium constant and selectivity, are compared for the different affinity membranes tested, as well as for available commercial membrane adsorbers. Exam of the impact of the improved affinity materials on industrial scale applications is also addressed.
The experimental data collected have been used also for the validation of a simulation model proposed. The model developed is based on species mass balance equation over the membrane column, coupled with a suitable kinetic equation which represents the interaction between the IgG target molecule and the ligand immobilized on the porous support. Model simulations are in good agreement with the experimental affinity cycles, demonstrating the accuracy of the model to describe the transport phenomena in the column and the adsorption binding mechanism.
On the basis of parameter values obtained for pure IgG solutions the model is able to predict the behaviour observed with a cell culture supernatant
Developments in membrane affinity chromatography for monoclonal antibody recovery
No full textThe great number of process development for monoclonal antibodies, presently in development stage, has emphasized the capability limits of the biotech industry. The recent improvements of fermentation technology, allow also to achieve high titers of monoclonal antibody in the supernatant production, and the present bottleneck for MABs’s production is associated to the downstream process required for the pure product recovery.
Bead-based affinity chromatography with Protein A is widely used in the primary capture stage. Membrane affinity chromatography has not yet experienced extensive application due to the lower capacity of membrane supports compared to chromatographic beads, yet it has several advantages deserving serious attention.
This work is focused on the purification of Immunoglobulin G (IgG) with affinity membranes. A new Protein A affinity membranes (Sartorius, Göettingen, Germany), as well as affinity membranes prepared with synthetic ligands have been characterized in detail in batch and dynamic experiments.
The membranes have been analysed by using pure solutions of polyclonal IgG, to determine their binding capacity, as well as a cell supernatant containing monoclonal IgG, to investigate their selectivity and general behavior. The influence of process conditions like flow rate and feed concentration on adsorption and elution have been studied to obtain indications for the optimal process conditions.
The affinity membrane purification process was also simulated with a mathematical model which was validated by using the experimental data obtained. The model can simulate adsorption, washing and elution steps by taking into account all the relevant transport phenomena
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