• Ensuring safety, efficacy, and consistency in biologics like MSCs and EVs requires robust analytical methods to meet regulatory standards and deliver reliable therapies to patients.
• Through a phase-appropriate approach, early qualification and later validation of assays align analytical methods with therapeutic goals while addressing critical quality attributes (CQAs) like potency, purity, and specificity.
• RoosterBio’s CD73 activity assay case study demonstrates the successful application of these principles, providing a foundation for advancing MSC-EV therapies into clinical applications.
• Trey Picou, PhD, and Madeline Cramer, PhD recently presented an information-packed webinar on these compelling items, “Preparing for the Clinic: Qualifying Analytical Methods.”

Why Should Drug Developers Prioritize Rigorous Analytical Methods?

Mesenchymal stem/stromal cells (MSCs) and their extracellular vesicles (EVs; aka “exosomes”) are no strangers to the rhetorical tug-of-war between “nature” and “nurture.” Although basic scientists and regenmed developers alike can weigh in from all points along this dialectic, clinical regulatory agencies try to reconcile these opposing forces to frame biologics drug identity with a single synthesis statement: “The Product is the Process.” Obviously, therapies based on MSCs and their EVs (MSC-EVs) are intrinsically complex, facing challenges unlike those seen with small molecules. [1, 2, 3, 4] Further, there is no FDA-approved EV/exosome product (yet!) and no guidance to navigate the route to commercialize an EV therapeutic. Thus, for any future MSC-EV drug, the imperative is to be proactive with understanding what is worth measuring, how to measure it, and what each measurement means in light of its quality threshold. [5]

At the outset, efforts to qualify MSC-EVs with analytical assays for right-fit critical quality attributes (CQAs) might thus seem a little tricky, if not daunting! To help bring clarity toward your quest to solve this perplexing dilemma, RoosterBio recently hosted a well-received webinar, presented by Trey Picou, PhD (Product Manager) and Madeline Cramer, PhD (Scientist in Analytical Development). You can sample a brief synopsis of it in this blog, but we highly recommend experiencing the entire production, “Preparing for the Clinic: Qualifying Analytical Methods.” [6] It will be well worth your time.

Dr. Trey Picou kicked off the webinar with a brief intro to RoosterBio and then, explained the gravity of how subtle changes in manufacturing processes can ramify and alter product quality, safety, or efficacy out of acceptable ranges. Because of this, regulatory agencies like the FDA require evidence that products maintain consistent CQAs that relate to identity, purity, potency, and safety across every batch. [7] Without validated analytical methods, it’s impossible to ensure the safety and efficacy of these therapies as they transition from research to clinical applications. By investing in fit-for-purpose assays and phase-appropriate validations, one can build the trust and reliability needed to translate cutting-edge science into impactful patient treatments.

How to Strategize Phase-appropriate Validation?

Let’s say that your lab has discovered a new MSC-EV drug candidate out of research-grade experiments, preparation methods, and raw materials.  When it’s time to manufacture this entity into human doses, developers might find that scale-up alters both activity assays and physiochemical properties, as cell behavior shifts with process changes. [8] Dr. Picou emphasized an iterative feedback loop: refine strategy, process, and analytical methods to ensure the product consistently meets intended quality attributes throughout the whole journey clinical development and manufacturing journey.

To begin first-in-human studies in the USA with MSC-EVs, an Investigational New Drug (IND) application must be prepared, filed, and approved by the FDA. Other regulatory jurisdictions may have similar guidelines. These applications must contain:

• Preclinical data to assess pharmacology and toxicology studies which is normally done through animal studies
• Manufacturing information such as chemistry, manufacturing, and controls (i.e., CMC), as well as composition and stability
• And detailed protocols for proposed clinical studies. (You need to inform the FDA regarding what exactly you want to do.)

Dr. Picou continued the webinar with some basic insights that pointed toward a validated quality analytics assay landscape, pursuant to a de-risked path into first-in-human trials and beyond. For MSC-EVs, relevant and assayable CQAs might be classified by Identity (e.g., particle size distribution, surface marker expression, cytosolic marker expression, lipid membrane composition, nucleic acid composition, morphology), Purity (e.g., endotoxin, residual proteins, etc.), Potency (e.g., cell-based assays, CD73 activity), and Safety (e.g., sterility, bioburden, mycoplasma, and viral agents; Figure 1, below). There are obviously myriad ways to credibly demonstrate identity, purity, potency, and safety across product manufacturing scale. But which methods are “fit for purpose” and hence practical—i.e., designed to directly address the question and product characteristic at hand?

Figure 1

Fit for purpose means that an assay needs appropriate reference materials, standards, and controls, shows well-defined dose responses, and aligns with intended ranges of specificity, accuracy, precision, sensitivity, robustness, sensitivity, and adaptability to scale. However, the concept doesn’t involve “overkill,” and will strive to reduce burden, with focus only on the validation parameters that are most relevant toward intended use. Trey emphatically states: “Choose what is right for your program at the right point in time.” Understand the purpose (the “Why”) before descending into the actions (the “How”).

With each deliberate assay purpose defined, the next step is execution, rooted in an iterative phase-appropriate strategy. First, there is Qualification in Early Development and Trial phases. These assays are designed to prove ability to reliably measure the linked CQAs even while processes and products evolve somewhat. This phase is about reducing risks—refining methods to align with the product’s needs without prematurely locking in rigid parameters. Method qualification can change with processes and products, but it’s also where one can nail down acceptance criteria. That said, during Qualification, there are reduced checked parameters by the Agency, with pre-testing often voluntary. Qualification is a work in progress. Yet, the operative word of “work-in-progress” is “work,” which means it’s unwise to delay it. [9] Be proactive from the very beginning of early preclinical research after candidate discovery. This will dramatically reduce the friction toward a successful IND launch.

Figure 2. Distinctions between assay Qualification and Validation stages. Table adapted from an outstanding review in blog by Dr. Anindya Ghosh Roy. [10]

Once processes stabilize, assay methods advance to Validation for Clinical and Commercial Stages (Figure 2, above). Validation is a regulatory requirement, based on thorough and expanded performance criteria such as those published by the ICH which is the International Council on Harmonization of technical requirements for registration of pharmaceuticals for human use. These guidelines, known as ICH Q2(R1) are the validation of analytical procedures, text and methodology. These must be in place before Phase III trials. The regulatory framework for Validation ensures that the quality assays and their data conform with stringent performance criteria, including specificity, precision, and robustness.

To effectively bridge Qualification and Validation processes, a qualification protocol designed to dovetail USP/ICH guidance on the key parameters is necessary. This is to be summarized in a qualification summary report document. It is an overview of the plan or process for validation and summarizes the results of right-fitting assay qualification activities. With this in hand, the Quality team will be able to report a body of work that is supported by a robust scientific and operational foundation.

As methods develop and mature, it will be important to hone key assay principles of interest to both your team…and to a regulatory agency. Concluding his section of the webinar, Dr. Picou briefly dissected each of these terms and how they matter: sensitivity, range, linearity, specificity, and accuracy & precision. This served as a good segue into Dr. Madeline Cramer’s section of the presentation, wherein her methodical efforts have enabled RoosterBio to progress toward a qualified MSC-EV potency assay via CD73 activity measurement.

What Can RoosterBio’s CD73 Assay Qualification Teach Us?

Dr. Madeline Cramer has been refining CD73 assay capabilities as a RoosterBio analytical service and has written on this topic in a recent blog article, published by RoosterBio. [11]  To demonstrate these principles in practice, Dr. Madeline Cramer presented a case study on the qualification of a CD73 activity assay. CD73, an enzyme found on MSC-derived EVs, converts extracellular AMP into adenosine. Extracellular nucleotides are alarm signals of cell damage and are pro-inflammatory, which CD73 dissipates in a cascade with CD39. In turn, the free adenosine metabolite of CD73 is a second messenger involved in a variety of downstream anti-inflammatory and pro-angiogenic effects. Thus, CD73 activity—which is enriched on MSC-EVs in particular—may be an important biomarker linked to the therapeutic potency of EVs. [11]

Figure 3

• Serial dilutions of AMP were used to generate a standard curve fit with 4-Parameter Logistic Regression (4PL)
• All points with a CV < 20% and a backfit between 80 – 120% were considered acceptable
• A quantitative range of 0.63 – 40µM AMP was identified for this assay

Dr. Cramer’s goal was to adapt an off-the-shelf AMP detection kit (Promega AMP-Glo™) into a robust assay for measuring CD73 bioactivity, thereby addressing a critical potency CQA for MSC-EV material. The kit measures AMP concentration via luminescence, where AMP consumption in the presence of CD73 is an indicator of dose-dependent CD73-selective activity. First, Dr. Cramer assessed the limits of quantification, looking at the key parameters of sensitivity and range, and generating a standard curve of luminescence vs. AMP concentration where the instrument could reliably read samples (or sample dilutions) in a range between 0.63 and 40mM AMP. (Figure 3, above). Next, she applied the curve model to demonstrate its linearity for both a recombinant CD73 enzyme control and also samples of purified MSC-EVs. The R² values for both were above 0.98, translating to a linear range for CD73 between 3 and 18mM. For the EV particles, a span of 0.2×10⁹ to 1 x 10⁹ could show linearity (Figure 4, below).

Figure 4

• CD73 enzyme and an EV sample were serially diluted and then CD73 activity was measured using the assay
• CD73 activity was linear in the range of 3 – 18µM, corresponding to a starting particle concentration between 2×108 and 1×109 P/mL
• Particle concentrations between 2×10⁸ and 1×10⁹ P/mL are within the linear range

What if loss of AMP signal was not specific to CD73? To show specificity, that CD73 was the genuine ingredient of recombinant protein (rCD73) or EV samples to metabolize AMP, APCP inhibitor was employed. APCP is a specific inhibitor of CD73, which blocked degradation of AMP in the CD73 activity assay (Figure 5, below).

Figure 5

• EV samples and CD73 enzyme showed CD73 activity when incubated with AMP
• In the presence of the CD73-specific inhibitor APCP, the CD73 activity was significantly decreased
• At least 80% of the activity was inhibited by APCP, showing the specificity of this assay

To assess accuracy, Dr. Cramer and her team measured the proximity between the expected value with the measured value of rCD73 and two MSC-EV samples. This was done by spiking in 10μM of AMP into rCD73 and EV samples with known AMP concentration and observing the commensurate bump in AMP concentration. Results of 112%-123% of expected spike recovery were measured, within the acceptable range of 70-130% for such assays (Figure 6, below).

Figure 6

• Samples were spiked with 10µM AMP prior to AMP concentration measurement
• The % recovery of the AMP spike was determined by comparing the AMP concentration with and without the spike
• Between 70 – 130% of the AMP spike was recovered for all samples which is considered acceptable

To show that the CD73 activity assay could be repeatable in different settings, Dr. Cramer demonstrated its reproducibility across multiple samples and experimental days, using EV samples from EVs collected from different MSC origin tissues and donors (bone marrow, adipose tissue, and umbilical cord). The readout was in AMP Consumption, or pmol/min per 1e9 EV particles. %CVs for all samples was less than 20% demonstrating this assay’s precision (Figure 7, below).

Figure 7

• The CD73 activity of MSC-derived EV samples from bone marrow (hBM), adipose (hAD) and umbilical cord (hUC) was evaluated
• Repeatability of the assay: variation within a single sample on a single day
• Intermediate precision: variation of the same sample across multiple days
• The %CVs were ≤ 20% for all samples, demonstrating the precision of this assay

Since the assay was observed to be functional in a defined linear range with specificity, accuracy, and reproducibility, it was possible to establish system suitability parameters to ensure sustained reliability of results. These can be used to benchmark the assay’s proper performance at the same level as shown in CD73 assay qualification. System suitability ensures that controls and assay specifications conform to expected standards, validating both proper assay execution and consistent performance over time. By setting precise acceptance criteria—such as CVs under 20% for triplicate measurements, R² above 0.98 for standard curves, and defined parameters for positive and negative controls—one can readily detect errors (e.g., pipetting or equipment issues) and maintain assay reliability. If criteria aren’t met, experiments can then be repeated to identify one-off issues or broader inconsistencies. This systematic approach ensures the assay remains robust and reproducible, even across multiple days and tissue sources. While this CD73 assay isn’t yet GMP-ready, this example qualification strategy could lay a foundation for future GMP testing, driving EVs closer to therapeutic application. (Figure 8, below).

Figure 8

• System suitability requires that all controls are conforming to assay specifications
• These parameters are used to ensure that the assay has been performed properly and at the same level as during its qualification

In short, Dr. Cramer qualified a CD73 activity assay according to CMC guidelines. It may serve as one possible readout of MSC-EV potency that is fit for its intended purpose, providing a robust tool for translation of prospective MSC-EV therapies into clinical applications. The remainder of the webinar involved a Q&A session that yielded additional insights in a lively dialog:

• Defining Criteria in Uncertain Terrain: Early acceptance criteria are often based on iterative testing and historical data, reflecting the evolving nature of biologics.
• Addressing Specificity in Novel Assays: How tools like recombinant proteins and targeted inhibitors ensure that assays measure only what they’re designed to measure.
• Navigating Complexity: Balancing sensitivity and precision remains a challenge in systems with inherent variability, requiring ongoing refinement.

So, please view the webinar for a fuller understanding of how analytics will affect your evolving clinical process development. Or view our related poster, presented at the ISEVxTech Meeting (Nov 2024, Baltimore). You might come to a similar resolution of the age-old question of “nature vs. nurture.” …No matter the “nature” of MSCs and their secreted EVs, or how they are “nurtured” during bioprocessing, the answer to either question is being answered by RoosterBio and other savvy operations: “MEASURE!”

 

References

1. Lembong, Josephine, et al. Bioreactor Parameters for Microcarrier-Based Human MSC Expansion under Xeno-Free Conditions in a Vertical-Wheel System. Bioengineering (Basel, Switzerland), 2020. 7, E73 DOI: 3390/bioengineering7030073.
2. Lenzini, Stephen. Big Effects in Small Packages: What Are Extracellular Vesicles, Exosomes, & Microvesicles & Why Are They En Route to the Clinic? 2021; Available from: https://www.roosterbio.com/blog/big-effects-in-small-packages-what-are-extracellular-vesicles-exosomes-microvesicles-why-are-they-en-route-to-the-clinic/.
3. Ng, K. S., et al., Bioprocess decision support tool for scalable manufacture of extracellular vesicles. Biotechnol Bioeng, 2019. 116(2): p. 307-319. 1002/bit.26809
4. Candiello, Joseph and Lim, Mayasari. Webinar: Best Practices in RegenMed Product & Process Development: Know your COGS. 2020; Available from: https://share.hsforms.com/1Ns3ZdJgFQKW05XATgdnfrQ3564o?
5. “Where Do We Come From? What Are We? Where Are We Going?” Know Your Process & Define Your Product with EV/Exosome Analytics. 2022; Available from: https://www.roosterbio.com/blog/where-do-we-come-from-what-are-we-where-are-we-going-know-your-process-define-your-product-with-ev-exosome-analytics/.
6. Cramer, Madeline, and Picou, Trey. Preparing for the Clinic: Qualifying Analytical Methods. RoosterBio Webinar 2024; Available from: https://tinyurl.com/RBI-analytics-cd73.
7. Campbell, A., et al., Concise Review: Process Development Considerations for Cell Therapy. Stem Cells Transl Med, 2015. 4(10): p. 1155-63. 5966/sctm.2014-0294
8. Kirian, R. Maintaining CQAs as Manufacturing Processes are Scaled from 2D to 3D Bioreactor Culture. RoosterBio Blog 2022; Available from: https://www.roosterbio.com/blog/maintaining-cqas-as-manufacturing-processes-are-scaled-from-2d-to-3d-bioreactor-culture/.
9. Williams, Kathy, and Hansen, Caitlin. Quality Begins at Inception. RoosterBio Blog 2020; Available from: https://www.roosterbio.com/blog/quality-begins-at-inception/.
10. Ghosh Roy, Anidya. What’s the difference between method qualification and method validation? Lösungsfabrik 2022; Available from: https://mpl.loesungsfabrik.de/en/english-blog/method-validation/qualification-versus-validation.
11. Cramer, Madeline. CD73: A Team Player Caught in the “AKT” of Wound Healing & Cell Survival via MSC Exosomes? RoosterBio Blog 2024; Available from: https://www.roosterbio.com/blog/cd73-a-team-player-caught-in-the-akt-of-wound-healing-cell-survival-via-msc-exosomes/.