Enthalpy contributions to adsorption of highly charged lysozyme onto a cation-exchanger under linear and overloaded conditions

Silva, G.L.; Marqués, Ferran; Thrash, Marvin E.; Dias-Cabral, A.C.

doi:10.1016/j.chroma.2014.05.049

Cited by 11 publications

(15 citation statements)

References 36 publications

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“…By reproducing lab‐scale conditions of a regular chromatography system, FMC is a powerful tool to characterize a chromatographic process with respect to the associated thermodynamics. We have already successfully investigated the energy profile in the purification of other biomolecules, namely lysozyme, plasmid DNA, and mAbs, and concluded that biomolecule purification in liquid chromatography is a complex process that should be carefully interpreted. The adsorption of a biomolecule to a ligand in a chromatographic process has different characteristics from a batch process both in terms of mass transfer properties and associated energy.…”

Section: Resultsmentioning

confidence: 99%

“…FMC, on the other hand, is able, to some extent, to dissect the subprocesses involved in the interaction between the biomolecule and the resin and as a consequence to discriminate between different energy sources. The technique has been used, with good results, to study the adsorption thermodynamics of biomolecules on ion exchange, hydrophobic interaction, and mixed‐mode chromatography . FMC simulates lab‐scale chromatography and implies that the system is in equilibrium.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Antibody Binding Heterogeneity of Protein A Resins

Silva

Plewka

Tscheließnig

et al. 2019

Biotechnology Journal

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Antibody Binding Heterogeneity of Protein A Resins

Silva

Plewka

Tscheließnig

et al. 2019

Biotechnology Journal

View full text Add to dashboard Cite

show abstract

“…B) differed significantly from that for BSA. The structure of LYZ is stable under the operating conditions used in this study ; hence, it is much less prone to unfold and aggregate than BSA. Therefore, LYZ exhibited typical for solid–liquid adsorption equilibrium a concave shape of the isotherm course, which could be described by the Langmuir isotherm equation (Eq.…”

Section: Results and Discusssionmentioning

confidence: 99%

A shortcut method for evaluation of protein deposition onto the membrane surface in crossflow ultrafiltration

Muca

Pia̧tkowski

Antos

2016

Engineering in Life Sciences

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In this study, a procedure for quantifying the surface deposition of proteins in crossflow ultrafiltration has been developed. The procedure consists of determining the protein adsorption behavior onto the membrane surface from a few dynamic measurements performed in a nonfiltration and a filtration mode, and evaluating the concentration polarization (CP) layer thickness based on the adsorption data. To predict the interdependence between the protein adsorption and CP, a simplified mathematical model has been formulated. The model was used to assess the protein adsorption and thus yield reduction in the ultrafiltration process at different protein concentration in the solution. As a case study, ultrafiltration of aqueous solutions of BSA and lysozyme (LYZ) was examined on a polyethersulfone membrane with the molecular weight cutoff of 10 or 100 kDa. The protein concentration in the solutions varied within a relatively low concentration range, i.e. below 10 mg mL−1, characteristic for solvent exchange between sequential operations of protein purification by chromatography and extraction. Both proteins markedly differed in the mechanism of surface deposition; for BSA hydrophobic interactions were suggested to be dominant, whereas in case of LYZ electrostatic interactions contributed the most to the deposition mechanism. The effect of additives of the protein solutions, i.e. inorganic salts, PEG, and urea depended on the adsorption mechanism and was also specific for each protein. Nevertheless, the proposed procedure performed well in the evaluation of surface deposition and yield reduction, regardless of the protein type and its solvent environment.

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“…Nevertheless, a deeper understanding of the phenomena involved in mAbs adsorption combined with the economic advantages of employing the PBA ligand, could change the paradigm in mAbs downstream processing, pushing the industry to adopt such chromatographic process either for mAb capture and/or polishing steps or even as a prechromatographic step for a longer lifetime of the costly protein A resin. Isothermal titration calorimetry (ITC), surface plasma resonance, confocal laser scanning microscopy, attenuated total reflectance–Fourier transform infrared spectroscopy, nuclear magnetic resonance, small‐angle X‐ray scattering, and flow microcalorimetry (FMC) are some of the techniques that have been employed to study the mechanisms behind the adsorption of biomolecules. However, from the above‐mentioned techniques, only few can operate in situ, i.e., in the chromatographic column, and consequently, account directly for the dynamics of the chromatographic process.…”

Section: Introductionmentioning

confidence: 99%

“…One of these techniques is FMC, which is able to simulate a packed‐bed chromatographic system at a microscale. The heat signal profile (magnitude and chronology of the thermal events) obtained can provide an overview of the driving forces of the adsorption of biomolecules, from small proteins to larger molecules such as mAbs and plasmid DNA …”

Section: Introductionmentioning

confidence: 99%

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

Rosa

Wagner

Silva

et al. 2019

Biotechnology Journal

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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 pK 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.

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Enthalpy contributions to adsorption of highly charged lysozyme onto a cation-exchanger under linear and overloaded conditions

Cited by 11 publications

References 36 publications

Antibody Binding Heterogeneity of Protein A Resins

Antibody Binding Heterogeneity of Protein A Resins

A shortcut method for evaluation of protein deposition onto the membrane surface in crossflow ultrafiltration

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

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