Samiul Amin

Detection of viscoelasticity in aggregating dilute protein solutions through dynamic light scattering-based optical microrheology

This study illustrates the applicability of dynamic light scattering (DLS)-based optical microrheology in generating new insights into the rheological response of dilute protein solutions as they start to form insoluble aggregates under the influence of a thermal stress. The technique is also shown to provide a quick method for measuring the viscosity in protein solutions. The optical microrheological technique, which is based on DLS with improved single scattering detection, is shown here to capture the rich dynamics in these systems, where traditional mechanical rheometry cannot be effectively employed due to low torque generation and high sample volume requirements and the more widely known diffusing wave spectroscopy microrheology technique is not desirable due to the required high probe particle concentrations The study illustrates the careful consideration which must be given to the tracer particle surface chemistry, tracer particle concentration and tracer particle size in order to extract out rheological responses that are truly representative of the underlying protein dynamics and microstructure. We outline a procedure for ensuring that the pitfalls inherent to this type of measurement are avoided.

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

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 %).

Combined Dynamic Light Scattering and Raman Spectroscopy Approach for Characterizing the Aggregation of Therapeutic Proteins

Determination of the physicochemical properties of protein therapeutics and their aggregates is critical for developing formulations that enhance product efficacy, stability, safety and manufacturability. Analytical challenges are compounded for materials: (1) that are formulated at high concentration, (2) that are formulated with a variety of excipients, and (3) that are available only in small volumes. In this article, a new instrument is described that measures protein secondary and tertiary structure, as well as molecular size, over a range of concentrations and formulation conditions of low volume samples. Specifically, characterization of colloidal and conformational stability is obtained through a combination of two well-established analytical techniques: dynamic light scattering (DLS) and Raman spectroscopy, respectively. As the data for these two analytical modalities are collected on the same sample at the same time, the technique enables direct correlation between them, in addition to the more straightforward benefit of minimizing sample usage by providing multiple analytical measurements on the same aliquot non-destructively. The ability to differentiate between unfolding and aggregation that the combination of these techniques provides enables insights into underlying protein aggregation mechanisms. The article will report on mechanistic insights for aggregation that have been obtained from the application of this technique to the characterization of lysozyme, which was evaluated as a function of concentration and pH.

Tracer microrheology study of a hydrophobically modified comblike associative polymer.

The viscoelastic properties of associative polymers are important not only for their use as rheology modifiers but also to understand their complex structure in aqueous media. In this study, the dynamics of comblike hydrophobically modified alkali swellable associative (HASE) polymers are probed using diffusing wave spectroscopy (DWS) based tracer microrheology. DWS-based tracer microrheology accurately probes the dynamics of HASE polymers, and the extracted microrheological moduli versus frequency profile obtained from this technique closely matches that obtained from rotational rheometry measurements. Quantitatively, however, the moduli extracted from DWS-based tracer microrheology measurements are slightly higher than those obtained using rotational rheometry. The creep compliance, elastic modulus, and relaxation time concentration scaling behavior exhibits a power-law dependence. The length scale associated with the elastic to glassy behavior change is obtained from the time-dependent diffusion coefficient. The Zimm-Rouse type scaling is recovered at high frequencies but shows a concentration effect switching from Zimm to more Rouse-like behavior at higher concentrations.

A novel combination of DLS-optical microrheology and low frequency Raman spectroscopy to reveal underlying biopolymer self-assembly and gelation mechanisms

The connectivity between gelation and increasing water confinement and structuring within nanopores of a thermally induced gel is demonstrated for the first time through low frequency Raman spectroscopy and optical microrheology measurements. Specifically, the work confirms that increased ordering of individual water molecules can be observed during the gelation of agarose upon cooling. More importantly, it illustrates the ability of the two techniques to provide new insights and a more direct link between intermolecular interactions/microstructure and evolving rheological response in gelling systems.

Structural Changes and Aggregation Mechanisms for Anti-Streptavidin IgG1 at Elevated Concentration

Non-native protein aggregation may occur during manufacturing and storage of protein therapeutics, and this may decrease drug efficacy or jeopardize patient safety. From a regulatory perspective, changes in higher order structure due to aggregation are of particular interest but can be difficult to monitor directly at elevated protein concentrations. The present report focuses on non-native aggregation of antistreptavidin (AS) IgG1 at 30 mg/mL under solution conditions that prior work at dilute concentrations (e.g., 1 mg/mL) indicated would result in different aggregation mechanisms. Time-dependent aggregation and structural changes were monitored in situ with dynamic light scattering, small-angle neutron scattering, and Raman scattering and ex situ with far-UV circular dichroism and second-derivative UV spectroscopy. The effects of adding 0.15 M (∼5 w/w %) sucrose were also assessed. The addition of sucrose decreased monomer loss rates but did not change protein-protein interactions, aggregation mechanism(s), or aggregate structure and morphology. Consistent with prior results, altering the pD or salt concentration had the primary effect of changing the aggregation mechanism. Overall, the results provide a comparison of aggregate structure and morphology created via different growth mechanisms using orthogonal techniques and show that the techniques agree at least qualitatively. Interestingly, AS-IgG1 aggregates created at pD 5.3 with no added salt formed the smallest aggregates but had the largest structural changes compared to other solution conditions. The observation that the larger aggregates were also those with less structural perturbation compared to folded AS-IgG1 might be expected to extend to other proteins if the same strong electrostatic repulsions that mediate aggregate growth also mediate structural changes of the constituent proteins within aggregates.

Passive optical mapping of structural evolution in complex fluids

Self-assembling complex systems exhibit properties that involve a broad spectrum of thermal, structural, morphological, and optical transitions. Various techniques have been used to assess different aspects of the phase transitions in these complex systems. However, because of inherent technical constraints, structural information is usually provided only within narrow ranges of concentrations and temperatures. We show here that by effectively suppressing multiple scattering, low-coherence dynamic light scattering permits assessing the aggregation dynamics of self-assembling systems in a completely passive manner and over ranges of concentration and temperatures well beyond the limits of traditional approaches. The power spectral analysis of scattered intensity fluctuations permits a reliable characterization of multiple relaxation times. We demonstrate that the entire phase diagram can be covered in a consistent way and structural phase transitions can be mapped over a broad optical regime from weak to strong scattering.

Microviscoelasticity of soft repulsive sphere dispersions: Tracer particle microrheology of triblock copolymer micellar liquids and soft crystals

Tracer particle microrheology using diffusing wave spectroscopy-based microrheology is demonstrated to be a useful method to study the dynamics of aqueous Pluronic™ F108 solutions, which are viewed as solutions of repulsive soft spheres. The measured zero-shear microviscosity of noncrystallizing micellar dispersions indicates micelle corona dehydration upon increasing temperature. Colloidal sphere thermal motion is shown to be exquisitely sensitive to the onset of crystallization in these micellar dispersions. High temperature dynamics are dominated by an apparent soft repulsive micelle-micelle interaction potential indicating the important role played by lubrication forces and ultimately micelle corona interpenetration and compression at sufficiently high concentrations. The measured microscopic viscoelastic storage and loss moduli are qualitatively similar to those experimentally observed in mechanical measurements on colloidal dispersions and crystals, and calculated from mode coupling theory of colloidal suspensions. The observation of subdiffusive colloidal sphere thermal motion at short time-scales is strong evidence that the observed microscopic viscoelastic properties reflect the dynamics of individual micelles rather than a dispersion of micellar crystallites.

Revealing New Structural Insights from Surfactant Micelles through DLS, Microrheology and Raman Spectroscopy

The correlation between molecular changes and microstructural evolution of rheological properties has been demonstrated for the first time in a mixed anionic/zwitterionic surfactant-based wormlike micellar system. Utilizing a novel combination of DLS-microrheology and Raman Spectroscopy, the effect of electrostatic screening on these properties of anionic (SLES) and zwitterionic (CapB) surfactant mixtures was studied by modulating the NaCl concentration. As Raman Spectroscopy delivers information about the molecular structure and DLS-microrheology characterizes viscoelastic properties, the combination of data delivered allows for a deeper understanding of the molecular changes underlying the viscoelastic ones. The high frequency viscoelastic response obtained through DLS-microrheology has shown the persistence of the Maxwell fluid response for low viscosity solutions at high NaCl concentrations. The intensity of the Raman band at 170 cm−1 exhibits very strong correlation with the viscosity variation. As this Raman band is assigned to hydrogen bonding, its variation with NaCl concentration additionally indicates differences in water structuring due to potential microstructural differences at low and high NaCl concentrations. The microstructural differences at low and high NaCl concentrations are further corroborated by persistence of a slow mode at the higher NaCl concentrations as seen through DLS measurements. The study illustrates the utility of the combined DLS, DLS-optical microrheology and Raman Spectroscopy in providing new molecular structural insights into the self-assembly process in complex fluids.

Colloidal Stability & Conformational Changes in β-Lactoglobulin: Unfolding to Self-Assembly.

A detailed understanding of the mechanism of unfolding, aggregation, and associated rheological changes is developed in this study for β-Lactoglobulin at different pH values through concomitant measurements utilizing dynamic light scattering (DLS), optical microrheology, Raman spectroscopy, and differential scanning calorimetry (DSC). The diffusion interaction parameter kD emerges as an accurate predictor of colloidal stability for this protein consistent with observed aggregation trends and rheology. Drastic aggregation and gelation were observed at pH 5.5. Under this condition, the protein’s secondary and tertiary structures changed simultaneously. At higher pH (7.0 and 8.5), oligomerizaton with no gel formation occurred. For these solutions, tertiary structure and secondary structure transitions were sequential. The low frequency Raman data, which is a good indicator of hydrogen bonding and structuring in water, has been shown to exhibit a strong correlation with the rheological evolution with temperature. This study has, for the first time, demonstrated that this low frequency Raman data, in conjunction with the DSC endotherm, can be been utilized to deconvolve protein unfolding and aggregation/gelation. These findings can have important implications for the development of protein-based biotherapeutics, where the formulation viscosity, aggregation, and stability strongly affects efficacy or in foods where protein structuring is critical for functional and sensory performance.