Talk given by Roland Drexel at the virtual NanoSafety 2020 conference highlighting the benefits of Electrical and Asymmetrical Flow Field-Flow Fractionation hyphenated with Multi-Angle Light Scattering and Nanoparticle Tracking Analysis for the analysis of exosomes and liposomes in complex biological media. The successful hyphenation of FFF and NTA is one of the major outcomes of the collaboration of Postnova Analytics with Malvern Panaalytical within the frame of the Horizon 2020 project ACEnano /

The technique of Field-Flow Fractionation (FFF) is already known for several decades. It was invented in 1966 by Prof. Calvin Giddings, at the University of Utah in Salt Lake City, USA. Since the foundation of Postnova Analytics GmbH in 1997 the company developed new Field-Flow Fractionation separation and detection techniques. Nowadays FFF is a reliable analytical tool for the separation and comprehensive characterization in various fields of applications. Field-Flow Fractionation (FFF) is a flow-based separation technique for analytes in the nano- and lower micrometer size range.

The principle of the FFF technique is a separation field that is perpendicular to the laminar eluent flow in a separation channel or hollow fiber. The separation field can be a cross flow, a temperature gradient or a centrifugal field. Recently, a new FFF technology, Electrical Asymmetrical Flow Field-Flow Fractionation (EAF4), has been introduced where, in addition to a cross flow, an electrical field is simultaneously applied to the FFF separation channel.After the separation process within the channel the eluent flow transports the sample fractions toward respective detectors. Depending on the kind of requested information these detectors can be a concentration detector like a Refractive Index, a UV or a Fluorescence detector, or Light Scattering Detectors, like a Multi-Angle Light Scattering (MALS) or a Dynamic Light Scattering (DLS) detector, for size and molecular weight determination. The hyphenation to a viscometer detector provides structural information of polymers and the coupling to ICP-MS instruments facilitates the chemical identification of the sample constituents.

In 2020, Postnova Analytics further launched a Raman Flow Cell which enables the hyphenation of FFF to Raman microspectroscopy for a size resolved chemical identification of for example plastic particles. Besides the progressive development of hardware, also software improvement is a decisive factor for obtaining good measurement results. The NovaAnalysis software, which has been launched in 2020, improves both FFF method development, and evaluation of the obtained data.

This presentation will give insights into the broad application range of multi-detector FFF highlighting the undisputable merits of the hyphenation of a powerful FFF platform with advanced detection techniques. The application areas of FFF include (Bio)Pharma/Nanomedicine, Environment, Polymers and Nanoparticles, Cosmetic, Food and Agriculture. One of the presented studies shows the hyphenation of Electrical Asymmetrical Flow Field-Flow Fractionation (EAF4) to Multi-Angle Light Scattering detection (MALS), which allows the determination of size and surface charge of liposomal encapsulated doxorubicin , a well-established chemotherapeutic agent for cancer treatment. Another application out of the field of Nanomedicine is the quality control of Adeno-Associated Viruses (AAV). The coupling of AF4 to UV and MALS detection allows the quantification of aggregates in AAV samples, which would be filtered out in column based techniques due to their size. A further study illustrates the size determination and chemical identification of a mixture of plastic particles by coupling Centrifugal Field-Flow Fractionation (CF3) to MALS and Raman Microspectroscopy. Moreover a comparison of single particle ICP-MS (spICP-MS) and the hyphenation of Asymmetrical Flow Field-Flow Fractionation (AF4) to MALS and ICP-MS will be presented. The element specific characterization of silica particles by AF4-MALS-ICP-MS is possible for particle sizes far below the detection limit of spICP-MS.

This webcast demonstrates that field-flow fractionation (FFF) interfaced with multi-angle light scattering (MALS) and other detectors is an ideal tool for characterization of different drug delivery systems, including liposomes and adeno-associated viruses.

In particular, we will discuss how:

• Properties like size and surface charge of liposomal doxorubicin can be measured by electrical asymmetrical flow FFF interfaced with a MALS detector in the course of few runs

• Different serotypes of adeno-associated viruses (AAV) and their aggregation behavior when they are exposed to heat can be studied using asymmetrical flow FFF-MALS

• Centrifugal FFF can be used to measure the mass difference between empty and filled liposomes

• Centrifugal FFF coupled to MALS can measure liposome density by measuring its mass and size simultaneously in a single run


Key Learning Objective:

• How electrical asymmetrical flow FFF works, and how it can reveal information about size and surface charge of liposomal drug delivery systems

• How different AAV serotypes aggregate when they are exposed to heat, and how flow FFF can separate aggregates and fragments from monomers

• How centrifugal FFF works, and how it can measure masses of empty and drug-loaded liposomes

A short introduction to Postnova’s Field-Flow Fractionation (FFF) platform. This includes: Asymmetric Flow FFF (AF4) which separates by size, Electrical AF4 (EAF4) which separates by size and charge, Centrifugal FFF (CF3) which separates by mass, and Thermal FFF (TF3) which separates by molecular weight and chemical composition. The size exclusion chromatography (SEC) option provides possibilities for orthogonal data. Detectors for FFF and SEC include refractive index, UV/Vis, multi-angle light scattering, dynamic light scattering, viscometry, inductively coupled plasma mass spectrometry, and more.

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