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 evaluation (NTA) has emerged to a vital and fast characterization technologies for exosomes, microvesicles or viruses. In mixture with fluorescence detection (F-NTA), NTA enables the user to perform biomarkers detection on the CD30 Proteins MedChemExpress single particle level, as a result enhancing true EV concentration measurement. Classic NTA instruments are equipped with a single laser, requiring phenotyping in sequence. Multi-fluorescence detection of four biomarkers in one sample by NTA is shown for the first time. Techniques: A four-laser NTA instrument (ZetaView PMX-420) equipped with excitation wavelengths of 405, 488, 520 and 640 nm and dedicated long-pass filters was evaluated. Concentration and particle size measurements were performed with fluorescent standard beads and proprietary labelled sub-micrometre sized vesicles. Phenotyping was performed on EVs from HCT116 cell line (HansaBioMed Life Sciences). Outcomes: The efficiencies on the individual laser channels were determined by fluorescently labelled vesicles. SOPs for conjugation of EVs had been optimized regarding antibody to vesicle ratio and incubation time. Phenotyping by single and multi-wavelength NTA for wash and no-wash methods were compared relating to background and efficiency. Summary/conclusion: Standardization of SOPs is a crucial to enhance repeatability for concentration measurements. Making use of four wavelengths, phenotyping of EVs was performed with four-fold reduction of sample quantity in shorter time in comparison to sequential one laser measurements. NTA delivers total particle count, biomarker count and/or vesicle count on 1 sample which includes size distributions. Cross-validation with complementary methods like ELISA and FC/ IFC becomes imperative.Introduction: The purification of Extracellular Vesicles (EVs) for industrial processes is still CD74 Proteins Formulation missing of reproducible, scalable and higher throughput system, applicable to various sources of material (cell conditioned media, biofluids, plant extracts). HansaBioMed Life Sciences (HBM-LS) has created a scalable EV purification course of action combining two tangential flow filtration actions followed by size exclusion chromatography. We set a standardized process which effortlessly permits the isolation and also the collection of large EVs (200 nm), the fluid concentration and the removal of tiny molecules ( 500 kDa) with minimal loss of EVs, lastly purified by SEC. The high-quality of vesicles has been assessed when it comes to particle size distribution, morphology, concentration, phenotyping and storage stability. Procedures: EVs have been isolated from cell conditioned media combining two 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. Outcomes: Analysing distinct purifications performed combining the double TFF and SEC we defined excellent parameters for EVs in term of size distribution, concentration.
