Lo GuazzibaParticle Metrix GmbH; bHansaBioMed Life GP-Ib alpha/CD42b Proteins Accession 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 crucial and rapid characterization technology for exosomes, microvesicles or viruses. In combination with fluorescence SIRP alpha Proteins site detection (F-NTA), NTA enables the user to carry out biomarkers detection around the single particle level, hence enhancing genuine EV concentration measurement. Classic NTA instruments are equipped with one particular 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 common beads and proprietary labelled sub-micrometre sized vesicles. Phenotyping was performed on EVs from HCT116 cell line (HansaBioMed Life Sciences). Results: The efficiencies on the person laser channels have been determined by fluorescently labelled vesicles. SOPs for conjugation of EVs were optimized regarding antibody to vesicle ratio and incubation time. Phenotyping by single and multi-wavelength NTA for wash and no-wash techniques have been compared concerning background and efficiency. Summary/conclusion: Standardization of SOPs is really a key to improve repeatability for concentration measurements. Employing 4 wavelengths, phenotyping of EVs was performed with four-fold reduction of sample amount in shorter time compared to sequential 1 laser measurements. NTA delivers total particle count, biomarker count and/or vesicle count on one particular sample which includes size distributions. Cross-validation with complementary methods like ELISA and FC/ IFC becomes crucial.Introduction: The purification of Extracellular Vesicles (EVs) for industrial processes is still missing of reproducible, scalable and high throughput strategy, applicable to numerous 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 along with the collection of significant EVs (200 nm), the fluid concentration as well as the removal of smaller molecules ( 500 kDa) with minimal loss of EVs, ultimately purified by SEC. The quality of vesicles has been assessed in terms of particle size distribution, morphology, concentration, phenotyping and storage stability. Methods: EVs have been isolated from cell conditioned media combining 2 TFF methods (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 various purifications performed combining the double TFF and SEC we defined high-quality parameters for EVs in term of size distribution, concentration.
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