Cell Therapy Manufacturing Research Articles

Applying the Functional Cell Library to Transform Cell Therapy Optimization and Manufacturing

Applying the Functional Cell Library to Transform Cell Therapy Optimization and Manufacturing

Assessment of Automated Flow Cytometry Data Analysis Tools within Cell and Gene Therapy Manufacturing

Flow cytometry is widely used within the manufacturing of cell and gene therapies to measure and characterise cells. Conventional manual data analysis relies heavily on operator judgement, presenting a major source of variation that can adversely impact the quality and predictive potential of therapies given to patients. Computational tools have the capacity to minimise operator variation and bias in flow cytometry data analysis; however, in many cases, confidence in these technologies has yet to be fully established mirrored by aspects of regulatory concern. Here, we employed synthetic flow cytometry datasets containing controlled population characteristics of separation, and normal/skew distributions to investigate the accuracy and reproducibility of six cell population identification tools, each of which implement different unsupervised clustering algorithms: Flock2, flowMeans, FlowSOM, PhenoGraph, SPADE3 and SWIFT (density-based, k-means, self-organising map, k-nearest neighbour, deterministic k-means, and model-based clustering, respectively). We found that outputs from software analysing the same reference synthetic dataset vary considerably and accuracy deteriorates as the cluster separation index falls below zero. Consequently, as clusters begin to merge, the flowMeans and Flock2 software platforms struggle to identify target clusters more than other platforms. Moreover, the presence of skewed cell populations resulted in poor performance from SWIFT, though FlowSOM, PhenoGraph and SPADE3 were relatively unaffected in comparison. These findings illustrate how novel flow cytometry synthetic datasets can be utilised to validate a range of automated cell identification methods, leading to enhanced confidence in the data quality of automated cell characterisations and enumerations.

Retrospective Analysis of Autologous Chondrocyte-Based Cytotherapy Production for Clinical Use: GMP Process-Based Manufacturing Optimization in a Swiss University Hospital.

Cultured autologous human articular chondrocyte (HAC) implantation has been extensively investigated for safe and effective promotion of structural and functional restoration of knee cartilage lesions. HAC-based cytotherapeutic products for clinical use must be manufactured under an appropriate quality assurance system and follow good manufacturing practices (GMP). A prospective clinical trial is ongoing in the Lausanne University Hospital, where the HAC manufacturing processes have been implemented internally. Following laboratory development and in-house GMP transposition of HAC cell therapy manufacturing, a total of 47 patients have been treated to date. The main aim of the present study was to retrospectively analyze the available manufacturing records of the produced HAC-based cytotherapeutic products, outlining the inter-individual variability existing among the 47 patients regarding standardized transplant product preparation. These data were used to ameliorate and to ensure the continued high quality of cytotherapeutic care in view of further clinical investigations, based on the synthetic analyses of existing GMP records. Therefore, a renewed risk analysis-based process definition was performed, with specific focus set on process parameters, controls, targets, and acceptance criteria. Overall, high importance of the interdisciplinary collaboration and of the manufacturing process robustness was underlined, considering the high variability (i.e., quantitative, functional) existing between the treated patients and between the derived primary HAC cell types.

Sterile Fluid Transfer for Cell Therapy Manufacturing—The Value of Multiple-Use Aseptic Connector

Sterile fluid transfer between vessels is a key step in cell therapy manufacturing and requires specialised devices to maintain sterility of both vessels before, during and after the transfer. This review introduces two main types of devices for sterile fluid transfer in cell therapy manufacturing, namely single-use sterile connectors and tube welders. While these are excellent devices for infrequent moderate to large volume transfers, a new multiple-use aseptic connector may fill the gap for frequent small volume fluid transfers that are particularly important for autologous cell therapy manufacturing.

Design of experiments as a decision tool for cell therapy manufacturing

Background aimsCell therapies are costlier to manufacture than small molecules and protein therapeutics because they require multiple manipulations and are often produced in an autologous manner. Strategies to lower the cost of goods to produce a cell therapy could make a significant impact on its total cost. MethodsBorrowing from the field of bioprocess development, the authors took a design of experiments (DoE)-based approach to understanding the manufacture of a cell therapy product in pre-clinical development, analyzing main cost factors in the production process. The cells used for these studies were autologous CD4+ T lymphocytes gene-edited using CRISPR/Cas9 and recombinant adeno-associated virus (AAV) to restore normal FOXP3 gene expression as a prospective investigational product for patients with immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome. ResultsUsing gene editing efficiency as the response variable, an initial screen was conducted for other variables that could influence the editing frequency. The multiplicity of infection (MOI) of AAV and amount of single guide RNA (sgRNA) were the significant factors used for the optimization step to generate a response contour plot. Cost analysis was done for multiple points in the design space to find cost drivers that could be reduced. For the range of values tested (50 000–750 000 vg/cell AAV and 0.8–4 μg sgRNA), editing with the highest MOI and sgRNA yielded the best gene editing frequency. However, cost analysis showed the optimal solution was gene editing at 193 000 vg/cell AAV and 1.78 μg sgRNA. ConclusionsThe authors used DoE to define key factors affecting the gene editing process for a potential investigational therapeutic, providing a novel and faster data-based approach to understanding factors driving complex biological processes. This approach could be applied in process development and aid in achieving more robust strategies for the manufacture of cellular therapeutics.

Magnetic selection for consistent cellular starting material in autologous cell therapy manufacture

An efficient cell selection method is crucial to deliver consistent autologous therapy products when the starting material received is highly variable. Whilst a variety of technologies are being adopted in the industry, there are few GMP-compliant options, and these technologies are often manual, semi-automated, and lack commercial viability. In this article, two experts share insights on obtaining highly purified cells using magnetically active cell selection in a flexible, closed manufacturing system. In addition, transitioning manual cell therapy production to scalable automated processes is discussed.

Assessing workforce needs for the emerging CAR-T cell therapy industry.

The complicated and labor-intensive manufacturing of novel cell and gene therapies requires efforts to improve workforce development programs for the nascent cell therapy industry broadly, and for cell manufacturing more specifically.

Systematic Design, Generation, and Application of Synthetic Datasets for Flow Cytometry.

Application of synthetic datasets in training and validation of analysis tools has led to improvements in many decision-making tasks in a range of domains from computer vision to digital pathology. Synthetic datasets overcome the constraints of real-world datasets, namely difficulties in collection and labeling, expense, time, and privacy concerns. In flow cytometry, real cell-based datasets are limited by properties such as size, number of parameters, distance between cell populations, and distributions and are often focused on a narrow range of disease or cell types. Researchers in some cases have designed these desired properties into synthetic datasets; however, operators have implemented them in inconsistent approaches, and there is a scarcity of publicly available, high-quality synthetic datasets. In this research, we propose a method to systematically design and generate flow cytometry synthetic datasets with highly controlled characteristics. We demonstrate the generation of two-cluster synthetic datasets with specific degrees of separation between cell populations, and of non-normal distributions with increasing levels of skewness and orientations of skew pairs. We apply our synthetic datasets to test the performance of a popular automated cell populations identification software, SPADE3, and define the region where the software performance decreases as the clusters get closer together. Application of the synthetic skewed dataset suggests the software is capable of processing non-normal data. We calculate the classification accuracy of SPADE3 with robustness not achievable with real-world datasets. Our approach aims to advance research toward generation of high-quality synthetic flow cytometry datasets and to increase their awareness among the community. The synthetic datasets can be used in benchmarking studies that critically evaluate cell population identification tools and help illustrate potential digital platform inconsistencies. These datasets have the potential to improve cell characterization workflows that integrate automated analysis in clinical diagnostics and cell therapy manufacturing.

POSC153 Long-Term Cost Efficiencies Linked to Automated vs. Manual Fill and Finish Process in Cell and Gene Therapy Manufacturing

Due to the high risk and variability in the final step of the cell manufacturing process, a large portion of the commercial segment is looking to automate. While there are several advantages to automation, this assessment aimed to explore the long-term cost efficiencies linked to this decision.

POSB297 Assessing the Cost and Value Drivers of Automation in Cell and Gene Therapy Manufacturing: A Decision Guide

Contrary to off-the shelf therapies, autologous solutions cannot benefit from economies of scale. One option to make the process more efficient is to automate different steps in the procedure. This assessment looks at the key parameters that should be considered when taking this decision.

Engineering approaches to adopt automation in tissue engineered and cell therapy product manufacture

<div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://bioinsights.blob.core.windows.net/uploads/Ioannis Profile copy.jpeg" data-image="1"></div><div class="author-det"> <b>Ioannis Papantoniou</b> is an Associate Professor head of the Tissue Engineering Lab of the Skeletal Biology and Engineering Research Centre, Department of Development and Regeneration, KU Leuven. He is also part of Prometheus, the translational division of the Leuven R&amp;D, KU Leuven. In addition he is also a visiting PI at the Institute of Chemical Engineering Sciences, Foundation of Research and Technology (ICEHT/FORTH) in Greece. He is the coordinator of the H2020 ‘Jointpromise’ project aiming at automating the biomanufacturing of organoid-based osteochondral implants and is also a participant and Board member of the recently launched H2020 project ‘AIDPATH’ aiming at the integration of Artificial Intelligence technologies in Cell Therapy Manufacturing. He has also obtained funding from European, regional and national sources and has coached many young researchers into obtaining personal Post-doctoral and PhD grants. He has been invited in several academic and industrial conferences to serve as session and track chair as well as invited panelist. He has provided numerous invited lectures in leading academic institutes across Europe. During his research activities multiple industrial collaborations have been carried out successfully with leading players in the ATMP manufacturing field. His main research focus is develop designed 3D cell-based products with built-in arranged quality attributes through high-precision bioengineering technologies for skeletal regeneration. In addition the integration of automation aspects in organoid based tissue products has been at the forefront of his interest. Recent research breakthroughs resulted in the development of cartilaginous organoids for the bioprinting of bone regenerating living implants. Their subsequent use in various contexts has been evaluated while their production for clinical application is under way at KU Leuven university hospitals. </div></div></div>

Understanding and improving cellular immunotherapies against cancer: From cell-manufacturing to tumor-immune models.

The tumor microenvironment (TME) is shaped by dynamic metabolic and immune interactions between precancerous and cancerous tumor cells and stromal cells like epithelial cells, fibroblasts, endothelial cells, and hematopoietically-derived immune cells. The metabolic states of the TME, including the hypoxic and acidic niches, influence the immunosuppressive phenotypes of the stromal and immune cells, which confers resistance to both host-mediated tumor killing and therapeutics. Numerous in vitro TME platforms for studying immunotherapies, including cell therapies, are being developed. However, we do not yet understand which immune and stromal components are most critical and how much model complexity is needed to answer specific questions. In addition, scalable sourcing and quality-control of appropriate TME cells for reproducibly manufacturing these platforms remain challenging. In this regard, lessons from the manufacturing of immunomodulatory cell therapies could provide helpful guidance. Although immune cell therapies have shown unprecedented results in hematological cancers and hold promise in solid tumors, their manufacture poses significant scale, cost, and quality control challenges. This review first provides an overview of the in vivo TME, discussing the most influential cell populations in the tumor-immune landscape. Next, we summarize current approaches for cell therapies against cancers and the relevant manufacturing platforms. We then evaluate current immune-tumor models ofthe TME and immunotherapies, highlighting the complexity, architecture, function, and cell sources. Finally, we present the technical and fundamental knowledge gaps in both cell manufacturing systems and immune-TME models that must be addressed to elucidate the interactions between endogenous tumor immunity and exogenous engineered immunity.

Acoustofluidic-mediated molecular delivery to human T cells with a three-dimensional-printed flow chamber.

Cell-based therapies have garnered significant interest to treat cancer and other diseases. Acoustofluidic technologies are in development to improve cell therapy manufacturing by facilitating rapid molecular delivery across the plasma membrane via ultrasound and microbubbles (MBs). In this study, a three-dimensional (3D) printed acoustofluidic device was used to deliver a fluorescent molecule, calcein, to human T cells. Intracellular delivery of calcein was assessed after varying parameters such as MB face charge, MB concentration, flow channel geometry, ultrasound pressure, and delivery time point after ultrasound treatment. MBs with a cationic surface charge caused statistically significant increases in calcein delivery during acoustofluidic treatment compared to MBs with a neutral surface charge (p < 0.001). Calcein delivery was significantly higher with a concentric spiral channel geometry compared to a rectilinear channel geometry (p < 0.001). Additionally, calcein delivery was significantly enhanced at increased ultrasound pressures of 5.1 MPa compared to lower ultrasound pressures between 0-3.8 MPa (p < 0.001). These results demonstrate that a 3D-printed acoustofluidic device can significantly enhance intracellular delivery of biomolecules to T cells, which may be a viable approach to advance cell-based therapies.

Process analytical technologies in cell therapy manufacturing: State‐of‐the‐art and future directions

AbstractCell therapies have the potential to effectively treat and even cure complex, currently untreatable diseases with unprecedented success. The cell therapy industry has been growing rapidly since the Food and Drug Administration approval of the first product in 2017. Despite tremendous promise, there are significant and unique challenges that must be overcome to make cell therapy manufacturing reproducible, scalable, high‐quality, and cost‐effective. Discovery and implementation of critical quality attributes (CQAs) and critical process parameters (CPPs) to the complex cell therapy manufacturing processes is one such grand challenge for the field. The role of process analytical technologies (PATs) in CQA/CPP discovery and eventual in‐process, or at‐process monitoring to maintain consistent process and product quality, is indispensable. Here we discuss the major challenges and the strategic framework for optimizing process development and related PATs for various cell therapies, with a focus on upstream processes. We introduce relevant approaches, such as quality‐by‐design (QbD), and the implementation of PATs to enable QbD in current biomanufacturing processes. We examine state‐of‐the‐art PAT implementation on standard physicochemical parameters in biopharmaceutical operations and consider potential cell therapy‐related parameters that may be instrumental in overcoming the challenges of the current cell therapy manufacturing landscape. Current innovations applied to the field, such as high‐throughput and high‐dimensional analyses, machine learning, and novel sensor technologies, are also discussed. We conclude that advances in PATs are necessary to identify CQAs and CPPs, overcome limitations in current operating processes, reduce overall product cost, and significantly accelerate the translation of laboratory discoveries into commercialized cell therapy products.

Understanding raw material performance: quality and consistency of cytokines for translation to the clinic

A deep understanding of origin, performance and quality of raw materials in cell and gene therapy development is crucial, but international standards for these raw materials are still missing in our industry. The challenge lies in the fact that raw biological materials are inherently variable, while batch-to-batch consistency is essential for successful and long-term commercialization of therapies. There is also the big debate of when to apply rigid standards, is commercial-scale manufacturing too late? One category of raw materials that require further standardization and characterization is cytokines, growth and differentiation factors (here named cytokines for simplicity). Cytokine quality and performance are directly linked to the clinical and commercial success of a therapy. However, there are important quality considerations to address during preclinical research to ensure your therapy is set up for regulatory and commercial success. Even cytokines that were originally developed for other uses, including those used as human therapeutics themselves, are not necessarily suited for use in cell and gene therapy manufacturing. Limited information on the potency and other critical attributes of the materials makes it difficult to define specifications for those reagents and to investigate the material. This article will explore ways of easing translation from preclinical development into the clinic, the importance of using animal-derived component-free (ADCF) cytokines, how to compare cytokines from different vendors and the value of international units of measurement.

Expert Roundtable: overcoming challenges in analytical assays for cellular therapies

<div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://bioinsights.blob.core.windows.net/uploads/Jara photo copy.jpg" data-image="1"></div><div class="author-det"><h3 class="author-name" style="color:#5D538A;">Jara Joedicke</h3><h5 class="author-title" style="color:#5D538A;">Senior Scientist, Max Delbrück Center</h5> Jara Joedicke is an immunologist with a passion for the development of novel cancer therapies. She has over 10 years molecular biology experience in both academia and industry. Throughout her career she has managed a variety of employees from students and postdoctoral researchers to technical staff. Her aim is to bring novel cell and gene therapies rapidly from the bench to the clinic, especially those utilizing CAR T cells. </div></div></div><div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://bioinsights.blob.core.windows.net/uploads/E Alonzo portrait-sq.jpg copy.png" data-image="1"></div><div class="author-det"><h3 class="author-name" style="color:#00A795;"><strong>Eric Alonzo</strong></h3><h5 class="author-title" style="color:#00A795;">Strategy Lead, Oncology, Analytical Development &amp; CMC Lead, bbT369 Program, bluebird bio</h5> Dr Alonzo serves as the CMC Lead for the bbT369, a pre-clinical program for the treatment of B-NHL and Analytical Strategy Lead for the Oncology Divsion at bluebird bio. Previously, Dr Alonzo led the Oncology Characterization Team where he focused on the development of analytical and characterization assays for CAR T/TCR cell programs. Prior to bluebird bio, Dr Alonzo lead efforts developing T-cell-based therapies at Agenus, and previous to his work at Agenus, he was a postdoctoral fellow at Harvard Medical School studying transcriptional and epigenetic dysregulation that leads to the development of T-cell acute lymphoblastic leukemia. Dr Alonzo received his PhD from the Cancer Biology Program at the Memorial Sloan-Kettering Cancer Center. </div></div></div><div class="authors authors-lg"><div class="author"><div class="author-img"><strong><strong><strong><img src="https://bioinsights.blob.core.windows.net/uploads/Marsilius Mues_Portrait copy.jpg" data-image="1"></strong></strong></strong></div><div class="author-det"><h3 class="author-name" style="color:#007589;"><strong><strong>Marsilius Mues</strong></strong></h3><h5 class="author-title" style="color:#007589;">R&amp;D Group Leader at Miltenyi Biotec</h5> Dr Mues joined the R&amp;D Department of Miltenyi Biotec in 2017. In his current role he leads the interdisciplinary research and development in the field of immuno-oncology and cellular therapeutics, with a focus on CAR T cell engineering for personalized immunotherapy. He oversees the establishment of multiparametric immune assays for functional characterization of engineered T cells, along with automated data acquisition and analysis tools for translational research, cell therapy manufacturing, and patient immune monitoring during clinical trials. He performed his PhD studies at the Max Planck Institute of Neurobiology on T cell signaling during autoimmune disease and completed his postdoctoral research at UCSF where he focused on novel combinatorial therapies using high-complexity lentiviral shRNA &amp; CRISPR/Cas9 libraries for T-cell leukemia. </div></div></div>

Bioreactor Technology for Cell Therapy Manufacturing in Regenerative Medicine

Bioreactor Technology for Cell Therapy Manufacturing in Regenerative Medicine

Are you finishing strong in cell therapy manufacturing? Tackling your final fill and finish challenges with automation

Fill and finish is a critical, high-value step in cell therapy manufacturing; failure is both costly for the manufacturer and potentially catastrophic for the patient waiting to receive therapy. This key step is frequently done manually, which poses a number of significant risks. In contrast, the Finia&#174;&#160;Fill and Finish System, designed to automate this process, may reduce contamination and labor costs while improving reproducibility and supporting GMP compliance.

The UK’s emerging regulatory framework for point-of-care manufacture: insights from a workshop on advanced therapies

Point-of-care (POC) manufacture can be defined as the production of therapies in clinical settings or units close to hospitals and patients. This approach is becoming increasingly viable due to the emergence of flexible manufacturing technologies. Expecting an increase in this kind of production, the UK’s regulatory agency, the Medicines and Healthcare products Regulatory Agency (MHRA) is proposing a regulatory framework specifically designed for POC manufacture. To discuss the challenges of POC manufacture and the MHRA’s proposal, the EPSRC Future Targeted Healthcare Manufacturing Hub (FTHMH) organized a workshop drawing insights from specialists in cell and gene therapy manufacture. Through presentations and discussion roundtables, the workshop highlighted the challenges for the UK and other countries implementing POC manufacture. The workshop attendees stressed four main issues: quality control; standardization and equipment use; availability of qualified personnel; and the challenges to be met by hospitals participating in POC manufacture systems. This commentary provides a summary of the points discussed in this workshop.

Industrializing viral vector manufacture

Daniel Teasley has worked at Merck for over 6 years, serving in product management and product strategy roles across multiple portfolios including custom cell line engineering services, regenerative medicine products for research, cell therapy manufacturing, and viral gene therapy manufacturing. For the past three years, he has been a Senior Product Manager responsible for the development, launch, and marketing of products and services that enable at-scale viral vector manufacturing. He also supports cross-business strategic initiatives in novel modality manufacturing. Dr. Teasley holds a PhD in Molecular Genetics and Genomics from Washington University in St. Louis and a BS in Biology from the College of William and Mary.Katherine (Katie) Schewe has worked at Merck for over 4 years in various roles from product management in Viral &amp; Gene Therapy to quality assurance of Active Pharmaceutical Ingredient manufacturing. Katie holds a MS in Chemistry from Indiana University Purdue University – Indianapolis and a BS in Chemistry from Southern Illinois University – Edwardsville.