Michihiro Nagao

Observation of Small Cluster Formation in Concentrated Monoclonal Antibody Solutions and Its Implications to Solution Viscosity

Monoclonal antibodies (mAbs) are a major class of biopharmaceuticals. It is hypothesized that some concentrated mAb solutions exhibit formation of a solution phase consisting of reversibly self-associated aggregates (or reversible clusters), which is speculated to be responsible for their distinct solution properties. Here, we report direct observation of reversible clusters in concentrated solutions of mAbs using neutron spin echo. Specifically, a stable mAb solution is studied across a transition from dispersed monomers in dilute solution to clustered states at more concentrated conditions, where clusters of a preferred size are observed. Once mAb clusters have formed, their size, in contrast to that observed in typical globular protein solutions, is observed to remain nearly constant over a wide range of concentrations. Our results not only conclusively establish a clear relationship between the undesirable high viscosity of some mAb solutions and the formation of reversible clusters with extended open structures, but also directly observe self-assembled mAb protein clusters of preferred small finite size similar to that in micelle formation that dominate the properties of concentrated mAb solutions.

Heterogeneous Slow Dynamics of Imidazolium-Based Ionic Liquids Studied by Neutron Spin Echo

We have investigated structure and relaxation phenomena for ionic liquids 1-octyl-3-methylimidazolium hexafluorophosphate (C8mimPF6) and bis(trifluoromethylsulfonyl)imide (C8mimTFSI) by means of neutron diffraction and neutron spin echo (NSE) techniques. The diffraction patterns show two distinct peaks appeared at scattering vectors Q of 0.3 and 1.0 Å(-1). The former originates from the nanoscale structure characteristic to ionic liquids and the latter due to the interionic correlations. Interestingly, the intensity of the low-Q peak drastically grows upon cooling and keeps growing even below the glass transition temperature. The NSE measurements have been performed at these two Q positions, to explore the time evolution of each correlation. The relaxation related to the ionic correlation (ionic diffusion) is of Arrhenius-type and exhibits nonexponential behavior. The activation energy (Ea) of the ionic diffusion, which is linked to viscosity, depends on the type of anion; the larger is the anion size, the smaller Ea becomes for most of anions. On the other hand, two kinds of relaxation processes, slower and faster ones, are found at the low-Q peak position. The most significant finding is that the fraction of the slower relaxation increases and that of the faster one decreases upon cooling. Combining the NSE data with the diffraction data, we conclude that there exist two parts in ILs: one with the ordered nanostructure exhibiting the slow relaxation, and the other with disordered structure showing faster relaxation. The structure and dynamics of ILs are heterogeneous in nature, and the fraction of each part changes with temperature.

Upgrade of the 32 m small-angle neutron scattering instrument SANS-U

The small-angle neutron scattering instrument, SANS-U, owned by the Institute for Solid State Physics, The University of Tokyo, has been upgraded. The SANS-U is a 32 m SANS instrument installed in 1991 at the guide hall of the JRR-3M research reactor of the Japan Atomic Energy Research Institute, and has been serving for inter-university cooperative research use since 1993. The major upgrades include (i) replacement of the two-dimensional area detector by a multi-wire type position-sensitive proportional counter, (ii) renewal of the operating system from a VAX and sequencers to an integrated PXI system controlled by LabVIEW-RT software, (iii) a focusing collimation system, and (iv) a variety of accessory equipment. These upgrades provide a wide dynamic range of neutron counting, user-friendly operation and real-time circular averaging of two-dimensional data.

Hierarchical structure and dynamics of an ionic liquid 1-octyl-3-methylimidazolium chloride

We have performed the heat capacity, neutron diffraction, and neutron quasielastic scattering measurements of an ionic liquid 1-octyl-3-methylimidazolium chloride (C8mimCl). The heat capacity data revealed that C8mimCl exhibits a glass transition with a large heat capacity jump at T(g) = 214 K, which is lower than T(g) of C4mimCl with a shorter alkyl-chain. In the neutron diffraction measurement for a deuterated analogue, d-C8mimCl, the peaks associated with the inter-domain, inter-ionic, and inter-alkyl-chain correlations appeared at (3, 11, and 14) nm(-1), respectively. The temperature dependence of these peaks indicates that the packing of the alkyl-chains becomes more compact and the domains become more vivid and larger as decreasing temperature. The quasielastic neutron scattering measurements using neutron spin echo and time-of-flight type instruments demonstrated that C8mimCl has faster relaxations probably owing to the alkyl-group and a slower relaxation owing to the ions. The latter relaxation, which is related to the glass transition, is of non-exponential as in the α relaxation of glass-forming molecular liquids. The relaxation of domains could not be observed in the present experiment but should have relaxation times longer than 100 ns. This is the first report to clarify temperature dependence of the hierarchical structure and relaxations simultaneously for a typical ionic liquid.

Neutron spin–echo investigations of membrane undulations in complex fluids involving amphiphiles

Abstract The intermediate functions I ( Q , t ) obtained from neutron spin–echo (NSE) experiments were well fitted to I(Q,t)=I(Q,0) exp [−(Γt) 2/3 ] for the bicontinuous microemulsion and the lamellar phases of the C 12 E 5 / n -octane/water system and also for the lamellar phase of the DPPC/water/CaCl 2 system. The relaxation rate Γ increased as Q 3 . These results support the theory presented by Zilman and Granek [Phys. Rev. Lett. 77 (1996) 4788]. Bending modulus of the membrane ϰ was estimated in the C 12 E 5 / n -octane/water system and in the DPPC/water/CaCl 2 system using their theory.

Pressure and temperature effects on the phase transition from a dense droplet to a lamellar structure in a ternary microemulsion

A small-angle x-ray scattering (SAXS) study of a ternary microemulsion composed of AOT [sodium bis(2-ethylhexyl) sulfosuccinate], water and n-decane was undertaken in order to clarify the phase behavior and the feature of the corresponding structural transition from a dense droplet to a lamellar structure with increasing pressure and temperature. The volume fractions of water and decane were fixed to be equal and the volume fraction of AOT against the whole volume (φs) was selected to be 0.209 and 0.230 in order to compare results with those obtained by small-angle neutron scattering (SANS). The pressure was varied between 1 and 800 bar under controlled temperature at 20, 25, 29, or 33 °C. Under all conditions applied, the phase transition from the droplet structure to the lamellar structure was observed. The results of analysis of the SAXS profiles indicated that the short-range adhesive potential between droplets becomes more intense with increasing pressure.

SAXS, SANS and NSE studies on unbound state in DPPC/water/CaCl2 system

The temperature and CaCl 2 concentration dependence of the lamellar repeat distance of a dipalmitoylphosphatidylcholine (DPPC) aqueous solution was investigated by small-angle X-ray and neutron scattering, and neutron spin echo. At certain CaCl 2 concentrations, the repeat distance in the liquid-crystalline phase tends to increase up to infinity while that in the gel phase has an upper limit. This behavior is attributable to the fact that the steric repulsion of lipid bilayers due to the membrane undulation in the liquid-crystalline phase is larger than that in the gel phase. The free-energy calculation suggested that the cooperation of the short-range electrostatic interaction and the long-range steric interaction is the origin of the increase in the repeat distance in the liquid-crystalline phase, and it can be the origin of an “unbinding transition”.

Bending modulus of lipid bilayers in a liquid-crystalline phase including an anomalous swelling regime estimated by neutron spin echo experiments

Abstract.Membrane fluctuations of dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) were investigated by neutron spin echo spectroscopy. The intermediate structure factor was analyzed in terms of the model proposed by Zilman and Granek (Phys. Rev. Lett. 77, 4788 (1996)), and the bending modulus of lipid bilayers was derived. The hardening of a lipid bilayer upon approaching the main transition point in the anomalous swelling regime was observed, which naturally connects the bending modulus in the gel phase below the main transition temperature.

2D-Ising-like critical behavior in mixtures of water and 3-methylpyridine including antagonistic salt or ionic surfactant

The effect of an antagonistic salt on the phase behavior and nanoscale structure of a mixture of D2O and 3-methylpyridine was investigated by visual inspection and small-angle neutron scattering (SANS). The addition of the antagonistic salt, namely sodium tetraphenylborate (NaBPh4), induces the shrinking of the two-phase region in contrast to the case in which a normal (hydrophilic) salt is added. Below the phase separation point, the SANS profiles cannot be described by the Ornstein–Zernike function owing to the existence of a long-range periodic structure. With increasing salt concentration, the critical exponents change from the values of 3D-Ising and approach those of 2D-Ising. These results suggest that the concentration fluctuation of the mixture of solvents is limited to a quasi two-dimensional space by the periodic structure induced by the adding the salt. The same behaviors were also observed in mixtures composed of water, 3-methylpyridine, and ionic surfactant.

Small-angle neutron scattering study of droplet density dependence of the water-in-oil droplet structure in a ternary microemulsion

Droplet density, O, dependence of the water-in-oil droplet structure of microemulsion was investigated by means of small-angle neutron scattering (SANS) at room temperature in a ternary system consisting of AOT (Aerosol-OT; dioctyl sulfosuccinate sodium salt), water, and n-decane. SANS profiles from 2 different contrast samples of the bulk contrast (ACT/D 2 O/C 10 H 22 ) and of the film contrast (ACT/D 2 O/C 10 D 22 ) were collected. By introducing a relative form factor as the ratio of scattering functions of the bulk and film contrast samples, we evaluated the O dependence of the shape and size of droplets without the influence of the structure factor, S(Q). The mean radii of droplet core and shell were constant, while the polydispersity index decreased for O ≤ O* ≃ 0.62. The evaluated structure factors S(Q) from the bulk-contrast samples were in good agreement with those from the film contrast. From the O dependence of S(Q), we confirmed that the droplet density fluctuations were dominant at low-O regime and the inter-droplet correlations at high-O regime. The crossover concentration of these two regimes was estimated to be about 0.25. It is demonstrated that this relative form factor method is quite useful for SANS experiments.