Thermodynamics of the adsorption of monoclonal antibodies in phenylboronate chromatography: Affinity versus multimodal interactions.
The aim of this work was to investigate the complex phenomena underlying the adsorption of an anti-human IL-8 (anti-IL8) monoclonal antibody (mAb) to m-aminophenylboronate (m-APBA) by Flow Microcalorimetry (FMC) and to understand the role of non-specific interactions in the adsorption process. FMC was exploited as a dynamic on-line method to measure instantaneous heat energy transfers in order to understand the surface phenomena underlying mAb's adsorption towards the synthetic ligand m-APBA under different pH (7.5, 8.5, 9.0, 9.5 and 10.0) and salt concentrations (0 and 150 mM NaCl). Results showed that the adsorption of anti-IL8 mAb to m-APBA is enthalpically driven (ΔHads<0), as expected for the predominant reversible esterification reaction between boronates and cis-diols-containing molecules. For all the pH conditions studied, thermograms presented a first exothermic peak, characteristic of the reversible esterification reaction between mAb (pI≥9.3) and m-APBA (pKa = 8.8), except at pH 9.0 in the presence of 150 mM NaCl, for which the thermogram presented a first endothermic peak. The heat of adsorption (ΔHads) obtained at conditions where cis-diol interactions were predominant was approximately -243 ± 38 kJ/mol against -82 ± 14 kJ/mol (p-value < 0.05) obtained at pH 9.0 with 150 mM NaCl. The observed shift in the thermogram profile at pH 9.0, 150 mM NaCl, and the consequent decrease of 60-70% in ΔHads were indicative of the promotion of electrostatic interactions between the protein and the ligand. Overall, and whereas the binding of the PBA ligand to mAb molecules has been described for decades as being affinity-based, our study demonstrates the multimodal behaviour of this interaction and contributes towards the understanding of the adsorption thermodynamics.
['Rosa|Sara A S L|SASL|', 'da Silva|C L|CL|', 'Aires-Barros|M Raquel|MR|', 'Dias-Cabral|A C|AC|', 'Azevedo|Ana M|AM|']
D000327:Adsorption D000911:Antibodies, Monoclonal D001897:Boronic Acids D002151:Calorimetry D002846:Chromatography, Affinity D006801:Humans D006860:Hydrogen Bonding D006863:Hydrogen-Ion Concentration D016209:Interleukin-8 D008024:Ligands D012212:Rheology D012492:Salts D013816:Thermodynamics
Monoclonal antibodies Phenylboronate chromatography Flow microcalorimetry Downstream processing Immunoglobulin G – IgG Protein adsorption