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Insoluble Aggregates (insoluble + aggregate)
Selected AbstractsEquilibrium 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] Small heat shock protein Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actinFEBS JOURNAL, Issue 22 2007Anastasia V. Pivovarova Previously, we have shown that the small heat shock protein with apparent molecular mass 27 kDa (Hsp27) does not affect the thermal unfolding of F-actin, but effectively prevents aggregation of thermally denatured F-actin [Pivovarova AV, Mikhailova VV, Chernik IS, Chebotareva NA, Levitsky DI & Gusev NB (2005) Biochem Biophys Res Commun331, 1548,1553], and supposed that Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actin. In the present work, we applied dynamic light scattering, analytical ultracentrifugation and size exclusion chromatography to examine the properties of complexes formed by denatured actin with a recombinant human Hsp27 mutant (Hsp27,3D) mimicking the naturally occurring phosphorylation of this protein at Ser15, Ser78, and Ser82. Our results show that formation of these complexes occurs upon heating and accompanies the F-actin thermal denaturation. All the methods show that the size of actin,Hsp27-3D complexes decreases with increasing Hsp27-3D concentration in the incubation mixture and that saturation occurs at approximately equimolar concentrations of Hsp27-3D and actin. Under these conditions, the complexes exhibit a hydrodynamic radius of ,,16 nm, a sedimentation coefficient of 17,20 S, and a molecular mass of about 2 MDa. It is supposed that Hsp27-3D binds to denatured actin monomers or short oligomers dissociated from actin filaments upon heating and protects them from aggregation by forming relatively small and highly soluble complexes. This mechanism might explain how small heat shock proteins prevent aggregation of denatured actin and by this means protect the cytoskeleton and the whole cell from damage caused by accumulation of large insoluble aggregates under heat shock conditions. [source] Potentiometric studies on the interaction between superoxide dismutase and hyaluronic acidJOURNAL OF APPLIED POLYMER SCIENCE, Issue 4 2009Yaofeng Fan Abstract The formation process of soluble complexes and insoluble aggregates between superoxide dismutase (SOD) and hyaluronic acid (HA) was studied using quasielastic light scattering and turbidimetric titration. The electrostatic binding between them was investigated in detail through potentiometric titration and turbidimetric titration carried out from high to low pH. Turbidimetric titration was used to determine the specific pH values at which soluble complex formation was initiated (pHc) and phase separation occurred (pH,). An increase of the ionic strength causes a decrease of pHc and pH,. With the increase of HA concentrations, pH, increases but pHc does not vary. The formed "salt bridges" between (SOD) and COO, (HA) result in the formation of stable SOD-HA complexes and even aggregates. The necessary condition of electrostatic binding was also given for protein-acidic polyelectrolyte systems. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009 [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] Characterization of antibody aggregation: Role of buried, unpaired cysteines in particle formationJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 2 2010Stephen R. Brych Abstract Proteins are susceptible to degradation upon exposure to a variety of stresses during product manufacturing, transportation and storage. In this study, we investigated the aggregation properties of a monoclonal antibody during agitation stress. Agitation exclusively led to insoluble aggregates, or particle formation. Removal or modification of the air,liquid interface with a surfactant (e.g., polysorbate) abrogated particle formation. The supernatant postagitation was analyzed using SE-HPLC, FTIR, and AUC analyses and revealed no changes in conformation and aggregation profile when compared to the nonagitated antibody sample. The antibody particles were comprised of a combination of nonnative intermolecular disulfide-linked covalent as well as noncovalent interactions. Analysis of the antibody's unpaired cysteines revealed that the nonnative intermolecular disulfide bonds were formed through buried cysteines, which suggested at least partial unfolding of the antibody domains. FTIR analysis indicated that the particulated antibody maintained significant native-like secondary structure suggesting that particle formation led to minimal structure changes, but capable of exposing free cysteines to solvent to form the nonnative intermolecular disulfide bonds. The results presented in this study indicate the importance of the interactions between the antibody and the air,liquid interface during agitation in the formation of particles and suggests that reduced disulfide bonds may play a significant role in the particulation reaction. This phenomenon can be applicable to other proteins with similar free cysteine and structural characteristics. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:764,781, 2010 [source] Simulation of pH-dependent edge strand rearrangement in human ,-2 microglobulinPROTEIN SCIENCE, Issue 1 2006Sheldon Park Abstract Amyloid fibrils formed from unrelated proteins often share morphological similarities, suggesting common biophysicalmechanisms for amyloidogenesis. Biochemical studies of human ,-2 microglobulin (,2M) have shown that its transition from a water-soluble protein to insoluble aggregates can be triggered by low pH. Additionally, biophysical measurements of ,2M using NMR have identified residues of the protein that participate in the formation of amyloid fibrils. The crystal structure of monomeric human ,2M determined at pH 5.7 shows that one of its edge ,-strands (strand D) adopts a conformation that differs from other structures of the same protein obtained at higher pH. This alternate ,-strand arrangement lacks a ,-bulge, which may facilitate protein aggregation through intermolecular ,-sheet association. To explore whether the pH change may yield the observed conformational difference, molecular dynamics simulations of ,2M were performed. The effects of pH were modeled by specifying the protonation states of Asp, Glu, and His, as well as the C terminus of the main chain. The bulged conformation of strand D is preferred at medium pH (pH 5,7), whereas at low pH (pH < 4) the straight conformation is observed. Therefore, low pH may stabilize the straight conformation of edge strand D and thus increase the amyloidogenicity of ,2M. [source] Conformations within soluble oligomers and insoluble aggregates revealed by resonance energy transferBIOPOLYMERS, Issue 4 2010Jyothi L. Digambaranath Abstract A fluorescently labeled 20-residue polyglutamic acid (polyE) peptide 20 amino acid length polyglutamic acid (E20) was used to study structural changes which occur in E20 as it co-aggregates with other unlabeled polyE peptides. Resonance energy transfer (RET) was performed using an o -aminobenzamide donor at the N-terminus and 3-nitrotyrosine acceptor at the C-terminus of E20. PolyE aggregates were not defined as amyloid, as they were nonfibrillar and did not bind congo red. Circular dichroism measurements indicate that polyE aggregation involves a transition from ,-helical monomers to aggregated ,-sheets. Soluble oligomers are also produced along with aggregates in the reaction, as determined through size exclusion chromatography. Time-resolved and steady-state RET measurements reveal four dominant E20 conformations: (1) a partially collapsed conformation (24 Å donor,acceptor distance) in monomers, (2) an extended conformation in soluble oligomers (>29 Å donor,acceptor distance), (3) a minor partially collapsed conformation (22 Å donor-acceptor distance) in aggregates, and (4) a major highly collapsed conformation (13 Å donor,acceptor distance) in aggregates. These findings demonstrate the use of RET as a means of determining angstrom-level structural details of soluble oligomer and aggregated states of proteins. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 299,317, 2010. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] Factors influencing antibody stability at solid,liquid interfaces in a high shear environmentBIOTECHNOLOGY PROGRESS, Issue 5 2009James G. Biddlecombe Abstract A rotating disk shear device was used to study the effect of interfacial shear on the structural integrity of human monoclonal antibodies of IgG4 isotype. Factors associated with the solution conditions (pH, ionic strength, surfactant concentration, temperature) and the interface (surface roughness) were studied for their effect on the rate of IgG4 monomer loss under high shear conditions. The structural integrity of the IgG4 was probed after exposure to interfacial shear effects by SDS-PAGE, IEF, dynamic light scattering, and peptide mapping by LC-MS. This analysis revealed that the main denaturation pathway of IgG4 exposed to these effects was the formation of large insoluble aggregates. Soluble aggregation, breakdown in primary structure, and chemical modifications were not detected. The dominant factors found to affect the rate of IgG4 monomer loss under interfacial shear conditions were found to be pH and the nanometer-scale surface roughness associated with the solid-liquid interface. Interestingly, temperature was not found to be a significant factor in the range tested (15,45°C). The addition of surfactant was found to have a significant stabilizing effect at concentrations up to 0.02% (w/v). Implications of these findings for the bioprocessing of this class of therapeutic protein are briefly discussed. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] | ||||||||||