Kinetics of Antibody Aggregation at Neutral pH and Ambient Temperatures Triggered by Temporal Exposure to Acid

The purification process of an antibody in manufacturing involves temporal exposure of the molecules to low pH followed by neutralizationpH-shift stresswhich causes aggregation. It remains unclear how aggregation triggered by pH-shift stress grows at neutral pH and how it depends on the temperature in an ambient range. We used static and dynamic light scattering to monitor the time-dependent evolution of the aggregate size of the pH-shift stressed antibody between 4.0 and 40.0 °C. A power-law relationship between the effective molecular weight and the effective hydrodynamic radius was found, indicating that the aggregates were fractal with a dimension of 1.98. We found that the aggregation kinetics in the lower-temperature range, 4.0–25.0 °C, were well described by the Smoluchowski aggregation equation. The temperature dependence of the effective aggregation rate constant gave 13 ± 1 kcal/mol of endothermic activation energy. Temporal acid exposure creates an enriched population of unfolded protein molecules that are competent of aggregating. Therefore, the energetically unfavorable unfolding step is not required and the aggregation proceeds faster. These findings provide a basis for predicting the growth of aggregates during storage under practical, ambient conditions

Hydration Promoted by a Methylene Group: A Volumetric Study on Alkynes in Water

Hydrocarbons including a methylene group are generally considered a hydrophobic building block, in the sense that the density of their hydration water is lower than that of bulk water. However, is the methylene group always hydrophobic? In this study, we experimentally determined the partial molar volume of a methylene group in water as 14.01 ± 0.46 cm³ mol^–1 for 1-alkyne, 9.83 ± 0.35 cm³ mol^–1 for 2-alkyne, and 11.39 ± 0.55 cm³ mol^–1 for 3-alkyne. These values are all unusually small compared to the ∼16 cm³ mol^–1 for model compounds from the literature. The subsequent volumetric analysis on the basis of the Kirkwood–Buff parameter indicates that the hydration water is enriched by the addition of a methylene group for 2-alkyne, while it is depleted for the reported model compounds that contain hydrophilic functional groups, 1-alkyne, and 3-alkyne. Our findings suggest that the triple bonded carbons in 2-alkyne that reduce hydration water act as a hydrophobic group in 2-alkyne. Thus, the methylene group should be called “hydrophilic” in this case because it actually recovers the hydration water when placed next to more hydrophobic groups. Therefore, we conclude that the hydrophobicity of a methylene group varies depending on its hydration environment due to other functional groups in the solute

Differences in the Structural Stability and Cooperativity between Monomeric Variants of Natural and de Novo Cro Proteins Revealed by High-Pressure Fourier Transform Infrared Spectroscopy

It is widely accepted that pressure affects the structure and dynamics of proteins; however, the underlying mechanism remains unresolved. Our previous studies have investigated the effects of pressure on fundamental secondary structural elements using model peptides, because these peptides represent a basis for understanding the effects of pressure on more complex structures. This study targeted monomeric variants of naturally occurring bacteriophage λ Cro (natural Cro) and de novo designed λ Cro (SN4m), which are α + β proteins. The sequence of SN4m is 75% different from that of natural Cro, but the structures are almost identical. Consequently, a comparison of the folding properties of these proteins is of interest. Pressure- and temperature-variable Fourier transform infrared spectroscopic analyses revealed that the α-helices and β-sheets of natural Cro are cooperatively and reversibly unfolded by pressure and temperature, whereas those of SN4m are not cooperatively unfolded by pressure; i.e., the α-helices of SN4m unfold at significantly higher pressures than the β-sheets and irreversibly unfold with increases in temperature. The higher unfolding pressure for the α-helices of SN4m indicates the presence of an intermediate structure of SN4m that does not retain β-sheet structure but does preserve the α-helices. These results demonstrate that the α-helices of natural Cro are stabilized by global tertiary contacts among the α-helices and the β-sheets, whereas the α-helices of SN4m are stabilized by local tertiary contacts between the α-helices

Conformational and Colloidal Stabilities of Human Immunoglobulin G Fc and Its Cyclized Variant: Independent and Compensatory Participation of Domains in Aggregation of Multidomain Proteins

Monoclonal immunoglobulin G (IgG) is a multidomain protein. It has been reported that the conformational and colloidal stabilities of each domain are different, and it is predicted that limited domains participate in IgG aggregation. In contrast, the influence of interdomain interactions on IgG aggregation remains unclear. The fragment crystallizable (Fc) region is also a multidomain protein consisting of two sets of C_H2 and C_H3 domains. Here, we have analyzed the conformational change and aggregate size of an aglycosylated Fc region induced by both acid and salt stresses and have elucidated the influence of interdomain interactions between C_H2 and C_H3 domains on the conformational and colloidal stabilities of the aglycosylated Fc region. Singular value decomposition analyses demonstrated that the C_H2 and C_H3 domains unfolded almost independently from each other in the aglycosylated Fc region. Meanwhile, the colloidal stabilities of the C_H2 and C_H3 domains affect the aggregation process of the unfolded aglycosylated Fc region in a compensatory way. Moreover, the influence of an additional interdomain disulfide bond, introduced at the C-terminal end of the C_H3 domains to produce the Fc variant, cyclized Fc, was evaluated. This interdomain disulfide bond increased the conformational stability of the C_H3 domain. The stabilization of the C_H3 domain in the cyclized Fc successfully improved aggregation tolerance following acid stress, although the sizes of aggregates produced were comparable to those of the aglycosylated Fc region

Asphaltene Aggregation Behavior in Bromobenzene Determined By Small-angle X‑ray Scattering

Small-angle X-ray scattering (SAXS) analyses of an asphaltene (a heptane-insoluble fraction in Canadian oil sand bitumen (CaAs)) at various concentrations in bromobenzene (BB) were performed at a synchrotron facility. BB is the first trial medium in which the aggregation behavior of asphaltenes has been elucidated, and is considered to be one of the “best” pure solvents for CaAs when determining the Hansen solubility parameters (HSP). Although the aggregation behavior of the CaAs in toluene (TL) and toluene–pentane mixed solvent (TL-PT10, containing 10% pentane on a volume basis) was confirmed to be similar to that reported in previous SAXS studies, the behavior in BB was markedly different. The results indicated that aggregates with a soft boundary of ∼30–60 Å in the radius of gyration (<i>R</i>_g), which were observable in TL and TL-PT10, disappeared in BB and larger aggregates with a clear boundary appeared simultaneously. This phenomenon supported a colloidal aggregation model, with HSP analyses suggesting that BB dispersed the colloid surface fraction at the molecular level and isolated the colloid core fraction, which led to the formation of a rigid aggregation of the core fraction. The HSP analyses enabled us to evaluate the aggregation behavior quantitatively, and the results obtained by SAXS were consistent with those obtained by Rayleigh scattering that we reported previously

Interaction Potential between Biological Sensing Nanoparticles Determined by Combining Small-Angle X‑ray Scattering and Model-Potential-Free Liquid Theory

Biological sensing technology utilizing nanoparticles extends through a diverse range of fields. The nanosensing is controlled using the assembly/disassembly of nanoparticles dominated by interaction forces between them. Although the interaction potential surface gives decisive information on the sensing mechanism, evaluating the quantitative profile has been impossible due to extremely complicated interactions of conjugated soft matter. In this study, a model-potential-free determination of the interaction potential surfaces was devised by combining small-angle scattering and liquid-state theory. The model-potential-free liquid theory was developed for colloidal nanoparticles inherently with strong van der Waals attraction forces by their nanoscopic size. The present method extracts interaction potential between nanoparticles even in systems with complicated interactions due to conjugated soft matter. By applying this determination method to a glutathione-triggered biosensing reaction, interaction potential curves between biosensing nanoparticles were realized for the first time. The analysis revealed peculiar potential surfaces of the sensing nanoparticles. The mechanism of colorimetric nanosensing function based on surface plasmon resonance is discussed from the viewpoint of the assembly/disassembly of nanoparticles in nanocomposites dominated by the interaction potential surfaces

An FT-IR Study on Packing Defects in Mixed β-Aggregates of Poly(l-glutamic acid) and Poly(d-glutamic acid): A High-Pressure Rescue from a Kinetic Trap

Under favorable conditions of pH and temperature, poly­(l-glutamic acid) (PLGA) adopts different types of secondary and quaternary structures, which include spiral assemblies of amyloid-like fibrils. Heating of acidified solutions of PLGA (or PDGA) triggers formation of β₂-type aggregates with morphological and tinctorial properties typical for amyloid fibrils. In contrast to regular antiparallel β-sheet (β₁), the amide I′ vibrational band of β₂-fibrils is unusually red-shifted below 1600 cm^–1, which has been attributed to bifurcated hydrogen bonds coupling CO and N–D groups of the main chains to glutamic acid side chains. However, unlike for pure PLGA, the amide I′ band of aggregates precipitating from racemic mixtures of PLGA and PDGA (β₁) is dominated by components at 1613 and 1685 cm^–1typically associated with intermolecular antiparallel β-sheets. The coaggregation of PLGA and PDGA chains is slower and biphasic and leads to less-structured assemblies of fibrils, which is reflected in scanning electron microscopy images, sedimentation properties, and fluorescence intensity after staining with thioflavin T. The β₁-type aggregates are metastable, and they slowly convert to fibrils with the infrared characteristics of β₂-type fibrils. The process is dramatically accelerated under high pressure. This implies the presence of void volumes within structural defects in racemic aggregates, preventing the precise alignment of main and side chains necessary to zip up ladders of bifurcated hydrogen bonds. As thermodynamic costs associated with maintaining void volumes within the racemic aggregate increase under high pressure, a hyperbaric treatment of misaligned chains leads to rectifying the packing defects and formation of the more compact form of fibrils