Physicochemical mechanisms of aggregation and fibril formation of α-synuclein and apolipoprotein A-I.

Deposition and accumulation of amyloid fibrils is a hallmark of a group of diseases called amyloidosis and neurodegenerative disorders. Although polypeptides potentially have a fibril-forming propensity, native proteins have evolved into proper functional conformations to avoid aggregation and fibril formation. Understanding the mechanism for regulation of fibril formation of native proteins provides clues for the rational design of molecules for inhibiting fibril formation. Although fibril formation is a complex multistep reaction, experimentally obtained fibril formation curves can be fitted with the Finke-Watzky (F-W) two-step model for homogeneous nucleation followed by autocatalytic fibril growth. The resultant F-W rate constants for nucleation and fibril formation provide information on the chemical kinetics of fibril formation. Using the F-W two-step model analysis, we investigated the physicochemical mechanisms of fibril formation of a Parkinson's disease protein α-synuclein (αS) and a systemic amyloidosis protein apolipoprotein A-I (apoA-I). The results indicate that the C-terminal region of αS enthalpically and entropically suppresses nucleation through the intramolecular interaction with the N-terminal region and the intermolecular interaction with existing fibrils. In contrast, the nucleation of the N-terminal fragment of apoA-I is entropically driven likely due to dehydration of large hydrophobic segments in the molecule. Based on our recent findings, we discuss the similarity and difference of the fibril formation mechanisms of αS and the N-terminal fragment of apoA-I from the physicochemical viewpoints.

Formation of Aggregate-Free Gold Nanoparticles in the Cyclodextrin-Tetrachloroaurate System Follows Finke-Watzky Kinetics.

Cyclodextrin-capped gold nanoparticles are promising drug-delivery vehicles, but the technique of their preparation without trace amounts of aggregates is still lacking, and the size-manipulation possibility is very limited. In the present study, gold nanoparticles were synthesized by means of 0.1% (w/w) tetrachloroauric acid reduction with cyclodextrins at room temperature, at cyclodextrin concentrations of 0.001 M, 0.002 M and 0.004 M, and pH values of 11, 11.5 and 12. The synthesized nanoparticles were characterized by dynamic light scattering in both back-scattering and forward-scattering modes, spectrophotometry, X-ray photoelectron spectroscopy, transmission electron microscopy and Fourier-transform infrared spectroscopy. These techniques revealed 14.9% Au1+ on their surfaces. The Finke-Watzky kinetics of the reaction was demonstrated, but the actual growth mechanism turned out to be multistage. The synthesis kinetics and the resulting particle-size distribution were pH-dependent. The reaction and centrifugation conditions for the recovery of aggregate-free nanoparticles with different size distributions were determined. The absorbances of the best preparations were 7.6 for α-cyclodextrin, 8.9 for ß-cyclodextrin and 7.5 for γ-cyclodextrin. Particle-size distribution by intensity was indicative of the complete absence of aggregates. The resulting preparations were ready to use without the need for concentration, filtration, or further purification. The synthesis meets the requirements of green chemistry.

Aggregation Kinetics for IgG1-Based Monoclonal Antibody Therapeutics.

Monoclonal antibodies (mAbs) as a class of therapeutic molecules are finding an increasing demand in the biotechnology industry for the treatment of diseases like cancer and multiple sclerosis. A key challenge associated to successful commercialization of mAbs is that from the various physical and chemical instabilities that are inherent to these molecules. Out of all probable instabilities, aggregation of mAbs has been a major problem that has been associated with a change in the protein structure and is a hurdle in various upstream and downstream processes. It can stimulate immune response causing protein misfolding having deleterious and harmful effects inside a cell. Also, the extra cost incurred to remove aggregated mAbs from the rest of the batch is huge. Size exclusion chromatography (SEC) is a major technique for characterizing aggregation in mAbs where change in the aggregates' size over time is estimated. The current project is an attempt to understand the rate and mechanism of formation of higher order oligomers when subjected to different environmental conditions such as buffer type, temperature, pH, and salt concentration. The results will be useful in avoiding the product exposure to conditions that can induce aggregation during upstream, downstream, and storage process. Extended Lumry-Eyring model (ELE), Lumry-Eyring Native Polymerization model (LENP), and Finke-Watzky model (F-W) have been employed in this work to fit the aggregation experimental data and results are compared to find the best fit model for mAb aggregation to connect the theoretical dots with the reality.

Kinetic studies of nucleation and growth of palladium nanoparticles.

In this paper, the kinetic studies of nucleation and growth processes of palladium nanoparticles formation are presented. As a palladium precursor the chloride complex of Pd(II) and as a reductant l-ascorbic acid, were used. Kinetic studies were conducted under different experimental conditions such as initial concentration of metal ions, ascorbic acid, chloride ions as well as at different temperature and ionic strength. Using Finke-Watzky model kinetic rate constants were established and discussed. The value of activation enthalpy and entropy have also been determined using Eyring-Polanyi equation. For all obtained colloids, plasmons and values of hydrodynamic radius were registered.

Effect of Mannitol on Nucleation and Crystal Growth of Amorphous Flavonoids: Implications on the Formation of Nanocrystalline Solid Dispersion.

In this work, we studied crystallization kinetics of amorphous hesperetin (HRN) and naringenin (NRN) alone, and in 1:1 proportion with mannitol at Tg + 15 K. Crystallization rate of NRN was found to be significantly higher than HRN. Mannitol accelerated crystallization of HRN as well as NRN. NRN exhibited higher crystallization rate than HRN, in presence of mannitol, as well. Finke-Watzky model was used to deconvolute the crystallization kinetics data into nucleation and crystal growth rate constant. HRN alone had 9.56 × 10(9) times faster nucleation rate and 1.88 times slower crystal growth than NRN alone. Mannitol increased nucleation and crystal growth rate of HRN as well as NRN. In presence of mannitol, HRN possessed 1.34 × 10(10) times faster nucleation rate and 1.70 times slower crystal growth rate than NRN. Differences in crystallization behavior of HRN and NRN were explained by their thermodynamic properties.

Fitting bevacizumab aggregation kinetic data with the Finke-Watzky two-step model: Effect of thermal and mechanical stress.

Size exclusion chromatography with light scattering detection (SEC-MALLS) was assessed as a means to characterize the type of bevacizumab aggregates that form under mechanical and thermal stress, quantitatively monitoring the aggregation kinetics. The analytical method was monitored and verified during routine use at two levels: (1) the "pre-study" validation shows that the method is specific, linear, accurate, precise, robust and stability indicating; (2) the "in-study" validation was verified by inserting quality control samples and the use of control charts, indicating that the analytical method is in statistical control and stable. The aggregation kinetics data were interpreted using a modified Lumry-Eyring model, but the quality of the fit can be considered poor (R(2)>0.96), especially at higher temperatures. This indicates that the order of the reaction could not be reliably determined, suggesting a different degradation mechanism. The kinetic data set also fit the minimalistic Finke-Watzky (F-W) 2-step model, with an excellent quality of fit (R(2)>0.99), yielding the first quantitative rate constant for the steps of nucleation and growth in bevacizumab aggregation. The bevacizumab pharmaceutical preparation contains (initially) dimers, approximately 1.6% of bevacizumab total concentration, and the effect on aggregation kinetics of seeding was analyzed using the F-W 2-step model assuming [B]0≠0 (for the seeded case). The results suggested that the seeding had no impact on aggregation kinetics. Furthermore, the Arrhenius equation cannot be used to extrapolate the shelf-life since no linear temperature dependence of the rate constant was found within the temperature range. Although the real-time stability data provides the basis for determining the product shelf-life, predictive methodologies such as Vogel-Tammann-Fulcher (VFT) or the Arrhenius approach can be misleading and result in overestimates of the product shelf-life. However, they can be successfully applied to fixing the lower and upper limits of the aggregation rate, i.e. the best and worst-case scenarios regarding the aggregation potential of the product. In conclusion, the present study evaluates the first application of the F-W 2-step model to fitting and interpretation of experimental aggregation data for bevacizumab pharmaceutical preparations, using SEC-MALLS in this context.