Now Amping Up QuickShip™-DSP

After Elvis’s 1958 departure for Army duty and a tragic plane crash in 1959, a musical cool season was felt by many who remember it. Did reactionary pop sensibilities then reflexively repress that rambunctious ruckus of rock’n’roll revolution by the early ’60s? Likewise, did the most radical biotech acts of the 2010s (e.g., CRISPR [1], CAR-T [2], CGTs [3]) also take a little bit of “quiet time?” soon after Tumultuous 2020? [4]

What might have lulled these bold biomedical technology platforms isn’t about what they can’t do; something else was determinative. Thus, we and others doggedly pursue an apropos question: “How can they be scaled and industrialized at reasonable cost for human medicine?” In the last decade, biotechnology process innovations [5, 6] have been quietly perfecting the practical infrastructure for a rapid “amping up” of advanced therapy bioprocesses. Thus, it won’t be long before exemplars such as RoosterBio’s AgentV-DSP [7] change the “tunes” we’ve been hearing of late, so you’d better bring your earplugs! To learn more (no earplugs required for now), check out our latest webinar on this. [8]

Stated simply, when a process innovation reaps tenfold (10x) greater efficiency of something, 1/10 raw materials are needed to provide the same product output. In terms of cellular therapies and bioproduction, higher efficiency means lower environmental impact. [9] 10x yield improvement also means that one log scale higher volume emerges from 1x raw materials.  That means lower cost for advanced therapies, finally enabling stadiums full of patients and who can afford the reimbursable “admissions prices.” We’ve seen this story play out before with monoclonal antibodies, [10] but what about new and promising therapies based on extracellular vesicles (EVs), exosomes, microvesicles, and microparticles? [11, 12, 13, 14]

Webinar

AgentV™-DSP can play an important role here. RoosterBio recently hosted an informative webinar unveiling Agent V-DSP, an innovative downstream processing reagent solution that addresses key bottlenecks in MSC-derived exosome (EV) manufacturing. Presented by Trey Picou, PhD (Product Manager) the session covered RoosterBio’s industry impact, the challenges in extracellular vesicle purification, and how AgentV- DSP significantly improves yield while maintaining quality. If you missed the live event, here’s a summary of the highlights to help you catch up.

Figure 1, above. There are challenges to manufacturing extracellular vesicles and exosomes into medicines and human doses. Two solutions are increasingly optimized. The third (downstream processing, DSP) has remained elusive until recently.

Like some older protype platforms now instrumental to biomedicine, extracellular vesicles/exosomes have faced hurdles (Figure 1, above). First, these include upstream optimization and scalability, such as transitioning from smaller-scale planar flask production to 3D bioreactors. Second, there has also been a lack of comprehensive analytical tools to enable standardized assays for the qualification and validation of extracellular vesicle product doses.

RoosterBio has accordingly forged ahead in recent years to devise EV-specific bioprocesses in both flasks and bioreactors, to optimize extracellular vesicle collection reagents and processes, and to lengthen the collection window of bioactive EVs from live producer cells like MSCs (mesenchymal stem/stromal cells). It has also adapted established biologics analytics methods directly to EVs, as well as employed new and specialized methods and instrumentation.

What’s remained as the final, third problem for extracellular vesicles has been DSP (downstream processing) of cell conditioned media into purified and concentrated particle solutions. Popular “benchtop” methods such as density gradient ultracentrifugation do not scale well. Yet even with scalable instrumentation unit operations borrowed from viral vector preparations [15] such as TFF, chromatography, and ion exchange, we were reacquainted with a common headache known to any Chemistry 101 Lab student:  the more you purify, the more you lose. We have noted that in a standard sequence of unit operations for EV preparation, loss of recovery is seen at key process steps of clarification, the first TFF step, and guard filtration. The culprit is filter fouling that necessitates costly filter swapping or the use of larger (and more costly) surface area filters. Even so, 100% particle yield may drop below 5% after a full DSP.

Figure 2, above. AgentV-DSP enables a quantum shift in extracellular vesicle purification efficiency as a convenient and simple additive to the bioprocess stream.

To remedy this “sticky” issue, RoosterBio developed a new product, AgentV-DSP, a downstream processing treatment that contains recombinant biological molecules that lead to DSP filtration improvements (Figure 2, above). AgentV-DSP prevents filter fouling, increases filter loading capacity, and enables greater EV particle yield throughout the downstream process steps. 1 mL of this solution is added to each liter of conditioned media, and the results may be dramatic for certain cell types. Using it, we now observe process runs unencumbered by overpressure events via conditioned media derived from either 2D (planar) or 3D bioreactor MSC culture systems.

Figure 3, above. Left, an extracellular vesicle downstream process in the absence of AgentV shows diminishing EV yield after each successive unit operation across clarification, TFF#1 concentration, guard filtration, TFF#2 and final 0.22mm. filtration and vialling (blue bars); right, in the presence of AgentV (orange-red bars), the process demonstrates far less loss across steps. Right, comparison of final yields between bioprocess in absence or presence of AgentV can amount to more than 10-fold.

In bake-off contests between identical DSP protocols except for AgentV-DSP variable (-/+), we regularly find that EV yields are approximately 50% plus reagent and barely ~5% without it—a 10x improvement in efficiency (Figure 3, above). We also note that there are no obvious differences (-/+ treatment) in the size distributions of the extracellular vesicle populations, nor in expression of canonical exosome tetraspanin markers as visualized by the Jess Automated Western Blot System (bio-techne). Further, ongoing experiments are demonstrating that these post-DSP, AgentV-treated samples retain biological activity in tissue culture cells [7] and in vivo, e.g., with quantifiable CD73 ecto-5’-nucleotidase and other attributes. [16] Notably, AgentV was reported for use as a key bioprocess reagent in a recent Nature Communications article authored by scientists from ShiftBio, Korea U.’s Institute of Science and Technology (KIST), Seoul National University, Dongguk U., and RoosterBio. This publication, titled “Dual-mode action of scalable, high-quality engineered stem cell-derived SIRPα-extracellular vesicles for treating acute liver failure,” shows that SIRPα-engineered EVs designed by ShiftBio and secreted from hMSC bioproducers demonstrate compelling features as a prospective, life-saving therapeutic reagent. [14]

Quick Ship DSP

AgentV-DSP fits seamlessly into RoosterBio’s end-to-end exosome manufacturing solution.  We offer everything needed for upstream manufacturing such as quality RoosterVial hMSCs and paired, high performance RoosterNourish expansion media. These media are tailor-made for both 2D and 3D processes and include RoosterReplenish bioreactor feeds, and are available in research, development, and GMP formats. We also offer low particulate extracellular vesicle harvesting media, called Rooster Collect, to maximize EV yields in absence of impurities and/or possible excipients. For smaller-scale R&D experiments or standards, we provide a catalog, off-the-shelf MSC-derived exosomes in vials produced using RoosterBio’s optimized 3D biomanufacturing process.

Figure 4, above. “Partial DSP” services can be offered by RoosterBio to provide a customer-supplied preparation of cell-conditioned medium, enriched and concentrated with extracellular vesicles and other factors. “Full DSP” services are available to customers to obtain both purified and concentrated EVs via additional process steps.

What if customers want much larger yields, e.g., for IND-enabling studies and/or in vivo work, yet lack the DSP know-how and/or CapEx? In part due to AgentV’s breakthrough, RoosterBio can now assist with routine, productized services that we’ve named QuickShip™-DSP (Figure 4, above).  QuickShip-DSP is presented in two flavors, Partial-DSP and Full-DSP. With both, the customer provides RoosterBio with any reasonable volume of conditioned media (CM) or valid biological fluid (e.g., milk, serum). In the Partial DSP, the CM is shipped to us, which is then clarified and concentrated with one TFF step, sterile filtered, vialed to customer specs, and frozen (-80^oC). In Full DSP, the CM is processed through multiple additional unit operations, also including a subsequent guard filtration, chromatography, a second TFF step, sterile filtration, vialing, and cryostorage.

Savings in Cost

Metric	Competitor (8x50L System)	RoosterBio (50L System with AgentV-DSP)
EV Yield	2.7×10¹⁴ particles	2.7×10¹⁴ particles
Bioreactor Size	8 x 50L runs	1 x 50L runs
GMP Suite Time	120 days	15 days
Cost per Dose (1×10¹¹ particles)	$2,000	<$250

Table 1, above. AgentV-DSP, combined with other RoosterBio media, primary cells, and optimized bioprocess, enables lower a per-dose cost of extracellular vesicles due to efficiency gains.

Powered by AgentV, clinical scale exosome yields can be approached at far lower cost than ever. We compared two extracellular vesicle processes, one from a competitor and the other, RoosterBio’s new method (Table 1, above). What does it take to manufacture 2700 doses at 10¹¹ EV particles per dose? This concentration is in the ballpark of engineered EVs administered to tail vein-injected mice in the recent study published with ShiftBio, [14] who used doses of 4 x 10⁹ per single injection. (If a mouse-to-human conversion factor of 12.3x is used, this roughly corresponds to about 0.5 x 10¹¹ EVs per human. [17, 18]) The competitor’s 3D-fed batch culture system uses 8 x 50L bioreactors, yielding single doses at a cost of approximately $2000 per dose, and taking up to four months of cumulative GMP suite time. RoosterBio’s 3D fed batch production system (employing high-performance media and cells and AgentV-DSP) cuts the media volume down to a single run with a 50L bioreactor, the GMP suite time down to 15 days, and the cost to below $250 per dose. This is within range of COGS for many commercial mAbs, today!

Questions

Dr. Trey Picou closed the 30-minute webinar by fielding a brief Q&A session. These questions related to GMP version availability, compatibility with conditioned media with non-MSC cell types, and the impact of Agent V-DSP on potency and CQAs of vialed EV product. To learn more, check out our webinar and listen to the answers.

One additional question came up: What exactly IS AgentV?

…At the risk of a non-linear reprise of this blog’s opening riff about what “volume” can mean… The short, punk answer is “I Can’t Explain.” It just so happens that within 10-15 years of music’s “quiet time,” the volume got really, REALLY turned up. When the Who famously sung that they Can’t Explain in 1976, they were maxing it out at over 120 decibels before thousands of London fans, a world record. Likewise, although RoosterBio would very much like to broadcast AgentV’s exploits at similar volume, good taste and sensibilities dictate otherwise. Wouldn’t it be better to allow AgentV’s 10x volume-per-EV output speak for itself—via its actions—from the safe insulation of a downstream bioprocess?

Cranked to 11x?

True enough, sometimes more is not better. Sometimes, what is thought to be “more” is actually the same (recall this scene from Spinal Tap). Yet sometimes, “more” means faster, cheaper, *and* better, i.e., “just right” for patients in need of advanced therapeutics and regenerative medicine cures.  Because of this, RoosterBio includes AgentV-DSP as part of a product and services ecosystem, that, when combined, enables final vialed product volumes of exosomes and/or extracellular vesicles “cranked to 11” and beyond. We hope you follow us along on our journey to even bolder new feats in the months and years to come.

 

References

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8. Picou, T. Introducing AgentV: A Scalable Solution for 10X Exosome Yield. RoosterBio Webinar 2025.
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