Generating MSC-EVs With A Scalable Manufacturing System

Authored by: Katrina Adlerz, PhD, Scientist, Analytical, Process & Product Development, RoosterBio

Scalability of EV Manufacturing is a Major Challenge

Previously, we discussed the emergence of MSC extracellular vesicles (EVs) as clinical therapies. However, a critical barrier in the development of MSC-EVs as a commercial therapy is generating the large amount of EVs that will be required per dose (1). A recent review estimated that the number of exosomes released from 2 million MSCs in 48 hours is equivalent to a single dose for a rodent (2), suggesting that similar to cell therapy, billions of cells will be required to manufacture an EV commercial therapy. In a recent survey, however, the majority of those working with EVs were working with less than 100mL of sample (3) indicating the lack of scalable manufacturing processes in the field. We have identified three keys that we believe are necessary to enable successful manufacturing of EVs for clinical therapies:

• Generating the billions of cells needed with a scalable manufacturing process
• Increasing the number of EVs generated per cell to maximize productivity
• Optimizing downstream EV purification to increase concentration with minimal processing loss (to be addressed in future blog)
• GMP quality supply chain takes years to develop. Starting with the right materials is critical.

More Cells Enable Production of More EVs

The first critical challenge to generating the EVs needed for product development and clinical therapies is growing the necessary numbers of cells. RoosterBio (RBI) high-volume cell formats and paired bioprocess growth medium are engineered for  scalable manufacturingto address this bottleneck. Figure 1 illustrates the ability of RBI systems to generate millions to billions of MSCs, which produce trillions of EVs, in an 8 to 10 day process. Our starting Working Cell Bank vial format and culture paradigm decrease manufacturing time and are scalable to yield the number of EVs required for clinical translation.

Scalable Process for MSC-EV Manufacturing

We recently introduced RoosterCollect^TM-EV, a low-particle medium that is engineered for EV collection. This medium is designed to be complementary to RoosterBio MSCs and the RoosterBio bioprocess growth medium RoosterNourish^TM. Together, these RBI products create a complete system for efficient cell growth and EV collection. 

Using these products, the optimized process (shown in Figure 2) is: 

1) Expand MSCs in RoosterNourish until at least 80% confluency 

2) Switch to RoosterCollect-EV 

3) Collect the conditioned medium

RoosterCollect-EV supports EV collection for at least two days with increasing particle concentration in the conditioned medium and collected particles in the size range expected for EVs (Figure 3).

This process works for both 2D flask culture and 3D bioreactor culture. Bioreactor culture allows for even greater MSC and EV yields with reductions in cost, labor, and time (see our poster presented at ISCT 2019). Also, conditioned media in 3L and 15L bioreactor culture had  greater particle concentration compared to the 2D flask system (Figure 4), which is at least partly explained by the increased cell density we can achieve in bioreactors (see our poster presented at ISEV 2019). 

Increased Productivity for More EVs

A complementary strategy to a scalable cell manufacturing process is increasing the EV productivity (i.e. the number of EVs produced per cell). Recently-introduced EV Boost^TM is a medium supplement that is designed as a tunable addition to RoosterCollect-EV medium. Depending on the number of EVs required, EV Boost can increase particle yield up to 5x and dramatically shorten collection times (Figure 5).

What’s Next in Scalable EV Production?

Optimizing EV yield will become increasingly important as EVs move to clinical therapies and EVs from billions of cells are required to satisfy dose requirements. RoosterBio’s complete system for MSC-EVs generates millions to billions of cells in 2D or bioreactor culture and trillions of EVs, this system also provides a scalable platform for increasing EV production. This, combined with optimized scalable downstream purification (a third key challenge in EV manufacturing, and subject of a future blog), will enable the success of EVs as clinical therapies. 

Rapid translation/transition of to cGMP production will drive the future of scalable EV production for years to come.

References:

1. Colao IL, Corteling R, Bracewell D, Wall I. Manufacturing Exosomes: A Promising Therapeutic Platform. Trends Mol Med. 2018;24(3):242-56.

2. Phinney DG, Pittenger MF. Concise Review: MSC-Derived Exosomes for Cell-Free Therapy. Stem Cells. 2017;35(4):851-8.

3. Gardiner C, Di Vizio D, Sahoo S, Théry C, Witwer KW, Wauben M, et al. Techniques used for the isolation and characterization of extracellular vesicles: results of a worldwide survey. J Extracell Vesicles. 2016;5:32945.

MSC-EVs Emerge as Clinical Therapies

Authored by: Katrina Adlerz, PhD, Scientist, RoosterBio and Divya Patel, PhD, Scientist, RoosterBio

MSC-EVs Emerge as a Cell-Free Therapy

Extracellular vesicle (EV) interest continues to increase as more evidence emerges about the ability of these lipid-bilayer membrane vesicles to elicit specific responses from recipient cells. EVs are secreted by most known cell types, including MSCs. Recently, many effects of MSC-based therapeutics have been attributed to their paracrine factors which includes MSC-derived EVs (1, 2). In particular, MSC-derived EVs have been shown to recapitulate therapeutic effects of MSCs in graft-versus-host disease (3)and myocardial ischemia (4), among others. Moreover, EVs derived from MSCs benefit from MSCs’ well-defined safety profile, with MSCs having been used in over 900 clinical trials. Given their therapeutic potential, EVs are on the rise as a novel clinical therapy for a broad range of applications. This interest is reflected in the high number of peer-reviewed publications in the past 10 years mentioning EVs (over 15,000), with 700 specifically on MSC-EVs (PubMed Search Results Oct 2019) and the larger presence of EVs at cell therapy conferences.

MSC-EVs as Drug Delivery Vehicles

In addition to their use as a cell-free therapy, there is also significant interest in using EVs as drug delivery vehicles. EVs are natural carriers of bioactive cargo such as proteins and RNA, which are protected by the lipid-bilayer membrane. Research efforts have focused on both exogenous loading of biological cargo and manipulating parent cells to engineer vesicles that contain cargo of interest. 

RoosterBio EVs

RoosterBio MSC-EVs were evaluated based on a guidance for defining EVs, as published by the International Society for Extracellular Vesicles (5). Particles collected from the conditioned medium of RoosterBio MSCs are in the EV size range, contain expected proteins and small RNAs, and have bioactivity in a wound healing assay (Figure 1).

Figure 1 A Particles collected from RoosterBio MSCs have diameters in the size range of 50 to 250 nm as measured by Nanosight and TEM. B Western Blot shows EVs express expected proteins: ALIX, TSG101, CD63, CD9, and CD81. C Collected EVs contain primarily small RNA. D Conditioned medium has bioactivity in a standard in vitro wound healing scratch test.

Challenges Facing the Field

While EVs hold much promise as a cell-free therapy or drug delivery vehicle, there are some key challenges in the translation of successful EV therapies, including generating the needed number of EVs. In our next blog we will discuss some of these key challenges that need to be addressed to enable the success of EV therapies and RoosterBio’s progress in meeting the needs for EV product development, clinical trials, and commercial therapies. 

References

1. Phinney DG, Pittenger MF. Concise Review: MSC-Derived Exosomes for Cell-Free Therapy. Stem Cells. 2017;35(4):851-8.

2. Caplan AI, Correa D. The MSC: an injury drugstore. Cell Stem Cell. 2011;9(1):11-5.

3. Kordelas L, Rebmann V, Ludwig AK, Radtke S, Ruesing J, Doeppner TR, et al. MSC-derived exosomes: a novel tool to treat therapy-refractory graft-versus-host disease. Leukemia. 2014;28(4):970-3.

4. Lai RC, Arslan F, Lee MM, Sze NS, Choo A, Chen TS, et al. Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury. Stem Cell Res. 2010;4(3):214-22.

5. Lötvall J, Hill AF, Hochberg F, Buzás EI, Di Vizio D, Gardiner C, et al. Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles. J Extracell Vesicles. 2014;3:26913.