Abstract
Gaining knowledge on the stability and viscosity of concentrated therapeutic protein solutions is of great relevance to the pharmaceutical industry. In this work, we borrow key concepts from colloid science to rationalize the impact of aggregate formation on the changes in viscosity of a concentrated monoclonal antibody solution. In particular, we monitor the kinetics of aggregate growth under thermal stress by static and dynamic light scattering, and we follow the rise in solution viscosity by measuring the diffusion coefficient of tracer nanoparticles with dynamic light scattering. Moreover, we characterize aggregate morphology in the frame of the fractal geometry. We show that the curves of the increase in viscosity with time monitored at three different protein concentrations collapse on one single master curve when the reaction profiles are normalized based on an effective volume fraction occupied by the aggregates, which depends on the aggregate size, concentration and morphology. Importantly, we find that the viscosity of an aggregate sample is lower than the viscosity of a monomeric sample of a similar occupied volume fraction due to the polydispersity of the aggregate distribution.
Highlights
Improving our understanding of protein stability in crowded environments is of primary importance in various research areas
We proved experimentally that the tracer nanoparticles are stable under the conditions of interest, and that they are not affecting the kinetics of monoclonal antibodies (mAbs) aggregation by successively verifying that: (i) the elevated temperature required to induce mAb aggregation does not destabilize the nanoparticles (Fig. S4(a), Electronic supplementary information (ESI)†); (ii) the nanoparticles are stable in the presence of mAb molecules under native conditions (Fig. S4(a), ESI†); (iii) the size of the nanoparticles is unchanged after mAb aggregation has been induced at high temperature (Fig. S4(b), ESI†); (iv) the measured viscosity is independent of the concentration of tracer particles (Fig. S5, ESI†)
We show that the increase in solution viscosity can be rationalized by using the concept of occupied volume fraction, which accounts for protein concentration, aggregate size and aggregate fractal dimension
Summary
Improving our understanding of protein stability in crowded environments is of primary importance in various research areas. We focus on the stability of monoclonal antibodies (mAbs), which represent promising therapeutics in the treatment of a number of human diseases and occupy the largest part of the biopharmaceutical market.[1] MAb-based drugs generally require administration at high protein concentrations, in the order of 80 g LÀ1 or more. Guaranteeing drug stability at such high concentration is challenging due to the strong aggregation propensity of proteins.[2] As the presence of protein aggregates may severely compromise drug efficacy and drug safety, there is a crucial need to gain fundamental knowledge on the mechanism of protein aggregation at high concentration.[3,4]
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