Kinetics of protein denaturation at gas—liquid interfaces

Donaldson, T.L.; Boonstra, Eric F; Hammond, John M

doi:10.1016/0021-9797(80)90213-1

Cited by 57 publications

(50 citation statements)

References 16 publications

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“…In both experiments, the air-liquid interface was present, yet this interfacial area alone was insufficient to drive aggregation upon milder agitation. We do not dispute the importance of the air-liquid interface in protein instability 32,33 but argue that the main factor determining protein aggregation in our experiments was the concentration of non-native species in solution. The observed disparity between the mild and rigorous agitation experiments can be explained by reversible changes in protein structure.…”

Section: Agitation-induced Mab Aggregationmentioning

confidence: 54%

The use of native cation‐exchange chromatography to study aggregation and phase separation of monoclonal antibodies

et al. 2010

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This study introduces a novel analytical approach for studying aggregation and phase separation of monoclonal antibodies (mAbs). The approach is based on using analytical scale cation-exchange chromatography (CEX) for measuring the loss of soluble monomer in the case of individual and mixed protein solutions. Native CEX outperforms traditional size-exclusion chromatography in separating complex protein mixtures, offering an easy way to assess mAb aggregation propensity. Different IgG1 and IgG2 molecules were tested individually and in mixtures consisting of up to four protein molecules. Antibody aggregation was induced by four different stress factors: high temperature, low pH, addition of fatty acids, and rigorous agitation. The extent of aggregation was determined from the amount of monomeric protein remaining in solution after stress. Consequently, it was possible to address the role of specific mAb regions in antibody aggregation by co-incubating Fab and Fc fragments with their respective full-length molecules. Our results revealed that the relative contribution of Fab and Fc regions in mAb aggregation is strongly dependent on pH and the stress factor applied. In addition, the CEX-based approach was used to study reversible protein precipitation due to phase separation, which demonstrated its use for a broader range of protein-protein association phenomena. In all cases, the role of Fab and Fc was clearly dissected, providing important information for engineering more stable mAb-based therapeutics.

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Section: Agitation-induced Mab Aggregationmentioning

confidence: 54%

The use of native cation‐exchange chromatography to study aggregation and phase separation of monoclonal antibodies

et al. 2010

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“…In support of this idea, acid phosphatase was denatured in laminar flows in the presence of a gas-liquid interface (Donaldson et al 1980) and that the denaturation of β-lactoglobulin by shaking its solution could be reduced to a very low level by the addition of surfactants or large polymers such as polyethylene glycol (Reese and Robbins 1981). Subsequently, Lee and Choo (1989) showed that the rate of denaturation of a lipase in a stirred tank reactor decreased significantly in the presence of polypropylene glycol or when the reactor was completely filled.…”

mentioning

confidence: 86%

Effects of shear on proteins in solution

2010

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The effects of "shear" on proteins in solution are described and discussed. Research on this topic covers many decades, beginning with investigations of possible denaturation of enzymes during processing, whilst more recent concerns are how the quality of therapeutic proteins might be affected by shear or shear related effects.The paradigm that emerges from most studies is that shear in the fluid mechanical sense is unlikely by itself to damage most proteins and that interfacial phenomena are critically important. In particular, moving gas-liquid interfaces can be very deleterious. Aggregation of therapeutic proteins on nanoparticles shed from solid surfaces is a recent concern because of potential consequences on patient safety. It is clear that labeling such damage as "shear" is a mistake as this inhibits clear investigations of, and thinking about, the true causes of damage to proteins in solution during processing.

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“…One of these processes is the possible unfolding of proteins at the interface [1][2][3]. That proteins may unfold at interfaces was hypothesized based on the observation that enzymes may loose their activity upon adsorption at the air-water interface [4,5]. A gain in free energy of the system would be obtained if the protein changes its conformation so that the polar residues are oriented towards the aqueous phase and the non-polar (or hydrophobic) residues to the air phase.…”

Section: Introductionmentioning

confidence: 99%

The adsorption and unfolding kinetics determines the folding state of proteins at the air–water interface and thereby the equation of state

Wierenga¹,

Egmond²,

Voragen³

et al. 2006

Journal of Colloid and Interface Science

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Unfolding of proteins has often been mentioned as an important factor during the adsorption process at air-water interfaces and in the increase of surface pressure at later stages of the adsorption process. This work focuses on the question whether the folding state of the adsorbed protein depends on the rate of adsorption to the interface, which can be controlled by bulk concentration. Therefore, the adsorption of proteins with varying structural stabilities at several protein concentrations was studied using ellipsometry and surface tensiometry. For β-lactoglobulin the adsorbed amount (Γ ) needed to reach a certain surface pressure (Π) decreased with decreasing bulk concentration. Ovalbumin showed no such dependence. To verify whether this difference in behavior is caused by the difference in structural stability, similar experiments were performed with cytochrome c and a destabilized variant of this protein. Both proteins showed identical Π-Γ , and no dependence on bulk concentration. From this work it was concluded that unfolding will only take place if the kinetics of adsorption is similar or slower than the kinetics of unfolding. The latter depends on the activation energy of unfolding (which is in the order of 100-300 kJ/mol), rather than the free energy of unfolding (typically 10-50 kJ/mol).

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Kinetics of protein denaturation at gas—liquid interfaces

Cited by 57 publications

References 16 publications

The use of native cation‐exchange chromatography to study aggregation and phase separation of monoclonal antibodies

The use of native cation‐exchange chromatography to study aggregation and phase separation of monoclonal antibodies

Effects of shear on proteins in solution

The adsorption and unfolding kinetics determines the folding state of proteins at the air–water interface and thereby the equation of state

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