Lo GuazzibaParticle Metrix GmbH; bHansaBioMed Life SciencesDouble tangential flow filtration and size RGS8 medchemexpress exclusion chromatography for scalable and reproducible EV isolation and size fractionation Elina Aleksejevaa, Julia Gavrilovaa, Maija Puhkab, Karina A. Barreiroc, Clemens Helmbrechtd and Paolo Guazziea HansaBioMed Life Sciences; bInstitute for Molecular Medicine Finland and EV Core, University of Helsinki; cInstitute for Molecular Medicine Finland (FIMM), University of Helsinki, Finland; dParticle Metrix; eHansaBioMed Life SciencesIntroduction: Nanoparticle tracking analysis (NTA) has emerged to a vital and quickly characterization technologies for exosomes, microvesicles or viruses. In combination with fluorescence detection (F-NTA), NTA enables the user to perform biomarkers detection on the single particle level, hence enhancing genuine EV mGluR7 site concentration measurement. Classic NTA instruments are equipped with a single laser, requiring phenotyping in sequence. Multi-fluorescence detection of 4 biomarkers in one sample by NTA is shown for the initial time. Techniques: A four-laser NTA instrument (ZetaView PMX-420) equipped with excitation wavelengths of 405, 488, 520 and 640 nm and committed long-pass filters was evaluated. Concentration and particle size measurements have been performed with fluorescent normal beads and proprietary labelled sub-micrometre sized vesicles. Phenotyping was performed on EVs from HCT116 cell line (HansaBioMed Life Sciences). Outcomes: The efficiencies from the individual laser channels had been determined by fluorescently labelled vesicles. SOPs for conjugation of EVs had been optimized with regards to antibody to vesicle ratio and incubation time. Phenotyping by single and multi-wavelength NTA for wash and no-wash methods have been compared with regards to background and efficiency. Summary/conclusion: Standardization of SOPs is often a essential to improve repeatability for concentration measurements. Utilizing 4 wavelengths, phenotyping of EVs was performed with four-fold reduction of sample quantity in shorter time when compared with sequential one laser measurements. NTA delivers total particle count, biomarker count and/or vesicle count on one sample which includes size distributions. Cross-validation with complementary procedures like ELISA and FC/ IFC becomes crucial.Introduction: The purification of Extracellular Vesicles (EVs) for industrial processes is still missing of reproducible, scalable and higher throughput system, applicable to numerous sources of material (cell conditioned media, biofluids, plant extracts). HansaBioMed Life Sciences (HBM-LS) has developed a scalable EV purification method combining two tangential flow filtration actions followed by size exclusion chromatography. We set a standardized process which effortlessly allows the isolation and also the collection of significant EVs (200 nm), the fluid concentration and the removal of small molecules ( 500 kDa) with minimal loss of EVs, lastly purified by SEC. The top quality of vesicles has been assessed when it comes to particle size distribution, morphology, concentration, phenotyping and storage stability. Strategies: EVs have been isolated from cell conditioned media combining 2 TFF steps (HBM-TFF: HBM-TFFMV) and SEC (maxiPURE-EVs HBM-LS). EV morphology and phenotype was analysed by NTA Zetaview (Particle Metrix), ExoTEST ELISA (HBMLS), and electron microscopy. Final results: Analysing distinct purifications performed combining the double TFF and SEC we defined top quality parameters for EVs in term of size distribution, concentration.