Lo GuazzibaParticle Metrix GmbH; bHansaBioMed Life SciencesDouble tangential flow filtration and size 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 essential and quick characterization technology for exosomes, microvesicles or viruses. In combination with fluorescence detection (F-NTA), NTA enables the user to execute biomarkers detection on the single particle level, thus enhancing genuine EV concentration measurement. Classic NTA instruments are equipped with 1 laser, requiring phenotyping in sequence. Multi-fluorescence detection of four biomarkers in 1 sample by NTA is shown for the initial time. Strategies: 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 had 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 with the person 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 techniques had been compared concerning background and efficiency. Summary/conclusion: Standardization of SOPs is usually a crucial to enhance repeatability for concentration measurements. Utilizing four wavelengths, phenotyping of EVs was performed with four-fold reduction of sample amount in shorter time in comparison to 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 tactics STAT6 Formulation including ELISA and FC/ IFC becomes imperative.Introduction: The RGS19 Storage & Stability purification of Extracellular Vesicles (EVs) for industrial processes continues to be missing of reproducible, scalable and higher throughput method, applicable to a number of sources of material (cell conditioned media, biofluids, plant extracts). HansaBioMed Life Sciences (HBM-LS) has created a scalable EV purification process combining two tangential flow filtration steps followed by size exclusion chromatography. We set a standardized procedure which effortlessly permits the isolation plus the collection of big EVs (200 nm), the fluid concentration and also the removal of tiny molecules ( 500 kDa) with minimal loss of EVs, finally purified by SEC. The high-quality of vesicles has been assessed in terms of particle size distribution, morphology, concentration, phenotyping and storage stability. Approaches: 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. Benefits: Analysing diverse purifications performed combining the double TFF and SEC we defined excellent parameters for EVs in term of size distribution, concentration.