Advance MSC-EV Processing From Lab to GMP

Application Note repligen.com © 2026 Repligen Corporation. All rights reserved. The trademarks mentioned herein are the property of Repligen Corporation and/or its affiliate(s) or their respective owners. TD0019_02MAR2026 RoosterBio provided scalable MSC expansion and EV production systems, while Repligen contributed advanced filtration platforms engineered for scalable purification of biologics and EVs. EV production at manufacturing scale is shown in Figure 1. Upstream production of EVs involved a structured workflow: 2D MSC seed train expansion, bioreactor-based MSC expansion, and bioreactor EV collection, all performed using RoosterBio’s high-quality MSC media platform, which includes both expansion and EV collection media. Downstream processing (DSP) began with harvest of the EV-rich conditioned media, followed by clarification using the Repligen KrosFlo® TFDF System. This step was optimized at lab development scale to identify process parameters that maximize recovery yield. Ongoing studies indicate that this clarification step can be scaled using the industrialized KrosFlo KTF+, an automated, recipe-driven ultrafiltration and diafiltration system suitable for GMP manufacturing as shown in Figure 1. After clarification, small-scale TFF development was performed with the intention to scale up to KrosFlo KTF+ TFF System for the industrial scale. Throughout the process, in-process quality checks were conducted using RoosterBio’s analytical tools to ensure robust and reproducible process parameters. Materials and Methods MSC-EVs were generated in a microcarrier-based bioreactor at 3 L development scale. Clarification was developed using a Repligen TFDF Lab system, followed by concentration and formulation using a TFF system. Table 1. RoosterBio Part Numbers Item Description Part Number RoosterVial™ hMSCs 10M Cell Vials MSC-030 RoosterNourish™- MSC-XF Expansion medium K82016 RoosterReplenish™- MSC-XF Bioreactor feed SU-023 RoosterCollect™-EV EV collection medium M2001 Development of a Scalable Manufacturing Therapeutic Platform for Extracellular Vesicles Derived from Mesenchymal Stromal Cells using Tangential Flow Depth Filtration® and Tangential Flow Filtration Summary Extracellular vesicles (EVs), including exosomes derived from mesenchymal stromal cells (MSC-EVs), are emerging as promising therapeutic agents in regenerative medicine and drug delivery; however, their inherent heterogeneity, fragility, and complex physicochemical properties pose significant manufacturing challenges, particularly at clinical and commercial scales. This collaborative study, conducted by RoosterBio® and Repligen, describes a scalable, closed, and automated process platform for MSC-EV production that integrates Tangential Flow Depth Filtration® (TFDF®) for clarification and Tangential Flow Filtration (TFF) for concentration and diafiltration. Results demonstrate robust scalability, high recovery yields, and preservation of EV identity and potency, providing a foundation for clinical-grade MSC-EV manufacturing. Introduction EVs mediate intercellular communication and exhibit therapeutic potential in multiple disease areas. MSCEVs have gained attention for their immunomodulatory and regenerative properties. To enable clinical translation, manufacturing platforms must support: • High cell density upstream production of EVrich conditioned media. • Scalable, low-shear downstream processing for clarification, concentration, and formulation. • Robust analytics to confirm EV identity, purity, and potency. 2 TD0019_02MAR2026 Application Note Development of a Scalable Manufacturing Therapeutic Platform for Extracellular Vesicles Derived from Mesenchymal Stromal Cells using Tangential Flow Depth Filtration® and Tangential Flow Filtration Figure 1. EV Upstream and Downstream Production From inoculation of seed train cell material to a final 3 liters of conditioned media, the total upstream process yields a single batch preparation across 14 days (Figure 2). This process proceeded along critical process parameters (CPPs) developed by RoosterBio, where after 4 days in 2D planar culture, cells were transferred into 3D expansion on microcarriers in a 3 L stirred-tank bioreactor. After 5 days of additional growth with the addition of RoosterReplenish-MSC-XF bioreactor feed, RoosterNourish growth medium was replaced with RoosterCollect-EV exosome collection medium for an additional 5 days. Conditioned medium at this stage is then ready for transfer to downstream processing. This EV collection medium is chemically defined and manufactured to be low in particulates so that the EVs are in a relatively pure state before proceeding into downstream processing. This upstream process can be fully supported by cGMP cell and media product formats for ready, scalable translation into clinical development. Figure 2. Development Scale of Upstream Culture of MSCs Enables Expansion into a 3D Mini Bioreactor. Clarification using KrosFlo TFDF Perfusion System in Clarification Mode A primary objective of the clarification step was to industrialize the downstream process by replacing the traditional harvest centrifugation of the harvest culture and with a fully closed, scalable clarification approach using the KrosFlo® TFDF® Lab-Scale System. TFDF technology enables rapid scalable, sterile clarification using a filter with large ID lumens and a thick outer wall. The TFDF filter acts as a depth filter with an average channel pore size of 2 – 5 µm, allowing high flux processing of complex cell culture fluids. A second objective was to eliminate 0.45 µm secondary filtration step, traditionally performed after clarification. During operation, the TFDF device was run in TFF mode, retaining intact cells and large debris within the retentante while transmitting large biomolecules (envelope viruses like lentivirus, AAV adenovirus, and EV particles) through the walls of the membrane to be collected in the permeate at development and manufacturing scale (Figure 3). Harvest clarification TFDF filters with 3 TD0019_02MAR2026 Application Note Development of a Scalable Manufacturing Therapeutic Platform for Extracellular Vesicles Derived from Mesenchymal Stromal Cells using Tangential Flow Depth Filtration® and Tangential Flow Filtration surface areas of 3 cm2 and 30 cm2 were used during the development study. Repligen and RoosterBio performed several development studies to identify CPPs for TFDF to show acceptable reproducibility and scalability (Figure 4). Figure 3. KrosFlo Lab-Scale TFDF used in Clarification Mode A: Lab-scale up to 40 L; B: KrosFlo KTF+ manufacturing-scale up to 2000 L; and C: A TFDF filter with 2 – 5 µm pore size tubular depth filter and varying depth filtration surface areas (polypropylene(PP)/polyethylene terephthalate (PET)) operated in TFF mode to easily transmit large, fragile molecules like EVs with a high flux rate. Figure 4. Clarification Study Design Three phases: TFDF lab scale, development, and reproducibility and scalability. The development studies in three phases were conducted to optimize TFDF clarification process parameters. In Phase 1, conditioned media from a 3 L bioreactor was used for three development runs using 1 – 1.2 L harvest load. The leading process step parameters were scaled up in Phase 2 with a load of 3 L harvest culture. Phase 3 demonstrated reproducibility of the 3-bioreactor run sequence. In Phase 1, several recirculation rates, initial concentration factors, and permeate fluxes were evaulated. The optimized flux recirculation rate and pressure profiles were identified for 1.2 L using 3 cm2 TFDF filters, 2.2 LPM and 650 LMH. An initial concentration factor of 1.3 and 0.49 diavolumes of diafiltration wash buffer were also selected. Secondary filtration studies were performed with a 0.8 μm capsule filter on post-TFDF clarified material. Product transmission across this filter step was poor; therefore, the step was eliminated to ensure improved yield. Subsequent downstream TFF steps were successful using TFDF 2 – 5 μm clarification without additional filtration prior to TFF loading, proving that the downstream process is simplified by eliminating the need to use a 0.45 µm filter between clarification and TFF. The goal of Phase 2 was to scale up by 3-fold to a larger flow path using 3 L EV-conditioned media, running the process under the parameters identified and optimized during Phase I. Lastly, the goal of Phase 3 was to demonstrate process robustness and reproducibility. 4 TD0019_02MAR2026 Application Note Development of a Scalable Manufacturing Therapeutic Platform for Extracellular Vesicles Derived from Mesenchymal Stromal Cells using Tangential Flow Depth Filtration® and Tangential Flow Filtration A turbidity reduction of over 60% was achieved by using the TFDF filter along with an EV recovery of 81% compared with the centrifugation control. Ultimately, this technology allows a reduction in step processing time and risk. Thus, it should be feasible to scale up manufacturing volumes with under two-hour processing time using a 2100 cm2 flow path (Table 1). Table 2. Scalability of TFDF for EVs: Identification of Small-Scale Process Parameters at Three Scales Filter Filter Area (cm2 ) Batch Size (L) Recirculation Rate (L/min) Throughput at 20% Expansion (L/m2 ) Permeate Flux (LMH) Permeate Flow Rate (L/min) Process Time (h) TFDF-30 30 3 3 1200 650 0.0325 1.85 TFDF-450 450 45 9 1200 650 0.488 1.85 TFDF-2100 2100 210 42 1200 650 2.2750 1.76 Concentration/Diafiltration: TFF1 using a KrosFlo KR2i TFF System The clarified harvest material post TFDF was concentrated and diafiltered using the bench-scale KrosFlo KR2i UF/DF (ultrafiltration/ diafiltration) system with hollow fiber filters for improved product quality and recovery (Figure 5). Figure 5. KrosFlo KR2i TFF System KrosFlo KR2i Tangential Flow Filtration (TFF) bench top system compatible with hollow fiber filters and flat sheet cassettes. The TFF system logs data and controls operation with KF Comm Software interacting with auxiliary scales and two additional pumps for automated process control. Pictured is the MidiKros™ Hollow Fiber Module used for MWCO screening. During Phase 1 of the TFF study, the effect of 300, 500, and 750 kDa molecular weight cutoff (MWCO) hollow fiber filters on the recovery yield of functional EV in the desired buffer solution was examined. A 115 cm2 surface area filter used for TFF1 small-scale development was scaled up to 790 cm2 for processing the larger scale confirmation studies. For all studies, the KR2i TFF system was used due to its flexible range of scales and embedded walkaway automation controls and the TFF process parameters included a 10X concentration (C), followed by a 5 diavolume (DV) diafiltration (D) and a second 10X concentration (Table 2). Phase 1 of the TFF study showed that 750 kDa obtained sufficient retention while also permitting a higher flux. Phase 2 validated the empirically derived process parameters at 0.5 L scale. The results were encouraging in that the TFF process was consistently completed in less than 3 hours. Due to the success of the prior clarification step, no filter fouling was observed, and over 90% of EVs were retained. The TFF concentration was scaled up for the Phase 2 study as shown in Figure 6. The filter was enlarged to accommodate full process batches of 3 – 4 liters, enabling generation of scale-up, reproducibility, and confirmation data. From these multi-liter batch prep 5 TD0019_02MAR2026 Application Note Development of a Scalable Manufacturing Therapeutic Platform for Extracellular Vesicles Derived from Mesenchymal Stromal Cells using Tangential Flow Depth Filtration® and Tangential Flow Filtration materials, three reproducibility runs were conducted, and all resulted in less than 1% permeate breakthrough with high recovery of EVs. Yields were approximately 4 – 7 trillion EVs per liter, which is within the range required for in vivo studies in larger animals; however, actual dosing depends heavily on species, body weight, administration route, therapeutic target, and dosing strategy. Figure 6. TFF Study Design Table 3. Parameters Identified at Small Scale were Scaled Up for Reproducibility Filter and Process Information Small-Scale Study Scale-Up Experiment 1 Scale-Up Experiment 2 Scale-Up Experiment 3 Type of Process UFDF (C/D/C) UFDF (C/D/C) Starting Turbidity 4.8 NTU 4.1 NTU 6.7 NTU Filter MWCO 750 kDa 750 kDa Filter Chemistry mPES mPES Filter Area 115 cm2 (0.0115 m2) 790 cm2 (0.079 m2) Volume Loaded 583 mL 3.26 L 3.68 L 3.98 L Loading Ratio 50.6 L/m2 41.2 L/m2 46.6 L/m2 37.7 L/m2 Shear 4000 s-1 4000 s-1 TMP Control 5 psi 5 psi Processing Steps C1: 10X D: 5 DV C2: 20X C1: 10X D: 5 DV C2: 20X Process Time 2.8 h 2.0 h 2.7 h 3.2 h For successful scale-up of the UF/DF TFF system, the KrosFlo KTF+ could process a batch of >200 liters of clarified harvest in less than two hours. Prospective scale-up steps via TFF instrumentation for ultrafiltration/diafiltration to concentrate EV samples are shown in Table 3. The lockdown parameter experiment readings are reported first, followed by the summary of the three experimental runs in a scale-up process, followed by prospective TFF plans to accommodate 240 or even 600 liters. 6 TD0019_02MAR2026 Application Note Development of a Scalable Manufacturing Therapeutic Platform for Extracellular Vesicles Derived from Mesenchymal Stromal Cells using Tangential Flow Depth Filtration® and Tangential Flow Filtration Table 4. Prospective TFF Scale-Up to >200 L of Clarified Harvest Filter Information and Process Parameters Small-Scale Experiment Scale-Up Experiment Scale-Up Plan Type of Process UFDF (C/D/C) UFDF (C/D/C) UFDF (C/D/C) Filter Spectrum® MidiKros (D02-E750-05-N) Spectrum MidiKros Sampler™ (S02-E750-05-N) Spectrum KrosFlo Max (X04-E750-05-N) Filter MWCO 750 kDa 750 kDa 750 kDa Filter Chemistry mPES mPES mPES Filter Area 0.0115 m2 0.079 m2 7.8 m2 Volume Loaded 583 mL 4 L 240 L Loading Ratio 50.6 L/m2 50.6 L/m2 30.8 L/m2 Process Time 2.8 h 2.8 h 1.8 h EV Analytics Characterization followed the recommendations of the Minimal Information for Studies of Extracellular Vesicles group (MISEV): • Identity: CD9, CD63, CD81 tetraspanins (western blot); lipid dye staining • Purity: Reduced protein and host DNA content; RNA maintained • Potency: In vitro wound healing assay: MSC-EVs promoted >80% wound closure. Critical Quality Attributes for EVs were evaluated following MISEV guidelines. RoosterBio established a panel of analytical assays to characterize EVs based on identity, purity, and bioactivity/potency. EVs were characterized at all stages of processing: (1) harvested conditioned media, (2) TFDF clarification, and (3) TFF concentration. EV identity was tested using canonical EV/exosome tetraspanins markers (CD9, CD63, CD81) via western blot. To confirm the lipid characteristics of the particles, samples were stained with a lipidspecific dye (MemGlow™, Cytoskeleton, Inc.) and quantified by percent of particles with positive staining. Clearance of impurities through processing was tested using assays developed to quantify protein, DNA, and RNA concentrations. Maintenance of EV potency was also tested using an in vitro scratch assay. Cells were grown on a well plate to ~80% confluency and then a scratch was manually created. The extent of wound closure post treatment with EVs was assessed. Results • TFDF replaced centrifugation with a continuous, closed, and scalable clarification step, preserving EV integrity and enabling rapid processing. o Average of 60.3% turbidity reduction using the TFDF clarification filter o EV recovery yield of 81% o Reducing processing time by reducing process steps at all scales, <2hrs for clarification step o Reduced risk by implementation of single-use solution for cell culture clarification • TFF provided robust concentration and buffer exchange with high yield and reproducibility. • Integrated process yielded 4 – 7 trillion EV/L with preserved bioactivity. • Scale-down/scale-up studies demonstrated strong parameter transferability. Clarification of EV using TFDF Cell Retention Technology Key process parameters were identified for 1 L harvest using the TFDF lab-scale system with a 3 cm2 TFDF clarification depth filter with a pore size of 2 – 5 µm and a recirculation rate of 2.0 – 2.2 LPM resulting in a high permeate flux of 650 LMH and EV recovery yield of 81%, which is comparable to the centrifugation control. Average turbidity reduction of over 60% was achieved. Turbidity reduction using the TFDF filter was 60.3%. 7 TD0019_02MAR2026 Application Note Development of a Scalable Manufacturing Therapeutic Platform for Extracellular Vesicles Derived from Mesenchymal Stromal Cells using Tangential Flow Depth Filtration® and Tangential Flow Filtration Identified parameters can be scaled to a 200 L scale bioreactor with adherent cells on microcarriers for EV production using KrosFlo TFDF with a TFDF filter of 0.21 m2 as shown in Table 1. Production of EV using suspension cells can be scaled to a 500 L bioreactor using 2 X 2100 cm2 TFDF filters with a throughput of 1400 L/m2 and a step time of less than 2.5 hours. EV Concentration using KrosFlo TFF Systems and Spectrum Hollow Fiber Filters For concentrating the clarified harvest post TFDF, flux excursions and retention experiments were performed to identify scalable operating conditions and the best membrane molecular weight cutoff. The small-scale development study using 115 cm2 was performed with various MWCO hollow-fiber filters under different conditions. The process flow strategy and parameters were identified as a 10X concentration, followed by a 5 volume diafiltration, and a final 20X concentration using hollow fiber filters with a MWCO of 750 kDa at an average flux of 28.5 LMH. A volume reduction of 20X with a high recovery yield of 92% and <1% of EVs in the permeate was achieved (Figure 7, Figure 8). Figure 7. Harvest Clarification with TFDF A 750 kDa membrane retained >99% EVs in the permeate during small development scale with loading 0.5 L (A) at average flux of 28.5 LMH (B). Figure 8. TFF1 EV Recovery Yield The recovery yield of EVs was high when using the scalable process conditions that were identified during the small-scale development. The development study was followed by scale-up runs using ~7X fold greater hollow fiber surface area (790 cm2 ) and the same operating conditions to demonstrate linear scalability and reproducibility (Figure 9). The identified optimal parameters yielded high A B 8 TD0019_02MAR2026 Application Note Development of a Scalable Manufacturing Therapeutic Platform for Extracellular Vesicles Derived from Mesenchymal Stromal Cells using Tangential Flow Depth Filtration® and Tangential Flow Filtration EV recovery, while maintaining MSC-EV identity and potency as demonstrated by lipid membrane dye staining, positive EV tetraspanin markers (CD81, CD63 and CD9), and the in vitro wound closure assay. This study demonstrated that integrating scalable, single use, closed system platforms operated at an early development step simplified and de-risked the manufacturing process at large scale with a high recovery yield, identity, and potency of the EVs. Figure 9. EV TFF Scale-Up and Reproducibility Scalability and reproducibility of TFF step using KR2i TFF system At all stages of processing, particles stained positive with the lipid dye MemGlow (~70%) which was manitained throughout processing. EVs generated from this process stained positive for EV specific tetraspanins markers (CD9, CD63, & CD81) using western blot, further confirming the EV identity (Figure 10). In an in vitro wound healing assay, wounds treated with EV samples generated through TFF induced >80% wound closure (compared to 100% positive control and ~40% negative control), indicating that the EVs generated in this process maintained their potency (Figure 11). The downstream process was successful in clearing impurity proteins and over 70% of impurity host DNA while maintaining RNA content. Figure 10. Identity and Potency Left: Representative western blots of canonical EV/exosome markers (CD9, CD63, and CD81) showing apparent enrichment in concentration across DSP steps of initial harvest of conditioned media (lane 1), samples after TFDF clarification (lane 2), samples after harvest, TFDF, and TFF (lane 3). Right: Experiment showing enhanced cell culture wound closure assay performance via addition of bioprocessed EVs/exosomes. (781) 250-0111 customerserviceUS@repligen.com 41 Seyon Street Waltham, MA, USA 02453 TD0019_02MAR2026 repligen.com © 2026 Repligen Corporation. All rights reserved. The trademarks mentioned herein are the property of Repligen Corporation and/or its affiliate(s) or their respective owners. Application Note Development of a Scalable Manufacturing Therapeutic Platform for Extracellular Vesicles Derived from Mesenchymal Stromal Cells using Tangential Flow Depth Filtration® and Tangential Flow Filtration Conclusion The integration of RoosterBio’s cell and media platform with the Repligen TFDF and TFF technologies establishes a scalable, GMPready manufacturing platform for MSC-derived EVs. This end to end platform supports the generation of high concentrations of EVs in 2D bioreactors while significantly simplifying the downstream clarification. Using TFDF, approximately 60% turbidity reduction, ~81% EV recovery were achieved relative to centrifugation controls, with clarification completed in less than 2.5 hours at scales of ≥200 L. Concentration and buffer exchange are similarly streamlined. Hollow fiber filters with a 750 kDa MWCO delivered up to 20-fold volume reduction, 92% EV retention, and less than 1% permeate breakthrough. Data were consistently reproducible in multi-liter runs, which were completed in under 3 hours per batch. The combined workflow substantially reduces manufacturing footprint and cost, achieving >4-fold reductions in media and nuclease usage at the 200 L scale. Bench-top predictive models accelerate development by enabling rapid CPP identification and direct scale-up to 200 L using a 2100 cm² TFDF module, further reducing reagent consumption relative to traditional batch processes. Overall, this integrated upstream and downstream platform, compatible with both 2D and 3D bioreactor formats, maintains EV identity, purity, and potency while enabling rapid, reproducible execution of each DSP unit operation. These results demonstrate how automated, closed-system technologies can accelerate development, mitigate risk, and support the industrialization and commercialization of EV-based therapeutics. References 1. Development of Manufacturing Therapeutic Platform for Extracellular Vesicles Derived from Mesenchymal Stromal Cell (MSC-EVs) Using Scalable Manufacturing Clarification & Concentration Diafiltration System platforms, The Tangential Flow Depth Filtration (TFDF) & the Tangential Flow Filtration (TFF) with Hollow Fibers. Elie Zakhem, Jae Jung, Cameron Garland, Michael Boychyn, Lauren Torres, Mario Sinani, Carl Breuning, Jeremy Neidert, Rachel Legmann. Presented at International Society for Cell & Gene Therapy (ISCT), 31-May to 3-June, Paris 2. Legmann R, Zakhem E, Jung J, Garland C, Boychyn M, Torres L, et al. Process Development and Manufacturing: Development of manufacturing therapeutic platform for extracellular vesicles derived from mesenchymal stromal cell (MSC-EVs) using tangential Flow Depth Filtration (TFDF) and Tangential Flow Filtration (TFF). Cytotherapy. 2023;25(6):S189; 10.1016/S1465-3249(23)00498-X. 3. Advanced Technology for Vector-Based Therapies Highlighting Stem Cell-Derived Exosomes Manufacturing Process, webinar Dec 7, 2023. 4. Highlighting a Manufacturing Process for Stem-Cell–Derived Exosomes, Elie Zakhem, Lauren Torres, Jeremy Neidert, BioProcess International, 22 (3) March 2024, www.bioprocessintl.com/category/webinars.