A Blog Summary of a Webinar Presentation by Dr. Michael Budiman, Downstream Application Leader at Cytiva, and Dr. Elie Zakhem, Senior Manager of Process Development at RoosterBio.

Exosomes and/or extracellular vesicles (EVs) are a promising therapeutic tool for a wide range of clinical conditions. However, scaling up their production and purification for clinical use presents challenges. To tackle these, RoosterBio and Cytiva have teamed up to develop and optimize a scalable exosome bioprocessing platform, designed to deliver high yields and purity at clinically relevant scales. [1, 2, 3]

This blog summarizes a recent webinar broadcast on September 18th, 2024 to highlight the notable progress of both teams and their ongoing collaboration. [4] It was co-presented by Michael Budiman, PhD, of Cytiva, and Elie Zakhem, PhD, PMP of RoosterBio. So, grab your coffee or favorite morning beverage and get ready to view some fascinating developments. Yes, there WILL be a quiz at the end of this blog regarding the material! Why don’t you give the presentation a watch… and then see how much you’ve learned?

Beginnings

Dr. Zakhem joined RoosterBio in 2021 after a role of Senior Scientist with the Intregra LifeSciences/ACell Team, where his work focused on developing, optimizing, and characterizing ECM-based materials for regenerative medicine applications. RoosterBio was founded in 2013 by Jon Rowley, PhD, with a mission to simplify and accelerate translation of advanced therapies cellular technologies into the clinic. [5] Initially, the focus was to fortify its customers with high performance culture media, ready-to-use mesenchymal stromal/stem cells (MSCs), and GMP-friendly bioprocess. Now RoosterBio rapidly builds its expertise and catalog to empower its customers and partners with even broader cell engineering capabilities, especially EV/exosome manufacture. [2, 3, 6, 7]

Dr. Budiman joined Cytiva in 2011 after serving as a post-doc at the Cleveland Clinic. Budiman held several roles before becoming a Downstream Application Leader in Genomic Medicine in 2023. Cytiva has a long heritage in providing expertise, services and solutions in research and commercial biomanufacturing from discovery to delivery of new therapeutics. It comprises Biotechnology business, having merged with Pall Life Sciences in 2023. Cytiva’s mission is to help its customers advance and accelerate therapeutics, both for pipeline development and already approved  therapies.

As webinar viewers can surmise, to this unique collaboration, each brings their own strengths. Cytiva provides advanced downstream processing expertise, including filtration, tangential flow filtration (TFF), and chromatography to enable high exosome yield, efficient impurity clearance, and scalability for clinical production. RoosterBio contributes optimized upstream MSC expansion systems and exosome production platforms, along with its proprietary Agent V™ technology [3, 8] to enhance exosome recovery and streamline the manufacturing process. Combining forces, they reported key breakthroughs after less than a year’s time.

Upstream Process (USP)

Elie Zakhem began the talk by introducing the What and Why of extracellular vesicles/exosomes, which are particles produced by cells, ranging in size from 40 to 1000 nanometers. Extracellular vesicles include exosomes, microvesicles, and apoptotic bodies. Their composition includes proteins, RNA, DNA, and lipids, which are delivered to recipient cells and play an essential role in their therapeutic impact. EVs also carry transmembrane proteins that act as identity markers of value to quality monitoring.

Extracellular vesicles derived from MSCs (MSC-EVs) are attractive due to their sourcing from cells that have been used safely in the clinic across human trials and approved regenerative medicines outside the USA. It’s plausible that we may hear of FDA-approved MSC therapies in the near-future. MSC-EVs may embody many of the therapeutic properties via their original live cell secretomes. [9, 10] However, unlike live cell therapies, exosome-based therapies could offer better cold chain stability for stockpiling and storage and a more straightforward regulatory profile. This makes them an attractive and engineerable alternative to living cell-based therapies from both a product and process angle. [11, 12, 13, 14]

Figure 1, above, process flow diagram for cell culture and upstream EV processing for scalable MSC-EV production using off-the-shelf reagents, consumables, and instruments.

Dr. Zakhem then introduced RoosterBio’s approach to standardization and industrialization of extracellular vesicle manufacturing. RoosterBio offers high-volume cell banks and xeno-free expansion media, enabling customers to scale MSC and EV production in bioreactors. [15] Their xeno-free EV production media enhances EV yield while maintaining clean EV collection without contaminants. RoosterBio also offers GMP-compliant products to help customers generate billions of MSCs, and hence, trillions of extracellular vesicles. A basic process flow diagram (see Figure 1, above) for upstream EV production via MSC cell culture was outlined. First, bone marrow-derived MSCs are cultured in a stirred-tank bioreactor using RoosterNourish™ media for five days without media exchange. After the growth phase, the cells are washed, and RoosterCollect™-EV media is added for extracellular vesicle collection, which takes place over five days. The conditioned media is harvested for downstream processing, which will be optimized to increase extracellular vesicle recovery and purity.

Downstream Process (DSP)

Figure 2, above, process flow diagram for extracellular vesicle downstream processing (DSP), beginning with AgentV™-DSP, for scalable MSC-EV production using off-the-shelf reagents, consumables and instruments.

The next section of the webinar was handed to Dr. Michael Budiman, where he outlined the downstream process (DSP) for exosome (EV) purification. The process begins with treating the MSC-conditioned media from the bioreactor with Agent V™, a drop-in RoosterBio product designed to increase EV yield and minimize pressure build-up during filtration steps. The media is then clarified to remove large debris using optimized filters that ensure maximal particle recovery while minimizing pressure.

Following clarification, the media undergoes tangential flow filtration (TFF) to concentrate the extracellular vesicles while maintaining pressure control. The next step is chromatography, where two types of resins were screened and optimized for extracellular vesicle recovery and impurity clearance. After purification, a second TFF step is conducted for buffer exchange, followed by sterile filtration and filling.

Dr. Budiman then detailed the systematic process optimization, broken down into three key areas: clarification, TFF, and chromatography. Several experiments were conducted using 3 × 3-liter bioreactor runs to optimize filtration, TFF, and chromatography parameters. Analytical controls were performed during and after the process to monitor EV recovery and purification efficiency, ensuring consistent quality throughout the downstream process.

After conducting multiple filter screenings, the 0.45 µm Fluorodyne™ DBL filter was identified as an optimal choice for clarifying exosome-conditioned media treated with Agent V™. The addition of Agent V resulted in a significant increase in EV yield and improved throughput during filtration. When a 5 µm Profile II prefilter was added before the DBL filter, the filter’s capacity improved by 50%, increasing throughput from 120 to 180 liters per meter squared, without compromising exosome yield. Additionally, the turbidity of the media was reduced from 11 to 3.4 NTU, ensuring cleaner filtrate for downstream processing.

In the tangential flow filtration (TFF) step, a hollow fiber format was chosen over a flat sheet format for better performance. The team screened different cut-off sizes and lumens, identifying the 750 kDa hollow fiber membrane (1mm lumen) as the best option. This setup achieved 75% exosome recovery, with a shear rate of 4000 per second and transmembrane pressure (TMP) of 7 PSI. The system also demonstrated effective impurity clearance, significantly reducing protein and DNA levels, as indicated by the results shown in the panel figures.

Looking next at the chromatography purification steps, Dr. Budiman showed results of performance testing to compare two kinds of resins: Capto™ Core 400 resin and superSEC resin. Each resin offers distinct advantages depending on the purification goals:

• Capto Core 400 resin: This multimodal resin acts as a scavenger, trapping small impurities while allowing larger molecules, like exosomes, to pass through. Capto Core 400 supports larger sample volumes, but the resin’s core ligands bind impurities tightly, which makes it difficult to clean and reuse.
• SuperSEC resin: A true size-exclusion resin, SuperSEC allows for simple cleaning and regeneration due to its non-binding nature. It enables the collection and fractionation of molecules, but the drawback is that it can handle only smaller sample volumes compared to Capto Core 400.

How did they perform?

• Capto Core 400 showed slightly better yield but lower protein clearance and particle-to-protein ratio than SuperSEC.
• SuperSEC resin achieved excellent protein clearance and a higher particle-to-protein ratio, making it more efficient in reducing impurities such as proteins and DNA.
• For albumin clearance, both resins were equally effective, reducing albumin levels below the lower limit of quantification.

Dr. Budiman concluded the Cytiva webinar section by addressing the scalability of the downstream processing (DSP) platform for exosome (EV) purification. He confirmed that the technologies presented—such as clarification, TFF, chromatography, and sterile filtration—are fully scalable from 3-liter to 200-liter bioreactor volumes. Cytiva’s products are designed to support scalability throughout the entire workflow, ensuring consistency and efficiency at larger production scales. Michael Budiman then transitioned the discussion back to Elie Zakhem, who reviewed ways to characterize the purified exosomes.

Characterization of EVs/Exosomes

Elie Zakhem resumed the webinar by discussing characterization of extracellular vesicles that exit the end-to-end purification process. RoosterBio has developed an analytical framework based on guidelines from MISEV (Minimal Information for Studies of Extracellular Vesicles) to assess the identity, purity, and bioactivity of the EVs. The purified extracellular vesicles were filled in vials at a concentration of 1.1 × 10¹⁰ particles per mL, with the particle size distribution within the expected range as measured by nanoparticle tracking analysis (NTA). The identity of the extracellular vesicles was confirmed using capillary western blot to detect tetraspanin markers (CD9, CD63, CD81), which are commonly used as markers for extracellular vesicles. The presence of these markers confirmed that the EV identity was maintained throughout the purification process.

Dr. Zakhem also demonstrated the efficient removal of impurities, such as proteins and nucleic acids, during the downstream process. Over 95% of the impurities were removed by the end of the DSP.  Finally, Zakhem discussed the maintenance of extracellular vesicle potency using a CD73 activity assay. CD73 is an enzyme that converts AMP (adenosine monophosphate) to adenosine, part of the classical pathway of extracellular adenosine production, which is a potent second messenger. [16] The assay confirmed that CD73 activity was maintained at various stages of downstream processing, indicating that the potency of the purified extracellular vesicles was preserved throughout the process. This may be important to ensure the sustained biological activity of the exosomes.

Conclusion

This webinar demonstrated that RoosterBio’s upstream processing platform is highly productive, and may be capable of generating biologically active MSC-derived extracellular vesicles. These were effectively processed using scalable downstream platforms from Cytiva, resulting in EV yields exceeding 75% at each step of the workflow. The implementation of ™-DSP [3] further improved exosome recovery and minimized pressure build-up during filtration. The work evaluated two chromatography platforms, providing flexibility depending on customer needs. The final analytics confirmed that there was no impact on EV identity or potency during the process. The entire platform was designed with scalability in mind, ensuring that the materials and methods used can be applied to clinical-scale production.

With this webinar extremely well-attended by hundreds, naturally there were many immediate questions (as many as 15-20) asked by the live audience, and subsequently too. This Q&A session covered various topics, including the impact of Agent V, the closed system question, resin choices, clarification methods, cell growth in different media, hollow fiber TFF modules, sample volumes, buffers, and more. Readers here can now freely follow along this lively discourse by logging in and tuning into the presentation.

We hope that there will be more questions about RoosterBio’s and Cytiva’s new streamlined process and broad capabilities to flexibly scale up for clinical translation.

Quiz Time

And now for that “pop quiz” we warned out about!!  Here are several multiple-choice questions to ensure that you were staying fully awake and engaged. The answers are below…

1. What was the primary purpose of using Agent V™ during the exosome purification process?

• A) To reduce cell death
• B) To improve the integrity of MSCs
• C) To increase exosome yield and minimize pressure build-up during filtration
• D) To enhance the growth of MSCs during expansion

2. What is the primary focus of the collaboration between RoosterBio and Cytiva, as discussed in the webinar?

• A) Developing MSC-based gene therapies
• B) Addressing challenges in exosome manufacturing
• C) Producing recombinant proteins
• D) Optimizing cancer cell therapies

3. Which resin provides higher protein clearance during exosome purification?

• A) Capto™ Core 400
• B) Profile II resin
• C) SuperSEC resin
• D) Sepharose resin

4. How does the use of Agent V™ affect the exosome recovery rate during the downstream process?

• A) It decreases exosome recovery by 10%
• B) It increases recovery up to 8- to 10-fold
• C) It has no effect on exosome recovery
• D) It increases recovery by only 10%

5. Which technique was used in Cytiva’s downstream process to clarify EV-conditioned media?

• A) Chromatography
• B) Density gradient ultracentrifugation
• C) Fluorodyne™ DBL filtration
• D) Centrifugation

6. Why is RoosterBio’s process for 3D bioreactor systems specifically recommended over 2D systems for exosome production?

• A) 3D systems allow for continuous media exchange
• B) 3D systems require less growth media
• C) 3D systems can collect exosomes for longer (up to 5 days) and thereby produce 8 to 10 times more EVs
• D) 3D systems have fewer contamination risks

7. What type of TFF (Tangential Flow Filtration) membrane did Cytiva use to achieve optimal exosome recovery?

• A) 1 kDa
• B) 10 kDa
• C) 750 kDa hollow fiber
• D) 1,000 kDa flat sheet

8. What storage buffer is currently being used by RoosterBio for exosome storage?

• A) PBS (Phosphate-buffered saline)
• B) DMEM (Dulbecco’s Modified Eagle Medium)
• C) Xeno-free serum
• D) Water

9. What was the result of impurity clearance (proteins and nucleic acids) at the end of the downstream process?

• A) 50% clearance
• B) 70% clearance
• C) Over 90% clearance
• D) Complete clearance (100%)

10. Which tetraspanin markers were confirmed to be present in the exosomes by RoosterBio’s analytical methods?

• A) CD4, CD8, and CD45
• B) CD9, CD63, and CD81
• C) CD19, CD34, and CD56
• D) CD1, CD20, and CD70

11. Why don’t skeletons fight each other?

• A) Because they don’t have the guts!
• B) Because they’re too bone-tired!
• C) Because they lack the heart for it!
• D) Because they’re always trying to keep it humerus!
• E) All of the above.

 

Quiz Answers (1-10)

1. Purpose of using Agent V™: (C) To increase exosome yield and minimize pressure build-up during filtration

2. Primary focus of the collaboration: (B) Addressing challenges in exosome manufacturing

3. Resin that provides higher protein clearance: (C) SuperSEC resin

4. Agent V™ effect on the exosome recovery rate: (B) It increases recovery up to 8 to 10-fold

5. Clarification technique used in Cytiva’s downstream process: (C) Fluorodyne™ DBL filtration

6. Why 3D bioreactor systems recommended over 2D systems: (C) 3D systems can collect exosomes for longer (up to 5 days) and produce 8 to 10 times more EVs

7. Type of TFF (Tangential Flow Filtration) membrane Cytiva used for optimal recovery: (C) 750 kDa hollow fiber

8. RoosterBio’s default exosome storage buffer: (A) PBS (Phosphate-buffered saline)

9. Impurity clearance final result: (C) Over 90% clearance

10. Tetraspanin markers validated as present: (B) CD9, CD63, and CD81

11. Skeletons and mortal conflict: (E) All of the above.

 

References

1. RoosterBio. Cytiva & RoosterBio Collaborate to Address Exosome Manufacturing Challenges. 2023; Available from: https://www.roosterbio.com/press_release/cytiva-roosterbio-collaborate-to-address.
2. Jung, J., et al., A COMPARABILITY STUDY OF CHROMATOGRAPHY RESINS SUITABLE FOR EV PURIFICATION FROM A HIGHLY PRODUCTIVE MSC BIOPROCESSING PLATFORM. Cytotherapy, 2024. 26(6): p. S87. 10.1016/j.jcyt.2024.03.163
3. Lenzini, S., et al., SCALABLE GMP-COMPATIBLE PROCESS SOLUTION FOR MSC-EV PURIFICATION WITH 10X YIELD IMPROVEMENTS. Cytotherapy, 2024. 26(6): p. S86. 10.1016/j.jcyt.2024.03.162
4. Cytiva, RoosterBio;. A Scalable Manufacturing Platform for the Purification of Stem Cell Derived Exosomes. 2024; Available from: https://share.hsforms.com/1LMGyCHQWSEmiuESLs_N8LQ3564o.
5. Olsen, T. R. and J. A. Rowley, Corporate profile: RoosterBio, Inc. Regen Med, 2018. 13(7): p. 753-757. 10.2217/rme-2018-0092
6. Willstaedt, T. M., A. Walde, and J. A. Rowley, A FED-BATCH CHEMICALLY DEFINED HMSC-EV BIOPROCESS MEDIUM ENABLING 2-4X EV YIELD IMPROVEMENTS IN BIOREACTOR CULTURE. Cytotherapy, 2024. 26(6): p. S59. 10.1016/j.jcyt.2024.03.105
7. Adlerz, K., et al., Strategies for scalable manufacturing and translation of MSC-derived extracellular vesicles. Stem Cell Res, 2020. 48: p. 101978. 10.1016/j.scr.2020.101978
8. RoosterBio. AgentV’s Virtues at the Vanguard of Extracellular Vesicles. 2024; Available from: https://www.roosterbio.com/blog/agentvs-virtues-at-the-vanguard-of-extracellular-vesicles/.
9. An, Seungwon, et al. Wound-Healing Effects of Mesenchymal Stromal Cell Secretome in the Cornea and the Role of Exosomes. Pharmaceutics, 2023. 15, 1486 DOI: 10.3390/pharmaceutics15051486.
10. Chiabotto, G., et al., Mesenchymal Stromal Cell-Derived Extracellular Vesicles for Reversing Hepatic Fibrosis in 3D Liver Spheroids. Biomedicines, 2024. 12(8). 10.3390/biomedicines12081849
11. Kalluri, R. and V. S. LeBleu, The biology, function, and biomedical applications of exosomes. Science, 2020. 367(6478). 10.1126/science.aau6977
12. Kojima, R., et al., Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for Parkinson’s disease treatment. Nat Commun, 2018. 9(1): p. 1305. 10.1038/s41467-018-03733-8
13. Chiang, C. L., et al., Dual targeted extracellular vesicles regulate oncogenic genes in advanced pancreatic cancer. Nat Commun, 2023. 14(1): p. 6692. 10.1038/s41467-023-42402-3
14. Fu, W., et al., CAR exosomes derived from effector CAR-T cells have potent antitumour effects and low toxicity. Nat Commun, 2019. 10(1): p. 4355. 10.1038/s41467-019-12321-3
15. Adlerz, K., et al., A scalable and xeno-free bioreactor system for biomanufacturing of hUC-MSCs. Cytotherapy, 2020. 22(5): p. S46-S47. 10.1016/j.jcyt.2020.03.052
16. Cramer, M. CD73: A Team Player Caught in the “AKT” of Wound Healing & Cell Survival via MSC Exosomes? 2024; Available from: https://www.roosterbio.com/blog/cd73-a-team-player-caught-in-the-akt-of-wound-healing-cell-survival-via-msc-exosomes/.