David Moolten, MD
Medical Director
American Red Cross
United States
Why the Quality and Make-up of Starting Material Matters
The familiar truism garbage in, garbage out can be ruinously accurate in the setting of cell and gene therapy production. What is in the bag at the beginning portends what will be there at the end. Rigorous characterization of starting material is thus a cornerstone of any successful process. Characterization allows the proper definition and inclusion of essential constituents that determine a drug's potency. As importantly, characterization facilitates the exclusion of those which corrupt the drug’s purity, may dilute its potency, and/or compromise its efficacy and even its safety. The presence of extraneous cells and/or substances can significantly add to the risk and severity of adverse events.
Poor characterization of starting material may result in outright failure of a process initially or at any phase of production. If the attributes of the starting material are not well enough defined, this variability is potentially multiplied throughout production, resulting in the increasingly difficult, if not impossible, task of adequately characterizing the product at the latter stages of the process and then correcting or controlling for deviations in the product’s attributes from desired norms. Similarly, good characterization of starting materials during development will drastically reduce the need to modify the production process as stringency increases during the lead-up to commercialization. The importance of this multiplier effect cannot be emphasized enough.
As an example, the presence of monocytes may suppress the activation and expansion of T cells essential to manufacturing CAR-T drugs, degrading their potency. Red blood cells and granulocytes can also have a negative impact on efforts to stimulate and expand T cells. This contamination not only attenuates potency but introduces inconsistency into the process and ultimately the therapeutic agent. The product will be both variable and reduced in potency. Since the complications of CAR-T therapy, such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), correlate with product potency, a variable product can be a dangerous product even when therapeutically robust. (1, 2, 3)
Therefore, having a starting material that keeps contaminants to a minimum and at the same time accurately quantifies their presence is critical. Of course, not all T cells are created equal, and T cell subset analysis in the starting material prior to further manufacture of CAR-T drugs crucially directs how that material must be processed, in some paradigms, for example, specifying a desired ratio of CD8+ cells (the primary target of CAR gene insertion) and CD4+ cells. The latter have a secondary role but provide essential support functions and independently contribute to tumor control. The presence of too few or too many CD4+ cells may attenuate CD8+ cell activity and/or encourage exhaustion. (4, 5, 6)
Quantifying the T-cell population of interest only tells half the story. The fitness of the cells, as measured by their expansion capability, the relative presence of less differentiated naïve and memory cells versus more differentiated and exhausted cells, dictates their actual potency. Further flow cytometric analysis allows discrimination. For example, CD45, a protein tyrosine phosphatase, appears broadly on the surface of white cells, whereas CD45RA represents an isoform found primarily on naïve T cells, CD45RO and isoform found on memory T cells. Intermediate CD45RO+ “stem-like” memory cells, sought after in some CAR-T paradigms, may possess both isoforms as well as the CCR7 homing marker. (7)
Blood is a diverse and highly complex mixture of cellular elements and small molecules. Starting material procured from patients is, by its nature, more variable and less robust, as it is subject to the attenuative impact of prior treatment, undermining the quality of the available T-cell population. While developers of autologous products can’t choose the source of their starting material, those devising drugs predicated on allogeneic cells from healthy individuals can. Defining optimal donor criteria for the intended drug, then identifying and recalling donors who meet them from an adequately large and diverse pool, hinges on sound characterization. Such characterization at the outset results in products of consistent and predictable composition, and a process under better control throughout development, potentially increasing its pace and lowering its costs.
The American Red Cross exemplifies how such principles can be applied at scale. The organization maintains a brick-and-mortar presence in all fifty states and the five territories (American Samoa, Guam, the Northern Mariana Islands, Puerto Rico, and the U.S. Virgin Islands), which includes hundreds of collection sites and dozens of strategically placed processing, testing, and storage facilities, all using a single set of common procedures governed by a single quality management system. Its healthy donor pool draws from a vast and varied geography featuring extensive size and demographic diversity. This donor pool and the broad scope of the organization’s operations allow great consistency and reproducibility in its processes, as well as statistical power in its testing and analyses. At the same time, the Red Cross can also furnish characterized plasma and cells obtained from individuals with a range of pathological conditions and disease states, understanding the value of such ancillary material as an investigational resource for developers.
The volume and scale of Red Cross’s activities create opportunities to partner with vendors of equipment and reagents used for cell collection, manufacturing, and storage to meet the specific needs of the organization. This volume allows the organization to affordably obtain products with altered specifications and to bring updated methods online quickly. Switching between the most basic tools and supplies can be non-trivial as a developer strives to keep an established process in control. This is particularly true for international developers looking to translate their activities to the U.S. The Red Cross has the ability to facilitate modifications as they become necessary over time.
The consistency and reliability of a well-characterized starting material begin with the same qualities in its supplier. The supplier's capabilities must be robust but also durable. The ability to return to that supplier and obtain the same predictably high-quality cellular product every time avoids costly troubleshooting and/or the need to find a new source and recalibrate the production process with costly delays as the developer moves down the path to commercialization. The Red Cross has formally existed since 1881; its blood collection activities date back to 1941, its involvement in cell therapy to 1999 (discounting bone marrow and blood stem cell processing and storage, which began earlier). (8)
The need to characterize starting material by quantifying the cell(s) of interest and determining their fitness while accounting for those the production process must ultimately exclude has been discussed; but it is also useful and potentially critical to have the ability to accomplish these tasks under different conditions: fresh, frozen, patient source, healthy donor source, in different media, and after various preliminary processing steps.
Returning to the CAR-T drug example, most but not all commercially available products rely on fresh apheresis leukopaks as their starting material, as opposed to frozen components containing cryoprotectant. Because the latter promises manufacturing flexibility, protection against transportation issues, and the ability to collect earlier when the patient (and their cells) is in a more optimal state of health, interest in frozen starting material continues to mount. The Red Cross not only collects over 1.1 million apheresis blood components each year but also has broad capabilities and long experience in the processing and cryopreservation of white blood cell products, such as those issued as leukopaks for the manufacture of therapeutic drugs or used as blood stem cells for transplantation. Moreover, the Red Cross has the logistics for transportation, processing, and storage of cryopreserved cellular products established in multiple geographies. (9)
Collecting over 4.5 million blood units annually throughout the country, the Red Cross features massive throughput for its rapid and highly standardized FDA-compliant testing while at the same time retaining the ability to track and trace each and every donor and blood component from source to final disposition, and brings this same capacity and granular management to its work in cell and gene therapy. (10)
In addition to requisite screening for pathogens, quantitative and qualitative data such as apheresis volume, donor demographics, donor physiologic parameters, run parameters, temperature, blood counts, white cell count and differential in the apheresis product, the Red Cross offers rapid enumeration of key lymphocyte populations such as T cells (CD3), B cells (CD19), natural killer cells (CD16/56), and monocytes (CD14). When more detail is needed, the Red Cross can provide comprehensive blood differential testing and flow cytometric evaluation of T-cell subsets such as CD4 helper and CD8 cytotoxic populations. If desired, the Red Cross can also run customized flow panels configured to protocols provided by the developer.
Additional donor testing available includes HLA and blood compatibility testing, KIR testing, rare donor characterization services, and release assays (e.g. sterility, endotoxin, Mycoplasma). The Red Cross will provide a certificate of analysis (COA) upon request, which serves as the definitive quality record verifying that the material meets all specified properties and relevant quality criteria. Such documentation is critical in cell and gene therapy, where product consistency and safety depend on thoroughly characterized starting material. During regulatory compliance assessments of clients, the FDA may review both vendor COAs, such as those provided by the Red Cross, and a company’s own testing of incoming material to ensure that all quality standards are met. While the future of the field is anyone's guess, the need for increasingly sophisticated assessments of starting material for an ever more diverse array of cell and gene therapies is certain, as is the ongoing dedication of the Red Cross to keep pace and continue to make available blood and the products derived from it, including those essential to such therapies.
References
1. Carniti C, Caldarelli NM, Agnelli L, Torelli T, Ljevar S, Jonnalagadda S, Zanirato G, Fardella E, Stella F, Lorenzini D, Brich S, Arienti F, Dodero A, Chiappella A, Magni M, Corradini P. Monocytes in leukapheresis products affect the outcome of CD19-targeted CAR T-cell therapy in patients with lymphoma. Blood Adv. 2024 Apr 23;8(8):1968-1980. doi: 10.1182/bloodadvances.2024012563. PMID: 38359407; PMCID: PMC11017285.
2. Long K, Meier C, Bernard A, Williams D, Davenport D, Woodward J. T-cell suppression by red blood cells is dependent on intact cells and is a consequence of blood bank processing. Transfusion. 2014 May;54(5):1340-7. doi: 10.1111/trf.12472. Epub 2013 Nov 4. PMID: 24188586; PMCID: PMC4344125.
3. Munder M, Schneider H, Luckner C, Giese T, Langhans CD, Fuentes JM, Kropf P, Mueller I, Kolb A, Modolell M, Ho AD. Suppression of T-cell functions by human granulocyte arginase. Blood. 2006 Sep 1;108(5):1627-34. doi: 10.1182/blood-2006-11-010389. Epub 2006 May 18. PMID: 16709924.
4. Galli E, Bellesi S, Pansini I, Di Cesare G, Iacovelli C, Malafronte R, Maiolo E, Chiusolo P, Sica S, Sorà F, Hohaus S. The CD4/CD8 ratio of infused CD19-CAR-T is a prognostic factor for efficacy and toxicity. Br J Haematol. 2023 Nov;203(4):564-570. doi: 10.1111/bjh.19117. Epub 2023 Oct 3. PMID: 37789569.
5. Melanie Ayala Ceja, Mobina Khericha, Caitlin M. Harris, Cristina Puig-Saus, Yvonne Y. Chen; CAR-T cell manufacturing: Major process parameters and next-generation strategies. J Exp Med 5 February 2024; 221 (2): e20230903. doi: https://doi.org/10.1084/jem.20230903
6. Wang, Y., Tong, C., Lu, Y. et al. Characteristics of premanufacture CD8+ T cells determine CAR-T efficacy in patients with diffuse large B-cell lymphoma. Sig Transduct Target Ther 8, 409 (2023). https://doi.org/10.1038/s41392-023-01659-2
7. Ploch W, Sadowski K, Olejarz W, Basak GW. Advancement and Challenges in Monitoring of CAR-T Cell Therapy: A Comprehensive Review of Parameters and Markers in Hematological Malignancies. Cancers (Basel). 2024 Sep 29;16(19):3339. doi: 10.3390/cancers16193339. PMID: 39409959; PMCID: PMC11475293.
8. https://www.redcross.org/about-us/who-we-are/history.html#:~:text=The%20Red%20Cross%20identified%20medical,for%20the%20International%20Red%20Cross.&text=Clarissa%20Harlowe%20Barton%E2%80%94teacher%2C%20government,23%20years%2C%20retiring%20in%201904.
9.https://www.redcross.org/donations/your-gift-matters/where-it-s-needed-most.html?srsltid=AfmBOoqD3DeMyn3lq7GV5eJg2TTlcqTcoqiSYEzvSB6WSnxB2wAf5i8z
10. https://www.redcross.org/donations/your-gift-matters/where-it-s-needed-most.html?srsltid=AfmBOoqD3DeMyn3lq7GV5eJg2TTlcqTcoqiSYEzvSB6WSnxB2wAf5i8z