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- Biophysical study of therapeutic antibody adsorption in affinity chromatographyPublication . Silva, Gonçalo Fradique Lopes da; Cabral, Ana Cristina Mendes Dias; Jungbauer, AloisThe monoclonal antibody market has been growing rapidly in the past decades, and the number of therapeutic areas where monoclonal antibodies (mAbs) are employed has been increasing, with cancer and autoimmune diseases being the most represented. There are already more than 50 approved products, representing a staggering $100 billion in global sales. Because of this high demand, antibody manufacturing has been in constant evolution and asking for new, more efficient, and more optimized methods to be applied both in the upstream and downstream processing. Protein A chromatography is step of choice of most of the pharmaceutical companies for the antibody capture in the downstream processing. It is a core unit operation that has been in constant evolution, with the new resins coming to the market having higher binding capacities than their predecessors and improved alkaline stability. Despite of this extensive improvement in Protein A resins, there are still some aspects that lack understanding and deep investigation, specifically the mechanism of interaction between the antibodies and the Protein A ligands, both under linear and overloaded conditions. This knowledge can be used for further enhancement of performance in the mAbs capture step. The knowledge accumulated during the last decades by studying chromatography for proteins bioprocess development has shed some light to the mechanistic understanding of protein–ligand interactions, though based on indirect measurements. Chromatography processes in general are characterized with online and offline sensors that probe the concentration (UV detector), purity (SEC-HPLC), potency (SPR), and structure (MALS, CD) of the product, as well as conductivity and pH that can be measured directly in the chromatography stations (ÄKTA). However, none of these probes operate in situ, i.e. in the chromatographic column where the interaction occurs. The online sensors tackle the elution peak, and the offline sensors analyse the sample afterwards. Therefore, this research consists in a biophysical study on the antibody adsorption to commercial Protein A resins with in-situ sensors, which resulted in an improved understanding of antibody–Protein A interactions, both under linear and overloaded conditions. Flow microcalorimetry was extensively used to retrieve the thermodynamic parameters during antibody adsorption. The microcalorimeter consists on a ID 6 mm × 6 mm column with two thermistors coupled on the column walls that are able to detect small changes in potential during a chromatographic process. The application of the technique to two commercial Protein A resins (MabSelect SuRe with a tetrameric Protein A ligand and TOYOPEARL AF-rProtein A HC with a hexameric Protein A ligand) showed an adsorption profile of exothermic nature with two sub-processes involved. The first and stronger moment was associated to the adsorption process itself. The second moment, less energetic, was associated either to reorganization of the antibody layer and the Protein A chain upon binding, or to antibody binding to a ligand where an antibody molecule would already be bound. These interpretations were reinforced by the small angle X-ray scattering (SAXS) studies. To characterize the changes in the antibody-Protein-A ligand complex and evaluate the influence of the surface topology on adsorption, SAXS was employed using a miniaturized, X-ray-transparent chromatography column packed with the resin. In this way, the protein absorption process could be followed and the formation of a protein layer on the chromatography resin fibres can be observed at the nanoscale and in a time-resolved manner. For the first time it was possible to directly correlate the nanostructure changes inside the column, upon adsorption and during elution. It was demonstrated the possibility of heterogeneous binding throughout the bead network depending on the resin saturation. By application of the broken rod model and under resin saturation it was proposed that an average of 1.2 antibodies adsorb per Protein A ligand in MabSelect SuRe at the outermost domains. Further investigation was performed at different surface concentrations in order to evaluate differences in the organization and stoichiometry in the different zones of the isotherm. The experimental data, analysed by the pearl necklace model, was compared with crystallographic structures of an IgG1 and a tetrameric chain of the B domain of Staphylococcal Protein A (the native form of the Protein A ligand present in MabSelect SuRe). It was found that at low isotherm concentrations the antibody to Protein A ratio was 1:1 and that at intermediate and high concentrations the 2:1 stoichiometry became favoured. The stoichiometry of 3:1 was also tested but was disregarded because of the strong steric effects. The offered approach in this thesis follows the adsorption process in situ, in the column and opens up new prospects to deeper investigation of all modes of chromatography of high industrial relevance, where the understanding of biomolecule–resin mechanism of interaction is of utmost importance.
- Understanding ion-exchange adsorption mechanism under overloaded conditionsPublication . Silva, Gonçalo Fradique Lopes da; Cabral, Ana Cristina Mendes DiasIon-exchange chromatography (IEC) is a powerful and widely used separation technique in the biotechnological industry. The greatest challenge of any chromatographic technique is predicting the adsorptive behaviour of biomolecules onto the chromatography resin. This investigation attempts to examine the complexity of protein adsorption onto ion-exchangers and the role of nonspecific effects in the establishment of the adsorptive process. Flow microcalorimetry (FMC) and adsorption isotherms measurements were used to illustrate lysozyme adsorption mechanism on carboxymethyl cellulose (CMC) at both absence and presence of salt (NaCl 50mM) at pH 5. FMC results show that under all the studied conditions the adsorptive process is, as expected in ion exchange, enthalpy driven. Direct correlation between microcalorimetry data and isotherm measurements is observed. Under linear protein concentrations, protein adsorption occurs in the same extension regardless salt concentration. However, when isotherm levelling point is reached, lysozyme reorientation in the presence of salt seems to be the leading mechanism to further adsorption. Under overloaded conditions in the presence of salt, with increasing surface concentration, as a new layer of protein molecules is formed, an expected decrease in the net heat of adsorption is observed, consistent with an energetic equilibrium towards the formation of the new layer. FMC experiments and isotherm measurements were also performed for Bovine Serum Albumin (BSA) adsorption onto Toyopearl® GigaCap Q-650M. The results showed a high overall exothermic process. Secondary adsorption of BSA to the surface, resulting from its alteration of conformation seems to be present. Also, at high protein surface concentrations, high repulsive interactions may occur. All these results confirm that FMC is a powerful technique to illustrate protein adsorption mechanisms in ion-exchange.