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This study focuses on the modulation of protein aggregation and immunogenicity. As a starting point for investigating long-range interactions within a non-native protein, the effects of perturbing denatured protein states on their aggregation, including the formation of amyloid fibrils, were evaluated. The effects of adducts, sugar modifications, and stabilization on protein aggregation were then examined. We also investigated how protein immunogenicity was affected by enhancing protein conformational stability and other factors.

The formation of neurotoxic aggregates by amyloid-β peptide (Aβ) is considered to be a key step in the onset of Alzheimer's disease. It is widely accepted that oligomers are more neurotoxic than amyloid fibrils in the aqueous-phase aggregation of Aβ. Membrane-mediated amyloidogenesis is also relevant to the pathology, although the relationship between the aggregate size and cytotoxicity has remained elusive. Fluorescence correlation spectroscopy (FCS) is a sensitive method to monitor molecular aggregation processes by measuring diffusion of fluorophore-labeled molecules. Here, we monitored the initial aggregation process of Aβ on cell membranes of SH-SY5Y neuroblastoma cells. For example, aggregation of Aβ-(1-42) was evaluated by using Aβ-(1-42) (5 μM) doped with fluorophore-Aβ-(1-42) (10 nM). A membrane-bound Aβ component was detected in FCS autocorrelation curve after 1-h incubation of the Aβs with the cells. Following incubation for ～10 h, Aβ-(1-42) formed oligomers composed of ～10 Aβ molecules. These Aβ oligomers formed on membranes did not induce activation of caspase-3, an effector caspase for apoptosis, therefore were not neurotoxic, in contrast to reported Aβ oligomers prepared in aqueous phase. Formation of larger Aβ fibrils on membranes was found to be critical for Aβ neurotoxicity. We also report that trace amounts of pyroglutaminated Aβ-(3-42), a minor species of Aβ, can enhance initial aggregation process of Aβ-(1-42) on the cell membranes. These results indicate the usefulness of FCS detection of small Aβ oligomers formed on cell surface, which can act as pathogenic seeds for amyloid fibrils responsible for neurotoxicity.

Biopharmaceuticals, including antibody drugs, are now popular because of their high specificity with low adverse effects, especially in the treatment of cancer and autoimmune diseases. However, because the active pharmaceutical ingredients of biopharmaceuticals are proteins, biophysical characterization of these therapeutic proteins should be required. In this manuscript, methods of chemical and physical characterization of therapeutic proteins are described. In terms of chemical characterization, analysis of chemical modifications of the constituent amino acids is explained. Physical characterization includes higher order structural analysis and assessment of protein aggregates. Quantification methods of aggregates with different sizes, recently encouraged by the U.S. Food and Drug Administration (FDA), are introduced. As for the stability of therapeutic proteins, the importance of chemical and physical stability is explained. Finally, the contribution of colloidal and structural stability to the production of an antibody drug less prone to aggregation is introduced.

Therapeutic monoclonal antibody (mAb) preparations are produced from cultured cells; therefore, detailed and multidimensional analyses of their heterogeneities are required. We analyzed five commercially available mAb preparations by high-temperature reversed-phase LC using a wide-pore core-shell column for pluralistic quality assessment. At a highly elevated column temperature, isopropanol with high eluotropic strength coefficients and a wide-pore core-shell type octyl column showed good peak resolution of the investigated mAbs and their related constituents. We used this method to estimate the residual rate of intact mAbs after a heat aggregation treatment and conducted fragmentation analysis by analyzing their pepsin digests. Each peak component was identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. All results were compared with those of reversed-phase and size exclusion analyses.

Although α-synuclein protein (αS) aggregates from a monomer to assemblies such as oligomer, protofibril and mature fibril, the early intermediate aggregate, that is, oligomer has been considered to be most toxic species in recent reports. While it was reported that αS concentration in cerebrospinal fluid was decreased significantly in the patients with Parkinson's disease (PD) and dementia with Lewy bodies, there were reports that αS oligomer concentration was elevated in cerebrospinal fluid of PD patients. Moreover, it was supposed that αS oligomer concentration was also elevated in blood of PD patients. Further studies of αS in cerebrospinal fluid and blood would lead to establishment of the significance of αS as a biomarker for α-synucleinopathies including PD.