SCALABLE GMP-COMPATIBLE PROCESS SOLUTION FOR MSC-EV PURIFICATION WITH 10X YIELD IMPROVEMENTS

Background & Aim Clinical 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 & Conclusion Human 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. Clinical 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. Human 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).