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.

ISCT 2018: MSC Biomanufacturing, Bioprocessing, Scale-Up, Analytics and Exosome Production Take Center Stage

Authored by Katrina Adlerz, Ph.D. Scientist, Analytical Development, RoosterBio Inc.

RoosterBio attended the International Society for Cell Therapy annual meeting, held May 2-5, 2018 in Montreal, which brought together leaders in the field including academic researchers, industry scientists, regulators, and clinicians. The society and conference focus on three key areas of translation: Academia, Regulatory, and Commercialization.

Six Roosters attended to hear the latest research and translational innovations at the scientific talks, present three different posters, and man the booth in the exhibit hall. Jon Rowley, founder and CTO of RoosterBio, also gave two talks discussing innovations to accelerate MSC Biomanufacturing, Bioprocessing and Scale-Up. He shared insights from RoosterBio’s path to GMP-manufactured cells and media as well as strategies for overcoming obstacles in his talk “Technologies for Radically Reducing Development Timelines of hMSC-based Therapeutic Products” during the Strategies for Commercialization Session. (New to MSCs? Read more about MSCs and biomanufacturing here, here, here and here.) For a copy of Jon's talk, email us.

RoosterBio Analytical, Process & Product Development efforts were well-represented with three posters.

- “A Xeno-Free Fed-Batch Microcarrier Suspension Bioreactor System for the Scalable and Economic Expansion of hBM-MSCs” showed that MSC critical quality attributes were maintained in 0.1L and 3L bioreactor culture. Poster.

- “Scalable Xeno-Free Manufacturing of Extracellular Vesicles Derived from Human Mesenchymal/Stromal Stem Cells” explained a process for generating a high yield of EVs/Exosomes from MSCs in a shortened time frame using RoosterNourish^TM-MSC-XF Media. Poster.

- “Development & Technology Transfer of a cGMP Potency Assay: Testing of an Ancillary Material for Stem Cell Manufacturing” outlined the steps in assay development, assay qualification/validation, and tech transfer for a custom potency assay based on cell expansion. Poster.

- In addition, the RoosterBio , BioLife Solutions and Brooks Life Science teams collaboratively presented a poster on MSC cryopreservation: "The Effect of Cryomedia Selection and Transient Warming Events on Post-Cryopreservation Human MSC Function". Poster. 

The posters gave presenting scientists the opportunity to talk with those doing similar work in process or assay development. It allowed us to learn from and share our expertise with the community.

The conference kicked off with a RoosterBio-sponsored workshop: Improving Mesenchymal Stem Cell Potency and Survival. Steven Bauer, Branch Chief of the Cellular and Tissue Therapy Branch  of the US FDA, and Head of the FDA's MSC Consortium, discussed his innovative work developing predictive assays for MSC potency by analyzing cell morphology in his talk “High Throughput Approaches to Assess MSC Function”.  You can find his blog here.  There were also a number of talks discussing clinical trials results. A common theme of the session was the need to develop analytical methods and assays that can predict MSC-based treatment efficacy in patients.

Good Manufacturing Practice for Cell and Cell-Based Therapies: Facilities & Quality Control

Novel cell and cell-based therapies require stringent manufacturing, testing and oversight to ensure integrity, function, and above all else, patient safety upon administration.  Tissue-derived cellular products are considered to be manufactured products and are regulated as such.  Thus, you must ensure that your cell manufacturing process is aligned to current Good Manufacturing Practice requirements.  (Note the “current”.  This means that these are evolving requirements, so you must stay up-to-date!)  In the United States, human cells, tissue and cellular- and tissue-based products (HCT/Ps) are regulated by the Center for Biologics Evaluation and Research (CBER), a division of the U.S. Food and Drug Administration (FDA).

When manufacturing cell and cell-based products, a production facility under strict Quality Control must be used.  Ideally, this facility includes the cell manufacturing suites, the storage space for raw and finished product and any laboratory/testing areas.  Thus, (1) facility design, access and maintenance, (2) equipment purchase, installation and operational qualification (I/OQ), use and maintenance and (3) raw material specifications, purchase, use and storage must all be carefully controlled.  For cell and cell-based therapies, terminal sterilization of the final product is often not possible.  As such, quality by design (QbD) is highly important in cell therapy, with stringent testing conducted on the tissue Donor (our next blog post in this series will cover this topic) to preclude risk of contamination at the source.  In addition, facility and equipment standards and monitoring must be instituted to ensure aseptic processing of cell and cell-based products.  To this end, closed systems and single-use disposables should be used whenever possible to minimize risk of contamination.  Therefore, a cGMP manufacturing facility must include clean rooms which control for temperature, humidity, pressure and air particulates, preventing any contamination of manufactured product due to the environment, materials, human handlers and cross-contamination from other products manufactured in the same facility.  As such, there should be uni-directional flow of materials and people through these areas and personnel must follow proper gowning procedures.  Facilities and equipment requirements are defined under US FDA 21CFR§211 and 21CFR§1271.

As mentioned above, stringent Quality Control systems must be in place to qualify all reagents and processes and to institute Standard Operating Procedures (SOPs) to ensure quality and consistency in manufacturing processes and the end product. 

Facility and Quality Control considerations for cGMP cell manufacturing.