Analytical Insights into Protein-Alum Interactions and Their Impact on Conformational Epitope.

Several alum-adjuvanted vaccines have been licensed in the past 40 years. Despite its extensive and continuous use, the immune mechanism of action of alum adjuvants is not yet completely understood. Many different variables during the formulation process have been assessed as critical for alum-adjuvanted vaccines, although most of them are still not yet fully understood. The absence of a clear understanding of all the possible variables regulating the mechanism of action and the behavior that alum adjuvant imposes on the protein antigen may also be related to analytical challenges. For this reason, there is an urgent need for a fast and simple tool that is possible without a preliminary sample manipulation and is able to control the amount and the degree of antigen adsorption levels and their consistency across different production processes. This work attempts to develop new analytical tools with the aim of directly quantifying and assessing both the content and/or the purity of formulated alum-adsorbed antigens, without any preliminary sample manipulation (e.g., antigen desorption) being reported. In addition, the different confirmation/behavior in terms of the response to specific monoclonal antibodies in the presence of different ratios of alum-OH adsorbent antigens have been investigated. As a proxy to develop new analytical tools, three recombinant protein adsorbed models were used as follows: Neisseria adhesin A (NadA), Neisserial Heparin Binding Antigen (NHBA), and factor H binding protein (fHbp) as antigens, as well as aluminum hydroxide (AH) as an adjuvant system. The selection of the adjuvanted system model was dictated due to the substantial quantity of the literature regarding the protein structure and immunological activities, meaning that they are well characterized, including their adhesion rate to alum. In conclusion, three different analytical tools were explored to quantify, detect, and study the behavior of antigens in the presence of the alum adjuvant.

Capillary Electrophoresis of Mono- and Oligosaccharides.

This chapter reports an overview of the recent advances in the analysis of mono- and oligosaccharides by capillary electrophoresis (CE); furthermore, relevant reviews and research articles recently published in the field are tabulated. Additionally, pretreatments and procedures applied to uncharged and acidic carbohydrates (i.e., monosaccharides and lower oligosaccharides carrying carboxylate, sulfate, or phosphate groups) are described.Representative examples of such procedures are reported in detail, upon describing robust methodologies for the study of (1) neutral oligosaccharides derivatized by reductive amination and by formation of glycosylamines; (2) sialic acid derivatized with 2-aminoacridone, released from human serum immunoglobulin G; (3) anomeric couples of neutral glycosides separated using borate-based buffers; (4) unsaturated, underivatized oligosaccharides from lyase-treated alginate.

Use of Capillary Electrophoresis for Polysaccharide Studies and Applications.

CE applications to charged polysaccharides are briefly reported. A simple procedure is presented to determine the esterification degree of a hyaluronan derivative. In this case the degree of substitution was as low as 14 %.The molecular weight distribution of mannuronic oligosaccharides mixture produced by hydrolysis of native polymannuronic is readily calculated from peak area of the species resolved by CE on the basis of a specific degree of polymerization.The influence of the applied electric field strength on the free solution mobility of hyaluronan samples is briefly addressed for molar masses of the order of 10(5) and 10(6) g/mol. The data are compared with the results obtained for a 50 % galactose substituted HA.Mobility data obtained as a function of buffer pH for a native HA sample as well as for two galactose-amide HA derivatives, having slightly different degrees of substitution, are presented and discussed in terms of the polymer charge density parameters ξ.In most cases, more questions than answers arise from the application of CE to charged polysaccharides. However, perspectives are disclosed for a further understanding of the reliability of CE applied for the structural elucidation of such macromolecules.

Terminal sterilization of BisGMA-TEGDMA thermoset materials and their bioactive surfaces by supercritical CO2.

The development of biomaterials endowed with bioactive features relies on a simultaneous insight into a proper terminal sterilization process. FDA recommendations on sterility of biomaterials are very strict: a sterility assurance level (SAL) of 10(-6) must be guaranteed for biomaterials to be used in human implants. In the present work, we have explored the potential of supercritical CO(2) (scCO(2)) in the presence of H(2)O(2) as a low-temperature sterilization process for thermoset materials and their bioactive surfaces. Different conditions allowing for terminal sterilization have been screened and a treatment time-amount of H(2)O(2) relationship proposed. The selected terminal sterilization conditions did not notably modify the mechanical properties of the thermoset nor of their fiber-reinforced composites. This was confirmed by µCT analyses performed prior to and after the treatment. On the contrary, terminal sterilization in the presence of H(2)O(2) induced a slight decrease in the surface hardness. The treatment of the thermoset material with scCO(2) led to a reduction in the residual unreacted monomers content, as determined by means of high performance liquid chromatography (HPLC) analyses. Finally, it was found that a thermoset coated with a polysaccharide layer containing silver nanoparticles maintained a very high antimicrobial efficacy even after the scCO(2)-based terminal sterilization.

Surface modification and polysaccharide deposition on BisGMA/TEGDMA thermoset.

Bisphenol A glycidylmethacrylate (BisGMA)/triethyleneglycol dimethacrylate (TEGDMA) thermosets and composites are well-known examples of biomaterials for dental applications that are receiving growing interest for orthopedic applications. While mechanical bulk properties are guaranteed by the presence of reinforcing fibers, in vitro and in vivo performances of these materials are ultimately driven by their ability to establish proper interactions between their surface and the surrounding tissues. Hence, the development of novel chemical processes enabling the introduction of bioactive molecules on the surface of these methacrylate-based thermosets is of particular interest. In the present work, we have devised a chemical strategy to expose carboxylic groups on the surface of the BisGMA/TEGDMA thermoset. The presence of negative charges was confirmed by Fourier transform infrared-attenuated total reflectance and by UV-vis spectrophotometry. Bulk mechanical properties and surface morphology of the thermoset were only slightly affected upon chemical functionalization. The activated material was further refined by the deposition of a lactose-modified chitosan (chitlac) driven by strong electrostatic interactions. The presence of the bioactive polysaccharide was confirmed by fluorescence spectroscopy and by confocal laser scanning microscopy measurements. Scratch tests were performed to evaluate the mechanical behavior of the coating. Finally, in vitro tests revealed that the presence of chitlac led to a slight enhancement of cell proliferation with respect to the unmodified BisGMA/TEGDMA thermoset. This effect was more pronounced when chitlac decorated with an arginine-glycine-aspartic acid (RGD) peptide was used in the preparation of the coating. In the latter case, the in vitro performance of the coated BisGMA/TEGDMA thermoset became comparable with that of clinically used roughened titanium.

Study of molar response of dextrans in electrochemical detection.

In this work, a methodological approach is reported, aimed at assessing the electrochemical response of some model gluco-oligosaccharides (dextrans). Such strategy is based on the complementary use of both anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and capillary zone electrophoresis coupled with UV detection (CZE-UV). Unlike HPAEC-PAD, CZE-UV required derivatization with a chromophoric dye (i.e., 8-aminonaphtalene-1,3,6-trisulphonic acid, ANTS) to enhance UV response and separation selectivity. From the comparison between chromophore response and PAD signal, the reliability of HPAEC-PAD for quantitative evaluation of dextran mixtures containing mainly oligomers with polymerization degree (DP) up to 18 could be proved, due to the fairly constant molar response. For higher DPs (up to 41), a maximum in the trend of the molar responses was observed followed by a steep decrease for DPs higher than about 30-35; indeed, an underestimation of weight-average molecular weight of dextran mixtures containing such oligomers was noticed.