Bethany Macleod

Sree Pulugulla is a Process Development Scientist at Lonza. He is passionate about analyzing and solving scientific problems, with expertise in immunotherapy, drug discovery, T-cell biology, and data analysis. Sree is skilled in laboratory investigations and is dedicated to advancing personalized medicine.

Bethany L. Macleod is an accomplished immunologist with over a decade of experience in biotech and academia. She specializes in immune responses, oncology, and cell therapy, with expertise in flow cytometry and human T cell culture. Bethany has a passion for leadership and mentorship, demonstrated by her guidance of students and contributions to innovative research techniques. She currently serves as a Process Development Scientist at Lonza. 

Aman Mehrotra is a Process Development Scientist at Lonza. He has a Master’s Degree in Microbiology and Immunology, and is skilled in multi-color flow cytometry and cell based assays.

Janet J. Sei, Ph.D., is a Process Development Manager at Lonza, specializing in immunology and immune responses. She is known for her strong problem-solving skills, adaptability, and leadership in collaborative environments. She utilizes her expertise in bioassay, process and product development.

Through this article, Pulugulla, et al. emphasize the critical role of bioassays in the process development of CAR-T cell therapies. Bioassays are essential for characterizing and optimizing the product throughout its development, starting from early-phase product characterization to aligning release criteria with regulatory guidelines.

Role of bioassay in process development for CAR-T 

In the current dynamic landscape of CAR-T cell therapy, the role of bioassays in process development is indispensable. As we navigate the complexities of manufacturing these life-saving therapies, it is critical to underscore the importance of integrating bioassays into the early development pipeline. Starting early is not only advantageous, but it is a necessity. The CAR-T development journey from bench to bedside requires a proactive approach, where assays and process development are not sequential but are rather interdependent. Analytical assays required for CAR-T cell product characterization are often complex and may necessitate developments that are specific to the unique requirements associated with a given product. By defining assays before process development and refining or modifying them as needed during process optimization, we can enable a streamlined trajectory from discovery to commercialization of the product. Early-phase product characterization should comprise a strategy that allows developers to comprehensively profile the product by understanding its phenotypic and functional characteristics. This proactive approach enables refining the process and anticipating challenges early in the development phase.  

As the product transitions from early-phase investigation to late-stage development, the focus should shift toward the release criteria. Aligning release assays with regulatory guidelines, particularly focusing on critical quality attributes (CQA), is paramount. Comprehensive early-phase product characterization assays could provide valuable insights into identifying CQA that should be assessed during product manufacturing and lot release. Beyond efficacy, current regulatory frameworks demand a holistic approach, encompassing CQA such as safety, identity, purity, and potency of the CAR-T drug product. 

"By defining assays prior to process development and refining or modifying them as needed during process optimization, we can ensure a streamlined trajectory from discovery to commercialization of the product."

Quality of product is more than just yield and viability

Meeting target dose levels is an important first step in producing a living drug product that is fit for patients. Indeed, cell counts, viability, and CAR expression are attributes that must be assessed for all products. Further, appropriately designed bioassays can provide considerable additional information that is beneficial during process development, which could support more robust products by enabling process development decisions that are shown via bioassay to increase fitness and potency. 

It is prudent to consider that while strong activation and cytokine stimuli may generate high numbers of T cells, there may be a functional cost to expansion, such as activation-induced cell death. Indeed, changes during process development could open the possibility of having undesired outcomes, such as high vector copy number (VCN), high pro-inflammatory and anti-inflammatory cytokine production, terminal differentiation, and exhaustion. Therefore, bioassays assessing activation, exhaustion, memory, cytotoxicity, persistence, VCN, metabolic fitness, and polyfunctional cytokines may help calibrate the balance between yield and functionality. As a final step in verifying the functionality of products from optimized manufacturing processes, in vivo studies could give additional insights into the quality of the products before the transition into the clinical phase.  

Recent research has highlighted the importance of metabolic fitness and ‘stem cell memory phenotype’ as excellent indicators that correlate with stronger CAR-T cell persistence. Consistent with this understanding, current advancements in CAR-T cell therapy have led to a shift in manufacturing from longer processes to rapid processes lasting 1-3 days. Younger products have been shown to possess stronger metabolic fitness, stem-like memory profile, and robust functionality, making them attractive products to infuse in patients. Reducing the overall manufacturing time also means shortening vein-to-vein time and reducing the overall production cost of the therapy.  

Furthermore, it’s not just about the end product. Irrespective of manufacturing process duration, guidance from regulatory agencies recommends that CGT developers should provide criteria for the input material. For example, high levels of red blood cells, platelets, or monocyte ‘impurities’ in blood-derived input materials have been shown to negatively impact the transduction and expansion of CAR-T cells, thus introducing variability in product manufacturing. Additionally, patient material is a variable that must be understood to ensure success in autologous manufacturing. Considering that patients may have co-morbidities and have often gone through prior lines of therapy, during process development, it is important to assess the performance of patient samples in addition to healthy donors to avoid unexpected process deficiencies during clinical manufacture.  

Bioassays from CAR-T and beyond 

While most FDA-approved and developing CGT products are CAR-T cell therapies, the landscape is rapidly expanding to include a diverse repertoire of cell-based therapeutic products such as CAR-Macrophages, Natural Killer (NK) cells, CAR-NK cells, Natural Killer T cells (NKT), TCR-engineered T cells (TCR-T) cells and Tumor Infiltrating Lymphocytes (TIL). Indeed, the bioassay considerations we have discussed herein apply to these various cell therapy modalities. Of note, implementing automated manufacturing platforms that incorporate in-line analytical monitoring of pH, dissolved oxygen, and CO₂ offers an additional level of control of the manufacturing process by providing dynamic feedback in assessing the health of the cell product during manufacture and superior ability to tailor processes to achieve consistent performance.