Development and applications of hollow fiber flow field-flow fractionation in the bioanalytical field. Studies of aggregation phenomena in complex protein samples

Recent advances in the fast growing area of therapeutic/diagnostic proteins and antibodies - novel and highly specific drugs - as well as the progress in the field of functional proteomics regarding the correlation between the aggregation of damaged proteins and (immuno) senescence or aging-related pathologies, underline the need for adequate analytical methods for the detection, separation, characterization and quantification of protein aggregates, regardless of the their origin or formation mechanism. Hollow fiber flow field-flow fractionation (HF5), the miniaturized version of FlowFFF and integral part of the Eclipse DUALTEC FFF separation system, was the focus of this research; this flow-based separation technique proved to be uniquely suited for the hydrodynamic size-based separation of proteins and protein aggregates in a very broad size and molecular weight (MW) range, often present at trace levels. HF5 has shown to be (a) highly selective in terms of protein diffusion coefficients, (b) versatile in terms of bio-compatible carrier solution choice, (c) able to preserve the biophysical properties/molecular conformation of the proteins/protein aggregates and (d) able to discriminate between different types of protein aggregates. Thanks to the miniaturization advantages and the online coupling with highly sensitive detection techniques (UV/Vis, intrinsic fluorescence and multi-angle light scattering), HF5 had very low detection/quantification limits for protein aggregates. Compared to size-exclusion chromatography (SEC), HF5 demonstrated superior selectivity and potential as orthogonal analytical method in the extended characterization assays, often required by therapeutic protein formulations. In addition, the developed HF5 methods have proven to be rapid, highly selective, sensitive and repeatable. HF5 was ideally suitable as first dimension of separation of aging-related protein aggregates from whole cell lysates (proteome pre-fractionation method) and, by HF5-(UV)-MALS online coupling, important biophysical information on the fractionated proteins and protein aggregates was gathered: size (rms radius and hydrodynamic radius), absolute MW and conformation.

Flow Field–Flow Fractionation for size analysis and characterization of nanoparticles for applications in Life Sciences

Nanotechnologies are rapidly expanding because of the opportunities that the new materials offer in many areas such as the manufacturing industry, food production, processing and preservation, and in the pharmaceutical and cosmetic industry. Size distribution of the nanoparticles determines their properties and is a fundamental parameter that needs to be monitored from the small-scale synthesis up to the bulk production and quality control of nanotech products on the market. A consequence of the increasing number of applications of nanomaterial is that the EU regulatory authorities are introducing the obligation for companies that make use of nanomaterials to acquire analytical platforms for the assessment of the size parameters of the nanomaterials. In this work, Asymmetrical Flow Field-Flow Fractionation (AF4) and Hollow Fiber F4 (HF5), hyphenated with Multiangle Light Scattering (MALS) are presented as tools for a deep functional characterization of nanoparticles. In particular, it is demonstrated the applicability of AF4-MALS for the characterization of liposomes in a wide series of mediums. Afterwards the technique is used to explore the functional features of a liposomal drug vector in terms of its biological and physical interaction with blood serum components: a comprehensive approach to understand the behavior of lipid vesicles in terms of drug release and fusion/interaction with other biological species is described, together with weaknesses and strength of the method. Afterwards the size characterization, size stability, and conjugation of azidothymidine drug molecules with a new generation of metastable drug vectors, the Metal Organic Frameworks, is discussed. Lastly, it is shown the applicability of HF5-ICP-MS for the rapid screening of samples of relevant nanorisk: rather than a deep and comprehensive characterization it this time shown a quick and smart methodology that within few steps provides qualitative information on the content of metallic nanoparticles in tattoo ink samples.

Carbonates and sulfides in orogenic garnet-bearing peridotites from the Ulten Zone as witnesses for Carbon and Sulfur mobility in orogenic settings

Convergent plate boundaries are sites of sustained chemical exchanges between the Earth’s surface and deep geochemical reservoirs, playing a major role in the global cycle of carbon and sulfur. However, carbon and sulfur recycling processes continue to be hotly debated. A critical gap in the knowledge of the whole subduction factory is given by the limited accessibility to the upper mantle residing above the subducting plate, the so-called mantle wedge. This thesis investigates the carbonate and sulfide metasomatism taking place during the whole metamorphic evolution of a mantle wedge involved in the Variscan continental collision. We integrate different detailed geochemical and petrological techniques to orogenic carbonated spinel and garnet peridotites from the Ulten Zone of the Eastern Italian Alps. Our data show that the Ulten Zone peridotite experienced multiple stages of addition and removal of carbon and sulfur throughout its metamorphic evolution, as follows: (1) The Variscan lithospheric mantle was initially depleted and sulfide-poor. It subsequently inherited a sulfur and carbon component during an early metasomatic stage, when hot, H2S-CO2-bearing melts leaving a subduction-modified source percolated the overlying spinel-facies peridotite in the mantle wedge; (2) Under peak eclogite-facies P-T conditions, pervasive carbonation and sulfidation occurred. Heterogeneous melt and fluid sources variably enriched in carbon, isotopically heavy sulfur and radiogenic Sr were involved; (3) Shortly after the attainment of peak-P conditions, peridotite bodies were incorporated in a tectonic mélange with the neighboring gneisses. Here, the Ulten Zone peridotite was exposed to channelized infiltration of hybridized C-O-H fluids that promoted the formation of veinlets of carbonates locally associated with sulfide grains. (4) Upon late retrogression, infiltration of serpentinizing fluids promoted C and S remobilization at shallow crustal levels.

Microwave and millimeter-wave spectroscopy of sulfur-containing molecules

The rotational spectroscopy of several sulfur bearing molecules and their 1:1 water complex, cysteamine, cysteamine monohydrate, 1-thioglycerol and 1-propanethiol were studied in the micro-wave and (or) millimeter-wave range. Precise laboratory spectra and conformational information were obtained. For cysteamine, the conformational space (at the B3LYP-GD3(BJ)/Def2-TZVP level) and the measurement and analysis of its rotational spectra in the 6 - 18 and 59.6 - 120 GHz are reported. The hyperfine structure of the rotational spectra was observed and analyzed for the first time. Based on the measured spectra, a search of the different conformers of cysteamine was performed toward the G+0.693-0.027 molecular cloud. We computed the upper limit of the ratio of ethanolamine to cysteamine, which is >0.8−5.3. For the cysteamine monohydrate, the conformational space was explored (at the B3LYP-GD3(BJ)/Def2-TZVP level). The rotational spectra of the cysteamine monohydrate complex have been assigned in the frequency range 6 – 18.5 GHz. The global minimum, Conf A1, was the only observed one. The 34S isotopologue of Conf A1 was observed in natural abundance, while 18O isotopologue was detected by introducing the H218O. In this conformer, the water molecule plays both proton donor and acceptor roles, forming a OHw···N interaction, a SH···Ow interaction and a CH···Ow interaction. The conformational space of 1-thioglycerol has been characterized by quantum mechanical calculation and its rotational spectrum has been recorded and analyzed in the frequency range 59.6 - 78.4 GHz. The global minimum of 1-thioglycerol is gTg’Gg’ and were detected together with gTg’Tg and gGgG’g, while the two detected conformers are g’G’gGg’ and tGgGg. The high-resolution rotational spectrum of 1-propanethiol in the frequency range 59.6 – 78.4 GHz was measured. Two conformers, Gg and Tg, were observed and their spectra were analyzed. Considering the overall conformational space calculated at the B3LYP-GD3(BJ)/Def2-TZVP level they are among the lowest energy conformers.