Yijia Jiang

BOTH FAMILIAL PARKINSON'S DISEASE MUTATIONS ACCELERATE ALPHA -SYNUCLEIN AGGREGATION

Parkinson’s disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies, the major component of which are filaments consisting of α-synuclein. Two recently identified point mutations in α-synuclein are the only known genetic causes of PD, but their pathogenic mechanism is not understood. Here we show that both wild type and mutant α-synuclein form insoluble fibrillar aggregates with antiparallel β-sheet structure upon incubation at physiological temperature in vitro. Importantly, aggregate formation is accelerated by both PD-linked mutations. Under the experimental conditions, the lag time for the formation of precipitable aggregates is about 280 h for the wild type protein, 180 h for the A30P mutant, and only 100 h for the A53T mutant protein. These data suggest that the formation of α-synuclein aggregates could be a critical step in PD pathogenesis, which is accelerated by the PD-linked mutations.

Parkinson's Disease-associated α-Synuclein Is More Fibrillogenic than β- and γ-Synuclein and Cannot Cross-seed Its Homologs

Parkinsons disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies. Recently, two point mutations in α-synuclein were found to be associated with familial PD, but as of yet no mutations have been described in the homologous genes β- and γ-synuclein. α-Synuclein forms the major fibrillar component of Lewy bodies, but these do not stain for β- or γ-synuclein. This result is very surprising, given the extent of sequence conservation and the high similarity in expression and subcellular localization, in particular between α- and β-synuclein. Here we compare in vitro fibrillogenesis of all three purified synucleins. We show that fresh solutions of α-, β-, and γ- synuclein show the same natively unfolded structure. While over time α-synuclein forms the previously described fibrils, no fibrils could be detected for β- and γ-synuclein under the same conditions. Most importantly, β- and γ-synuclein could not be cross-seeded with α-synuclein fibrils. However, under conditions that drastically accelerate aggregation, γ-synuclein can form fibrils with a lag phase roughly three times longer than α-synuclein. These results indicate that β- and γ-synuclein are intrinsically less fibrillogenic than α-synuclein and cannot form mixed fibrils with α-synuclein, which may explain why they do not appear in the pathological hallmarks of PD, although they are closely related to α-synuclein and are also abundant in brain.

A Critical Review of Analytical Methods for Subvisible and Visible Particles

The subvisible and visible particles present in a solution are often classified based on size, and are quantified by the actual number of particles present rather than by weight or molar amounts. The analysis of these particles in protein therapeutics are governed by compendial methods and the regulatory agencies, and the methods available to measure them originally evolved focusing on potential safety issues, including capillary occlusion and immunogenicity, that might arise from their presence. Ultracentrifugation, size exclusion chromatography, etc., discussed in previous articles, can be used to analyze aggregates of less than 0.10 microns. This article will focus on methods for analyzing and quantitating sub visible particles (SbVP) of 2 microns or larger. At the present time there is no routine method for quantitating sub visible particles (SbVP) between 0.1 microns and 2 microns. The most common technique for quantitating the amount of subvisible particles between 2 and 100 microns is the light obscuration method. This technique can determine size and amount of particles, but cannot differentiate between the types of particles, such as protein particles, foreign material, micro bubbles or silicone oil droplets, that can be present in protein solutions. The difficulties in adapting this method, originally developed for small molecule drugs for IV administration, to protein therapeutics delivered subcutaneously is discussed. The flow imaging techniques can determine morphology and optical characteristics of the particles, but still not identify the chemical composition. Other methods that can also be used, but are applicable for characterization purposes only, are discussed. The primary method for quantitating visible particles is visual inspection, a method that can be subjective and relies on adequate training of the human inspectors. Automated methods for visible particle determination are being developed. Identification of the chemical composition of isolated particles greater than about 50 microns is possible using several micro-spectroscopic methods, and these will also be discussed.

Tungsten-induced protein aggregation: Solution behavior

Tungsten has been associated with protein aggregation in prefilled syringes (PFSs). This study probed the relationship between PFSs, tungsten, visible particles, and protein aggregates. Experiments were carried out spiking solutions of two different model proteins with tungsten species obtained from the extraction of tungsten pins typically used in syringe manufacturing processes. These results were compared to those obtained with various soluble tungsten species from commercial sources. Although visible protein particles and aggregates were induced by tungsten from both sources, the extract from tungsten pins was more effective at inducing the formation of the soluble protein aggregates than the tungsten from other sources. Furthermore, our studies showed that the effect of tungsten on protein aggregation is dependent on the pH of the buffer used, the tungsten species, and the tungsten concentration present. The lower pH and increased tungsten concentration induced more protein aggregation. The protein molecules in the tungsten-induced aggregates had mostly nativelike structure, and aggregation was at least partly reversible. The aggregation was dependent on tungsten and protein concentration, and the ratio of these two and appears to arise through electrostatic interaction between protein and tungsten molecules. The level of tungsten required from the various sources was different, but in all cases it was at least an order of magnitude greater than the typical soluble tungsten levels measured in commercial PFS.

Qualification of FTIR spectroscopic method for protein secondary structural analysis

Fourier transform infrared (FTIR) spectroscopy is widely used to study protein secondary structure both in solution and in the solid state. The FTIR spectroscopic method has also been employed as a characterization method by the biopharmaceutical industry to determine the higher order structure of protein therapeutics, and to determine if any changes in protein conformation have occurred as a result of changes to process, formulation, manufacture, and storage conditions. The results of these studies are often included in regulatory filings; when comparability is assessed, the comparison is often qualitative. To demonstrate that the method can be quantitative, and is suitable for these intended purposes, the precision and sensitivity of the FTIR method were evaluated. The results show that FTIR spectroscopic analysis is reproducible with suitable method precision, that is, spectral similarity of replicate measurements is greater than 90%. The method can detect secondary structural changes caused by pH and denaturant. The sensitivity of the method in detecting structural changes depends on the extent of the changes and their impact on the resulting spectral similarity and characteristic FTIR bands. The results of these assessments are described in this paper.

Applications of differential scanning calorimetry for thermal stability analysis of proteins: Qualification of DSC

Differential scanning calorimetry (DSC) has been used to characterize protein thermal stability, overall conformation, and domain folding integrity by the biopharmaceutical industry. Recently, there have been increased requests from regulatory agencies for the qualification of characterization methods including DSC. Understanding the method precision can help determine what differences between samples are significant and also establish the acceptance criteria for comparability and other characterization studies. In this study, we identify the parameters for the qualification of DSC for thermal stability analysis of proteins. We use these parameters to assess the precision and sensitivity of DSC and demonstrate that DSC is suitable for protein thermal stability analysis for these purposes. Several molecules from different structural families were studied. The experiments and data analyses were performed by different analysts using different instruments at different sites. The results show that the (apparent) thermal transition midpoint (T(m)) values obtained for the same protein by same and different instruments and/or analysts are quite reproducible, and the profile similarity values obtained for the same protein from the same instrument are also high. DSC is an appropriate method for assessing protein thermal stability and conformational changes.

Applications of circular dichroism (CD) for structural analysis of proteins: qualification of near‐ and far‐UV CD for protein higher order structural analysis

Circular dichroism (CD) spectroscopy is routinely used in the biopharmaceutical industry to study the effects of manufacturing, formulation, and storage conditions on protein conformation and stability, and these results are often included in regulatory filings. In this context, the purpose of CD spectroscopy is often to verify that a change in the formulation or manufacturing process of a product has not produced a change in the conformation of a protein. A comparison of two or more spectra is often required to confirm that the proteins structure has been maintained. Traditionally, such comparisons have been qualitative in nature, based on visually inspecting the overlaid spectra. However, visual assessment is inherently subjective and therefore prone to error. Furthermore, recent requests from regulatory agencies to demonstrate the suitability of the CD spectroscopic method for the purpose of comparing spectra have highlighted the need to appropriately qualify CD spectroscopy for characterization of biopharmaceutical protein products. In this study, we use a numerical spectral comparison approach to establish the precision of the CD spectroscopic method and to demonstrate that it is suitable for protein structural characterization in numerous biopharmaceutical applications.

Separation and characterization of protein aggregates and particles by field flow fractionation.

Field flow fractionation (FFF) is a technique that holds great promise for the analysis and characterization of protein aggregates and particles, due to its wide dynamic range and matrix-free separation mechanism. FFF can be routinely used to achieve good monomer-oligomer separation and quantification for a variety of protein types, and is a reasonable choice for an orthogonal method for size exclusion chromatography and analytical ultracentrifugation. Quantifying sub-micrometer particles in protein therapeutics is a potential of the FFF technique that is yet to be realized, due to the lack of detection with sufficient sensitivity. In this article the effect of several important parameters on the optimization of FFF analyses are explored, and the strengths, weaknesses, and potential new applications of the technique are discussed.

Physical and biophysical effects of polysorbate 20 and 80 on darbepoetin alfa.

We studied the physical and biophysical affects of the nonionic surfactants polysorbate 20 and 80 and their mechanism of interaction using darbepoetin alfa, a 4-helix bundle protein, as the exemplary protein. Differences were observed between the abilities of the polysorbates to prevent surface loss/aggregation and correlated with each polysorbates initiation of micelle formation prior to the critical micelle concentration (CMC). The biophysical properties monitored by far-UV circular dichroism (CD) and tryptophan (Trp) fluorescence showed effects due to polysorbates, but were not correlated with their CMC. At a constant protein concentration PS-80 induced alpha-helix in the protein with a maximal effect at 15:1 molar ratio of PS-80/protein. PS-20 initially induced alpha-helix with a maximal effect at 1.5:1 ratio followed by a decrease in the alpha-helix content. PS-80 had no effect on near-UV CD but increased Trp fluorescence only at the 150:1 polysorbate/protein ratio. PS-20 decreased the near-UV CD and Trp fluorescence. Thermodynamic studies by isothermal titration calorimetry (ITC) demonstrated that the protein interacts with monomeric polysorbate, but not with polysorbate micelles. The data suggest that the polysorbates differentially interact with the protein and that the biophysical effects are dependent on the structure of the polysorbate and the polysorbate to protein ratio.

Precision of protein aggregation measurements by sedimentation velocity analytical ultracentrifugation in biopharmaceutical applications.

Sedimentation velocity analytical ultracentrifugation (SV-AUC) is routinely applied in biopharmaceutical development to measure levels of protein aggregation in protein products. SV-AUC is free from many limitations intrinsic to size exclusion chromatography (SEC) such as mobile phase and column interaction effects on protein self-association. Despite these clear advantages, SV-AUC exhibits lower precision measurements than corresponding measurements by SEC. The precision of SV-AUC is influenced by numerous factors, including sample characteristics, cell alignment, centerpiece quality, and data analysis approaches. In this study, we evaluate the precision of SV-AUC in its current practice utilizing a multilaboratory, multiproduct intermediate precision study. We then explore experimental approaches to improve SV-AUC measurement precision, with emphasis on utilization of high quality centerpieces.