Aggregate structure, morphology and the effect of aggregation mechanisms on viscosity at elevated protein concentrations

Abstract

Non-native aggregation is a common issue in a number of degenerative diseases and during manufacturing of protein-based therapeutics. There is a growing interest to monitor protein stability at intermediate to high protein concentrations, which are required for therapeutic dosing of subcutaneous injections. An understanding of the impact of protein structural changes and interactions on the protein aggregation mechanisms and resulting aggregate size and morphology may lead to improved strategies to reduce aggregation and solution viscosity. This report investigates non-native aggregation of a model protein, α-chymotrypsinogen, under accelerated conditions at elevated protein concentrations. Far-UV circular dichroism and Raman scattering show structural changes during aggregation. Size exclusion chromatography and laser light scattering are used to monitor the progression of aggregate growth and monomer loss. Monomer loss is concomitant with increased β-sheet structures as monomers are added to aggregates, which illustrate a transition from a native monomeric state to an aggregate state. Aggregates grow predominantly through monomer-addition, resulting in a semi-flexible polymer morphology. Analysis of aggregation growth kinetics shows that pH strongly affects the characteristic timescales for nucleation (τn) and growth (τg), while the initial protein concentration has only minor effects on τn or τg. Low-shear viscosity measurements follow a common scaling relationship between average aggregate molecular weight (Mw(agg)) and concentration (σ), which is consistent with semi-dilute polymer-solution theory. The results establish a link between aggregate growth mechanisms, which couple Mw(agg) and σ, to increases in solution viscosity even at these intermediate protein concentrations (less than 3w/v %).

Parallel chromatography and in situ scattering to interrogate competing protein aggregation pathways

“…As seen in Figure (B), these variations are not observed at early times, and all the temperature conditions show linear behavior for M w / M 1 values below approximately 2, which is consistent with previous work for aggregation of monoclonal antibodies . The upward curvature at later times in Figure (A) is expected, as aggregates grow to larger sizes and likely also coalesce with each other . The linear regime obtained up to M w / M 1 ∼ 2 [cf., Fig.…”

“…S1(A)]. This qualitative behavior was also consistent with results for other proteins . As noted in Methods, attempts to purify dimer via chromatography for seeding experiments were unsuccessful, potentially because of issues with column interactions that limited resolution of monomer and aggregate species that are being investigated as part of future work.…”

Molecular basis of high viscosity in concentrated antibody solutions: Strategies for high concentration drug product development

“…Nicoud et al 48 show that the aggregate fractal dimension of a mAb increases as concentration rises. Barnett et al 49 used the protein a-chymotrypsinogen as a model system to illustrate that the 3D structure of the protein changes with increasing concentration. In this case, monomer loss is followed by increase in viscosity.…”

Recent applications of light scattering measurement in the biological and biopharmaceutical sciences

“…The use of MALS and/or DLS, sometimes in conjunction with other methods of macromolecular characterization (e.g., CD, fluorescence or Raman spectroscopy, SAXS, SANS), to monitor the progress of irreversible aggregation following an initiating event provides information about the time evolution of aggregate size, which has been used to develop models for the mechanism of aggregate formation (see for example [21, 22]). Measurement of effective charge via ELS has also been found to correlate with the rate of irreversible aggregation of glycosylated and non-glycosylated Fc-1 antibody fragments [23]; the tendency to aggregate diminishes with increasing effective charge.…”