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Soluble Aggregates (soluble + aggregate)
Selected AbstractsBiophysical studies of the development of amyloid fibrils from a peptide fragment of cold shock protein BFEBS JOURNAL, Issue 9 2000Deborah K. Wilkins The peptide CspB-1, which represents residues 1,22 of the cold shock protein CspB from Bacillus subtilis, has been shown to form amyloid fibrils when solutions containing this peptide in aqueous (50%) acetonitrile are diluted in water [M. Großet al. (1999) Protein Science8, 1350,1357] We established conditions in which reproducible kinetic steps associated with the formation of these fibrils can be observed. Studies combining these conditions with a range of biophysical methods reveal that a variety of distinct events occurs during the process that results in amyloid fibrils. A CD spectrum indicative of ,,structure is observed within 1 min of the solvent shift, and its intensity increases on a longer timescale in at least two kinetic phases. The characteristic wavelength shift of the amyloid-binding dye Congo Red is established within 30 min of the initiation of the aggregation process and corresponds to one of the phases observed by CD and to changes in the Fourier transform-infrared spectrum indicative of ,,structure. Short fibrillar structures begin to be visible under the electron microscope after these events, and longer, well-defined amyloid fibrils are established on a timescale of hours. NMR spectroscopy shows that there are no significant changes in the concentration of monomeric species in solution during the events leading to fibril formation, but that soluble aggregates too large to be visible in NMR spectra are present throughout the process. A model for amyloid formation by this peptide is presented which is consistent with these kinetic data and with published work on a variety of disease-related systems. These findings support the concept that the ability to form amyloid fibrils is a generic property of polypeptide chains, and that the mechanism of their formation is similar for different peptides and proteins. [source] Physicochemical characterization of carrageenans,A critical reinvestigationJOURNAL OF APPLIED POLYMER SCIENCE, Issue 6 2008Gisela Berth Abstract Kappa-, iota-, and lambda-carrageenan (food grade) were analyzed by static light scattering (MALS in batch mode) in 0.1M NaNO3 at 25 and 60°C, earlier heated up to 90°C or not. At 25°C, there was a strong tendency for a concentration-dependent aggregation in the order lambda < kappa < iota. At 60°C, all samples were molecularly dispersed. The strongly temperature-dependent refractive index increments (equilibrium dialysis) differ. Data interpretation in terms of the wormlike chain model using the Skolnik-Odijk-Fixman approach led to an intrinsic persistence length around 3 to 4 nm and expansion factors as high as 1.5 and above in a thermodynamically good solvent for all three types. Triple-detector HPSEC (DRI, MALS, viscometry) on the three commercial samples plus a degraded (by acidic hydrolysis) kappa-carrageenan in the same solvent/eluant at 60°C yielded a uniform and slightly curved [,]- M relationship for 5 × 103 , M/(g mol) , 3 × 106 and a nearly identical molar mass dependence of the radius of gyration. HPSEC at 25°C on kappa-carrageenan confirmed formation of soluble aggregates. Special emphasis was put on analytical and methodological aspects. The reliability of the experimental data was demonstrated by analogous measurements on dextran calibration standards. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008 [source] Equilibrium studies of protein aggregates and homogeneous nucleation in protein formulationJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 2 2010Sylvia Kiese Abstract Shaking or heat stress may induce protein aggregates. Aggregation behavior of an IgG1 stressed by shaking or heat following static storage at 5 and 25°C was investigated to determine whether protein aggregates exist in equilibrium. Aggregates were detected using different analytical methods including visual inspection, turbidity, light obscuration, size exclusion chromatography, and dynamic light scattering. Significant differences were evident between shaken and heated samples upon storage. Visible and subvisible particles (insoluble aggregates), turbidity and z -average diameter decreased whilst soluble aggregate content increased in shaken samples over time. Insoluble aggregates were considered to be reversible and dissociate into soluble aggregates and both aggregate types existed in equilibrium. Heat-induced aggregates had a denatured protein structure and upon static storage, no significant change in insoluble aggregates content was shown, whilst changes in soluble aggregates content occurred. This suggested that heat-induced insoluble aggregates were irreversible and not in equilibrium with soluble aggregates. Additionally, the aggregation behavior of unstressed IgG1 after spiking with heavily aggregated material (shaken or heat stressed) was studied. The aggregation behavior was not significantly altered, independent of the spiking concentration over time. Thus, neither mechanically stressed native nor temperature-induced denatured aggregates were involved in nucleating or propagating aggregation. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:632,644, 2010 [source] Self-buffering antibody formulationsJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 8 2008Yatin R. Gokarn Abstract Monoclonal antibodies (mAbs) often require the development of high-concentration formulations. In such cases, and when it is desirable to formulate a mAb around pH 5.0, we explored a novel approach of controlling the formulation pH by harnessing the ability of mAbs to "self-buffer." Buffer capacities of four representative IgG2 molecules (designated mAb1 through mAb4) were measured in the pH 4,6 range. The buffer capacity results indicated that the mAbs possessed a significant amount of buffer capacity, which increased linearly with concentration. By 60,80 mg/mL, the mAb buffer capacities surpassed that of 10 mM acetate, which is commonly employed in formulations for buffering in the pH 4,6 range. Accelerated high temperature stability studies (50°C over 3 weeks) conducted with a representative antibody in a self-buffered formulation (50 mg/mL mAb1 in 5.25% sorbitol, pH 5.0) and with solutions formulated using conventional buffers (50 mg/mL mAb1 in 5.25% sorbitol, 25 or 50 mM acetate, glutamate or succinate, also at pH 5.0) indicated that mAb1 was most resistant to the formation of soluble aggregates in the self-buffered formulation. Increased soluble aggregate levels were observed in all the conventionally buffered (acetate, glutamate, and succinate) formulations, which further increased with increasing buffer strength. The long-term stability of the self-buffered liquid mAb1 formulation (60 mg/mL in 5% sorbitol, 0.01% polysorbate 20, pH 5.2) was comparable to the conventionally buffered (60 mg/mL in 10 mM acetate or glutamate, 5.25% sorbitol, 0.01% polysorbate 20, pH 5.2) formulations. No significant change in pH was observed after 12 months of storage at 37 and 4°C for the self-buffered formulation. The 60 mg/mL self-buffered formulation of mAb1 was also observed to be stable to freeze-thaw cycling (five cycles, ,20°C,,,room temperature). Self-buffered formulations may be a better alternative for the development of high-concentration antibody and protein dosage forms. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97: 3051,3066, 2008 [source] Increased aggregation propensity of IgG2 subclass over IgG1: Role of conformational changes and covalent character in isolated aggregatesPROTEIN SCIENCE, Issue 9 2010Heather Franey Abstract Aggregation of human therapeutic antibodies represents a significant hurdle to product development. In a test across multiple antibodies, it was observed that IgG1 antibodies aggregated less, on average, than IgG2 antibodies under physiological pH and mildly elevated temperature. This phenomenon was also observed for IgG1 and IgG2 subclasses of anti-streptavidin, which shared 95% sequence identity but varied in interchain disulfide connectivity. To investigate the structural and covalent changes associated with greater aggregation in IgG2 subclasses, soluble aggregates from the two forms of anti-streptavidin were isolated and characterized. Sedimentation velocity analytical ultracentrifugation (SV-AUC) measurements confirmed that the aggregates were present in solution, and revealed that the IgG1 aggregate was composed of a predominant species, whereas the IgG2 aggregate was heterogeneous. Tertiary structural changes accompanied antibody aggregation as evidenced by greater ANS (8-Anilino-1-naphthalene sulfonic acid) binding to the aggregates over monomer, and differences in disulfide character and tryptophan environments between monomer, oligomer and aggregate species, as observed by near-UV circular dichroism (CD). Differences between subclasses were observed in the secondary structural changes that accompanied aggregation, particularly in the intermolecular ,-sheet and turn structures between the monomer and aggregate species. Free thiol determination showed ,2.4-fold lower quantity of free cysteines in the IgG1 subclass, consistent with the 2.4-fold reduction in aggregation of the IgG1 form when compared with IgG2 under these conditions. These observations suggested an important role for disulfide bond formation, as well as secondary and tertiary structural transitions, during antibody aggregation. Such degradations may be minimized using appropriate formulation conditions. [source] Deamidation destabilizes and triggers aggregation of a lens protein, ,A3-crystallinPROTEIN SCIENCE, Issue 9 2008Takumi Takata Abstract Protein aggregation is a hallmark of several neurodegenerative diseases and also of cataracts. The major proteins in the lens of the eye are crystallins, which accumulate throughout life and are extensively modified. Deamidation is the major modification in the lens during aging and cataracts. Among the crystallins, the ,A3-subunit has been found to have multiple sites of deamidation associated with the insoluble proteins in vivo. Several sites were predicted to be exposed on the surface of ,A3 and were investigated in this study. Deamidation was mimicked by site-directed mutagenesis at Q42 and N54 on the N-terminal domain, N133 and N155 on the C-terminal domain, and N120 in the peptide connecting the domains. Deamidation altered the tertiary structure without disrupting the secondary structure or the dimer formation of ,A3. Deamidations in the C-terminal domain and in the connecting peptide decreased stability to a greater extent than deamidations in the N-terminal domain. Deamidation at N54 and N155 also disrupted the association with the ,B1-subunit. Sedimentation velocity experiments integrated with high-resolution analysis detected soluble aggregates at 15%,20% in all deamidated proteins, but not in wild-type ,A3. These aggregates had elevated frictional ratios, suggesting that they were elongated. The detection of aggregates in vitro strongly suggests that deamidation may contribute to protein aggregation in the lens. A potential mechanism may include decreased stability and/or altered interactions with other ,-subunits. Understanding the role of deamidation in the long-lived crystallins has important implications in other aggregation diseases. [source] Modulation of protein aggregation by polyethylene glycol conjugation: GCSF as a case studyPROTEIN SCIENCE, Issue 5 2006Rahul S. Rajan Abstract Polyethylene glycol (PEG) conjugation to proteins has emerged as an important technology to produce drug molecules with sustained duration in the body. However, the implications of PEG conjugation to protein aggregation have not been well understood. In this study, conducted under physiological pH and temperature, N-terminal attachment of a 20 kDa PEG moiety to GCSF had the ability to (1) prevent protein precipitation by rendering the aggregates soluble, and (2) slow the rate of aggregation relative to GCSF. Our data suggest that PEG-GCSF solubility was mediated by favorable solvation of water molecules around the PEG group. PEG-GCSF appeared to aggregate on the same pathway as that of GCSF, as evidenced by (a) almost identical secondary structural transitions accompanying aggregation, (b) almost identical covalent character in the aggregates, and (c) the ability of PEG-GCSF to rescue GCSF precipitation. To understand the role of PEG length, the aggregation properties of free GCSF were compared to 5kPEG-GCSF and 20kPEG-GCSF. It was observed that even 5kPEG-GCSF avoided precipitation by forming soluble aggregates, and the stability toward aggregation was vastly improved compared to GCSF, but only marginally less stable than the 20kPEG-GCSF. Biological activity measurements demonstrated that both 5kPEG-GCSF and 20kPEG-GCSF retained greater activity after incubation at physiological conditions than free GCSF, consistent with the stability measurements. The data is most compatible with a model where PEG conjugation preserves the mechanism underlying protein aggregation in GCSF, steric hindrance by PEG influences aggregation rate, while aqueous solubility is mediated by polar PEG groups on the aggregate surface. [source] Impact of freezing on pH of buffered solutions and consequences for monoclonal antibody aggregationBIOTECHNOLOGY PROGRESS, Issue 3 2010Parag Kolhe Abstract Freezing of biologic drug substance at large scale is an important unit operation that enables manufacturing flexibility and increased use-period for the material. Stability of the biologic in frozen solutions is associated with a number of issues including potentially destabilizing pH changes. The pH changes arise from temperature-associated change in the pKas, solubility limitations, eutectic crystallization, and cryoconcentration. The pH changes for most of the common protein formulation buffers in the frozen state have not been systematically measured. Sodium phosphate buffer, a well-studied system, shows the greatest change in pH when going from +25 to ,30°C. Among the other buffers, histidine hydrochloride, sodium acetate, histidine acetate, citrate, and succinate, less than 1 pH unit change (increase) was observed over the temperature range from +25 to ,30°C, whereas Tris-hydrochloride had an ,1.2 pH unit increase. In general, a steady increase in pH was observed for all these buffers once cooled below 0°C. A formulated IgG2 monoclonal antibody in histidine buffer with added trehalose showed the same pH behavior as the buffer itself. This antibody in various formulations was subject to freeze/thaw cycling representing a wide process (phase transition) time range, reflective of practical situations. Measurement of soluble aggregates after repeated freeze,thaw cycles shows that the change in pH was not a factor for aggregate formation in this case, which instead is governed by the presence or absence of noncrystallizing cryoprotective excipients. In the absence of a cryoprotectant, longer phase transition times lead to higher aggregation. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] | ||||||||||