Cell and Gene Therapies: The Evolution of Advanced Biologics

CELL AND GENE THERAPIES: The Evolution of Advanced Biologics SPONSORED BY Transforming Healthcare: What’s on the Horizon for Cell Therapies? Addressing Development Challenges for RNA Therapeutics The Move From Protein‑Based to Cell‑Based Therapies Credit: iStock/Jian Fan CONTENTS 5 The Move From Protein‑Based to Cell-Based Therapies 9 Manipulating Tumor-Infiltrating Lymphocytes for Cancer Therapy 15 Host Cell Protein Analysis in Biologics Manufacturing 20 Addressing Development Challenges for RNA Therapeutics 24 Techniques and Advancements in Recombinant Adeno‑Associated Virus Purification for Gene Therapies 28 Integrating Phenotypic Readouts Into CAR T Production 36 Transforming Healthcare: What’s on the Horizon for Cell Therapies? CELL AND GENE THERAPIES: THE EVOLUTION OF ADVANCED BIOLOGICS 3 TECHNOLOGYNETWORKS.COM FOREWORD Cell and gene therapies are shifting treatment strategies from managing symptoms to addressing disease at its biological roots. As these approaches move from the lab to the clinic, they bring both extraordinary potential and complex challenges. This eBook explores advances in cell-based therapies, RNA medicines, immune cell therapies, and gene delivery, alongside the manufacturing, analytical, and regulatory considerations that underpin the success of these modalities. Together, these expertled perspectives highlight how innovation at the bench must be matched by progress in scalability, quality, and control. Whether you are developing, analyzing, or enabling these next-generation medicines, this eBook offers perspective on a field redefining what is possible in healthcare. The Technology Networks editorial team Unmatched End-to-End RNA Capabilities: Integration from Discovery to Commercialization Aldevron streamlines RNA therapeutics with an end-to-end workflow, combining plasmid DNA, mRNA synthesis, and encapsulation. Backed by expertise from Aldevron and partnerships with IDT and Cytiva, we accelerate your path from discovery to delivery. Our consolidated approach offers: Speed: Eliminates the friction of managing multiple vendors. Scalability: Efficiently scales from clinical trials to commercial launch. Risk Mitigation: Internal analytics and dedicated program management ensure quality and reliability. Don’t delay your path to the clinic. Only Aldevron combines demonstrated expertise and innovative solutions with a fully integrated workflow to accelerate your program. Explore Aldevron’s Capabilites ald.bio/rna-tech-network ADVANCE BEYOND THE TEMPLATE AM-RP-1964-v1 5 CELL AND GENE THERAPIES The Move From Protein‑Based to Cell-Based Therapies Aron Gyorgypal, PhD The biotherapeutic market is currently dominated by protein-based biologics. These drugs are produced by cells that are programmed to over-express a protein that can engage the immune system to fight against disease. Antibody-based therapeutics are the most popular example to explain the biologics market, where antibodies are produced with a certain antigen-binding domain that can target, for example, a cancer marker, which then signals the immune system to react. Though this model of immunotherapy works and has been quite successful for certain indications, in some scenarios the immune system may be impaired, or the disease type may not be responsive to antibody-based therapies, such as with poor tumor penetration, immunosuppressive tumor microenvironments, or refractory autoimmune disease. In these disease models, cell therapies could become a viable approach. A clear example is with triple-class refractory multiple myeloma in which patients no longer respond to proteasome inhibitors, immunomodulatory drugs, or anti-CD38 monoclonal antibodies. In this case, patients are given B-cell maturation antigen (BCMA) targeting chimeric antigen receptor (CAR) T-cell therapy. In this cell therapy, and similarly with others, a patient's own T cells are purified and genetically engineered to recognize and attack a certain target. Here, the use of CAR T-cell therapy showed an 81% overall response rate in 16 patients who received the highest dose of the BCMA-directed therapy. Though the idea of producing cell therapies to target diseases that do not respond to traditional proteinbased therapies is widely considered the next evolution in targeting advanced complex diseases, current technical hurdles exist in terms of developability and manufacturability. This article looks at the key hurdles and innovations that are shaping the field of cell therapy. Credit: iStock/koto_feja TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: THE EVOLUTION OF ADVANCED BIOLOGICS 6 Inconsistency in cell therapy manufacturing Unlike other biologics, such as monoclonal antibodies, where the product is produced and stabilized in formulation to allow for storage, cell therapies are living cells. These cells cannot be terminally sterilized and are sensitive to their environment, such as fluctuations in pH and temperature, causing heterogeneity between batches. This causes a unique relationship between the manufacturing process and product quality, where the process doesn't make the product; instead, the process is the product. Indeed, the early commercial CAR T-cell therapies validated this concern, with some products having manufacturing failures upwards of 10%. While this may not sound high, even a 0.1% failure rate in manufacturing is considered atypical for medical device or therapeutic companies, resulting in failure in treatment, which may lead to patient death. Timing also adds another layer of complexity to the production supply chain. For autologous cell therapies, where the therapeutic is added to a patient's own cells, tight coordination is required between patient apheresis, manufacturing, and reinfusion. A 2023 nationwide survey examining CD19-targeting cell therapy, tisagenlecleucel, found a 7.4% failure rate, 30 of 408 patients with diffuse large B-cell lymphoma, of which many did not survive long enough for a second attempt at treatment. These failures in manufacturing showcase the need for better production methodologies, namely real-time product monitoring and adaptive control strategies, rather than final product quality measurement reflecting the same shifts seen in revolutionizing biologic-based production in the biopharma 4.0 model. Autologous approach to cell therapy As briefly touched upon prior, autologous cell therapies rely on using a patient's own cells to produce the therapeutic, which creates an individualized product for each recipient. Here, the patients' cells are removed through apheresis, the cells are processed, genetically modified and expanded prior to returning to the same patient. This personalized model mitigates risks associated with rejection, eliminating the concerns of graft-versus-host disease. Yet, autologous manufacturing presents challenges, such as the time between patient cell sourcing and engineered cell re-infusion. The typical time period from vein to vein can take anywhere from 9–14 days, with some therapies taking over 49 days on average. This timeline produces high-stakes pressure on manufacturing, especially in cases where patients are diagnosed with aggressive cancers. New technologies have been in the works to curb this timeline; for example, Ghassemi and colleagues at Penn Medicine published a study in Nature Biomedical Engineering demonstrating the generation of functional CAR T cells within 24 hours from T cells derived from peripheral blood without T-cell activation or ex vivo expansion. Similarly, a team at Hebei Yanda Lu Daopei Hospital developed a similar manufacturing platform, named FasTCAR-T, described in a study published in Blood Cancer Journal. This FasTCAR-T platform has recently been shown to induce disease remission in refractory systemic lupus erythematosus patients while also showing a favorable safety profile in a Phase 1b clinical trial. While these strides are being made to bring down time from vein to vein, the economics of autologous cell therapy are another challenge. Manufacturing cost alone ranges upwards of $100,000 to $220,000 per patient, as production costs require dedicated equipment, consumables, and personnel time. The other option for cell therapy is to produce a therapeutic that can be used universally, known as allogeneic cell therapies. Manufacturing universal cell therapies Allogeneic cell therapies use healthy donor cells to create standardized products for patients in advance TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: THE EVOLUTION OF ADVANCED BIOLOGICS 7 and cryopreserve them until required, allowing for larger batch manufacturing. These standardized cell therapies, in theory, would allow for universal infusion to donors for specific diseases without the lead time associated with autologous cell therapy approaches. For example, a Phase 1 study of CAR T-cell therapy for relapsed/refractory multiple myeloma showed an overall response rate of 90%. Similarly, an allogeneic CAR T-cell therapy for hematologic malignancies, Orca-T, saw an overall survival rate of 94% versus 83% with conventional allogeneic transplant, along with a relapse-free survival of 76% in the Phase 1b clinical trial. Economics favor allogeneic cell therapies at scale, given that a single donor batch can treat upwards of 100 patients, dramatically decreasing the cost per dose. With centralized manufacturing, similar to how standard protein-based biologics are produced, the autologous approach cannot compete at scale. However, allogeneic therapies still face technical hurdles, such as the risk of graft-versus-host disease, and require further genetic editing to reduce immunogenicity. Even with modifications, the risk of rejection is unavoidable, and the additional gene edits add manufacturing complexity and regulatory scrutiny. Building quality in cell therapy manufacturing To advance cell therapies further, new methodologies are being developed to make manufacturing faster and cheaper. Genetic modification of T cells is typically done through viral transduction with either a lentiviral or retroviral vector to insert CAR constructs. However, recently, non-viral alternatives have gained traction to avoid vector and integration costs, using electroporation or lipid nanoparticles (LNPs). With some data suggesting that LNPs even outperform electroporation, as LNP-derived CAR T cells provide prolonged functionality in vitro due to extended CARmRNA persistence in the cell. Another viable approach to consider is in vivo CARTcell induction, in which the lentiviral vector or LNPs are introduced directly into the patient and directed to introduce the CAR-encoding genetic material into endogenous T cells, omitting ex vivo transduction. Though current limitations still need to be assessed, such as for T-cell exhaustion, senescence in patientderived cells and risk of toxicity. Furthermore, culture time and duration are known to influence T-cell phenotype, critically affecting product quality. Interestingly, the 24-hour manufacturing processes that have been developed by multiple groups in the past years have all consistently shown production of higher percentages of naïve and stem-cell-like T cells. These platforms correlate with improved cytotoxic activity and enhanced clinical persistence. On another note, the use of bioreactors has been indispensable for cell therapy manufacturing. Modern bioreactors incorporate in-line sensors for process readouts, such as pH, dissolved oxygen, and cell density. This should, in theory, allow for advanced process control and increased automation. Automated systems allow for enhanced data capture and traceability, which help document parameters throughout production. Enhanced data capture also would allow for dynamic monitoring of the process to ensure quality, aided by process analytical technology (PAT). Besides typical temperature, pH, and dissolved oxygen monitoring, these systems could be integrated with in-line flow cytometry systems to measure CAR expression over time. PAT would also allow for the creation of machine learning algorithms to augment the bioprocess further and predict product quality or enhance process control. These technologies are attainable, but implementation may remain challenging and should be further researched. Navigating the regulatory landscape TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: THE EVOLUTION OF ADVANCED BIOLOGICS 8 The FDA's framework emphasizes the need for robust preclinical data and clinical trials to ensure the safety and effectiveness of stem cell products. The FDA has adapted its current regular frameworks as of September 2025. The regenerative medicine advanced therapy designation provides expedited development pathways for promising treatments. Autologous cell therapies face a unique regulatory challenge as each batch serves a single patient, which makes statistical process control difficult. Given variability in patient material, deviations in processing and the final product are inevitable. The road ahead The development and manufacturing of cell therapies have come remarkably far in the past decade. Today, 46 cell and gene therapies have been FDA-approved. New technologies are being produced and integrated into cell therapy-based applications. Yet barriers persist, as manufacturing costs remain high, which do not allow for widespread access. Supply chain logistics also present an ongoing challenge for autologous therapies, and quality control methods require refinement. 9 CELL AND GENE THERAPIES Manipulating TumorInfiltrating Lymphocytes for Cancer Therapy Katie Brighton Cancer immunotherapy continues to evolve beyond first-generation approaches, as researchers seek to overcome the limitations of existing treatments in solid tumors. While therapies such as CAR T cells have demonstrated remarkable success in hematological malignancies, their translation into solid tumor settings remains challenging due to antigen heterogeneity and immunosuppressive microenvironments. Tumor-infiltrating lymphocytes (TILs)—and more recently, CRISPR-edited TILs—are emerging as a promising alternative. Dr. Beau Webber, an associate professor at the University of Minnesota, is working to develop geneedited TIL therapies to enhance anti-tumor immunity. His research explores how genome engineering can improve T-cell function, persistence, and therapeutic efficacy. Technology Networks spoke with Webber to discuss how TILs differ from other cell therapies, how CRISPR editing is reshaping their capabilities, and what recent clinical findings reveal about their future potential. Credit: iStock/Meletios Verras TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: THE EVOLUTION OF ADVANCED BIOLOGICS 10 Broad antigen recognition vs single-target engineering in cell therapies How do TILs and gene-edited TILs differ from other immune cell-based therapies, like CAR T-cell therapy? CAR T-cell therapy is built around a highly targeted strategy: patient T cells are genetically engineered to express a synthetic receptor directed at a single tumorassociated antigen. This precision has driven successes in cancers where the target is uniformly expressed, such as CD19-positive B cell malignancies. However, this same specificity limits their application in solid tumors, where antigen expression is often heterogeneous. By contrast, TILs are derived directly from a patient’s tumor, providing an inherently diverse and polyclonal population of T cells. These cells recognize multiple tumor-specific neoantigens via their native T-cell receptors (TCRs), allowing them to respond to a broader array of cancer targets. This characteristic is particularly important in solid tumors, where antigen variability can undermine single-target approaches. “Gene-edited TILs represent the next evolution,” explained Webber. “While traditional TIL therapy relies on the cells' natural fitness, we use CRISPR to ‘upgrade’ them.” Rather than introducing new receptors, genome editing is used to enhance the intrinsic capabilities of these cells. “By knocking out inhibitory genes like CISH, we remove the molecular brakes that tumors use to shut down or evade immune responses,” said Webber. “This creates a therapy that combines the broad recognition capabilities of natural TILs with the enhanced persistence and potency afforded by genome editing.” Key differences between TILs and CAR T-cell therapies: ∙ TILs recognize multiple neoantigens, while CAR T-cells typically target a single surface antigen ∙ Polyclonal TIL populations are better suited to heterogeneous solid tumors ∙ CRISPR editing enhances natural TIL function rather than adding synthetic receptors Engineering TIL fitness through CRISPR-mediated checkpoint removal How do you approach engineering TILs to reshape their activation, persistence, or metabolic fitness in the tumor microenvironment? The tumor microenvironment imposes a range of suppressive signals that dampen T-cell activity. One strategy to counteract this involves targeting intracellular checkpoint pathways that regulate TCR signaling. Webber’s group focuses on the CISH gene, which encodes a protein that acts as a negative regulator of T-cell activation. Using CRISPR/Cas9, the team deletes CISH from TILs, effectively removing an internal “brake” that limits their responsiveness. This intervention amplifies TCR signaling, enabling T cells to respond more robustly to tumor antigens. The result is a marked increase in functional avidity—the ability of T cells to detect and respond to even low levels of antigen. This heightened sensitivity translates into improved anti-tumor activity. Edited TILs demonstrate stronger cytokine production and enhanced cytotoxicity, key features for effective tumor clearance. “Essentially, CISH-deleted TILs become ‘hypersensitive’ to the tumor in the best way possible,” said Webber. TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: THE EVOLUTION OF ADVANCED BIOLOGICS 11 CRISPR strategies enhancing TIL function: ∙ Knockout of CISH removes intracellular inhibitory signaling ∙ Increased functional avidity improves detection of low antigen levels ∙ Enhanced cytokine production and cytolytic activity boost tumor killing First-in-human CRISPR-edited TIL trial demonstrates safety and early efficacy Can you tell us more about the CRISPRedited TILs trialed in humans for advanced gastrointestinal cancer? What were the key takeaways from this trial? Translating CRISPR-edited TILs into the clinic represents a significant milestone in cell therapy. In a trial involving patients with advanced gastrointestinal cancers who had exhausted standard treatments, Webber’s team evaluated the safety and feasibility of CISH-edited TILs. “This was a trial of multiple firsts,” he said. “This was the first time that CRISPR had been deployed in a TIL therapy, and the first time that CISH knockout T cells had been tested in humans.” One achievement from the trial was demonstrating that CRISPR editing could be successfully integrated into TIL manufacturing without compromising cell viability. This was a critical step, as the production process is already complex and highly individualized. Encouraging signs of clinical activity were also observed. Half of the patients experienced disease stabilization, and notably, one patient achieved a durable complete response that has persisted for three years. While early-stage, these results underscore the therapeutic potential of engineered TILs in difficult-totreat cancers. Clinical outcomes and implications: ∙ CRISPR editing integrated into TIL manufacturing successfully ∙ First-in-human demonstration of CISH knockout T cells ∙ Evidence of disease stabilization and durable response in advanced cancer Overcoming manufacturing and scalability barriers in TIL therapy What challenges are associated with using TILs for cancer therapy, and what innovations do you see as key for overcoming these hurdles? Despite their potential, TIL therapies face significant logistical and scalability challenges. The process requires tumor resection, ex vivo expansion, and, in some cases, neoantigen selection before large-scale expansion and reinfusion. “The long 'vein-to-vein' time can be critical for patients with rapidly progressing disease,” noted Webber. Another limitation lies in the variability of tumor samples. Not all biopsies yield sufficient numbers of high-quality, tumor-reactive TILs, making consistent manufacturing difficult. These challenges have historically constrained the scalability of TILbased therapies. To address these issues, researchers are developing faster and more efficient manufacturing methods, such as accelerated expansion protocols and techniques to enrich for neoantigen-reactive T cells without laborintensive screening. “We are also implementing multiplex, non-viral engineering methods to further enhance the TIL to increase their persistence and resilience in the tumor microenvironment,” said Webber. TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: THE EVOLUTION OF ADVANCED BIOLOGICS 12 Innovations improving TIL scalability: ∙ Accelerated manufacturing protocols reduce production timelines ∙ Enrichment strategies improve yield of tumorreactive TILs ∙ Multiplex and non-viral engineering enhance cell durability Combining CRISPR-edited TILs with checkpoint inhibitors to target “cold” tumors Looking ahead, how might CRISPR‑engineered TILs integrate with existing therapies, and are there combinations that you think have potential to unlock responses in historically “cold” tumors? Combination therapies are likely to play a central role in maximizing the efficacy of CRISPR-engineered TILs. One particularly promising strategy involves pairing these cells with immune checkpoint inhibitors (ICIs), such as PD-1 blockade therapies. “While ICIs like pembrolizumab release the ‘external’ brakes, our gene-edited TILs have their ‘internal’ brakes removed. Together, this creates a dual-layered assault that is much harder for the tumor to suppress.”— Dr. Beau Webber. Preclinical studies from the group have already demonstrated synergy between CISH-edited TILs and PD-1 inhibitors. Such combinations could be especially valuable in “cold” tumors, which typically lack sufficient immune infiltration and are resistant to immunotherapy alone. Combination strategies advancing immunotherapy: ∙ CRISPR edits remove internal T cell inhibition ∙ Checkpoint inhibitors release external immune suppression ∙ Synergistic effects may improve responses in immunologically “cold” tumors Advancing multiplex genome editing in next-generation TIL therapies At the American Society of Gene & Cell Therapy meeting 2026, Webber presented insights from translating CRISPR-edited TILs into clinical practice. His talk reflected both the successes and the challenges encountered during the development of firstin-human therapies. Beyond single-gene editing, his group has now advanced into multiplex genome engineering, enabling simultaneous modification of multiple targets within T cells. This approach opens new possibilities for refining TIL function, improving persistence and overcoming multiple suppressive pathways at once. These developments signal a shift towards increasingly sophisticated cell therapies that integrate multiple layers of engineering to enhance efficacy. Next-generation TIL engineering trends: ∙ Transition from single-gene to multiplex CRISPR editing ∙ Focus on improving multiple aspects of T-cell performance simultaneously ∙ Ongoing clinical translation informing next-stage innovation TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: THE EVOLUTION OF ADVANCED BIOLOGICS 13 CRISPR-engineered tumor-infiltrating lymphocytes represent a significant evolution in cancer immunotherapy, combining the natural tumor-recognition capabilities of TILs with enhanced functional performance. By targeting intracellular checkpoints such as CISH, researchers can boost T-cell sensitivity and persistence in challenging tumor environments. Early clinical results have demonstrated both safety and promising therapeutic activity. As manufacturing processes improve and combination strategies emerge, CRISPR-edited TILs may help overcome longstanding barriers in treating solid tumors. Advances in multiplex genome editing further point to a future of increasingly precise and potent cell-based therapies. Key takeaways ∙ TILs provide broad antigen recognition compared to single-target CAR T cells ∙ CRISPR editing enhances T cell activity by removing inhibitory pathways ∙ Early clinical trials show feasibility, safety, and encouraging patient outcomes ∙ Combination therapies with checkpoint inhibitors may unlock responses in “cold” tumors MEET THE INTERVIEWEE: Beau R. Webber, PhD, is an associate professor in the Department of Pediatrics, Division of Hematology and Oncology at the University of Minnesota. Webber led early efforts implementing targeted nucleases for gene editing in primary human T cells and published seminal studies using Cas9 base editors for highly efficient, multiplex editing in primary human T cells. Webber’s laboratory is currently focused on synergizing genome engineering, stem cell biology, and adoptive cellular therapy to develop novel treatments for genetic disease and cancer. High quality plasmids. Scalable gene therapies. In cell and gene therapy everything starts with plasmid quality. Scale with confidence. The foundation of scalable gene therapies starts with • Biotage® PhyPrep Maxi - Mega - Giga scalability High purity and yield for vectors Reproducible batch performance Explore the automated system to learn more. 15 CELL AND GENE THERAPIES Host Cell Protein Analysis in Biologics Manufacturing Neeta Ratanghayra, MPharm Impurity control is a fundamental step in biologics manufacturing, crucial for safeguarding the safety, efficacy, and consistency of therapeutic products. A critical component of this process is the removal of impurities originating from the hosT cells. These impurities—such as host cell DNA, lipids, protein aggregates, and host cell proteins (HCPs)—can hamper drug purity, disrupt manufacturing consistency and, most importantly, compromise patient safety. HCPs, endogenous proteins expressed by the production cell line, are vital for cellular processes like gene transcription, protein synthesis, cell growth, proliferation, and survival. However, during fermentation, cell death and lysis can cause these proteins to be released into the product stream. Even trace levels of HCPs can have significant consequences. They may degrade the therapeutic products or their excipients, provoke unwanted immune responses in patients and ultimately hamper the quality, stability, and shelf life of the final biologic product. Because of these risks, regulatory authorities require that HCPs be rigorously identified, quantified, and controlled throughout the biomanufacturing process. Tracking and measuring HCPs is a major challenge Accurately detecting and quantifying HCPs is critical for supporting process development and mitigating risks. This requires analytical methods that are not only highly sensitive but also offer rapid turnaround times. However, identifying and measuring HCPs is a major challenge. HCPs are typically present at very low concentrations (1 to 100 parts per million) and exhibit wide variability in molecular mass, isoelectric point, Credit: iStock/Maxiphoto TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: THE EVOLUTION OF ADVANCED BIOLOGICS 16 hydrophobicity, and structural characteristics. Some possess protease activity, which can alter the protein composition in the culture supernatant and interfere with downstream purification. Furthermore, posttranslational modifications can complicate analysis, making quantification and characterization difficult. In an ideal scenario, a HCP assay should detect the majority of HCPs early in the production process, particularly those likely to persist through purification, as well as trace levels of residual protein in the final product. The assay should also be sensitive enough to identify changes in the HCP profile resulting from process failures, such as a leaking chromatography column. Similarly, if manufacturing processes are modified, the assay must be capable of detecting any newly introduced or altered HCPs. How testing methods have evolved HCPs represent a heterogeneous group of impurities, and as such, a range of analytical technologies and regulatory expectations have emerged to support their detection and characterization. Traditionally, the industry has used a threshold of 100 ng/mg of therapeutic protein as an acceptable limit for residual HCPs. However, the goal is always to minimize HCP levels as much as possible, since the most significant risks are often linked to specific individual proteins rather than total levels. Importantly, the HCP profile can be influenced by several upstream factors, including cell culture duration, feeding strategies, culture temperature, and process scale-up during commercial manufacturing. This variability highlights the importance of continuous and precise monitoring of HCPs throughout all stages of the production process. Enzyme-linked immunosorbent assays (ELISAs) are widely used for HCP analysis due to their sensitivity, scalability, and high throughput. However, ELISAs have certain limitations. They only provide a total concentration of HCPs without identifying the individual proteins present, and their antibody-based detection does not offer complete coverage. Since the potential for immunogenicity or degradation of monoclonal antibodies often results from specific HCPs, regardless of the overall level, there is a growing need for alternative methods. As a result, mass spectrometry (MS) has emerged as a promising tool for HCP monitoring, enabling the identification and quantification of individual HCPs for more accurate risk assessment through an unbiased, protein-specific analysis. “HCP analysis has significantly evolved over the past few decades, primarily driven by the change in regulatory expectations on the topic,” Dr. Anurag Rathore, professor in the Department of Chemical Engineering at the Indian Institute of Technology, Delhi, said. “From the time when just showing the HCPs were < 10 ppm using a generic ELISA, to now where a more “As important as instrumentation is to invest in, developing process and productspecific methods using the appropriate reference standards that better reflect the actual HCP profile should also be explored." - Dr. Jared Auclair TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: THE EVOLUTION OF ADVANCED BIOLOGICS 17 detailed analysis is expected to be able to elucidate the amounts and identity of the most copious HCPs. Given the enormous diversity in HCPs that are present, there is room for innovation both in analytical tools and approaches,” he continued. “The most common method for detecting HCPs in biopharmaceuticals today is ELISAs, which remains the industry standard due to its high throughput and established regulatory acceptance,” said Dr. Jared Auclair, Dean, College of Professional Studies and Director, Bioinnovation at Northeastern University. “That said, over the last several years, MS has emerged as a powerful alternative due to its superior sensitivity and ability to identify the specific (problematic) HCPs. One must consider the trade-offs in sensitivity, specificity, throughput, and cost when determining the appropriate tool. For example, ELISA will be faster and cheaper at the expense of sensitivity and specificity,” he added. MS offers a significant advantage in its ability to simultaneously detect and identify multiple protein analytes within a single sample, enabling rapid and high-throughput analysis. However, achieving absolute quantification across a broad range of proteins remains a technical challenge. Despite this, MS allows researchers to move beyond total HCP quantification, providing detailed insight into the identity and relative abundance of individual proteins, including lowabundance proteins. As a result, liquid chromatographytandem mass spectrometry (LC-MS/MS) has become a valuable tool for the in-depth and efficient monitoring of HCPs. What do regulators expect? In recent years, regulatory agencies have placed increasing emphasis on the detection and control of process-related impurities, particularly HCPs. Key guidance documents from the US Pharmacopeia and the European Pharmacopeia outline recommended strategies for monitoring HCPs, alongside guidance from the International Council for Harmonisation and regulatory bodies such as the US Food and Drug Administration and the European Medicines Agency. These guidelines do not establish a fixed numerical limit for acceptable HCP levels, as the risk associated with HCPs is highly contextual. Factors such as dosage, route of administration, frequency of exposure, disease indication, patient population, and the specific nature of the HCP impurity all contribute to the risk. Regulatory agencies stress the importance of establishing a robust HCP assay early in process “A deeper understanding of the HCPs that are present in the final product— both identify and quantity—is expected from industry. While large companies may have the expertise and means to do this, smaller companies are likely to need support from analytical CMOs.” - Dr. Anurag Rathore TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: THE EVOLUTION OF ADVANCED BIOLOGICS 18 development. If the assay lacks sufficient coverage or fails to detect the dominant HCPs in the final product, this can lead to significant delays in later development stages. To enhance detection, regulators recommend using orthogonal analytical techniques, such as LC-MS, electrophoresis, high-performance liquid chromatography, and western blotting. These methods help validate HCP-ELISA results and support the characterization of profiles throughout the manufacturing process. Best practices for HCP testing in biopharmaceutical development As regulatory expectations increase and analytical technologies advance, biopharmaceutical companies are reassessing their strategies for HCP testing. Experts in the field emphasize the importance of more tailored and comprehensive approaches. “A multi-method approach that combines immunoassays (i.e., ELISA) with orthogonal techniques (i.e., MS) to overcome the limitations of any single method is recommended,” Auclair said. “As important as instrumentation is to invest in, developing processand product-specific methods using the appropriate reference standards that better reflect the actual HCP profile should also be explored. Lastly, stay current with regulatory guidance, as expectations for HCP characterization continue to evolve toward more comprehensive identification and risk assessment of individual proteins, rather than just total HCP content.” “Measurement via generic ELISA may not be enough for getting regulatory approval for your product,” Rathore added. “A deeper understanding of the HCPs that are present in the final product—both identity and quantity—is expected from industry. While large companies may have the expertise and means to do this, smaller companies are likely to need support from analytical CMOs,” he concluded. A clearer path to safer biologics As biologics become more sophisticated, the demand for precise impurity control, especially for HCPs continues to rise. Advances in analytical technologies, such as MS, are reshaping how manufacturers understand and manage impurity profiles. Rather than relying solely on total HCP counts, developers can identify and quantify individual proteins, track how they co-purify and evaluate their potential risk to product quality and patient safety. This shift represents a critical step forward in biological drug development. MEET THE INTERVIEWEES: Anurag S. Rathore is a professor and head of the Department of Chemical Engineering at IIT Delhi. He also serves as Coordinator of the DBT Center of Excellence for Biopharmaceutical Technology. At IIT Delhi, Dr. Rathore leads the Bioprocessing and Bioseparations Laboratory. His research aims to develop robust and economical processes for the manufacturing of safe and efficacious drugs. A Yale PhD and former industry leader, Dr. Rathore has authored over 700 publications and is widely recognized for his contributions to biopharmaceutical innovation. Jared R. Auclair is currently the dean at the College of Professional Studies and the director of bioinnovation at Northeastern University. Dr. Auclair directs the Biopharmaceutical Analysis Training Laboratory and oversees the Asia-Pacific Economic Cooperation Center of Regulatory Excellence in Biotherapeutics. His research expertise includes analytical chemistry, protein biochemistry, and regulatory science, with a focus on biopharmaceutical development and analysis. He holds a bachelor’s degree in biotechnology from Worcester Polytechnic Institute and a PhD in biomedical science from the University of Massachusetts Medical. MS in the biotherapeutic development pipeline MS is used at multiple points during biopharmaceutical development, from the early research stages through to post-approval manufacturing. To further characterize samples, MS is often combined with other analytical tools such as liquid chromatography (LC-MS), capillary electrophoresis (CE-MS). MS can also be combined with imaging using matrix-assisted laser desorption ionization (MALDI) imaging. Click here to view the full infographic Structural characterization TECHNIQUE Amino acid sequence profiling MS USE LC-MS can be used to determine the amino acid and terminal amino acid sequences, to characterize the product, compare to the desired product, and investigate batch-tobatch consistency. USED DURING Drug discovery, production for clinical trials, and post-approval manufacture. TECHNIQUE Carbohydrate structure MS USE If the therapeutic is a glycoprotein, carbohydrate content (e.g., glycosylation sites, carbohydrate chains) must be characterized. This can be done using LC-MS, MALDI-MS or CE-MS. USED DURING Production for clinical trials, and post-approval manufacture. TECHNIQUE Protein structure mapping MS USE LC-MS is used to confirm the product has the appropriate structure, with the correct tertiary structure, such as disulphide bridges. Proteomics MS methods – particularly MS/MS coupling - can use sequential fragmentation to determine structure. USED DURING Drug discovery, production for clinical trials, and post-approval manufacture. 20 CELL AND GENE THERAPIES Addressing Development Challenges for RNA Therapeutics Joanna Owens, PhD When the concept of RNA interference was first introduced in the early 2000s, it raised the tantalizing possibility of using RNA molecules to modify human gene expression.Since then, a wide range of RNA molecules have been identified, and several RNA therapeutics have been approved with many more in development. Yet the road to approval for clinical application remains challenging for these biological molecules. This article explores how challenges such as sequence optimization, immune activation, and off-target effects are being addressed to enhance the safety, durability, and efficacy of RNA therapeutics. Types of RNA therapeutics A growing understanding of RNA molecules and their roles in controlling gene activity and protein synthesis has led to a wide array of new treatment modalities. These range from short, antisense oligonucleotides, to more structurally complex RNA molecules, each optimized in different formulations for effective delivery. RNA molecules are by their nature short-lived, synthesized to perform a specific task and then degraded. To develop effective, safe, and durable therapeutics, researchers need first to understand the cellular and tissue dynamics of RNA molecules in detail. “For any given RNA, for example, encoding a viral spike protein, there are millions of potential different variants for the same spike protein, so the design space is almost Credit: iStock/Christoph Burgstedt TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: THE EVOLUTION OF ADVANCED BIOLOGICS 21 infinite,” said Professor Anne Willis, director of the MRC Toxicology Unit at the University of Cambridge. “Working out which is the most efficacious sequence for a therapeutic is challenging as it depends on so many variables.” These include which tissue you want the therapeutic to target, how to select coding regions to give optimal translation elongation rates while preventing ribosome collisions along the mRNA, and choosing the best 5’ and 3’ untranslated region (UTR). These can all influence translation efficiency and protein stability and mitigate potential safety issues in different tissues. Willis has been working with colleagues at the University of Kent on experiments to address such questions, such as determining the optimum 3’ UTR sequences in liver, muscle, and brain and the decoding speeds of individual transfer RNAs (tRNAs). These data have been used to develop a computational algorithm that evaluates RNA sequences based on the desired properties of the resulting RNA and generates a shortlist of RNA sequences that can be explored experimentally. “We performed a head-to-head comparison with the design strategy of an existing mRNA vaccine and found that our sequence gives a 20-fold better B-cell response and a five-fold increase in T-cell response,” explained Willis. “This means we could reduce the RNA dose in each vaccine, making it not only cheaper but also safer, by reducing effects intrinsic to RNAs like immunogenicity.” A single RNA therapeutic for multiple diseases Research underpinning the mRNA vaccines for COVID-19 had paved the way for new RNA therapeutic modalities to move from idea to reality. One example is the “basket approach”, in which multiple mRNAs are designed to cover viral evolution over time. A similar approach is being used by Professor Zoya Ignatova, director of the Institute of Biochemistry and Molecular Biology, University of Hamburg, who is developing suppressor tRNAs for rare monogenic disorders caused by nonsense mutations. “In our case, the basket approach rather comes from developing a single entity to address multiple pathologies caused by multiple genes,” she explained. Around 11% of all genetic diseases are caused by nonsense mutations and are usually connected with devastating phenotypes because the resulting proteins are not just misfolded but their translation is terminated entirely by the introduction of premature stop codons. Ignatova’s research focuses on designing variants of tRNAs that act as suppressor tRNAs and can specifically find those nonsense mutation-induced stop codons and restore protein synthesis. “One of the problems with using an mRNA library replacement approach to monogenic diseases is the packaging capacity of the engineered non-toxic viruses used to deliver them,” said Ignatova. “Most genes that are usually mutated or linked to a genetic disease are very large and this hinders the ability of the virus to deliver them.” By contrast, tRNAs are very small, usually between 70 and 90 nucleotides, and can be packaged easily. “Working out which is the most efficacious sequence for a therapeutic is challenging as it depends on so many variables.” - Prof. Anne Willis TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: THE EVOLUTION OF ADVANCED BIOLOGICS 22 “Moreover, there are only three stop codons, so we can design a small set of suppressor tRNAs to be used for the same type of mutation across many different genes, and multiple diseases”. However, care must be taken with designing suppressor tRNAs, because the nonsense mutation-induced stop codon is identical to the natural stop of every gene, and so off-target effects of suppressor tRNAs could potentially stop the termination of normal proteins, leading them to be mis-manufactured and misfolded. Ignatova’s team has worked out how to manipulate tRNAs to prevent off-target effects and leave normal stop codons untouched, “but this will have to be shown for every tRNA put on the market.” she cautioned. Mitigating off-target effects Off-target effects can be tissue-dependent too because, despite their uniform genetic information, tissues have very different concentrations of protein translation machinery components that may lead to toxicity in one tissue but none in another. “We must measure the natural resources of the cell precisely, from the enzymes used to charge tRNAs with the amino acids to the ribosomal molecular machinery needed to synthesize the proteins,” said Ignatova. “If we are introducing something new, we try not to perturb this equilibrium in every cell.” This also applies to RNA therapeutics beyond tRNAs. Willis’ team is exploring the idea of protein replacement therapy, but getting the product to the right tissue is essential. “In terms of delivery, if you need to replace a protein in the liver, that's really easy because lipid nanoparticles containing mRNAs will migrate to the liver anyway,” she said. “But if we want protein replacement in the heart and not in the liver, that's more of a challenge. There’s some fantastic research focusing on exploring new lipidbased particles and nanostructures that can target the cargo to the right organ.” Other off-target effects can include unintentional changes to reading frames during protein translation, expression in non-target tissues, or blocking translation of essential proteins. Research from Willis’ team showed that certain modifications to mRNA vaccines can cause the ribosome to switch reading frames, or “frameshift.” “The off-target effect was surprising, but we know the sequence that causes a frameshift so it’s possible to modify the mRNA to take those sites out,” said Willis. Immunogenicity Immunogenicity is another common challenge in developing RNA therapeutics, because often when RNA is introduced into a cell, the cell thinks it is being attacked by a virus and triggers the innate immune response. “Immunogenicity turns off protein synthesis, which is the one thing you're trying to achieve,” said Willis, “and it also induces inflammatory cytokines which, in the worst-case scenario, can result in a cytokine storm.” Researchers commonly make modifications to RNA sequences to avoid this immunogenicity, but care is needed to avoid destroying the physiological structure and function of the RNA. “When we administer an already synthesized tRNA, the secondary structure of tRNA molecules usually helps decrease immunogenicity, and yet they can still be immunogenic,” explained Ignatova. “We have found positions in one or two places to introduce natural modifications that increase the efficacy of the tRNA and silence the immunogenicity completely.” Future challenges Despite rapid progress in this field, getting some of these innovative therapies to patients will require time and working closely with regulatory authorities. For suppressor tRNAs, the challenges include conducting trials in such rare diseases and gathering preclinical evidence when few reliable models exist. TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: THE EVOLUTION OF ADVANCED BIOLOGICS 23 “Individual patient trials will really be the rolling stone in the whole process showing that it works and then we can start to recruit other patients with the same type of mutation,” said Ignatova. “Even two years ago, regulatory authorities weren’t prepared for this and would have required a standard clinical trial set up with a test arm. But now, they are moving forward with the field and endorsing models such as patientderived materials (e.g., fibroblasts and epithelial cells) or induced pluripotent stem cells, which can provide disease models in a matter of months rather than years.” For Willis’ field, one of the outstanding challenges is to understand the relationship between protein structure and translation and find the sweet spot between getting enough protein made with the desired rate of turnover. “For certain structures, you want your protein to be translated quite slowly to ensure it folds correctly, especially for large molecules such as membrane proteins,” she said. “We've worked on translation forever, so it’s nice to use that knowledge to translate the science and help others working in this area with diseases that we couldn't even think of treating before.” MEET THE INTERVIEWEES: Anne Willis is director of the MRC Toxicology Unit at the University of Cambridge. She obtained a PhD in Biochemistry from the University of London while working in the Imperial Cancer Research Fund laboratories (now CRUK) on DNA repair with Dr Tomas Lindahl. In 2004, she was appointed Director of Cancer Research Nottingham and Chair of Cancer Cell Biology, where she was based in the School of Pharmacy, before becoming Director of the MRC Toxicology Unit in 2010 where her research is directed towards understanding the role of posttranscriptional control in response to toxic injury with a focus on RNA-binding proteins, regulatory RNA motifs, and tRNAs. Zoya Ignatova is a professor of RNA Biology and Managing director of the Institute of Biochemistry and Molecular Biology at the University of Hamburg (UHH), Germany. She received her PhD in 2001 from the Technical University of Hamburg and was a postdoctoral fellow at University of Massachusetts. Ignatova has made fundamental discoveries on the role of tRNAs in modulating translation kinetics and protein folding and function and her group’s work now focuses on addressing the fundamental biological processes underlying a range of severe, currently incurable conditions, ranging from monogenic disorders to complex genetic and non-genetic conditions. 24 CELL AND GENE THERAPIES Techniques and Advancements in Recombinant Adeno‑Associated Virus Purification for Gene Therapies Frank Charlton, PhD Adeno-associated viruses (AAVs) have emerged in recent years as a crucial tool in clinical gene therapies, offering low pathogenicity and long-lived gene expression in target cells. AAVs are simple nonenveloped viruses with a single-stranded DNA (ssDNA) genome. Recombinant adeno-associated viruses (rAAVs) are structurally similar to wild-type AAVs; however, the Rep and Cap genes are removed and replaced with a transgene—the genetic cargo that can repair or replace a faulty gene. rAAVs are increasingly important in viral-based therapies, and at present, there are seven rAAV therapies approved by the European Medicines Agency and the US Food and Drug Administration for the treatment of genetic diseases ranging from congenital blindness to muscular dystrophy and hemophilia. Over 200 clinical trials have been undertaken to bring new rAAV therapies to the clinic; however, manufacturing innovation has lagged behind. Dr. Guangping Gao, professor of microbiology and physiological systems and Penelope Booth Rockwell Professor in Biomedical Research at UMass Chan Medical School, explained some of the issues we currently face: “As a field, we are experiencing several obstacles. Credit: iStock/quantic69 TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: THE EVOLUTION OF ADVANCED BIOLOGICS 25 First, we need more efficient and target-specific capsids, which are the essence of any therapy using AAVs. Secondly, manufacturing is a major hurdle, which is part of the reason why rAAV therapies are so expensive.” This article explores advances in the purification of rAAVs to meet current clinical and research needs. How are rAAVs made? AAVs and rAAVs cannot replicate independently and require co-infection with a helper virus—typically adenoviruses (AdVs) or herpes simplex virus (HSV)— to amplify in a host. rAAVs offer an even safer profile, as they lack viral genes and cannot replicate even with a helper virus present. However, their inability to self-amplify restricts sustained rAAV production within a batch. rAAVs are typically produced in suitable host cells through the coordinated expression of Rep and Cap components, together with helper genes or a helper virus, and the desired transgene. There are several options for production, as noted by Dr. Jessica Whelan, lecturer and assistant professor at University College Dublin. “There is not a clearly preferred production system. Transient transfection, producer cell lines, and baculovirus-based insect cell production systems are used—each with their advantages and disadvantages. Depending on the production system, the characteristics of the material to be purified vary, which impacts downstream process design.” Traditional rAAV production has relied on transient transfection of Human Embryonic Kidney 293 cells with plasmid DNA encoding the transgene, AAV Rep/ Cap genes and AdV helper genes. While this approach offers tunability, it remains inefficient and scaledup suspension cultures can often produce ≥80% empty capsids, necessitating extensive downstream purification to separate empty capsids from full ones. An alternative is the Baculovirus expression vector system using Spodoptera frugiperda (Sf9) insect cells, which was employed to manufacture Glybera®, the first approved rAAV therapy (now discontinued). This scalable platform is widely used in protein production but tends to generate higher levels of defective or non-transducing particles. Another strategy uses recombinant herpes simplex virus (rHSV), where one rHSV delivers AAV Rep and Cap genes to producer cells and another delivers the transgene. HSV’s natural role as a helper virus enables high-quality rAAV production at high titers. However, scalability is limited by the complexity of generating starting materials, and rigorous purification is required to eliminate residual rHSV, which is neurotropic and neurotoxic. Stable producer cell lines offer further scalability and reproducibility. In this system, cells stably expressing Rep and Cap are stimulated with recombinant AdV to drive rAAV production. Although effective, this approach again requires stringent purification to remove residual AdV and oncogenic DNA derived from transformed cell lines. Across all viral-based production systems, scalability is offset by the burden of downstream purification. As Gao emphasized, “When you use viral systems for production, the key challenge is removing the helper virus—at the virus, DNA, and protein level. This requires more stringent purification, as mammalian helper viruses are much more immunogenic than AAV itself.” Purification of rAAVs The choice of upstream methods for rAAV production has a direct and significant impact on downstream processing requirements. Both Gao and Whelan emphasized this connection. “The upstream and downstream processes are highly interconnected,” explained Gao. “What you have in your upstream processes will determine the downstream output.” Whelan echoed this, noting, “depending on the production system, the characteristics of the material to be purified vary, which impacts downstream process design.” Each production method introduces process-related contaminants and product-related impurities, such as empty or partially full capsids. These by-products TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: THE EVOLUTION OF ADVANCED BIOLOGICS 26 must be removed, but each purification step can lead to product losses and sequential processing may significantly reduce the final yield. “Purification remains challenging for a multiplicity of reasons,” Whelan explained. “These include low AAV concentrations, the difficulty of balancing yield and purity given the similarity between full capsids and empty or partially filled capsids, the lack of a standardized downstream platform comparable to that for monoclonal antibodies and the need for improved analytical tools and methods.” The main steps of purification are broadly broken down in the following sections. Cell harvest and lysis rAAVs must first be harvested from producer cells. For small research volumes (<10 mL), freeze-thaw is suitable, but larger scales require chemical or physical cell disruption. Microfluidic disruption is increasingly recognized as scalable and current good manufacturing practice-compliant, using high-pressure shear forces to lyse cells while simultaneously shearing DNA, reducing viscosity, and aiding clarification. Clarification After lysis, clarification removes cellular debris. Initial low-speed centrifugation removes large particles, while enhanced depth filtration offers scalable, gentle separation of rAAVs from crude lysates. Tangential flow filtration is now widely used as a filtration method, concentrating rAAV lysates ~10-fold and removing soluble impurities without clogging. Capsid capture Clarified lysates still contain impurities such as proteins, nucleic acids, and empty capsids. Early methods like iodixanol or cesium chloride (CsCl) gradient ultracentrifugation exploited physical differences but were labor-intensive, low-throughput, and unsuitable for scale, with CsCl particularly damaging to capsids. Affinity chromatography has largely replaced these methods, capturing rAAV via serotype-specific ligands or newer cross-serotype resins (e.g., AVB Sepharose, POROS™ CaptureSelect™) using nanobody-derived ligands. These offer high yield and purity, but at high cost. Emerging ligands include AAVX, which binds serotypes 1–9, and AAV receptor, a biologically relevant receptor candidate. Computationally designed peptide ligands also show promise, offering gentle elution and lower production costs, potentially enabling broad serotypeagnostic resin development. “Relative to commercial resins for antibodies, resins for AAVs have lower binding capacities, short lifespans and are often serotype-specific,” noted Whelan. “However, significant progress is being made.” Aqueous two-phase systems (or aqueous biphasic systems) partition molecules based on physicochemical properties, offering gentle separation without harsh solvents. While design complexity limits scalability, well-optimized systems show strong potential. Steric exclusion chromatography (SXC) uses PEG-based molecular crowding and hydrophilic membranes to bind AAVs, providing up to 10-fold greater binding capacity than affinity resins while maintaining physiological conditions. Polishing – ion exchange chromatography The final stage of downstream purification involves polishing the final product to remove product-related impurities and any residual process-related ones. “Your downstream processing is the key,” emphasized Gao. “It doesn't matter which production method is used; one of the major issues is removing empty particles. This is a major challenge because you need product-specific downstream processing, particularly polishing, to remove empty particles.” Ion (cation/anion) exchange chromatography is widely used, often sequentially, to enhance yields. Ultracentrifugation remains an option, but it is low-yield and unscalable. “Sedimentation processes may or may not be perfect for clinical vector manufacturing or scaling up, and there TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: THE EVOLUTION OF ADVANCED BIOLOGICS 27 is a push for scaling up of column chromatography,” said Gao. “Affinity columns are good for capture of serotype-specific particles but cannot differentiate between empty and full capsids. If you wish to remove gradient-based sedimentation, you need a polishing step that captures minor differences based on surface charge, ionic strength or anything that can distinguish empty from full or partial from full.” Recent advances include monolithic columns, which speed processing by improving flow through a continuous porous phase, and membrane-based chromatography, where modified anion exchange membranes deliver high yields and preserve infectivity. Outlook While large-scale rAAV production is still developing, both experts expressed optimism. Gao highlighted recent progress: “From ~10% full particle recovery in the first clinical trials in 2007/2008, we can now reach 60% owing to chromatography improvements.” He added, “different technologies have emerged in recent years, but they remain small-scale. I’m watching their development and migration to larger scales, as well as improvements in reproducibility.” Whelan emphasized lessons from therapeutic proteins: “Leveraging knowledge and experience from the therapeutic protein space, chromatography and membrane-based filtration are the current focus for scalable, commercial manufacturing.” She remained positive about the field’s outlook: “While there is a distance to go, the growing demand for AAVs is driving intense innovation, with regular advances in resins, analytical techniques and process intensification. Despite AAV-specific challenges, high innovation levels and lessons from other modalities point to a promising future.” MEET THE INTERVIEWEES: Dr. Guangping Gao is an internationally recognized researcher in the field of gene therapy and AAVs. He serves as director of the Li Weibo Institute for Rare Diseases Research, director of the Horae Gene Therapy Centre and Viral Vector Core, Professor of Microbiology and Physiological Systems and Penelope Booth Rockwell Professor in Biomedical Research at UMass Chan Medical School. Dr. Jessica Whelan is an accomplished scientist in bioprocess engineering with a strong track record in both academic and industrial applications. Dr. Whelan worked as an engineer for MSD before completing a PhD and postdoctoral training in bioprocessing, PAT and advanced control. She has since worked as the Director of Technical Operations for APC, Associate Director of MS&T upstream at MBS and is currently an Assistant Professor in the Biomanufacturing Research Group at UCD, as well as Head of School in the School of Chemical and Bioprocess Engineering. “While there is a distance to go, the growing demand for AAVs is driving intense innovation, with regular advances in resins, analytical techniques and process intensification. Despite AAV-specific challenges, high innovation levels and lessons from other modalities point to a promising future.” - Dr. Jessica Whelan 28 CELL AND GENE THERAPIES Integrating Phenotypic Readouts Into CAR T Production Chimeric antigen receptor T-cell (CAR T) therapies have changed the treatment landscape for several hematological malignancies. Yet manufacturing remains a major barrier to broader clinical impact. Workflows are often slow and costly, particularly in autologous settings where each batch starts with patient-specific material that varies in quality. Expanding into new indications and allogeneic formats has only added to those pressures. A collaboration between University College London (UCL) and Sartorius has addressed these challenges through a series of studies on stirred-tank bioreactor (STR)-based CAR T manufacturing. STRs are well established in large-scale biologics production and offer proven scalability, flexible operation, and strong regulatory prior knowledge. However, concerns about shear sensitivity historically limited their use with T cells, and early CAR T expansion work was largely confined to small volumes. The studies reviewed here progressively moved from establishing that CAR T cells can be expanded reliably in STRs without loss of quality or function, to asking how that process could be optimized to produce more cells, faster, while keeping the phenotype that makes a product clinically viable. Producing cells in an STR is not the same as knowing those cells are potent enough to work clinically. Traditional process readouts such as fold expansion, viability, and metabolite consumption are essential, but they do not reveal whether a manufacturing change has helped or compromised product quality. In this collaboration, that question is answered by combining STR bioprocessing with live-cell functional analysis and high-throughput screening (HTS) by cytometry. Incucyte® Live-Cell Imaging and Analysis enables kinetic assessment of tumor cell killing, while iQue® HTS cytometry supports rapid multiplex measurement Credit: iStock/ALIOUI Mohammed Elamine TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: THE EVOLUTION OF ADVANCED BIOLOGICS 29 of cytokine secretion and, in several studies, activation, exhaustion, and phenotypic marker expression. Together, these tools address a real gap in CAR T process development: connecting manufacturing choices to evidence of product function and phenotype. The studies reviewed here show how this approach played out across process optimization, perfusion intensification, serum-free adaptation, and scale-up. Establishing the baseline: Seed train and activation parameters set the quality ceiling The first question in any process development effort is which variables matter most. In the earliest study in this series, Hood et al. applied a quality-by-design (QbD) framework to investigate how key expansion variables shaped CAR T yield and quality. Using a design of experiments (DOE) approach in gas-permeable culture plates, followed by validation in Ambr® 250 High Throughput STR, the study examined the effects of activation number, seed train duration, seeding density, and IL-2 concentration. The findings were clear. Repeated activation and a longer seed train negatively impacted both growth and quality. A single activation step with a shorter, 3-day seed train produced substantially higher T-cell yields than two activations with a 7-day seed train. The optimized condition also reduced exhaustion marker expression and improved metabolic efficiency. Cells from less favorable conditions consumed more glucose, produced more lactate, and showed higher levels of T-cell exhaustion markers PD-1 and LAG-3. Separately, conventional flow cytometry also confirmed that both conditions maintained a predominantly less differentiated central memory phenotype, a finding that is clinically significant, as less differentiated T cells are associated with greater CAR T efficacy and persistence in vivo. Productivity Improve eciency through automation & process optimization Scalability Successfully scale up or out to clinical and commercial volumes to meet patient demand Supply Secure a consistent supply of high-quality raw materials Process Control Demonstrate process reproducibility to meet tightened regulatory standards Quality Ensure the consistency, purity, and safety of the final therapeutic FIGURE 1: KEY DRIVERS IMPACTING CELL AND GENE THERAPY TIME-TO-MARKET AND COST OF GOODS. CREDIT: SARTORIUS. TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: THE EVOLUTION OF ADVANCED BIOLOGICS 30 This study established a key process principle for everything that follows: early decisions about how to prepare cells shape not only yield, but the functional quality of the final product, and those effects carry through into the STR environment. Functional testing strengthened that conclusion. Tumor killing assays performed on the Incucyte® live-cell system showed that CAR T cells from both conditions eliminated CD19+ NALM6 cells in vitro (a B-cell leukemia line expressing the CAR Target antigen) with cells from the optimized process killing more effectively. Kinetic imaging confirmed near-complete targeT-cell clearance, with CAR+ cells displaying the elongated morphology characteristic of activated T cells. That level of detail, both the rate of killing and the cellular behavior driving it, is uniquely provided by live-cell imaging. Cytokine profiling with iQue® HTS cytometry added a further layer of resolution: while interferon-gamma (IFN-γ) secretion, a key indicator of T-cell effector activity, was comparable between conditions, tumor necrosis factor-alpha (TNF-α) was significantly lower in the preoptimized condition. This is important because TNF-α decline is an early sign of T cell exhaustion, preceding loss of IFN-γ and further functional deterioration. The cytometry data did not simply confirm cytokine secretion; it identified a difference that growth metrics alone would have missed, and in doing so connected the upstream process choices directly to product function. Intensifying expansion without sacrificing function Once the seed train and activation strategy had been improved, the next challenge was pushing yields higher within a scalable expansion platform. Hood et al. addressed this by examining perfusion in stirred-tank bioreactors using a QbD approach in the Ambr® 250 High Throughput Perfusion system. The goal was to find perfusion parameters that increased output without compromising quality. 0123456 7 0 20 40 60 80 100 120 140 Day from seed Cell Fold 3 Day, 1 Activation 7 Day, 2 Activations 100 120 140 3 Day, 1 Activation 7 Day, 2 Activations 0 25 50 75 100 125 150 Cell Fold CART cell growth during and at the end of the 7-day culture in T-flasks Activation and exhaustion marker expression 0 20 40 60 80 100 %CD3+CD69+ 0 20 40 60 80 100 %CD3+PD1+LAG3+ CD69 [%] PD-1 + Lag-3 [%] Dierentiation marker expression upon 7-day culture in T-flasks 0 20 40 60 80 100 %CD8+CD45RO-CCR7+(naïve) Naive 0 20 40 60 80 100 %CD8+CD45RO-CCR7+ (CM) Central Memory 0 20 40 60 80 100 %CD8+CD45RO-CCR7+ (EM) EŠector Memory 0 20 40 60 80 100 % CD8+CD45RO-CCR7+ (E) EŠector Less diŠerentiated Terminally diŠerentiated 2 D0 D1 D2 D3 D4 D5 D6 D7 D8 D9 7 Day Culture (T-flasks) 2nd Activation* Every 2nd day: feed by diluting 7 Day, 2 Activations Thaw Activation* Transduction Seed train 7 Day Culture (T-flasks) 3 Day, 1 Activation Every 2nd day: feed by diluting Seed Train FIGURE 2: BENEFICIAL EFFECTS OF REDUCTION IN LENGTH OF SEED TRAIN ON EXPANSION AND MARKER EXPRESSION. FOR ALL GRAPHS TEAL DATA REPRESENTS THREE-DAY SEED TRAIN WITH ONE ACTIVATION, GREY DATA SEVEN DAY WITH TWO ACTIVATIONS, DIFFERENT SYMBOLS REPRESENT DIFFERENT DONORS. CREDIT: SARTORIUS. TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: THE EVOLUTION OF ADVANCED BIOLOGICS 31 A DOE study looked at when perfusion started, how fast it ran, and donor-to-donor variation. Earlier initiation and higher perfusion rates produced the strongest growth, with the optimized process delivering a nearly 3-fold increase in cell density over fed-batch culture and approximately 30-fold over static flask expansion, while maintaining comparable phenotype and exhaustion profiles across all three processes. Those results needed to be tested functionally. Higher cell densities raise concern about exhaustion or reduced potency, but the data did not bear that out. Critically, live-cell cytotoxicity assays were applied not only to the optimized condition but across all 15 perfusion conditions tested in the DOE. Effective killing of CD19+ NALM6 target cells was confirmed in every case, with NALM6 cells essentially absent at the end of the 2-day co-culture. Kinetic imaging again showed CAR+ cells in the elongated morphology of activated T cells, showing what growth numbers alone cannot. Cytokine profiling with HTS cytometry showed that IFN-γ and TNF-α secretion from perfusion-expanded cells was comparable to flask controls, with both significantly exceeding non-transduced controls. The finding that high-density perfusion culture did not suppress cytokine output is the key result here: it demonstrates that process intensification left effector function intact. Functional assessment across the full DOE design space, rather than only on the winning condition, confirmed that the functional gains were not an artefact of cherry-picking. Every condition that supported strong growth also supported effective killing and cytokine output, which justifies moving a process forward with confidence. Serum-free and adaptive: Building a clinically transferable process iQue® HTS Platform Multiplexed cell marker and cytokine profiling of T-cell activation, exhaustion, and killing Incucyte® Live-Cell Analysis System CAR T cell functional characterization through quantitive live-cell imaging and analysis of T-cell killing Specific killing upon co-culture of CAR T cells with target cells 0 5 10 15 20 25 30 35 40 45 0 2 4 6 8 Elapsed Time (hours) Normalised NALM6 Count NALM6 3 Day, 1 Activation CAR+ 3 Day, 1 Activation Non-transduced 7 Day, 2 Activations CAR+ 7 Day, 2 Activations Non-transduced 0 2 4 6 8 Normalised NALM6 Count NALM6 Nontransduced CAR+ Specific cytokine secretion by CART cells upon co-culture with target cells 0 20 40 60 80 100 120 140 IFNγ (pg/mL) Nontransduced CAR+ 0 5 10 15 20 25 TNFα (pg/mL) Nontransduced CAR+ 0 5 10 15 20 25 30 35 40 45 0 2 4 6 8 Elapsed Time (hours) Normalised NALM6 Count NALM6 3 Day, 1 Activation CAR+ 3 Day, 1 Activation Non-transduced 7 Day, 2 Activations CAR+ 7 Day, 2 Activations Non-transduced Target Cells NALM6 Add CAR T Cells Co-culture FIGURE 3: SEED TRAIN OPTIMIZATION EFFECTS ON TUMOR CLEARANCE AND CYTOKINE PROFILES. FOR ALL GRAPHS TEAL DATA REPRESENTS THREE-DAY SEED TRAIN WITH ONE ACTIVATION, GREY DATA SEVEN DAY WITH TWO ACTIVATIONS, DIFFERENT SYMBOLS REPRESENT DIFFERENT DONORS. DATA SHOWN FOR BOTH NONTRANSFECTED AND CAR POSITIVE CELLS. CREDIT: SARTORIUS. TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: THE EVOLUTION OF ADVANCED BIOLOGICS 32 The next challenge was making this process more clinically relevant. Serum-containing workflows can support robust growth but introduce variability, supply concerns, and added safety considerations. Springuel et al. adapted the perfusion strategy to 4Cell® Nutri-T GMP, a xeno-free, serum-free medium and again used DOE to look at perfusion start time, rate, and donor variability. The core trends held. Earlier perfusion initiation and higher perfusion rates supported stronger growth and viability. The best-performing condition, initiated early with daily medium exchange, reached a representative first CAR T dose by 3.5 days compared to 7 days for fed-batch, and produced 4.5 times the total dose yield. In autologous manufacturing, where the expansion phase is among the longest and most time-sensitive steps, compressing that timeline by more than half is a clinically meaningful gain. The study also explored adaptive perfusion. Because CAR T growth and metabolic demand decline over culture time, the team tested a strategy in which perfusion rates were reduced gradually after peak growth. This reduced medium consumption while maintaining comparable final yield, phenotype, and cytotoxicity, demonstrating that intensification can be refined for efficiency as well as output. Both instruments contributed meaningfully here. The HTS cytometry was used with dedicated activation and exhaustion kits, profiling CD69, CD25, and HLA-DR alongside PD-1, LAG-3, and TIM-3. This panel captured dynamics that single-marker readouts would miss: early activation markers declined over culture while HLA-DR expression rose, and triple exhaustion marker co-expression fell markedly from inoculation to Day 7 across all conditions, including the highest-density perfusion cultures. Here again, conventional flow cytometry confirmed that the memory phenotype and CD4/CD8 ratio were preserved consistently across all perfusion conditions. More than 90% of harvested CD8+ cells maintained Dierentiation marker expression upon 7-day culture in Ambr® 250 0 20 40 60 80 100 % CD8+CD45RO-CCR7+ Naive Central Memory E
ector Memory E
ector 0 20 40 60 80 100 %CD8+CCR7+CD45RO+ 0 20 40 60 80 100 %CD8+CD45RO+CCR7- 0 20 40 60 80 100 %CD8+CCR7-CD45ROLess di
erentiated Terminally di
erentiated Activation and exhaustion marker expression Inoculation Harvest 0 20 40 60 80 100 %CD8+ CD69+ *** CD69 [%] CD25 [%] HLA-DR [%] PD-1 + Lag-3 + Tim-3 [%] Inoculation Harvest 0 20 40 60 80 100 %CD8+ CD25+ *** Inoculation Harvest 0 20 40 60 80 100 %CD8+ HLA-DR+ *** Inoculation Harvest 0 20 40 60 80 100 %CD8+PD-1+LAG3+TIM3+ *** Perfusion Parameter A 1 2 3 Fed-batch FIGURE 4: PHENOTYPE MARKER EXPRESSION ACROSS PERFUSION PARAMETERS AND DONORS. COLOR REPRESENTS CHANGES IN PERFUSION PARAMETER A, SYMBOL SIZE PARAMETER B AND SHAPE DIFFERENT DONORS. CREDIT: SARTORIUS TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: THE EVOLUTION OF ADVANCED BIOLOGICS 33 naïve and central memory profiles, while the expected shift toward CD8+ predominance progressed throughout culture, neither outcome was significantly influenced by perfusion rate or start time. Live-cell cytotoxicity assays were run at both Day 7 and Day 12 harvest points. Kinetic imaging confirmed that CAR+ cells retained the elongated morphology of activated T cells at both timepoints, and killing capacity was preserved across the extended adaptive perfusion culture. Cytokine analysis by HTS cytometry added further resolution, revealing inter-process differences in IFN-γ and TNF-α at Day 7 that diminished by Day 12. That temporal pattern would not have been visible with a single endpoint readout. This study integrates key process needs: serum-free media optimization, processes robust to donor variability, adaptive process control, and functional characterization at multiple timepoints. The case it makes is simple: getting to a dose faster is not enough if the cells are not up to the job. From bench to batch: Scale-up with functional continuity The final study takes the process to a larger scale. Springuel et al. ran the perfusion-based serum-free process in a 2 L single-use STR, with parallel cultures in the 250 mL Ambr® 250 system as a scaledown model. The 2 L process consistently reached around 30 × 10⁶ cells/mL and yielded 113 ± 7 CAR T doses per batch, a precise and reproducible result that is a meaningful step toward large-scale allogeneic production. FIGURE 5: CONSISTENCY IN CAR T CELL PHENOTYPE ACROSS VARIOUS SCALES Naive Central Memory Eector Memory Eector Inoculum Univessel 2L 0 10 20 30 40 50 %CAR+ of CD3+ Ambr® 250 CAR expression CD4/CD8 Ratio Exhaustion Memory Phenotype Inoculum Univessel 2L 0 20 40 60 80 100 %CD4+/CD8+ CD4+ CD8+ Ambr® 250 Inoculum Univessel 2L 0 20 40 60 80 100 %+PD1+LAG3+TIM3+ of CD3+ Ambr® 250 Inoculum Univessel 2L 0 25 50 75 100 %CD8+ memory subset Naive Central Memory Eector Memory Eector Ambr® 250 Non-transduced Ambr® 250 Univessel 2 L 0 12 24 36 48 60 72 0 1 2 3 4 Time (hours) Normalised green object count Ambr® 250 Univessel 2 L Non-transduced Specific killing of target cells upon co-culture with CAR T cells TNF-α secretion Non-transduced Univessel 2L 0 1 2 3 Normalised GFP+NALM6:CAR T at 70hours Ambr® 250 Non-transduced Ambr® 250 Univessel 2L 0 200 400 600 800 TNF-α (pg/mL) 0 12 24 36 48 60 72 0 1 2 3 4 Time (hours) Normalised green object count Ambr® 250 Univessel 2 L Non-transduced FIGURE 6: CONSISTENT TUMOR CYTOTOXICITY OF HARVESTED CAR T CELLS ACROSS VARIOUS SCALES. CREDIT: SARTORIUS. TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: THE EVOLUTION OF ADVANCED BIOLOGICS 34 Scale-up was not judged by cell growth alone. The 250 mL and 2 L systems were compared across growth, phenotype, cytotoxicity, and cytokine secretion, with principal component analysis (PCA) confirming close clustering of quality attributes between scales. This validates the Ambr® 250 as a predictive scale-down model (SDM) for future allogeneic process development. The study also integrated multifrequency capacitance sensing in the 2 L bioreactor, achieving R² = 0.98 against offline cell counts. A single-frequency approach would not have worked here: T cells change size after activation, which throws off a fixed-frequency signal. Scanning across multiple frequencies, analyzed with multivariate tools, resolved that problem and opens a path toward automated, capacitance-driven perfusion control. For downstream processing, the Ksep® 400 automated harvest, concentration, and washing of the full bioreactor culture, achieving greater than 90% cell recovery and a 9-fold volume reduction. These are scalable, clinically meaningful outcomes, not simply proof-of-concept figures. Functional characterization tracked the product across every step. Live-cell cytotoxicity assays ran for 3 days across all three platforms, confirming equivalent targetspecific killing from 250 mL and 2 L bioreactors and well-plate controls. HTS cytometry was again used with both the Activation and Exhaustion kits across the same marker panel, confirming comparable expression levels and consistent phenotypic trajectories between scales. Critically, both instruments were also applied to directly compare manually harvested and Ksep® 400-processed cells. Automated harvesting did not alter cytotoxicity, IFN-γ or TNF-α secretion, CAR expression, or exhaustion marker levels. This is the most demanding test of functional continuity in the series: it shows that Incucyte® and iQue® HTS systems serve as quality gates at the downstream processing step, not only during expansion, making them integral checkpoints across the full manufacturing workflow. Taken together, these studies show how CAR T process development improves when it is connected to functional product characterization. Across seed train optimization, perfusion intensification, serum-free media adaptation, and 2 L scale-up, the UCL–Sartorius collaboration paired each manufacturing step with readouts that connect it to product function. Incucyte® Live-Cell Analysis played a central role by showing how process changes affected the kinetics of CAR T target cell killing in real time. Concurrently, iQue® HTS cytometry complemented that by enabling rapid cytokine and phenotypic profiling at high throughout. Together, these tools moved the workflow beyond simple expansion metrics toward a more complete assessment of product quality. The ultimate goal in CAR T manufacturing is real-time product release: cutting the week-long assay burden that follows expansion by building characterization into the process itself, so that it supports release decisions rather than delaying them. As the field moves toward allogeneic formats and larger patient populations, that shift will matter more than ever—and this body of work shows what it takes to get there. REFERENCES: 1. Hood T, Slingsby F, Sandner V, et al. A quality-by-design approach to improve process understanding and optimise the production and quality of CAR-T cells in automated stirred-tank bioreactors. Front Immunol. 2024;15. doi: 10.3389/fimmu.2024.1335932 2. Hood T, Springuel P, Slingsby F, et al. Establishing a scalable perfusion strategy for the manufacture of CAR-T cells in stirredtank bioreactors using a quality-by-design approach. Bioeng Transl Med. 2025;10(3):e10753. doi: 10.1002/btm2.10753 3. Springuel P, Hood T, Slingsby F, et al. Optimising and adapting perfusion feeds in serum-free medium to intensify CAR-T cell expansion in stirred-tank bioreactors. Front Bioeng Biotechnol. 2025;13. doi: 10.3389/fbioe.2025.1593895 4. Springuel P, Silva Couto P, Stibbs DJ, et al. Scalable CAR-T production in a 2-litre perfusion stirred-tank bioreactor with automated harvesting and scale-down model characterisation. Front Bioeng Biotechnol. 2026;13. doi: 10.3389/fbioe.2025.1694134 Achieve Reliable Transfection with Polyplus Reagents Try a Free Sample and See the Difference: High-Performance Reagents for Better Results Achieving successful transfection in your life science research can be challenging, especially when working with hard-to-transfect cells. 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Life Science Research (in vitro) Reagent Molecule Delivered Targeted Cell Recommended Use Plasmid DNA mRNA SiRNA | miRNA Protein Adherent Suspension Specific jetOPTIMUS® ++++ + All cells; including primary and stem cells Gene expression, Genome editing, R&D virus production jetPRIME® +++ Gene expression, RNA interference INTERFERIN® +++ +++ RNA interference jetMESSENGER® +++ +++ Hard-to-transfect cells Genome expression, Genome editing jetPEI® ++ + Genome expression, HTS PULSin® +++ +++ Proteofection Specifications subject to change without notice. ©️2025. All rights reserved. All names of Sartorius products are registered trademarks and the property of Sartorius AG an/or one of its affiliated companies Transfection-Applications-Flyer-Sartorius-en Version: 11 | 2025 Find the Best Reagent for Your Application Your Sartorius Sales Specialist: 36 CELL AND GENE THERAPIES Transforming Healthcare: What’s on the Horizon for Cell Therapies? Kerry Taylor-Smith Cell therapy is a promising, rapidly advancing field with the potential to transform treatments across many diseases. It is primarily used to treat cancer but is expanding into incurable diseases such as autoimmune disease and heart fibrosis. “Cell therapies have enormous potential to treat patients with a disease or injury that requires replacement or repair,” explained Dr. April Pyle, professor and vice chair of microbiology, immunology and molecular genetics at the University of California, Los Angeles’ (UCLA's) Broad Stem Cell Research Center. “They can be used to directly deliver new cells to patients in need of cells in a particular tissue or organ system.” “These cells could be isolated from a matched donor (allogeneic), or isolated from a patient, then processed in the lab and reinfused to the same patient (autologous)," added Dr. Ahmed Gad, a postdoctoral associate in pediatric hematology, oncology, and cell and gene therapy at Baylor College of Medicine. This broad definition means blood transfusions and bone marrow transplants are considered types of cell therapy , but more recently, the term is used to refer to the transfer of a particular cell population, including stem cells and immune cells. Cell-based therapies, such as stem cell-based therapies and CAR T-cell therapies, are transforming healthcare, but what does the future hold for them and how can we overcome the barriers to their expansion? Credit: iStock/anusorn nakdee TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: THE EVOLUTION OF ADVANCED BIOLOGICS 37 Immune cell therapies Immune cell therapies isolate and process a particular immune cell population and use it for therapeutic intervention. The first to receive US Food and Drug Administration (FDA) approval was sipuleucel-T, a dendritic cell (DC) therapy for men with advanced prostate cancer that had not responded to hormone therapy. Gad describes sipuleucel-T as “kind of a hack to the system” with DCs acting as “professional antigenpresenting cells” that detect foreign antigens originating from pathogens, mutated proteins or foreign tissues. DCs travel to an injured site, ingest proteins, digest them into small peptides (epitopes), and present them on their surface with human leukocytic antigen (HLA), before moving to the lymph nodes where T cells and B cells reside. Human leukocytic antigen (HLA) HLA molecules are cell surface proteins whose primary function is to present endogenous and exogenous antigens to T cells for recognition. In other words, they help the body's immune system identify foreign cells. During treatment, a patient’s DCs are isolated and incubated in the lab with synthetic prostate cancer proteins. This is then reinfused into the patient to activate T cells—what Gad calls "professional assassins”—that seek out and destroy cancerous cells displaying HLA and foreign antigens specific to the prostate cancer proteins. The FDA has since approved multiple T-cell therapy products, including those with transgenic T-cell receptors (tgTCRs) or chimeric antigen receptors (CARs). Transgenic T-cell receptors (tgTCRs) tgTCRs are artificial receptors, resembling the natural T-cell receptor (TCR) structure, targeting cancer antigens, which are introduced into patient-derived T cells using viral vectors. CAR T-cell therapy and cancer Gad’s lab focuses on CAR T-cell therapy, “a form of engineered T-cell therapy that brings the best of two worlds together”. Used mainly in oncology, patientderived T cells are “engineered to express a synthetic surface receptor that has the antigen binding domain of an antibody fused to the TCR signaling machinery; this fusion is what gives it the name ‘chimeric’,” he explained. Antibodies have “the superb ability to identify proteins in their intact form with no need for digestion and presentation on HLA,” Gad continued. When a CAR's antibody-derived antigen-binding domain identifies a tumor cell's surface antigen, it forms an immune synapse—an interaction site between the CAR T cell and the tumor cell. This triggers downstream signaling, recruiting co-activatory, co-inhibitory, and adhesion molecules to the immune synapse. These interactions lead CAR T cells to recruit lethal mechanisms to the immune synapse to kill the cancer cell. Gad said CAR T-cell therapy is “compatible with any patient's cancer and can be easily engineered against any tumor-associated surface protein.” His lab pioneers the development of HER2 CAR T-cell therapy for treating brain and bone/sarcoma cancers. To better understand the determinants of CAR T-cell success on the microscopic level, Gad’s research focuses on the CAR immune synapse (CARIS), the “battlefront between a CAR T cell and a cancer cell”, and the events taking place there. This offers a better TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: THE EVOLUTION OF ADVANCED BIOLOGICS 38 insight into the tactics used by both the CAR T cells and tumor cells during their fight and can drive the engineering of CAR T cells with better therapeutic capacities. In their recent research paper, Gad and colleagues found that the two most common designs of CAR T cell—CD28 signaling and 41BB signaling CAR T cells—use two different killing strategies. “CD28 CAR T cells are serial killers,” Gad explained. “They have the ability to kill a cancer cell fast and move to kill others.” They succeed because they have brief synapse interactions, “in which they can converge toxic granules ‘grenades’ toward the synapse and release them onto the tumor cell. These grenades make pores in the cell membrane of the tumor cell through which toxic proteins get in to kill it.” The second design, 41BB CAR T cells, Gad describes as ‘cooperative killers’: “When they see the tumor, they expand into more CAR T cells. Multiple CAR T cells then bind to a single cancer cell. They have a more protracted interaction that rely on a more chronic killing machinery, which is the chronic interaction between Fas ligands on CAR-T with Fas receptors on the surface of tumor cells.” On the molecular level, Gad found that CAR T cells use special membrane microdomains called lipid rafts to recruit functional receptors to the CARIS. These rigid islands that float on the surface of the fluid cell membrane provide structural and functional support at the immune synapse. Gad’s research found that the dynamics of CAR interaction with lipid rafts influence the cancer-killing behavior of CAR T cells. “For example, the brief cellular interactions that CD28 CAR T cells have with cancer cells are a reflection of the brief molecular interactions between CD28 CAR molecules and CAR T cell membrane lipid rafts,” Gad explained. “In the same sense, 41BB CAR T-cell protracted CARIS correlates with protracted interactions between 41BB CAR molecules and CAR T cell membrane lipid rafts. This knowledge can guide us to control the dynamics of CAR recruitment to the CARIS, and accordingly, the killing strategy used by CAR T cells.” Stem cell-based therapies Stem cells are unique in that they can differentiate into the cell type of interest, meaning they are of great use in cell therapies. “There are two different types of stem cells: multipotent, which can give rise to cells from a particular tissue (i.e., blood stem cells); or pluripotent, which can give rise to all three major lineages of the body (i.e., mesoderm, ectoderm and endoderm),” explained Pyle. “Pluripotent stem cells are powerful in that they can generate most if not all cell types in a dish and can even be generated from each patient to enable personalized medicine approaches to treat disease,” Pyle said. Stem cell therapy, or regenerative medicine, uses stem cells or their derivatives to promote the repair response of diseased, dysfunctional or injured tissue. Stem cells are cultivated in the laboratory and manipulated to develop into specific types of cells, which can then be infused into a patient. Pyle studies human pluripotent stem cells (hPSCs) and their ability to differentiate into one of the mesoderm lineages called skeletal muscle. “Skeletal muscle is endowed with a stem cell called a muscle stem cell that can regenerate after injury, but is defective in aging, muscle loss and in various genetic diseases,” said Pyle. “We have shown that hPSCderived skeletal muscle cells can repopulate regions of muscle with the potential to restore function in patients with muscle disease.” Pyle and her colleagues have created a first-of-itskind roadmap of the development of human skeletal muscle. They identified various cell types, but focused on muscle progenitor cells, which form muscles before birth, and muscle stem cells, which aid muscle formation after birth and regeneration from injury throughout life. The researchers charted how the cells' gene networks changed as cells mature, locating the precise networks present in muscle progenitor and stem cells across TECHNOLOGYNETWORKS.COM CELL AND GENE THERAPIES: THE EVOLUTION OF ADVANCED BIOLOGICS 39 development. Their work is essential to developing methods to create such cells in a dish to treat muscle disorders like muscular dystrophies and sarcopenia, the age-related loss of muscle mass and strength The future of cell therapy Pyle says the potential for stem cell-based cell therapy is enormous. “Not only can we generate most specialized cell types from pluripotent stem cells now in the lab, but many in the field have also begun to translate these to patients in need, including for diabetes and heart disease, among many others.” “The future of stem cells is bright and offers groundbreaking therapies for many developmental and adult disorders and diseases,” said Pyle. Gad is just as enthusiastic about the future of CAR T-cell therapies. Most targeted biological therapies and CAR T-cell therapies are in the field of oncology, “as they can laser-target tumor cells with much less side effects compared to chemo- and radiotherapy,” Gad said. While many CAR T-cell therapies target solid tumors in the clinical pipeline, “the complexity of the disease renders these therapeutic modalities less successful compared to those against hematological malignancies.” Gad foresees CAR T-cell therapy moving “toward more sophisticated designs that address the resistance mechanisms used by cancer to evade immune response.” He also believes CAR T-cell therapy could target with high precision other incurable diseases such as autoimmune disease and heart fibrosis. “Accordingly, CAR T-cell therapy can transform the healthcare system, given that the system can adapt to their [patients] very different needs.” But there are barriers to overcome, which Gad believes are likely to be industrial rather than scientific. Personalized therapies like CAR T-cell therapy and tgTCR are manufactured from the blood of each single patient and processed separately, then delivered to the hospital where the patient is being treated. “This makes the production much slower, requires more sophisticated shipping and introduces more variables to the process,” Gad said. Some companies have developed point-of-care manufacturing where benchtop machines manufacture CAR T-cell therapy at the hospital, “reducing the veinto-vein time, by limiting the wait time that comes with production in large-scale industrial facilities, shipping and delivery,” explained Gad. Another option is for “off-the-shelf" CAR T-cell therapy, to try to delete immune recognition genes from CAR T cells and make them compatible with all patients rather than a single patient, to avoid rejection by the patient's immune system. “This would make larger-scale production more feasible. Together with more training for healthcare personnel, cell therapies can be more affordable and accessible,” Gad said. Pyle believes that, depending on the type of therapy needed, the ability to expand cells to the levels needed for clinical avenues can be challenging. “However, many scientists, including those at UCLA, have been working on approaches to improve both scalability and safety in clinical good manufacturing settings. With training, hard work and dedicated teams working on these projects, I am certain we will be able to overcome these barriers.” MEET THE INTERVIEWEES: Dr. April Pyle is a professor and vice chair of microbiology, immunology and molecular genetics and a member of the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA. Her research focuses on the biology of muscle stem cells and human pluripotent stem cells and the differentiation of these cells into other cell types for use in regenerative medicine. Dr. Ahmed Gad is a postdoctoral associate in pediatric hematology, oncology, cell and gene therapy at Baylor College of Medicine. His research focuses on applying concepts of pharmacology to assess and improve CAR T-cell therapies for the treatment of solid and blood cancers. CELL AND GENE THERAPIES: THE EVOLUTION OF ADVANCED BIOLOGICS 40 TECHNOLOGYNETWORKS.COM CONTRIBUTORS Aron Gyorgypal, PhD Dr. Aron Gyorgypal is a postdoctoral research fellow at Harvard Medical School and Massachusetts General Hospital in Boston, Massachusetts. Following the completion of his PhD, he joined the Anthony Lab at Harvard Medical School and Massachusetts General Hospital in the immunology department, where he leverages his engineering skillset to deepen the understanding of immunological disorders and develop therapeutic solutions against such conditions. Katie Brighton Katie joined Technology Networks in January 2022 as a scientific copywriter. She holds a master’s by research degree in molecular and cellular biology and a bachelor's degree in biochemistry from the University of Leeds. Frank Charlton, PhD Dr. Frank Charlton obtained his PhD in molecular and cellular biology at the University of Leeds, where he studied the potential of exploiting host cell ion channels as an antiviral target for emerging viral diseases. Charlton then embarked on a postdoc at EPFL in Lausanne, Switzerland, where he worked as the resident virologist in a materials science group that develops nanoparticles as broad-spectrum antivirals. Joanna Owens, PhD Joanna Owens holds a PhD in molecular toxicology from the University of Surrey. She has over 20 years’ experience writing about a wide range of scientific topics in biosciences, pharmaceuticals and biotechnology. Kerry Taylor-Smith Kerry Taylor-Smith is a science writer who covers everything from climate change and the environment, to health and wellness, to material science, astronomy and space. She has a BSc in Natural Sciences from the University of Bath and over a decade of experience writing for various scientific and consumer publications. Neeta Ratanghayra, MPharm Neeta Ratanghayra is a freelance medical writer specializing in developing content for the pharma, biotech, and healthcare industries. She has a Master’s degree in Pharmacy and has worked on a variety of medical writing deliverables for global pharma companies and healthcare organizations. Before delving into medical writing, Neeta worked in the clinical research industry, where she handled multiple clinical research projects intended for various regulatory submissions.