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Biotechnol J ; 14(8): e1800632, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30945463

RESUMO

Protein A affinity chromatography is a core unit operation in antibody manufacturing. Nevertheless, there is not enough understanding of in-column antibody adsorption in the Protein A capture step. This work aims to investigate in situ the establishment of an antibody (trastuzumab) layer during Protein A chromatography both in terms of energetic contributions and uptake kinetics. Flow microcalorimetry is employed as a technique with an in situ operating detector, which provides an understanding of the thermodynamics of the adsorption process. In addition, the antibody uptake rate is also investigated in order to establish a correlation between its diffusion on the stationary phase and the associated thermodynamics. Two resins with different particle size, intraparticle porosity, and a Protein A ligand structure are studied: the synthetically engineered B-domain tetrameric MabSelect SuRe and the synthetically engineered C-domain hexameric TOYOPEARL AF-rProtein A HC. The uptake rate follows a pore diffusion model at low equilibrium time, showing a slower diffusivity after a certain time because of the heterogeneous binding nature of these two resins. In addition, the microcalorimetric studies show that adsorption enthalpy is highly favourable at low isotherm concentrations and evolves toward an equilibrium with increasing surface concentration. These data suggest that the relationship between adsorption enthalpy and the establishment of the antibody layer in the Protein A chain is consistent with heterogeneous adsorption.


Assuntos
Anticorpos/metabolismo , Proteína Estafilocócica A/metabolismo , Resinas de Troca Aniônica , Sítios de Ligação , Calorimetria , Cromatografia de Afinidade/métodos , Cinética , Ligantes , Trastuzumab/metabolismo
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