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Castel Guelfo di Bologna, Italy

Anand R.,CNR Institute for Organic Syntheses and Photoreactivity | Borghi F.,University of Bologna | Manoli F.,CNR Institute for Organic Syntheses and Photoreactivity | Manet I.,CNR Institute for Organic Syntheses and Photoreactivity | And 5 more authors.
Journal of Physical Chemistry B | Year: 2014

Doxorubicin (DOX) entrapment in porous Fe(III)-trimesate metal organic frameworks (MIL-100(Fe)) nanoparticles was investigated in neutral Tris buffer via UV-vis absorption, circular dichroism (CD), and fluorescence. The binding constants and the absolute spectra of the DOX-MIL-100(Fe) complexes were determined via absorption and fluorescence titrations. A binding model where DOX associates as monomer to the dehydrated Fe3O (OH)(H 2O)2 [(C6H3)(CO2) 3]2 structural unit in 1:1 stoichiometry, with apparent association constant of (1.1 to 1.8) × 104 M-1, was found to reasonably fit the experimental data. Spectroscopic data indicate that DOX binding occurs via the formation of highly stable coordination bonds between one or both deprotonated hydroxyl groups of the aglycone moiety and coordinatively unsaturated Fe(III) centers. Complete quenching of the DOX fluorescence and remarkable thermal and photochemical stability were observed for DOX incorporated in the MIL-100(Fe) framework. © 2014 American Chemical Society. Source

Johann C.,Wyatt Technology Europe GmbH | Elsenberg S.,Wyatt Technology Europe GmbH | Roesch U.,Wyatt Technology Europe GmbH | Rambaldi D.C.,Wyatt Technology Europe GmbH | And 2 more authors.
Journal of Chromatography A | Year: 2011

A new system design and setup are proposed for the combined use of asymmetrical flow field-flow fractionation (AF4) and hollow-fiber flow field-flow fractionation (HF5) within the same instrumentation. To this purpose, three innovations are presented: (a) a new flow control scheme where focusing flow rates are measured in real time allowing to adjust the flow rate ratio as desired; (b) a new HF5 channel design consisting of two sets of ferrule, gasket and cap nut used to mount the fiber inside a tube. This design provides a mechanism for effective and straightforward sealing of the fiber; (c) a new AF4 channel design with only two fluid connections on the upper plate. Only one pump is needed to deliver the necessary flow rates. In the focusing/relaxation step the two parts of the focusing flow and a bypass flow flushing the detectors are created with two splits of the flow from the pump. In the elution mode the cross-flow is measured and controlled with a flow controller device. This leads to reduced pressure pulsations in the channel and improves signal to noise ratio in the detectors. Experimental results of the separation of bovine serum albumin (BSA) and of a mix of four proteins demonstrate a significant improvement in the HF5 separation performance, in terms of efficiency, resolution, and run-to-run reproducibility compared to what has been reported in the literature. Separation performance in HF5 mode is shown to be comparable to the performance in AF4 mode using a channel with two connections in the upper plate. © 2010 Elsevier B.V. Source

Reschiglian P.,University of Bologna | Reschiglian P.,ByFlow srl | Roda B.,University of Bologna | Roda B.,ByFlow srl | And 5 more authors.
Analytical and Bioanalytical Chemistry | Year: 2014

The rapid development of protein-based pharmaceuticals highlights the need for robust analytical methods to ensure their quality and stability. Among proteins used in pharmaceutical applications, an important and ever increasing role is represented by monoclonal antibodies and large proteins, which are often modified to enhance their activity or stability when used as drugs. The bioactivity and the stability of those proteins are closely related to the maintenance of their complex structure, which however are influenced by many external factors that can cause degradation and/or aggregation. The presence of aggregates in these drugs could reduce their bioactivity and bioavailability, and induce immunogenicity. The choice of the proper analytical method for the analysis of aggregates is fundamental to understand their (size) dimensional range, their amount, and if they are present in the sample as generated by an aggregation or as an artifact due to the method itself. Size exclusion chromatography is one of the most important techniques for the quality control of pharmaceutical proteins; however, its application is limited to relatively low molar mass aggregates. Among the techniques for the size characterization of proteins, field-flow fractionation (FFF) represents a competitive choice because of its soft mechanism due to the absence of a stationary phase and application in a broader size range, from nanometer- to micrometer-sized analytes. In this paper, the microcolumn variant of FFF, the hollow-fiber flow FFF, was online coupled with multi-angle light scattering, and a method for the characterization of aggregates with high reproducibility and low limit of detection was demonstrated employing an avidin derivate as sample model. [Figure not available: see fulltext.] © 2013 Springer-Verlag Berlin Heidelberg. Source

Melucci M.,CNR Institute for Organic Syntheses and Photoreactivity | Melucci M.,CNR Institute of Chemistry of organometallic Compounds | Zambianchi M.,CNR Institute for Organic Syntheses and Photoreactivity | Barbarella G.,CNR Institute for Organic Syntheses and Photoreactivity | And 11 more authors.
Journal of Materials Chemistry | Year: 2010

Oligothiophenes (TFs) with blue, green and orange emission have been used for the first time as doping fluorophores of silica nanoparticles (SiO 2NPs). High purification of the new nanoparticles (TFsSiO 2NPs) from free molecular fluorophores was achieved by means of asymmetrical flow field-flow fractionation on-line combined with multi-angle light scattering and fluorescent detection (AF4-MALS-FD). The synthesis, structural, compositional and optical characterizations of the new TFsSiO 2NPs are reported. We show that the tailored co-assembly of TFs in bi- and tricomponent TFsSiO2NPs allows for the fine-tuning of the emission of the nanoparticles from blue to white by means of FRET processes between adjacent TFs. These unique optical signatures make TFsSiO2NPs potentially effective tools for fluorescent sensing and labeling. © 2010 The Royal Society of Chemistry. Source

Marassi V.,University of Bologna | Roda B.,University of Bologna | Roda B.,ByFlow srl | Zattoni A.,University of Bologna | And 4 more authors.
Journal of Chromatography A | Year: 2014

Monoclonal antibodies (mAbs) are promising reagents both for the manufacture of drug substances and for their employment as a drug themselves, but to be approved for utilization, according to FDA recommendations and WHO guidelines, they have to undergo verifications regarding their purity, stability and percentage of aggregates. Moreover, stability tests of lots have to be performed in order to verify molecular size distribution over time and lot-to-lot consistency. Recent works in literature have highlighted the need for suitable, sensitive and reliable complementary analytical techniques for the characterization of mAbs and quantification of aggregates. Size-exclusion chromatography (SEC) is the reference technique in the biopharmaceutical industry for its robustness, high performance and simple use; however it presents some limitations especially toward the separation and detection of aggregates with high molecular weight. On the other hand, flow field-flow fractionation (F4) in its miniaturized version (hollow fiber flow field-flow fractionation, HF5) shows comparable performances with interesting additional advantages: a broad size range, gentle separation mechanism with low dilution factor and higher sensitivity. To propose HF5 as a complementary technique for evaluating aggregates' content in mAbs samples, a comparative study of both SEC and HF5 performances has been made. In this work, SEC and HF5 were coupled with UV and multi-angle light scattering detection and employed first in separating standard samples of proteins mixture used as a sample model. Then, a screening of mobile phases and an evaluation of separation performances was performed on a therapeutic mAbs formulation, demonstrating the complementarities between SEC and HF5 and their possible use as a separative platform approach for the characterization and quality control of protein drugs. © 2014 Elsevier B.V. Source

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