Part II of a Blog Series on CQAs & CPPs.

<< Read Part I

Mesenchymal stromal/stem cells (MSCs) have comprised the cellular source material for over 1,000 clinical trials worldwide in the last 10 years, dosed into a planned enrollment of over 70,000 patients to date. [1] These trials span tens of different indications across therapeutic classes ranging from cartilage regeneration to anti-oncology. Although the safety record across two decades has been outstanding thus far, MSCs can be a highly diverse modality.  With variable tissue sourcing, bioproduction methods, formulations, route of administration, and patient monitoring, the myth that each Critical Quality Attribute (CQA) will be the same for every MSC clinical product has long been dispelled. In reality, some CQAs will be the same— and others will be very different. Our goal in this post is to outline what quality attributes may be similar across products and which will be different, as well as highlight the important differentiation of what is a Critical Quality Attribute vs a quality attribute.

“Critical Quality Attribute: physical, chemical, biological, or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality”

-ICH Guideline Pharmaceutical Development Q8(R2)

This definition of Critical Quality Attribute is taken directly from the ICH Guideline for Pharmaceutical Development [2]. Here we use MSCs as a case study for understanding CQAs as they apply to Cell Therapy, and we look at the CQAs of MSCs including their importance and their current limitations.

Current State of MSC (Critical) Quality Attributes

Critical Quality Attributes as defined in the US Code of Federal Regulations (21CFR610) include: Safety, Purity, Identity, and Potency. Safety CQAs are fairly standard across the cell therapy field and often can rely on compendial methods for testing things like sterility and mycoplasma. In defining the other CQAs, however, cell therapy is challenged by the inherent biological variability and lack of clarity around CQA definitions. [3] MSCs, for example, can be isolated from different tissue sources using a number of methods. Historically, various assessments have been used to evaluate and define MSCs. Therefore, in 2006, the International Society for Cell and Gene Therapy (ISCT) proposed three minimal criteria for defining an MSC [4]:

• adherence to plastic
• expression of a panel of surface markers and absence of a panel of surface markers
• trilineage differentiation potential into osteoblasts, adipocytes, and chondroblasts

These characteristics have been widely adopted by the field as MSC Critical Quality Attributes in both research as well as in early phase clinical trials [5]. While these assessments may point to the purity and identity of the cell product, functional assessments are also critical, and each MSC therapy must define a specific potency CQA. A potency CQA is a characteristic that is related to the therapeutic product’s mechanism of action and correlated to clinical efficacy [6]. Therefore, a potency assay for one clinical indication is likely not appropriate for a different indication. For instance, an MSC treatment for Graft vs. Host Disease may include a potency assay measuring the specific aspect of immunomodulation that is key to the product’s efficacy. A genetically modified MSC product being used for delivery of a therapeutic molecule, on the other hand, may instead have a potency assay to measure the production of a specific protein, for example.

RoosterBio, as a raw material supplier, investigates the following broad functional assessments of MSC bioactivity which may apply to many different therapeutic applications:

• expansion potential
• immunomodulatory activity by IDO
• angiogenic cytokine secretion

While not potency measurements, these functional attributes may help in the initial selection of donors to screen for a given therapeutic indication, capturing some of the inherent biological variability due to differences in starting material from the different donor sources.

Current Limitations of Critical Quality Attributes

There continues to be discussion about the utility of the 2006 ISCT-defined characteristics (adherence to plastic, surface marker expression, and trilineage differentiation) as MSC CQAs. Questions have been raised about whether the identified attributes and corresponding in vitro assays are good predictors of in vivo function. [7] A study by Steve Bauer’s group showed differences even among in vitro assays. They showed that while MSC expansion capacity and cell size changed as the population doubling level (PDL) increased, surface marker expression was maintained [8] (further discussed in a legacy blog on Current Bottlenecks in MSC Research). Although surface marker expression is currently suggested by ISCT as an MSC identity attribute, this raises the question of whether the panel of positive and negative surface markers used for MSCs is a good indication that the cells are maintaining an MSC identity, and furthermore, brings into question whether surface marker expression is even an indication of a consistent cell product.

The assays used to evaluate Critical Quality Attributes also may lack sensitivity to capture shifts in cell function. For example, trilineage differentiation potential is historically based on histological staining of differentiated cells which is subjective and qualitative. These limitations in not only identifying CQAs but also in being able to accurately measure them were previously discussed in Part I of this blog series: “Known Unknowns” Affect Critical Process Parameters & Critical Quality Attributes in the Bioproduction Cycle, [9] where we also discussed a new definition of CQA as proposed in an editorial by Dr. Mark Witcher: Why Controlling CQAs Isn’t Good Enough For Gene & Cell Therapies. Dr. Witcher proposed broadening the current definition of CQAs to include not just measurable properties and characteristics but also unmeasurable ones to better suit the current state of cell and gene therapy [10].

Process Development & Critical Quality Attributes

“The product is the process” is a common phrase heard in the cell therapy field. This phrase holds true because of the lack of understanding about which Critical Quality Attributes are important in cell therapy and the limitations in our ability to measure them. In turn, this means that once a process is developed, the process must operate within a narrow range of process parameters for subsequent manufacturing lots.

Process development, therefore, becomes about identifying these process parameters and by necessity must focus on finding parameters that maintain the measurable CQAs, i.e., the process must be optimized and process parameters selected such that the maintenance of these CQAs is ensured. Then, once the process parameters are selected, they must not change for fear of altering the unmeasurable CQAs.

For example, as we have scaled our bioreactor process, we have demonstrated that CQAs are maintained between flasks and bioreactors as well as across bioreactor scales by comparing hMSC surface marker expression, trilineage differentiation, cytokine secretion, IDO activity, and expansion potential in a cell growth assay. [11] The bioreactor process now operates within a range of Critical Process Parameters as we will detail in a future blog.

Future of Critical Quality Attributes & Process Development

This adage that the “product is the process” and this proposed definition of Critical Quality Attributes as both measurable and unmeasurable characteristics will be needed in the short term. Ultimately, however, the field must develop a better understanding of which CQAs are truly important to “ensure the desired product quality” as stated in the ICH Q8(R2) Guideline [6]. This also necessitates developing more sensitive assays.

The FDA decision on Mesoblast’s remestemcel-L product for the treatment of Graft vs Host Disease highlights the critical need and importance of understanding CQAs, especially potency assays as MSC therapies approach commercial therapy. The FDA recently issued a Complete Response Letter to Mesoblast that asked remestemcel-L to undergo an additional clinical trial in order to show efficacy and also “identified a need for further scientific rationale to demonstrate the relationship of potency measurements to the product’s biologic activity.” The press release from Mesoblast goes on to say, “Assays measuring the potency of remestemcel-L will continue to be refined to provide further scientific rationale for its use in severe inflammatory diseases with high mortality risk, such as SR-aGVHD and COVID-19 ARDS.”

Developing more sensitive CQA assessments is broadly recognized as both a challenge and an opportunity by the field. In fact, the NSF Engineering Research Center for Cell Manufacturing Technologies and partners identified CQA and in-line Critical Process Parameter (CPP) monitoring systems as a high-priority activity in their Cell Manufacturing Roadmap to 2030 first released in 2016 and last updated in 2019 [12]:

“Cell Critical Quality Attribute and Critical Process Parameter Measurement

• Develop standardized, modular platform technologies and high-throughput assays or surrogates to ensure lot-to-lot consistency in terms of phenotype, functionality, quality, and potency over a range of time frames across the product lifecycle
• Develop real-time CQA and CPP monitoring systems (e.g., with smart sensors and real-time in-line or at-line PAT and controls) that non-destructively gather and transmit CQA data and adjust process parameters to drive cell populations to the desired functional state”

Once we have a clear understanding of true Critical Quality Attributes and assays sensitive enough to evaluate them, the field can begin to advance to a Quality By Design approach where the process operates within a range and the CQAs, not the process, truly define the product [13]. At this point, however, we must define the product by controlling the process and adhering to defined CPPs. This will be discussed in Part III of this blog series: Identify & Define Your Cell Therapy’s Biomanufacturing Approach for Critical Process Parameters (CPPs).

Finally, while this blog focused on MSC CQAs, similar ideas also apply to cell therapy ancillary materials (e.g. cell expansion media) as well as extracellular vesicles derived from MSCs. For media, some of the CQAs might include chemical properties like pH and osmolality. While extracellular vesicles are at a similar early stage to MSCs, the CQAs, as well as assays to measure them, are just being defined.

At RoosterBio, we offer a wide range of development services that will help you understand the “known unknowns” that affect your CPPs & CQAs.

Read Part III >>

References

1. Cell Trials Data. https://celltrials.org/cells-data Accessed.
2. Guideline ICHHT. Pharmaceutical development. Q8 (2R) As revised in August. 2009.
3. Karanu F, Ott L, Webster DA, Stehno-Bittel L. Improved harmonization of critical characterization assays across cell therapies. Regen Med. 2020;15(5):1661-78; doi: 10.2217/rme-2020-0003.
4. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8(4):315-7; doi: 10.1080/14653240600855905.
5. Mendicino M, Bailey AM, Wonnacott K, Puri RK, Bauer SR. MSC-based product characterization for clinical trials: an FDA perspective. Cell Stem Cell. 2014;14(2):141-5; doi: 10.1016/j.stem.2014.01.013.
6. Administration FaD. Guidance for industry: Potency tests for cellular and gene therapy products. Center for Drug Evaluation and Research (CDER). 2011; doi: https://bit.ly/3q8yKQB.
7. Phinney DG, Sensebe L. Mesenchymal stromal cells: misconceptions and evolving concepts. Cytotherapy. 2013;15(2):140-5; doi: 10.1016/j.jcyt.2012.11.005.
8. Lo Surdo JL, Millis BA, Bauer SR. Automated microscopy as a quantitative method to measure differences in adipogenic differentiation in preparations of human mesenchymal stromal cells. Cytotherapy. 2013;15(12):1527-40; doi: 10.1016/j.jcyt.2013.04.010.
9. Carson J. “Known Unknowns” Affect Critical Process Parameters & Critical Quality Attributes in the Bioproduction Cycle. https://www.roosterbio.com/blog/known-unknowns-affect-critical-process-parameters-critical-quality-attributes-in-the-bioproduction-cycle (2021). Accessed.
10. Witcher M. Why Controlling CQAs Isn’t Good Enough For Gene & Cell Therapies. https://bit.ly/2xBvCHj (2020). Accessed.
11. Kirian RD, Wang D, Takacs J, Tsai A, Cruz K, Rosello F, et al. Scaling a xeno-free fed-batch microcarrier suspension bioreactor system from development to production scale for manufacturing XF hMSCs. Cytotherapy. 2019;21(5, Supplement):S71-S2; doi: https://doi.org/10.1016/j.jcyt.2019.03.464.
12. Manufacturing CNERCfCMTGTMCfTCCaMNC. Cell Manufacturing Roadmap to 2030. http://www.cellmanufacturingusa.org/sites/default/files/Cell-Manufacturing-Roadmap-to-2030_ForWeb_110819.pdf (2019). Accessed.
13. Yu LX, Amidon G, Khan MA, Hoag SW, Polli J, Raju GK, et al. Understanding pharmaceutical quality by design. AAPS J. 2014;16(4):771-83; doi: 10.1208/s12248-014-9598-3.