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Mobile‐Phase Modulators as Salt Tolerance Enhancers in Phenylboronate Chromatography: Thermodynamic Evaluation of the Mechanisms Underlying the Adsorption of Monoclonal Antibodies
Author(s) -
Rosa Sara A. S. L.,
Wagner Alexandra,
da Silva Cláudia L.,
AiresBarros Maria R.,
Azevedo Ana M.,
DiasCabral Ana C.
Publication year - 2019
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201800586
Subject(s) - chemistry , adsorption , ligand (biochemistry) , monoclonal antibody , boronic acid , phase (matter) , salt (chemistry) , chromatography , combinatorial chemistry , isothermal microcalorimetry , organic chemistry , biochemistry , antibody , receptor , physics , quantum mechanics , enthalpy , immunology , biology
Phenylboronate chromatography has been employed for bioseparation applications though details concerning the mechanisms of interaction between the ligand and macromolecules remain widely unknown. Here, the phenomena underlying the adsorption of an anti‐human interleukin‐8 (anti‐IL8) monoclonal antibody (mAb) onto an m ‐aminophenylboronic acid ( m ‐APBA) ligand in the presence of different mobile‐phase modulators (NaF/MgCl 2 /(NH 4 ) 2 SO 4 ) and under different pH values (7.5/8.5/9.0) is investigated. Flow microcalorimetry (FMC) is applied to measure instantaneous heat energy transfer, providing insights about the role of specific and nonspecific interactions involved in the adsorptive process. Results show that the adsorption of anti‐IL8 mAb to m ‐APBA is enthalpically driven, corroborating the presence of the reversible esterification reaction between boronic acid or boronates and cis ‐diol‐containing molecules. Nevertheless, for all mobile‐phase modulators studied, changes in thermogram profiles are observed as well as reductions in the net heat of adsorption when increasing the pH. Overall, FMC and parallel chromatographic experiments data suggest that ligand salt tolerance could be enhanced using mobile‐phase modulators, with all salts studied promoting the specific cis ‐diol interactions and reducing nonspecific interactions. The last feature is more noticeable at pH values above ligand's p K a , mainly due to the ability of NaF and (NH 4 ) 2 SO 4 to diminish electrostatic interactions when compared to the commonly used NaCl.