L'attività di ricerca è dedicata ai processi di separazione a membrana, processi cromatografici e, in
senso allargato, alle bioseparazioni. L'attività di ricerca
riguarda la separazione di biomolecole quali proteine e organelli cellulari. Lo studio consiste nell'immobilizzazione su
membrane microporose e altri supporti cromatografici di ligandi
naturali o sintetici che hanno affinità per la proteina di
interesse. Le membrane, o i supporti, di affinità così
ottenuti sono caratterizzati sperimentalmente in modo da
determinare i parametri termodinamici e cinetici del sistema
proteina-membrana di affinità considerato e di comprendere quali
sono i meccanismi fondamentali alla base della separazione. Il
processo cromatografico viene studiato sia dal punto di vista
sperimentale, sia dal punto di vista fisico-matematico con
l'obiettivo di ottenere un modello matematico previsionale per
diversi sistemi studiati.
Negli ultimi anni l'attività di ricerca coinvolge anche processi di
aferesi per rimozione di tossine da sangue umano e lo studio di
processo di sistemi cromatografici industriali.
The research is carried out along the following themes:
- Affinity membranes for biomolecules purification
- Mathematical modeling of convective chromatographic processes
- Biomedical applications
- Affinity membranes for biomolecules purification
Different target proteins have been identified and purified with affinity membrane technology, among those fusion proteins with the MBP affinity tag, lectins and immunoglobulins G and M of human origin that are of particular interest for their possible use in the production of therapeutic agents.
The purification of immunoglobulins has been studied within the AIMs (Advanced Interactive Materials by design) project part of the EU 6th Framework Program. Objective of AIMs was the development of new materials and methods for the purification of monoclonal antibodies as alternative to Protein A packed column chromatography. The Department of Chemical Engineering of the University of Bologna, under the scientific guidance of Prof. Giulio Sarti, was responsible of the sub-project “membranes”. The preparation and characterization of affinity membranes, based on new materials (membrane supports and ligands), the mathematical model of the membrane process as well as the coordination of the membrane group were part of the work performed.
Mimetic ligands, namely D-PAM, A2P and B14, which mimic the interaction between the Fc fragments of an antibody and Protein A were utilized using different spacer arms. In collaboration with Prof. Carlo Cavallotti of Politecnico of Milano, the interaction mechanisms protein-ligand and membrane-ligand was studied with the aid of a molecular dynamics model that has been applied to the experimental results obtained in Bologna and tuned according to the experimental evidence. From this collaboration came the idea to design a synthetic ligand for IgG purification and to immobilize this new ligand on a membrane surface. The project was the subject of a project, PRIN 2008 titled “Sviluppo di membrane di affinità per anticorpi monoclonali”. During this project the interactions and the effects of spacer arms were investigated in detail. As a result, a couple of possible ligands were identified and one of those has been recently synthetized to be immobilized on membrane surfaces.
ii) Mathematical modelling of convective chromatographic processes
In parallel to the experimental activity, my research involves the understanding of the fundamental mechanisms that govern the adsorption of proteins on affinity membranes. From a study of the characteristics time of the process it can be observed that axial dispersion needs to be considered in all cases, on the contrary, the kinetic of adsorption and elution is a characteristic of the specific protein/ligand system and needs to be identified experimentally. For instance, for the system IgG and A2P ligand and for the system IgG/B14 ligand it has been observed that a bi-Langmuir kinetics describes very well the phenomena involved, whilst for the system IgG/protein A ligand a classic Langmuir type kinetics is more suitable.
The results of these studies have been implemented in a model for the description of the whole chromatographic cycle: adsorption, washing and elution. Model validation has been undertaken with data obtained for the different affinity systems. The mathematical model describes very well the adsorption and washing step, while for the elution step some deviations are observed which are more or less pronounced depending on the affinity system. The model has only one fitting parameter for the kinetic of adsorption and one for the kinetic of elution. Once the kinetic parameter has been determined for pure components, the model can be used in a predictive way to describe the adsorption of IgG in a complex mixture, like a cell culture supernatant, with no fitting parameters.
Scale-up studies have been performed in order to use the model as a predictive tool for bioprocess design. The model has been tested to study the influence of kinetics on the performance of industrial scale membrane adsorbers. The results showed that the identification of the correct kinetic is paramount for a correct process description, the use of different models can bring to differences up to 40% between the breakthrough curves obtained with Langmuir and bi-Langmuir equations.
The model has been successfully used with monoliths with immobilized protein A, indeed monoliths are continue chromatographic stationary phases in which the predominant transport phenomena is convective flow, for this reason, the considerations in terms of characteristic times that are in order for membranes hold also for these systems.
iii) Biomedical applications
The high specificity of affinity interactions makes them particularly attractive for biomedical separations. Among the separations considered is the purification of proteins for therapeutic use and selective apheresis; within the first class is the purification of plasminogen from human serum for ophthalmology applications. The aim of this study is to isolate pure plasminogen to be converted to plasmin, enzyme used for vitrectomy. To this aim lysine, amino acid which has affinity for plasminogen, was successfully immobilized on cellulosic membranes that were used to isolate plasminogen from human serum, which was converted in vitro to plasmin using streptokinase.
In the second class, the objective is to remove toxins of high molecular weight such has lipoproteins, fibrinogen and immunoglobulin light chains which, due to chronical conditions, our body is not able to eliminate. The project, aims at the functionalization of membranes to be used in adsorptive mode for the removal of the target toxins during apheresis.