Kinetics of Monoclonal Antibody Aggregation from Dilute toward Concentrated Conditions

Abstract

Gaining understanding on the aggregation behavior of proteins under concentrated conditions is of both fundamental and industrial relevance. Here, we study the aggregation kinetics of a model monoclonal antibody (mAb) under thermal stress over a wide range of protein concentrations in various buffer solutions. We follow experimentally the monomer depletion and the aggregate growth by size exclusion chromatography with inline light scattering. We describe the experimental results in the frame of a kinetic model based on population balance equations, which allows one to discriminate the contributions of the conformational and of the colloidal stabilities to the global aggregation rate. Finally, we propose an expression for the aggregation rate constant, which accounts for solution viscosity, protein-protein interactions, as well as aggregate compactness. All these effects can be quantified by light scattering techniques. It is found that the model describes well the experimental data under dilute conditions. Under concentrated conditions, good model predictions are obtained when the solution pH is far below the isoelectric point (pI) of the mAb. However, peculiar effects arise when the solution pH is increased toward the mAb pI, and possible explanations are discussed.

Characterisation of protein aggregation with the Smoluchowski coagulation approach for use in biopharmaceuticals

“…Because the conformational change is a unimolecular process and coagulation is a binary process, this ratio is also affected by protein concentration, as also described by Nicoud et al 22 The resulting sigmoidal shape of the monomer concentration and the apparent lag time in the concentration evolution of conformational isomers and dimers display a power-law dependence on concentration. But the power law is not only valid for a broad range of concentrations, as shown in Fig.…”

“…On contact, the particles coalesce with a finite probability, described by the Fuchs stability ratio W and the exponent of the product kernel g. The Fuchs ratio can be computed from the total interaction potential between the primary particles, but is usually determined from the fit due to the complexity of protein-protein interactions. 22 The parameter g describes the sticking probability for two colliding aggregates and is proportional to the total number of primary particle interactions upon their contact. Since the number of primary particles located on the external surface of a fractal aggregate scales as i 1À1/df , g can be roughly estimated as…”

“…A fractal dimension of anywhere between 1.5 and 2.6 has been reported for protein aggregates of various sizes, formed under different conditions. [19][20][21][22][23][24] Note that even a small difference in the estimation of fractal dimension brings rather major differences in the particle size. For example, a globular aggregate built from 1 million primary particles of size 10 nm measures 1 mm in size, while the same aggregate behaving as a polymer chain is forty times larger, at 40 mm.…”

“…Its basic principle has been applied to polymerization, coagulation of aerosols and flocculation. Recently, application of the same kinetics to biopharmaceuticals has been proven successful by the ground-breaking work of Arosio and Nicoud et al [18][19][20][21][22] Aggregation of human immunoglobulin G under severe stress conditions has been described in terms of population balance modelling, identifying dominant molecular mechanisms based on bulk aggregation measurements of smaller oligomers under various stress conditions, such as exposure to acid 23,24 and severe temperature. The effect of various excipients and conditions on the model parameters was also studied.…”

Novel chemical degradation pathways of proteins mediated by tryptophan oxidation: tryptophan side chain fragmentation

“…Protein pharmaceuticals can degrade via multiple chemical and physical processes, and detailed knowledge of these processes is critical to prevent degradation and to maintain stability of formulations . The last years have witnessed significant progress in our understanding of physical degradation processes, for example leading to aggregation and particle formation, in part due to the development of new analytical methodology and the critical evaluation of its potential and limitations . In contrast, the characterization of chemical degradation processes is far from complete, for the most part due to the continuous discovery of new reaction pathways and products, which can be unique to specific protein sequences.…”