Sugar molecules as a physical barrier separating therapeutic antibody proteins in freeze-dried formulations: small-angle X-ray scattering insights

In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique where X-rays are elastically scattered by an inhomogeneous sample in the nm-range at small angles (typically 0.1 - 5°) and wide angles (typically > 5°). This technique provides information about the shape, size, and distribution of macromolecules, characteristic distances of partially ordered materials, pore sizes, and surface-to-volume ratio. Small Angle X-ray Scattering (SAXS) is capable of delivering structural information of macromolecules between 1 and 200 nm, whereas Wide Angle X-ray Scattering (WAXS) can resolve even smaller Bragg spacing of samples between 0.33 nm and 0.49 nm based on the specific system setup and detector. The spacing is determined from Bragg's law and is dependent on the wavelength and incident angle. In a SWAXS experiment, the materials can be solid or liquid and may contain solid, liquid or gaseous domains (so-called particles) of the same or another material in any combination. SWAXS applications are very broad and include colloids of all types: metals, composites, cement, oil, polymers, plastics, proteins, foods, and pharmaceuticals. For solid samples, the thickness is limited to approximately 5 mm. Usage of a lab-based SWAXS instrument is detailed in this paper. With the available software (e.g., GNOM-ATSAS 2.3 package by D. Svergun EMBL-Hamburg and EasySWAXS software) for the SWAXS system, an experiment can be conducted to determine certain parameters of interest for the given sample. One example of a biological macromolecule experiment is the analysis of 2 wt% lysozyme in a water-based aqueous buffer which can be chosen and prepared through numerous methods. The preparation of the sample follows the guidelines below in the Preparation of the Sample section. Through SWAXS experimentation, important structural parameters of lysozyme, e.g. the radius of gyration, can be analyzed.

In this paper, we present a study of the structural and self-assembling properties of a new family of bolaamphiphiles. These bolaamphiphiles are unsymmetrical, having one sugar polar head at one side and one glycine betaine polar head at the other side. The variations that we introduced concern the length of the main bridging chain that connects the two polar heads as well as the length of the side chain linked at the anomeric position of the sugar moiety. Another variation concerns the introduction of a diacetylenic unit into the main chain in order to rigidify it. We have performed small angle X-ray scattering (SAXS) and wide angle X-ray scattering (WAXS) on the dry compounds as a function of temperature and observed the lamellar structures. We also measured the SAXS and WAXS spectra of aqueous solutions of these compounds that have shown various lamellar structures. The hydrocarbon chain fluidity and, as a consequence, the interlamellar distance varied as a function of temperature. The obtained SAXS and WAXS results are compared with the polarized optical microscopy measurements.

Structure formation during the isothermal crystallization of a linear polyethylene (Mw=32 100 g/mol, Mw/Mn=1.1) has been monitored by simultaneous real time small-angle x-ray scattering (SAXS) and wide angle x-ray scattering (WAXS) with synchrotron radiation. Changes in the crystalline and amorphous scattering in WAXS occur simultaneously with the changes observed in the SAXS intensity profile, suggesting that the resulting scattering is a consequence of forming crystals. At the early stage of crystal growth, the SAXS intensity at q<0.02 Å−1 increases while that at higher wave vectors remains constant. Meanwhile, the apparent crystallinity from WAXS increases to about 10% before two peaks in the SAXS intensity profile can be resolved. As lamellar stacks develop, the period corresponding to the first SAXS peak, L1, decreases initially. After reaching a minimum, L1 further increases with crystallization time. On the other hand, the period corresponding to the second SAXS peak increases with crystallization time. At late times, the rate of increase is very slow. Changes in the crystallinity obtained from WAXS and the total scattering power from SAXS were evaluated and compared with a model for the crystallizing system which accounts for changes in the fraction of lamellar aggregates. The relative importance of two secondary crystallization processes, namely the formation of new lamellar stacks and increase in the crystallinity within lamellae, were evaluated during and after primary crystallization.

Comprehensive characterization of nanostructured lipid carriers using laboratory and synchrotron X-ray scattering and diffraction

The effect of polydispersity on structure development in linear polyethylenes is studied by simultaneous real time small-angle x-ray scattering (SAXS) and wide-angle x-ray scattering (WAXS). Our measurements show that below 128 °C, changes in the SAXS and WAXS intensity profiles for a broad molecular-weight distribution sample occur simultaneously as previously reported for a narrow molecular-weight distribution sample. We demonstrate that variations in the SAXS profile can be correlated with the morphological changes associated with the evolution of single chain folded crystal lamellae to lamellar aggregates. At 128 °C and higher temperatures, changes in the SAXS intensity profile for the broad molecular weight distribution sample occur much earlier than the onset of lamellar crystal growth evidenced in the WAXS profile. For the period before crystal growth is manifest in the WAXS scattering, the SAXS intensity at low angles increases while the domain size remains constant. The low-angle intensity uptake in SAXS can be best described by the Guinier approximation. On the other hand, the temperature dependence of the variation in the average crystal thickness during isothermal crystal growth is a function of the relative rates of primary crystallization and thickening at a given temperature. We discuss the effect of a broad distribution in chain length on the various stages in the development of the morphology.

Effect of process variables on the Small and Wide Angle X-ray Scattering (SWAXS) patterns of powders, granules and pharmaceutical tablets

New Cr X-ray absorption fine structure (XAFS) data have been combined with the results of small angle X-ray scattering (SAXS) and wide angle X-ray scattering (WAXS) experiments to probe in detail the crystallisation mechanism in cordierite (Mg2Al4Si5O18) glass doped with 0.34 mol% Cr2O3. By direct comparison with chromo-aluminate spinels (MgCr2xAl2(1 - x)O4) Cr XAFS is used to determine the composition of the devitrified Cr species. This is identified as MgCr(0.18)Al(1.82)O4, which can be directly related to the Cr content in the starting glass and as a result the total crystalline volume in the fully developed ceramic is predicted to be 4%. In situ WAXS not only reveals the presence of the spinel phase but also a silica-rich stuffed quartz phase. This grows independently of the spinel and is probably nucleated from the glass surface. From our knowledge of the compositions of both crystalline phases we are able to deduce that the SAXS contrast between the surrounding glass and the spinel crystallites is 30 times greater than that between the quartz crystallites and the glass matrix, and therefore dominates the measured scattered intensity and the SAXS invariant that is derived from it. As a consequence we are able to show that the spinel crystalline volume fraction inherent in the SAXS is in close agreement with the 4% value obtained from the Cr XAFS. Furthermore in situ SAXS reveals the gradual development of the spinel particle size and shape during heat treatment. This is conducted in the super-cooled region just above the glass transition temperature, Tg. By employing a two-step annealing process nucleation can be separated from growth and from time-resolved SAXS measurements the alumino-chromate nanocrystals are found to be closely monodispersed. Over a total time course of 600 min they grow from rough crystallites to smooth spherical particles of radius 21 +/- 2 nm, with a final density of (1.2 +/- 0.4) x 10(21) m(-3). As the process of ceramic formation takes place in the viscous melt, growth is indeed found to be limited by diffusion and is complete when all the Cr is exhausted. We use this comprehensive in situ study of crystallisation in cordierite glass to demonstrate the advantages of combining SAXS, WAXS and XAFS for probing the time-resolved chemistry, the microstructure and its development from nucleation sites, that underpins the processing of nanoparticle ceramics.

ABSTRACTThe morphology of ionomers, e.g., poly(ethylene-methacrylic acid) (EMA) lead salts (EMA/Pb) and lead sulfide compounds (EMA/PbS), has been studied by using the techniques of small angle x-ray scattering (SAXS), anomalous SAXS (ASAXS), wide angle x-ray scattering (WAXS), and differential scanning calorimetry (DSC). EMA/Pb containing less than 5 wt% of lead exhibited two characteristic SAXS peaks which corresponded to the lamellar structure of the partially crystalline polymer matrix and the ionic structure of the lead aggregates that were present in the amorphous regions. The lead aggregates were not distributed uniformly and increased in packing density with increasing lead content. Both DSC and WAXS showed that the crystalline phase was present for all EMA/Pb samples and that the crystallinity decreased slightly with increasing lead content. ASAXS near the L3 absorption edge of lead permitted the extraction of the scattered intensity of lead ions from the SAXS patterns of the superimposed crystalline and ionic structures. Correlation function analysis revealed that the ionic aggregates of the EMA/Pb containing 5 wt% of lead could be described by a liquid-like model with a short range order of 2-4 nm. EMA/PbS samples were made by a reaction of EMA/Pb ionomers with hydrogen sulfide. Instead of an ionic peak as shown by EMA/Pb samples, the SAXS patterns of EMA/PbS showed a broad diffraction peak located at the same q value as the lamellar peak of the EMA in acid form. The (lamellar) peak could be attributed to the interference between the PbS crystallites in the neighboring lamellae.

Nanoparticles (mean size around 7 nm) of the standard pyrogenic Aerosil 1380 (Degussa) pregrafted by γ-irradiation with styrene were melt-compounded with the general purpose isotactic polypropylene homopolymer (PP) to prepare nanocomposites with filler volume contents up to 4.68%. Solid-state properties of the nanocomposites were characterized by wide-angle x-ray scattering (WAXS), small-angle x-ray scattering (SAXS), differential scanning calorimetry, and stretching calorimetry. The nearly identical overall patterns, the angular positions of the crystalline reflections on the WAXS diagrams as well as the WAXS degrees of crystallinity of both the neat polymer PP-0 and all nanocomposites suggested that the structure of the crystalline PP lamellae remained unchanged, irrespective of the filler content. However, a well-resolved SAXS reflection seen for PP-0 was not detectable on the SAXS curves of nanocomposites with low filler contents due to the sharp increase of SAXS intensity in the same range of scattering vectors. These results implied a significant increase in structural heterogeneity due to the appearance of new and strongly scattering entities (presumably polymer–nanoparticle interfaces and microvoids) with a broad distribution of sizes. In contrast to the basically composition-invariant WAXS crystallinities for nanocomposites, higher the filler volume content, the calorimetric crystallinities for the polymer matrix tended to increase, while the apparent densities of the polymer matrix decreased. Moreover, the Young's moduli of nanocomposites were considerably in excess of, whereas thermal expansivities, limiting strains for elastic behavior, and breaking strains, were much below the reasonable theoretical predictions. These experimental observations were explained by a model assuming that a nonnegligible portion of PP chains in the melt state would be anchored by each end to the available absorption-active sites of two different neighboring nanoparticles. The restricted chain mobility in these sites should facilitate the crystal nucleation in the undercooled PP melt; hence, the same PP chain might be involved in two nucleation events at the surfaces of two adjacent nanoparticles. Presumably, subsequent crystallization in the undercooled melts of both neat PP and nanocomposites would proceed via the usual growth of chain-folded lamellae (therefore, the WAXS patterns should be similar). However, the tie-chains in the interlamellar space of the neat PP are expected to remain in the relaxed, coiled state, whereas in the latter case, a simultaneous lamellar growth at fixed positions of the same PP chains on adjacent nanoparticles would end up with not only a considerable extension of tie-chains but also with a concomitant fall in the local packing density in the interlamellar space. *Dedicated to Prof. F. J. Baltá Calleja on the occasion of his 65th birthday.

We studied seasonal dynamics of carbohydrate storage in red spruce (Picea rubens Sarg.) seedlings by measuring starch and sugar concentrations of old needles (>/= one year old), new needles (< one year old), stems, and roots in two stands in the Green Mountains of Vermont. Although the two stands differed in many site characteristics including percent slope, aspect, soil type, drainage, and 564 m in elevation, concentrations and seasonal patterns of carbohydrates were similar for the two stands. For all tissues, starch concentrations peaked in late spring, declined through summer, and reached a minimum in winter. Sugar concentrations were greater than starch concentrations in all months except May and June. Sugar concentrations peaked in winter, and old needles showed a significant increase in sugar concentration between February and March. This increase in sugar concentration occurred without any reduction in localized starch concentrations or reductions in sugar or starch concentrations in new needles, stems or roots. Because March measurements were made toward the end of a prolonged thaw, a time when increases in photosynthesis have been documented for red spruce, it is likely that the March increase in sugar concentrations resulted from photosynthesis during the thaw. Compared with stems and roots, needles generally contained the highest concentration of carbohydrates and exhibited the greatest seasonal change in carbohydrate concentration. Needles were also the largest reservoir of carbohydrates throughout the year, especially during winter. Because of the critical roles of needles in photosynthesis and storage of carbohydrates, we conclude that any factors that disrupt the accumulation or availability of carbohydrates in red spruce needles will greatly alter plant carbon relations.

Experiment 21 - X-ray diffraction

In fibre production, draw induced crystallisation and orientation are the dominant factors that influence the properties of the final end use article. These properties depend on processing conditions; in particular, the rate of temperature change, which affects crystallinity, and the stress applied during the drawing process, which affects orientation and may also facilitate crystallisation. Polymer crystalline regions can be characterised by wide angle X-ray scattering (WAXS) and small angle X-ray scattering (SAXS). An X-ray system capable of in situ determination of fibre structure as it develops during processing has been designed and installed at Clemson University, allowing simultaneous measurement in the WAXS and SAXS regions from the extruder exit and down the spinline. Results from the in situ study of isotactic poly(propylene) with determinations of crystallinity and crystalline orientation from WAXS patterns, and lamellar long period from SAXS patterns, diameter, temperature and velocity, at varying distances from the exit of the extruder are presented.

Improved water dispersibility and photostability in folic acid nanoparticles with transglycosylated naringin using combined processes of wet-milling and freeze-drying.

Atherosclerosis is often described as a single disease entity however the size and composition of each plaque is unique to the individual. Thus, the field currently lacks a technique that can discriminate stable form unstable plaques to identify those being at risk of suffering a thromboembolic event in the near future. There exists a perfect opportunity to develop a non-invasive imaging technique or device that would be able to discriminate between different plaques at multiple locations in an artery. Such a device requires extensive chemical, structural, and spatial information available on the different plaque components to define the stability of the plaque. With informed consent and approved ethics, human carotid endarterectomy specimens were collected, fixed and mounted in Kapton tape, which is an ideal substrate for use with x-rays. Small-angle x-ray scattering was used to gather information on the size, shape and structure of the constituents of a plaque such as collagen and lipid-based materials. Then wide-angle x-ray scattering was used to readily determine the crystalline materials such as crystalline cholesterol and hydroxyapatite. Results currently indicate that this technique can discriminate between crystalline cholesterol, cholesteryl esters, phospholipids, collagen, elastin, hydroxyapatite and other calcium-based salts. This technique can map across the face of the plaque indicating the co-location of materials. In particular, for the crystalline materials, the crystalline planes can be indexed showing how the material grows in the plaque. Relating this information back to patient data can help identify features of a vulnerable plaque. Funding sources Medical Research Council, UK.

Shear-induced crystallization of isotactic polypropylene with different molecular weight distributions: in situ small- and wide-angle X-ray scattering studies