“V” for Value

Emerging new therapeutic agents based on extracellular vesicle and/or exosome modalities can offer great hope against unmet clinical needs. ^{[1, 2, 3, 4, 5, 6]}  Nevertheless, it’s imperative that this nascent drug product class exceeds defined parameters of safety, identity, strength, purity, and quality (SISPQ) during development. ^{[7, 8]} With COGS (Cost On Goods Sold) ^{[9, 10]} as the other major driver of biologics sale price, EV/exosome medicines also must be manufacturable and affordable. Applying the historical example of monoclonal antibody (mAb) market expansion to exosomes, we have reason for optimism.  This blog is not just about “hindcasting” EVs however. Here, RoosterBio proudly announces an actual breakthrough that will begin to blaze a path toward a bright future for exosomes. Voila, AgentV™!

Vastly Vaulted Process Vitality Viewed Via a Venerable Biotech Venture

Can we briefly revisit mAbs as a historical platform technology that’s analogous to EVs? Today, approximately 20-25% of sales are directly consumed by the mAb’s manufacture for market. In addition, process development and clinical manufacturing may add up to 40-60% of development costs. ^[11]  Despite this, the global market value of mAbs continues to grow each year, even with early blockbusters now meeting their patent cliffs and emerging biosimilars. There have been notable dramatic improvements in production efficiency (Figure 1). Taken together, innovations have increased mAb titer via CHO production cells more than 10-fold—in some cases exceeding 6 g/L or even 10 g/L. ^{[12, 13, 14]} Moreover, supply chain industrialization for standardized downstream processing (DSP) based on Protein A resins and high-volume instruments for clarification, concentration, purification, and polishing have further contributed to some COGS reduction, with downstream yields improving as much as 2x.

Average Titer vs Global Market Value Over Time

Figure 1, above. Co-plot of average titer of monoclonal antibody harvests (blue bars) ^[12] per year adjacent to plot of global market value of mAbs (orange bars). ^[15] Disruptive improvement in production efficiency does not necessarily hinder profitable market expansion when prior profit margins can multiply the funding of future pipeline shots on goal.

EV/exosome COGS reduction could follow a similar route to mAbs in the coming years. As EV products get approved for patients with large prevalence indications, we can readily expect similar upgrades in both upstream (USP) and downstream (DSP) processing. Yes, these breakthoughs will promote efficiency gains, but this is not to be a zero-sum game. With early platform adopters winning greater confidence amongst more risk-averse (yet cash-rich) pharma players, and new pipelines priming for more EV/exosome shots on goal, overall market size can grow. This accelerating demand for greater product volume will outpace the dropping drug price as per many mAbs. Reduced “toil and trouble” to manufacture doesn’t automatically drive a “race to the bottom” (Figure 1). Patients in need, biotech developers, and a health system’s reimbursement capacity all can “win.”  This is a vision RoosterBio works towards, one that is fundamental to its core mission.

Veto the Vexing Vulnerability to Vandalized Vesicle Bioproduction

Nearly three-quarters of EV clinical trials employ mesenchymal stem/stromal cells (MSCs) as bioproduction hosts. ^[16] This is because of MSCs’ long track record of safety as the dominant allogeneic cell type used for regenerative medicine cell therapy trials. ^[17] MSCs are also, conveniently, prolific producers of EVs. ^{[18, 19]} Companies such as RoosterBio have optimized standard culture procedures, cell banks, application paired media, and genetic engineering reagents for bioprocess that is scalable to many tens of liters. ^[20] Along this leading edge of expertise, however, some investigators have contended with unexpected challenges. As they began to employ systems designed for larger-scale EV harvesting via MSC-conditioned media, RoosterBio has been empowered to solve several of these issues for its customers in recent years. ^[21] First, we developed specialized RoosterCollect™ medium products for processes that enable a longer period of EV collection time into conditioned media of fed-batch cultures. Next, together with RoosterBio’s high-performance expansion media, we optimized microcarrier and cell densities to enable maximum cells per mL of bioreactor volume.  However, the ultimate challenge—poor recovery of EV product yield during downstream processing (DSP) unit operations—originally seemed daunting (Figure 2) ^[22].

Figure 2, above.  Left, a standard purification scheme for EVs across clarification, concentration, filtrations, chromatography, and polishing results in >90% clearance of both EV-associated and EV-unassociated protein from final vialed EV product. Right, along with rigorous protein clearance, there is commensurate loss of EV particles yielded from the process during this same sequence of operations.

In RoosterBio demo process runs, it was noted that much of the EV material was lost at filtration steps, i.e., chokepoints with reduced membrane penetration of the sample fluid. These steps faced frequent overpressure events due to filter fouling and increased pressure load capacity (Figure 3). Overpressure issues are not merely expensive via direct loss of product. They’re also a “monkey wrench” thrown into the whole process, wherein a haphazard detour of operator time and expense is necessary to swap in a new filter unit.

Figure 3, above.  Top, a standard purification scheme to isolate EVs from MSC-conditioned media across clarification, concentration, purification, polishing, and fill/finish. Bottom left, example filter overpressure event during a large-pore size depth filter step that required a process delay involving a filter swap. Bottom right, membrane blockage due to filter fouling leads to massive EV loss during processing, resulting in less than 5% recovery.

To solve this challenge, RoosterBio recently introduced its Secret (chemical) Agent to its customers as a launched product: “AgentV-DSP.” To prevent filter fouling, increase filter loading capacity, and enable greater EV yield throughout the end-to-end DSP, one simply drops in the solution after microcarrier removal from the conditioned medium. One mL of AgentV-DSP effectively treats one liter of conditioned medium (1:1000 dilution).

For Validation’s Sake, Vicariously View Valorized Vesicles’ Velocity into Vials

What can one expect when using AgentV with an EV/exosome purification process? During development of this new product for MSC-conditioned media from 3D bioreactor runs, RoosterBio consistently observed huge gains in particle yield. Whereas legacy process DSP yields amounted to 5-10% of the input, 35-70% particle recovery is now commonplace with AgentV, an approximately tenfold increase (Figure 4). This additive product clearly alleviates filter fouling and permits an increased load capacity of incoming secretome-enriched media. The bottom line? When RoosterBio combines its novel upstream process (USP) innovations with DSP gains achieved from AgentV, the costs per process development batch decrease more than 20-fold, ultimately driving EV dose costs via live MSC secretomes down towards commercially relevant levels.

Figure 4, above left. In the presence of AgentV, cumulative recovery of EV particle material is greatly increased across each step of a DSP operation, compared with a parallel “legacy” EV purification process that does not employ AgentV. Above right, total EV particles from preps via 3L of MSC-conditioned media, comparing NTA reading from “upstream” unprocessed medium at time of harvest, a particle count from the bioprocess run with no AgentV, and a particle count from the run performed in the presence of AgentV. Recoveries of >50% with AgentV are common, in contrast with other process runs that only obtain ~5% of EVs from the original volumes of conditioned media.

RoosterBio was fortunate to visit the ISCT Annual Meeting this year (2024) ^[23] to present its work to develop AgentV in greater detail in the form of a poster, Scalable GMP-compatible Process Solution for MSC-EV Purification with 10X Yield Improvements. ^[24] To learn more details about how this reagent can help one’s EV manufacturing process with no apparent detriment to critical quality attributes like EV size distribution, CD73 activity, or tetraspanins expression, this is an excellent resource. You can also download the AgentV protocol. Readers may be struck by how simple AgentV is to adopt, including its broad working temperature between room temperature and 37^oC.

Verdict

What’s next for AgentV? Be advised, RoosterBio has explored (and is now in the process of formulating) the material for other unique extracellular vesicle product formats! But until then, please do keep this tongue-twister in mind:

Verily, this vintage blog author vehemently vows penitence for the verbose, yet vivacious, V’s volleyed in the vicinity of the various vocal vindications for the verisimilitude of AgentV’s value.

 

References

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