Background & Aims Extracellular vesicles (EVs) derived from human mesenchymal stromal cells (hMSC-EVs) have been studied in over 200 preclinical applications and dozens of human clinical trials, underscoring the need for scalable production processes compatible with GMP environments. Most existing 2D and 3D Bioreactor hMSC-EV production processes require a cell expansion stage utilizing undefined components, followed by a wash and medium exchange to remove expansion medium impurities prior to an EV collection phase in a defined medium. Simplifying this 3D process to include cell expansion and EV collection in one medium requires chemically defined growth conditions, a fed-batch medium design, and an efficient process to maximize cell and EV yield, and final product quality. We have developed a chemically defined, scalable fed-batch bioreactor production medium to enable the streamlined and highly efficient production of hMSC-EVs. Methodology This study evaluates hMSC-EV production and EV quality across multiple donors and tissues in 0.1L microcarrier spinner flask cultures using a traditional cell expansion, wash, collect process vs the single-step production process, including scale-up to a 3L stirred tank bioreactor. MSC-EVs were produced from hBM- and hUC-MSCs in either RoosterNourish-MSC-XF/RoosterReplenish/RoosterCollect-EV or the new highly productive, chemically defined fed-batch system (HD-EV) and collected at set times. Results HD-EV cultures led to increased EV production on days 5, 7, 10 and 12 of culture, while maintaining healthy viable cell profiles. The fed-batch process for hMSC-EV production increased the EV collection window from healthy hMSCs resulting in 2-4x increase in hMSC-EV yield over traditional EV production processes. Elimination of the medium exchange and wash steps resulted in utilization of fewer raw materials, retention of EVs produced during cell growth, and significant reductions in total media used and total cost per billion EVs. Additionally, EV Quality Attributes including size distribution, tetraspanin expression and lipid content are preserved in the HD-EV system. Scale up in 3L Eppendorf bioreactor showed comparable results to those observed in 0.1L spinner cultures. Conclusion This highly productive chemically defined EV medium is a simplified, time and cost saving solution for the large-scale production of higher purity hMSC-EVs necessary for extensive clinical investigations.

Background & AimClinical trials investigating extracellular vesicles (EVs) from mesenchymal stromal cells (MSCs) have rapidly increased in recent years. Scalable GMP-compatible production of MSC-EVs requires bioreactor expansion and downstream processing (DSP) for purification. We have published MSC-EV bioreactor processes from 3L to 50L with DSP operations including clarification, tangential flow filtration (TFF), chromatography, and formulation/fill. Cumulative post-DSP yields of MSC-EVs are often <10%, representing a major challenge in total EVs per lot and increased cost of goods. Recently, we discovered a novel reagent (Agent V, patent pending) that streamlines DSP and greatly increases total EV yield while maintaining MSC-EV critical quality attributes (CQAs). The goal of this study was to optimize the concentration of Agent V in a bioreactor feedstream of harvested conditioned medium (CM) that maximizes DSP yield while maintaining EV CQAs.Methods, Results & ConclusionHuman bone marrow MSC-EVs were produced in a 3L bioreactor (Eppendorf) using RoosterCollect™-EV for a 5-day collection as described previously. Agent V (RoosterBio) was added to harvested CM at 1, 0.1, and 0.01 U/mL for at least 15 minutes and MSC-EV yield though clarification filters (Sartorius) was quantified by NTA (Particle Metrix). The optimal Agent V dose (0.1 U/mL) was chosen to treat CM. MSC-EV yield and purity (particles/mg protein) for each additional unit operation (TFF, Repligen; chromatography, Cytiva) were measured +/- Agent V treatment. Purified MSC-EVs were tested for MSC-EV identity (CD63/CD9/CD81/ALIX/TSG101 via ProteinSimple Jess) and function (CD73 activity).MSC-EV yield following DSP is often <10%, with ∼50-60% loss at the initial clarification step. Treating CM with Agent V led to a dose-dependent yield improvement through a 5µm clarification filter. Treated and untreated CM were processed sequentially through each DSP unit operation, demonstrating that treatment leads to significant cumulative yield improvements (∼5% vs. ∼50%, a 10-fold increase). Furthermore, MSC-EV markers were maintained (ProteinSimple Jess), and purified MSC-EVs from the Agent V treated group showed ∼3X greater CD73 activity per particle.Agent V is a novel, GMP compatible reagent that simplifies and streamlines EV purification by increasing MSC-EV DSP yield 10-fold while maintaining CQAs and possibly enhancing functionality.

Background & AimBackground & Aim: The number of clinical trials investigating extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) as a therapeutic agent has rapidly increased in recent years. However, methods to purify the EVs are still considered as one of the main challenges during processing. The goal of this study was to evaluate EV yield and purity using different chromatography resins for EV purification.Methods, Results & Conclusion: MethodsHuman MSC-EV conditioned media was produced in a bioreactor using RoosterCollect™-EV medium as described previously. The media was harvested, clarified and concentrated through TFF. Concentrated media was then split into aliquots which were used to load onto different chromatography resins (CaptoCore 400 and SuperSEC EV columns). Pre-column pressures, EV recovery and Purity (measured as particles/mg of protein) were assessed. EV Critical Quality Attributes (CQAs) were measured at the end of the process to confirm EV purity, identity and bioactivity.Results9.1E9 EVs/ml were generated using xeno-free (XF)-RoosterVial-hBM MSCs, RoosterNourish-MSC-XF expansion media and RoosterCollect-EV medium. Two chromatography modalities were evaluated; a commercially available resin (multimodal ligands with size-exclusion – Capto Core 400) and an EV-specific size exclusion-based resin (SuperSEC). There was minimal pre-column pressure rise during the process, indicating adequate loading of the columns and their compatibility with the feed. Both resins achieved high EV recovery (97% recovery with Capto Core 400 vs. 89% recovery with SuperSEC). Both columns achieved high protein impurity clearance, leading to increase in purity level compared to harvest. At harvest, purity was at 9E+10 particles/mg of protein. Capto Core 400 increased purity to 6E+11 particles/mg protein while SuperSEC increased purity to 1.4E+12 particles/mg protein. EVs generated in this process maintained critical quality attributes of EV identity (expression of EV specific tetraspanins CD63, CD9 and CD81).ConclusionIn this work, we generated a high concentration of clinically relevant EVs and compared two chromatography resins for EV purification. Both resins showed similar high EV recovery and increase in purity, indicating that most of the impurities were cleared through those resins. Both resins showed promising results as purification platforms for EV application.

Background & Aim: A consortium of leading human mesenchymal stem/stromal cell (hMSC) therapy developers, Sentien Biotechnologies, GenCure Biomanufacturing and RoosterBio, are developing a large-scale hMSC biomanufacturing process with a deep Quality focus, culminating in a potency assay qualified with human clinical samples. Methods, Results & Conclusion: A Xeno-Free (XF), fed-batch, microcarrier-based bioreactor process for hMSC manufacturing had been developed and optimized [1], and scaled to a 50L process, with demonstrated comparability between the hMSC critical quality attributes (CQAs) from the bioreactor process and from 2D control cells of similar population doubling (PDL) [2]. Based on this previous process development, GenCure and RoosterBio are leading the first stage of biomanufacturing through upstream (2D seed train & bioreactor expansion) and downstream process (continuous counterflow centrigufation, formulation & fill, and cryopreservation). Bioreactor runs at the 50 L scale were performed using the most commonly used hMSC sources in regenerative medicine: bone marrow (BM-MSCs), umbilical cord (UC-MSCs) and adipose (AD-MSCs). Critical process parameters (CPPs) are defined and critical quality attributes (CQAs) of the harvested cell product are characterized. An initial production run produced over 33billion BM-MSCs that passed the ISCT minimal criteria for MSCs. The subsequent expansion of UC- and AD-MSCs will be presented. Sentien's proprietary platform was used to ask questions on how the MSCs reacted to different stimuli with results showing that BM-MSCs were able to sense and respond with different secretomes to inflammatory stimuli. Our data also showed that BMMSCs induced changes in CD4, CD8 and CD19 cells and significantly reduced TNF-a levels in activated PBMCs, demonstrating their immunomodulatory capabilities. The same assays will be performed using the resulting MSCs from umbilical cord and adipose tissue expanded in the 50L bioreactor. Sentien, which has treated 16 subjects with acute kidney injury (AKI, open IND) and is currently running a trial in severe COVID-19 patients with AKI, will contribute biomarker data from the clinical-scale bioreactor and patient samples. Testing the in vitro developed potency hypothesis against the clinical samples will form the basis of a true potency assay. Through this work, the consortium will develop a generalized quality framework for large scale MSC manufacturing and potency assay development for broad use in regenerative medicine.

Human mesenchymal stem/stromal cells (hMSCs) have been investigated and proven to be a well-tolerated, safe therapy for a variety of indications, as shown by over 900 registered hMSC-based clinical trials. To meet the commercial demand for clinical manufacturing of hMSCs, production requires a scale that can achieve a lot size of ~100B cells, which requires innovative manufacturing technologies such as 3D bioreactors. A robust suspension bioreactor process that can be scaled-up to the relevant scale is therefore crucial. In this study, we developed a fed-batch, microcarrier-based bioreactor process, which enhances media productivity and drives a cost-effective and less labor-intensive hMSC expansion process. We determined parameter settings for various stages of the culture: inoculation, bioreactor culture, and harvest. Addition of a bioreactor feed, using a fed-batch approach, was necessary to replenish the mitogenic factors that were depleted from the media within the first 3 days of culture. Our study resulted in an optimized hMSC culture protocol that consistently achieved hMSC densities between 2 × 105–6 × 105 cells/mL within 5 days with no media exchange, maintaining the final cell population doubling level (PDL) at 16–20. Using multiple hMSC donors, we showed that this process was robust and yielded hMSCs that maintained expansion, phenotypic characteristic, and functional properties. The developed process in a vertical-wheel suspension bioreactor can be scaled to the levels needed to meet commercial demand of hMSCs.

Background & Aim Due to their potential as a key bioactive agent in regenerative medicine applications, MSC-derived extracellular vesicles (MSC-EVs) are increasingly being investigated as a clinical therapy. Manufacturing that generates enough EVs for product development and clinical doses is currently a limitation in the field and clearly a scalable manufacturing solution will be necessary for successful translation. Moreover, a complementary approach that increases the EV productivity, i.e. the number of EVs produced per cell, could further help to accelerate the development of MSC-EVs as a therapy. Methods, Results & Conclusion We developed a process that leverages a series of new cell culture reagents to couple to our established cell-media system for scalable manufacturing of MSC-EVs. Briefly, human bone marrow- or umbilical cord-derived MSCs were rapidly expanded under xeno-free conditions, i.e. >150X expansion within 10 days. Cultures were then switched to our proprietary EV collection medium and EVs were harvested for up to three additional days. To increase the productivity of MSCs, two medium supplements were developed that increased EV yield by either increasing the number of EVs generated per cell in a shortened culture process or increasing the number of collected EVs by lengthening the EV collection culture period. At the end of culture, the EVs in the conditioned media were concentrated using a tangential flow filtration (TFF) system. This scalable MSC-EV manufacturing method was implemented in both 2D flask and 3D bioreactor culture and generated over 2,000 particles per cell in 2D and over 4,000 particles per cell in 3D. With the addition of a medium supplement to increase EVs produced per cell, the EV productivity was increased >2x after 24hrs. Alternatively, EV productivity was also increased >2x by addition of the medium supplement that extended EV collection culture period. MSC-EV success in clinical translation will be reliant on a manufacturing method that can scalably and reliably generate large amounts of EVs. These results present one such solution. Furthermore, increasing EV productivity, for instance by medium supplements that increase EVs per cell or lengthen culture times could further address the limitation of generating the EVs required for development and translation of clinical therapies. Due to their potential as a key bioactive agent in regenerative medicine applications, MSC-derived extracellular vesicles (MSC-EVs) are increasingly being investigated as a clinical therapy. Manufacturing that generates enough EVs for product development and clinical doses is currently a limitation in the field and clearly a scalable manufacturing solution will be necessary for successful translation. Moreover, a complementary approach that increases the EV productivity, i.e. the number of EVs produced per cell, could further help to accelerate the development of MSC-EVs as a therapy. We developed a process that leverages a series of new cell culture reagents to couple to our established cell-media system for scalable manufacturing of MSC-EVs. Briefly, human bone marrow- or umbilical cord-derived MSCs were rapidly expanded under xeno-free conditions, i.e. >150X expansion within 10 days. Cultures were then switched to our proprietary EV collection medium and EVs were harvested for up to three additional days. To increase the productivity of MSCs, two medium supplements were developed that increased EV yield by either increasing the number of EVs generated per cell in a shortened culture process or increasing the number of collected EVs by lengthening the EV collection culture period. At the end of culture, the EVs in the conditioned media were concentrated using a tangential flow filtration (TFF) system.

Mesenchymal Stem/Stromal Cells (MSCs) are a well-studied cellular therapy with many clinical trials over the last few decades to treat a range of therapeutic indications. Recently, extracellular vesicles secreted by MSCs (MSC-EVs) have been shown to recapitulate many of the therapeutic effects of the MSCs themselves. While research in MSC-EVs has exploded, it is still early in their development towards a clinical therapy. One of the main challenges in cellular therapy, which will clearly also be a challenge in MSC-EV manufacturing, is developing a scalable, cGMP-compatible manufacturing paradigm. Therefore, the focus of this review is to identify some key MSC-EV manufacturing considerations such as the selection of critical raw materials, manufacturing platforms, and critical quality attribute assays. Addressing these issues early in research and development will accelerate clinical product development, clinical trials, and commercial therapies of MSC-EVs.

Background & Aim Mesenchymal stem/stromal cells (MSCs) are a prominent cell type in cellular therapies with over 900 registered clinical trials. A critical bottleneck in using MSCs, however, is generating the large number of cells required for both clinical trials and commercial therapies. Many commercial therapies will require 100's of millions to billions of cells. While 2D cell culture may be able to generate sufficient cells for development and early clinical trials, later clinical trials and commercial therapies will require 3D bioreactors in a scalable manufacturing platform. Thus for consistency in the therapeutic product from the development stage through clinical trials and commercial therapies, it is necessary that the state of the cells be comparable across the transition from 2D to 3D culture and then maintained with scale-up in 3D. Here, we establish a scalable 3D manufacturing process using MSCs isolated from human umbilical cord tissue (hUC-MSCs) to generate cells at a consistent PDL that maintain comparable critical quality attributes (CQAs) to 2D flask culture. Methods, Results & Conclusion hUC-MSCs were isolated from the perivascular region of human umbilical cords. Using a xeno-free culture system (medium, reagents and materials), cells were cryopreserved to create a Working Cell Bank (WCB). WCB vials were expanded either in 2D cell stacks or on microcarriers in a small-scale (0.1L) or larger scale (15L) bioreactor. Cells were then assessed for CQAs: final PDL, typical MSC surface marker expression (positive for CD90 and CD166, negative for CD34 and CD45), trilineage differentiation potential (osteogenesis, adipogenesis, and chondrogenesis), and functional properties including IDO secretion and angiogenic cytokine secretion (VEGF, IL-8, bFGF, HGF, TIMP1 and TIMP2). hUC-MSCs were successfully expanded in 2D flasks, a 0.1L bioreactor, and a 15L bioreactor. Cells maintained all the tested CQAs across both 2D and 3D manufacturing platforms as well as with increases in scale in 3D. Therefore, this study establishes a scalable xeno-free manufacturing paradigm to reproducibly generate populations of hUC-MSCs that have consistent properties. Repeatedly generating cell populations with similar attributes is critical for well-designed studies during the development of cellular therapy approaches and as these approaches move towards clinical trials and commercial therapies. Mesenchymal stem/stromal cells (MSCs) are a prominent cell type in cellular therapies with over 900 registered clinical trials. A critical bottleneck in using MSCs, however, is generating the large number of cells required for both clinical trials and commercial therapies. Many commercial therapies will require 100's of millions to billions of cells. While 2D cell culture may be able to generate sufficient cells for development and early clinical trials, later clinical trials and commercial therapies will require 3D bioreactors in a scalable manufacturing platform. Thus for consistency in the therapeutic product from the development stage through clinical trials and commercial therapies, it is necessary that the state of the cells be comparable across the transition from 2D to 3D culture and then maintained with scale-up in 3D. Here, we establish a scalable 3D manufacturing process using MSCs isolated from human umbilical cord tissue (hUC-MSCs) to generate cells at a consistent PDL that maintain comparable critical quality attributes (CQAs) to 2D flask culture. hUC-MSCs were isolated from the perivascular region of human umbilical cords. Using a xeno-free culture system (medium, reagents and materials), cells were cryopreserved to create a Working Cell Bank (WCB). WCB vials were expanded either in 2D cell stacks or on microcarriers in a small-scale (0.1L) or larger scale (15L) bioreactor. Cells were then assessed for CQAs: final PDL, typical MSC surface marker expression (positive for CD90 and CD166, negative for CD34 and CD45), trilineage differentiation potential (osteogenesis, adipogenesis, and chondrogenesis), and functional properties including IDO secretion and angiogenic cytokine secretion (VEGF, IL-8, bFGF, HGF, TIMP1 and TIMP2).