Meet Manufacturing Demand With Gentle, Size-Based Exosome Purification

A280 A490 Cond Fraction 500 450 400 350 300 250 200 150 100 50 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Time (min) Time (min) A280, A490 (mAU) F7 F8 F9 F10 F11 F12 F13 F14 F15 F16 F17 F18 F19 F20 F21 F22 F23 A280 A490 Cond Fraction 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 A280, A490 (mAU) F20 F21 F22 F23 F24 F25 F26 F27 F28 F29 F30 F31 F32 F33 F34 F35 F36 F37 F38 F39 F40 F41 F42 F43 F44 F45 F46 F47 F48 F49 F50 F51 F52 F53 F54 F55 F56 F57 F58 F59 F60 F61 F62 F63 F64 F65 F66 F67 4.0 × 1010 3.5 × 1010 3.0 × 1010 2.5 × 1010 2.0 × 1010 1.5 x 1010 1.0 × 1010 9.5 × 109 0 F7 F8 F9 F10 F11 F12 F13 F14 F15 Exosome particles/mL 6 × 1010 5 × 1010 4 × 1010 3 × 1010 2 × 1010 1 × 1010 0 F20 F20-31 F32 F33-37 F38-42 F43-47 F48-52 F53-57 F58-62 F63-67 Exosome particles/mL Fractions Fractions A280 Cond Fraction 2.4 2.0 1.6 1.2 0.8 0.4 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 Time (min) A280 (mAU) Exosome particles/mL F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 Exosome particles/mL Protein (µg/mL) 2.0 × 109 1.8 × 109 1.6 × 109 1.4 × 109 1.2 × 109 1.0 × 109 8.0 × 108 6.0 × 108 4.0 × 108 2.0 × 108 0 1.2 1.0 0.8 0.6 0.4 0.2 0 superSEC resin in HiScale™ column F2-3 F4-5 F6-7 F8-9 F10-11 F12 Fractions Purifying exosomes to meet manufacturing demand using a gentle, size-based, and scalable purification solution Jagan Billakanti2 , Jon Lundqvist1 , Peter Guterstam1 , and David Haylock3 1 Cytiva Björkgatan 30, 751 84 Uppsala, Sweden, 2 Cytiva, Australia, 3 Vivazome Melbourne, Australia Results: scalable chromatographic purification of exosomes with superSEC resin — faster elution than Sepharose™ CL-2B resin Introduction Exosomes are a subset of extracellular vesicles (EVs) that play vital roles in intercellular communication and various patho/ physiological processes. Isolation, purification and concentration of exosomes from biological samples are essential for studying their biological functions to enable their clinical utilization including drug delivery, diagnostics, and therapeutic applications. However, the isolation, purification, and concentration of exosomes from cell culture supernatants (CCS) containing a mixture of nano-size EVs as well as media, serum, and host cell components (e.g., proteins, DNA, lipids etc.) is a major challenge for exosome biomanufacturing. We show that by combining tangential flow filtration (TFF) and size exclusion chromatography (SEC), we can effectively remove most sample contaminants including media-derived and host-cell proteins (HCP), DNA, and serum components. Furthermore, this combined approach ensures minimal loss of EVs during the isolation process, resulting in high EV recovery and preserving the biological activity of the purified exosomes. Here, we present a scalable workflow for the isolation and purification of exosomes using a proprietary methodology that utilizes TFF and an innovative SEC resin — Cytiva™ superSEC resin (Fig 1). Our results demonstrate that the workflow effectively and rapidly isolates exosomes allowing high-purity EV preparations. Importantly, superSEC resin enables reduced purification times and the process can be readily scaled up. Materials and methods Bioproduction of exosomes Exosomes were isolated from keratinocytes, amniocytes and stem cells (HEK293, CAP™, hMSC) at 10 L scale (titer: 109 to 1011 particles/mL). Harvest and clarification Cell debris was removed by centrifugation (2500 × g for 10 min). As an alternative approach, depth filtration can be used. Latex particle size range superSEC vs Sepharose CL-2B HiScreen column (10 cm b.h., 7.7 mm diameter, 4.7 mL). Both Sepharose CL-2B and superSEC resins have suitable fractionation range for large entities. Latex particle size separation range is 50 to 70 nm. Purification on superSEC vs Capto Core 700 resins Purification of exosomes from human mesenchymal stem (hMSC) cell line Purification of exosomes from Cytiva amniocyte production cell line (CAP) producing GFP exosomes provided by Evox Conclusions The combined TFF and SEC workflow for exosome sample preparation: 1. Enables effective, rapid, scalable, and high-throughput isolation and purification of exosomes. 2. Allows the high recovery of exosomes with excellent purity. 3. Can be used for various biological samples including cell culture media, blood, saliva, urine, milk, etc. Here, we demonstrated that the use of the novel superSEC resin and TFF offers a new and alternative way to isolate and purify exosomes from the biological milieu. Our method is ‘gentle’ and highly effective and can be utilized for both fundamental research* and industrial-scale production of EVs for clinical or commercial applications. For instance, purification of a 50 L exosome feed takes only 3 to 4 h and will require a column packed with 5 L of superSEC resin and 6 to 7 purification cycles. * superSEC resin allows for all components in the load samples to be fractionated and analyzed without loss on the resin. Fig 5. Chromatograms from the CAP CCS run on (A) 2 × HiScreen packed with superSEC resin in series (9.4 mL bed volume) and (B) superSEC resin packed in HiScale column (106 mL bed volume). Sample: TFF retented GFP- exosomes. The GFP-exosomes was detected at A490 (dark blue curve). (C) SEM images before and after purification of GFP-exosomes from CAP with the HiScale column run. Fig 3. Representative chromatograms from purification runs of hMSC with Tricorn™ 10/200 (17 mL bed volume) column packed with (A) Sepharose CL-2B and (B) superSEC resins, respectively. Sample load 2 mL. F = fractions of 4 mL. Left peaks contained hMSC-exosomes while protein and DNA impurities were found in the righthand peaks. Particle and protein analysis of fractions collected from runs are shown with (C) Sepharose CL-2B and (D) superSEC resins, respectively. (A) (A) (B) (B) (C) (A) (C) (D) (B) (C) Fig 1. TFF/SEC workflow for exosome purification presented in this study (orange dashed line). Fig 4. (A) Chromatogram from superSEC resin packed in HiScale 26/40 column (106 mL bed volume). Sample load 12 mL. (B) Particle concentration and protein concentration in each fraction. (C) TEM image showing intact hMSC-exosomes (magnification 20 000×). Fig 2. (A) selectivity curve latex particles for superSEC and Sepharose CL-2B packed in HiScreen column format, V0 = void volume, VT = total volume. (B) overlay chromatograms for latex particles 67 nm (blue curve) and 51 nm (orange curve) sizes, conductivity curve (dark blue). (A) (B) * The data in this application note has been generated in collaboration with VivaZome Therapeutics Pty Ltd in Australia and Evox Therapeutics Ltd in the UK. cytiva.com Cytiva and the Drop logo are trademarks of Life Sciences IP Holdings Corporation or an affiliate doing business as Cytiva. CAP, Capto, CEVEC, HiScale, HiScreen, Sepharose, and Tricorn are trademarks of Global Life Sciences Solutions USA LLC or an affiliate doing business as Cytiva. Any other third-party trademarks are the property of their respective owners. © 2024 Cytiva. For local office contact information, visit cytiva.com/contact. CY44496-05May24-PO Table 1. Comparison of purification with superSEC (2 × HiScreen, 20 cm b.h., 7.7 mm diameter, 9.4 mL) and Capto Core 700 (1 × HiScreen, 10 cm b.h., 7.7 mm diameter, 4.7 mL) columns. Recovery of GFP-exosomes, protein, and DNA residues removal were analyzed. Sample: TFF retentate Resin Recovery exosomes (%) Protein removal* (%) DNA removal* (%) Capto Core 700 64 6.1 (total 98.3) 79 (total 99.5) superSEC 56 7.3 (total 99.6) 95 (total 99.9) * Related to retentate from TFF (hollow-fiber membrane with 750 kDa cut-off) including degrading host DNA with benzonase before load on the resin. Within bracket total removal (including both TFF and resin) of protein and DNA. Benefits with each resin Capto Core 700 resin • Exosome yield • Sample volumes SuperSEC resin • Impurity removal Method: HiScreen column packed with superSEC resin • Sample load: 1 mL TFF rentate with GFP-exosomes (CAP) • Wash/elution: 10 mM PBS pH 7.4 • Flow rate: 1 mL/min Exosome isolation, concentration and buffer exchange with TFF membrane cartridge with 750 kDa cut-off to remove most of host cell proteins and DNA. 1. 10-fold concentration and 5-fold diafiltration at pH 7.4, phosphate-buffered saline (PBS). 2. Benzonase treatment for 2 h to degrade host cell DNA to low molecular weight fragments. 3. 10-fold diafiltration with PBS using 750 kDa cut-off cartridge to further remove HCP and degraded DNA. Chromatographic purification Removal of remaining HCP and DNA residues using either (A) superSEC or (B) Capto Core 700 resins (depending on volumes). Physical, chemical and molecular characterization • UV detection at 260, 280, and 490 nm in chromatograms. • Nanoparticle tracking to determine particle concentration of exosomes. • ELISA CD63 assay to detect exosomes. • Total protein concentration by BCA assay kit. • DNA content by ssDNA assay kit. • Western blotting for canonical exosome proteins (CD9, CD63, CD81, ALIX, TSG101, syntein-1). • Transmission electron microcopy (TEM) to assess vesicle integrity and aggregation. • Scanning electron microscopy (SEM) to examine exosome morphology and size. • The results from three CCS instances (HEK293 not shown) validated that superSEC resin is capable of efficiently purifying exosomes. As a result, it serves as an excellent exosome purification step following TFF. • The introduction of superSEC with enhanced pressure-flow characteristics, facilitates convenient resin packing in large-scale columns and enables rapid SEC cycle times. Method: HiScreen™ Capto Core 700 column • Sample load: 1 mL TFF rentate with GFP-exosomes (CAP) • Wash/elution: 10 mM PBS pH 7.4 • CIP: 30% isopropanol/1 M NaOH • Flow rate: 1 mL/min (4.7 min residence time) Upstream Harvest and clarification Concentration and buffer exchange Chromatography purification Sterile filtration Secondary concentration and formulation Fill and finish Harvest material Conc. 10× Wash 5× Benzonase 2 h Wash 10× Retentate A280 Cond Fraction 4.4 4.0 3.6 3.2 2.8 2.4 2.0 1.6 1.2 0.8 0.4 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 Time (min) A280 (mAU) Protein (µg/mL) Exosome particles/mL Exosome particles/mL Exosome particles/mL Protein (µg/mL) 6 × 108 5 × 108 4 × 108 3 × 108 2 × 108 1 × 108 0 Fractions Fractions F2 F3 F4 F5 F6 F7 Exosome particles/mL Protein (µg/mL) 3 × 109 2.5 × 108 2 × 108 1.5 × 108 1 × 108 5 × 108 0 8 7 6 5 4 3 2 1 0 F2 F3 F4 F5 F6 F7 4.0 3.0 2.0 1.0 0 F2 F3 F4 F5 F6 F7 A280 Cond Fraction 3.8 3.4 3.0 2.6 2.2 1.8 1.4 1.0 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 Time (min) A280 (mAU) F2 F3 F4 F5 F6 F7 After TFF After superSEC resin Sample load: 15 mL superSEC resin Chromatography cycle time is 50 min Sepharose CL-2B Chromatography cycle time is 170 min Sample load: 1 mL 6 5 4 3 2 1 0 0 20 40 60 80 100 120 superSEC Sepharose CL-2B Retention volume (mL) Absorbance (mAU) Latex particle size (nm) Volume (mL) Selectivity curves V0 VT 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0 0 1 2 3 4 5 6 7 8