Mobile-Phase Modulators as Salt Tolerance Enhancers in Phenylboronate Chromatography: Thermodynamic Evaluation of the Mechanisms Underlying the Adsorption of Monoclonal Antibodies.

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 /(NH ) SO ) and under different pH values (7.5/8.

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.

High-Throughput Nanoliter-Scale Analysis and Optimization of Multimodal Chromatography for the Capture of Monoclonal Antibodies.

Multimodal ligands are synthetic molecules comprising multiple types of interactions that have been increasingly used for the capture of different biopharmaceutical compounds within complex biological mixtures. For monoclonal antibodies (mAbs) in particular, these ligands have shown the possibility of direct capture from cell culture supernatants in native conditions, as well as enhanced selectivity and affinity compared to traditional single-mode ligands. However, performing the capture of a target mAb using multimodal chromatography comes with the need for extensive optimization of the operating conditions, due to the multitude of interactions that can be promoted in parallel.

Phenylboronic acid as a multi-modal ligand for the capture of monoclonal antibodies: development and optimization of a washing step.

In this work, phenylboronic acid (PBA) was thoroughly investigated as a synthetic ligand for the purification of an immunoglobulin G (IgG) from a clarified cell supernatant from Chinese Hamster Ovary (CHO) cell cultures. In particular, the study was focused on the development of a washing step and in the optimization of the elution step using a serum containing supernatant. From the different conditions tested, best recoveries - 99% - and purifications - protein purity of 81% and a purification factor of 16 out of a maximum of 20 - were achieved using 100mM d-sorbitol in 10mM Tris-HCl as washing buffer and 0.

Magnetic aqueous two phase fishing: a hybrid process technology for antibody purification.

The potential to combine aqueous two-phase extraction (ATPE) with magnetic separation was here investigated with the aim of developing a selective non-chromatographic method for the purification of antibodies from cell culture supernatants. Aqueous two-phase systems (ATPS) composed of polyethylene glycol (PEG) and dextran were supplemented with several surface modified magnetic particles (MPs) at distinct salt concentrations. The partition of pure human IgG in the upper and lower phases as well as the amount adsorbed at the MPs surface was investigated, indicating that MPs coated with dextran and gum Arabic established the lowest amount of non-specific interactions.