Gene Therapy Manufacturing Research Articles

Next-generation DMSO-Free & serum-free cryopreservation for cell and gene therapy manufacturing and processing

Next-generation DMSO-Free & serum-free cryopreservation for cell and gene therapy manufacturing and processing

Device for automated aseptic sampling: Automated sampling solution for future cell and gene manufacturing.

Cell sampling is a key step performed regularly throughout the cell manufacturing process to gather cell samples for cell growth, progress, and characteristics analysis. While the current method of sampling by pipetting in a biosafety cabinet is commonly used, it is labour-intensive and susceptible to contamination risks. We have developed Device for Automated Aseptic Sampling (DAAS), to enable automated, small volume (0.02-1.00mL) aseptic sampling with minimal dead volume primarily for cell and gene therapy manufacturing. The aim of DAAS is to enable an accurate and consistent sampling process, with minimal contamination risks and interruption to the cells in culture. DAAS can potentially interface with other automated solutions to enable automated and streamlined cell manufacturing workflow and reduce overall manufacturing costs. DAAS has been verified as an aseptic sampling solution via repeated microbial ingression tests. It has also been tested for achieving comparable cell density and viability compared to manual pipetting, with negligible cross-sample carryover when used to sample Jurkat cells of different cell concentrations. The application of using DAAS to sample cell periodically and monitor cell growth and viability continuously for prolonged cell culture was successfully demonstrated with Jurkat cell culture in a static culture flask and donor T cell culture in an automated bioreactor system over a culture duration of 10days in a Biosafety Level-2 laboratory. Overall, DAAS presents great potential as an automated and aseptic sampling solution, offering cell and gene therapy manufacturers easier and more frequent access to cell samples with minimal interruptions to the cell culture. This enables close monitoring of cell culture and a more automated, connected and cost-effect cell and gene therapy manufacturing process.

Single-use technologies in bio manufacturing: benefits and implementation challenges

Single-use technologies (SUT) have revolutionized the bio manufacturing industry by offering flexibility, cost efficiency, and improved contamination control. Initially adopted for simpler processes, SUT has expanded to complex bioprocessing operations, driven by the growing demand for biologics, vaccines, and personalized medicines. This review explores the benefits of SUT, including reduced capital and operational expenditures, faster setup and turnaround times, and enhanced product safety. The scalability of SUT allows for rapid adaptation to market demands, significantly accelerating the time-to-market for critical therapies. However, the implementation of SUT is not without challenges. Material compatibility, leachables, waste management, and supply chain reliability pose significant hurdles. Moreover, regulatory and validation challenges complicate the adoption of these technologies in large-scale production. Case studies, including the rapid deployment of COVID-19 vaccines and the production of monoclonal antibodies, illustrate SUT's practical applications and benefits. The review also examines future trends, highlighting advances in materials, automation, and digital integration, as well as the expanding applications of SUT in cell and gene therapy manufacturing. As the bio manufacturing landscape continues to evolve, SUT will play a crucial role in meeting the industry's growing needs, provided the challenges associated with its implementation are effectively managed.

Particulates in CGT Guidance Survey Results – Cell and Gene Therapy Industry Knowledge Awareness

The ISCT Particulates Working Group has surveyed the global industry on commonly used regulatory guidance documents pertaining to particulates across various cell and gene therapy (CGT) product-based chemistry and manufacturing controls. Responses were collated from academic and industrially based individuals involved in, or making decisions for, manufacturing, quality control, and analytics. Two surveys covering therapy developers and manufacturing suppliers were created and distributed via email, through focus groups, and via social media platforms. In total, 97 responses were received from CGT professionals at different career levels, which varied from technicians to corporate executives. The responses represent a snapshot of the different CGT industries, with a bias in cell therapy developers, due to the distribution of respondents. Overall, we observed a widespread use of common guidance and legislative documents with specific testing requirements pinned to certain cell and gene therapy product types. Cell-based combination drugs and devices are not well addressed and may require other guidance that were not referenced. These results indicate that CGT particulate risk management should be clarified by collating various particulate guidelines into an easy-to-use guide that brings awareness to the various legislative and guidance documents pertaining to new and novel therapies, their classification, route of administration, and regulatory jurisdiction under which they are handled. The group also recommends clearly outlining the differences and similarities in particulate management regulations across the globe. As the CGT industry is undergoing maturation, the risk analysis methods are also maturing. With such a broad range in product types, routes of administration, source materials, and processing methods, standards of risk analysis and control need to be specific to the risk profile associated with any given drug product. The manufacturing strategy of novel drug types represented under the advanced therapy umbrella will require careful interpretation and implementation of the existing guidance identified in this survey, as not one guidance covers all manufacturing challenges with particulates.

Serotype-agnostic affinity purification of adeno-associated virus (AAV) via peptide-functionalized chromatographic resins

Adeno-associated viruses (AAVs) have emerged as a prominent family of vectors for gene delivery, providing therapeutic options to diseases once deemed incurable. At the same time, they necessitate efficient and affordable purification methods that can be platformed to serve all AAV serotypes. Current chromatographic tools, while affording high product purity, fail to bind certain serotypes, provide limited yield and lifetime, and impose harsh elution conditions that can compromise the vector's activity and safety. Addressing these challenges, this work demonstrates the application of new peptide ligands as the first serotype-agnostic technology for AAV purification by affinity chromatography. Our study reveals a pH-dependent affinity interaction: AAV2, AAV3, AAV6, AAV9, and AAVrh.10 are effectively captured at neutral pH, while binding AAV1, AAV5, AAV7, and AAV8 is stronger in a slightly acidic environment. The elution of bound AAVs was achieved using magnesium chloride at neutral pH for all serotypes, consistently affording capsid yields above 50% and genome yields above 80%, together with a >100-fold reduction in host cell proteins and nucleic acids. In particular, peptide ligand A10 exhibited remarkable binding capacity (> 1014 vp per mL of resin) and purification performance for all AAV serotypes, demonstrating broad applicability for gene therapy manufacturing. Finally, this work introduces novel alkaline-stable variants of A10 and demonstrates their use as the first affinity ligands capable of performing multiple cycles of AAV2, AAV8, and AAV9 purification with intermediate caustic cleaning without loss of capacity or product quality. Collectively, these results demonstrate the promise of this technology to further the impact and affordability of gene therapy.

Computational fluid dynamics based digital twins of fixed bed bioreactors validate scaling principles for recombinant adeno-associated virus gene therapy manufacturing.

Gene therapy using recombinant adeno-associated virus (rAAV) as delivery vehicles has garnered much interest in recent years. There are still significant gaps in our fundamental understanding of the manufacturing processes to deliver sufficient products. Manufacturing efforts of rAAV using HEK293 cells have commonly relied on fixed bed falling film bioreactors like the iCELLis®. We used computational fluid dynamics (CFD) to validate the operating conditions required for a predictive iCELLis® 500 scale-down model. The small-scale and at-scale systems have different flow paths causing validation of the corresponding agitation rates required to achieve the same linear flow through the fixed bed across scales to be non-trivial. Therefore, we used CFD to predict the theoretical scaling relationship. In addition, CFD could predict kLa differences between the two systems and the operating conditions required to match kLa between scales. We also confirmed that the location of DO control must be the same in both systems to achieve proper scaling. Experimental runs confirming the validity of the novel scale-down model showed that based on the modifications to the iCELLis® Nano system, we achieved similar DO, key metabolite, pH, and GC titer trends in both systems.

Trends in innovation for downstream process intensification, product yield, single use closed systems, and gentle ATMP bioprocessing: a KOL panel discussing 2024 insights in gene therapy manufacturing

Trends in innovation for downstream process intensification, product yield, single use closed systems, and gentle ATMP bioprocessing: a KOL panel discussing 2024 insights in gene therapy manufacturing

Cell and Gene Therapy Manufacturing and Development Trends

Cell and Gene Therapy Manufacturing and Development Trends

Case study: becoming the first GMP-certified cell and gene therapy manufacturing facility in Thailand

Case study: becoming the first GMP-certified cell and gene therapy manufacturing facility in Thailand

The evolution of gene therapy manufacturing

The evolution of gene therapy manufacturing

Investigation of CD83 As a Novel Human Hematopoietic Stem Cell Inflammatory Activation Marker

Hematopoietic stem cells (HSC) are responsible for life-long blood production and must balance homeostasis with sudden demand situations such as infections. However, the heterogeneity of molecular programs engaged by acute inflammatory stimuli to activate emergency hematopoiesis at the level of HSC are poorly understood. In a recent study linking human HSC heterogeneity to aging, recovery from severe COVID-19 infection, and clonal hematopoiesis (CH), we discovered two HSC subsets through multiomic analysis, denoted HSC-I and -II, in a xenograft model of inflammation-recovery. While HSC-I had few inflammation-driven molecular changes, HSC-II showed sustained epigenetic and transcriptional alterations in response to dual tumor necrosis factor α (TNFα) or lipopolysaccharide (LPS) challenges despite a 2.5-month recovery period. Importantly, a signature of long-term memory T cells (Akondy et al 2017) was enriched in HSC-II (Zeng, Nagree, et al, in submission). We interrogated the human HSC multiomic signatures uncovered from this human xenograft model to identify potential regulators associated with HSC response to and recovery from acute inflammation. We identified CD83 as a candidate that is differentially expressed between our HSC subsets. Similarly, we found CD83 expression to be enriched in a subset of adult-derived HSCs from patients where CH status was established and enrichment of an HSC-II signature observed (Jakobsen et al, submitted). CD83, first identified as a dendritic cell marker, is expressed on many immune cells, with linkage to controlling murine T regulatory (T-Reg) vs memory cell fate (Doebbeler et al, 2018), but has not previously been shown to be relevant for human HSC biology. Flow cytometry analysis of cord blood (CB) hematopoietic stem and progenitor cells (HSPC) showed CD83 to be low or absent in HSC (Figure A) and their downstream progeny at steady state. However, robust CD83 expression was observed in multiple HSPC subpopulations including long-term (LT-) and short-term (ST-) HSC within 16 hours of ex vivo culture, with highest expression in LT-HSC (Figure A). Importantly, CD83 surface expression on LT-HSC was enhanced following culture with TNFα (Figure A). Bystander effects were excluded by demonstrating similar findings after culture of purified HSC populations. Acute in vivo LPS treatment of mice xenografted with CB-derived HSC was sufficient to upregulate CD83 surface expression after 16 hours on LT-HSC/HSPC. To elucidate the potential function of CD83-expressing HSC/multipotent progenitors (MPP), CD19-CD34+CD38-CD45RA- cells were sorted from xenografts following acute inflammatory challenge for low and high CD83 expression and subjected to bulk RNA sequencing. We found enrichment of a gene expression signature specific to activated human HSCs (Garcia-Prat et al, 2021) within CD83-positive HSC/MPP (Figure B). Strikingly, enrichment of a signature of stimulated vs resting T cells (Galetti et al, 2020) and of effector T cells (Akondy et al 2017) was found in CD83-positive compared to CD83-negative HSC/MPP (Figure B). T cell effector function is in part dictated by metabolic reprogramming. We thus examined reactive oxygen species (ROS) levels in LT-HSC following ex vivo culture with TNFα and found lower ROS levels with CD83 expression. We therefore hypothesize that CD83 may be an ‘effector’ marker defining a distinct HSC subset during homeostatic or inflammatory activation that is subsequently fated to become memory-like HSC-II. We are undertaking studies to determine the mechanistic role of CD83 in HSC function and HSC-II formation via Cas9-sgRNA-mediated knockout or lentivirus-mediated overexpression of CD83. In summary, CD83 represents a long-sought marker of inflammatory activation at the HSC level. CD83 may have significant clinical potential as a biomarker of aberrant activation in gene therapy manufacturing protocols or for HSC transplantation. Future studies will shed light on whether CD83 can functionally regulate the molecular programs underlying human HSC heterogeneity.

Significant Differences in Capsid Properties and Potency Between Adeno-Associated Virus Vectors Produced in Sf9 and HEK293 Cells.

For successful vector-based gene therapy manufacturing, the selected adeno-associated virus (AAV) vector production system must produce vector at sufficient scale. However, concerns have arisen regarding the quality of vector produced using different systems. In this study, we compared AAV serotypes 1, 8, and 9 produced by two different systems (Sf9/baculovirus and HEK293/transfection) and purified by two separate processes. We evaluated capsid properties, including protein composition, post-translational modification, particle content profiles, and in vitro and in vivo vector potency. Vectors produced in the Sf9/baculovirus system displayed reduced incorporation of viral protein 1 and 2 into the capsid, increased capsid protein deamidation, increased empty and partially packaged particles in vector preparations, and an overall reduced potency. The differences observed were largely independent of the harvest method and purification process. These findings illustrate the need for careful consideration when choosing an AAV vector production system for clinical production.

Uncovering Manufacturing Challenges Behind Cell and Gene Therapy

Uncovering Manufacturing Challenges Behind Cell and Gene Therapy

Overcoming Obstacles in Gene Therapy Manufacturing

Genetic Engineering & Biotechnology NewsVol. 43, No. 5 Sponsored ContentOvercoming Obstacles in Gene Therapy ManufacturingJohn Kerwin and Patrick StarremansJohn KerwinSearch for more papers by this author and Patrick StarremansSearch for more papers by this authorPublished Online:8 May 2023https://doi.org/10.1089/gen.43.05.13AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetails Volume 43Issue 5May 2023 Information© 2023 by GEN PublishingTo cite this article:John Kerwin and Patrick Starremans.Overcoming Obstacles in Gene Therapy Manufacturing.Genetic Engineering & Biotechnology News.May 2023.39-39.http://doi.org/10.1089/gen.43.05.13Published in Volume: 43 Issue 5: May 8, 2023PDF download

Adding Consistency to Gene Therapy Manufacturing

Genetic Engineering & Biotechnology NewsVol. 43, No. 5 Sponsored ContentAdding Consistency to Gene Therapy ManufacturingViralgen developed a dedicated platform and a designed-for-use cell line, which combine to improve efficiency and reduce costsPublished Online:8 May 2023https://doi.org/10.1089/gen.43.05.06AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetails Volume 43Issue 5May 2023 Information© 2023 by GEN PublishingTo cite this article:Adding Consistency to Gene Therapy Manufacturing.Genetic Engineering & Biotechnology News.May 2023.21-21.http://doi.org/10.1089/gen.43.05.06Published in Volume: 43 Issue 5: May 8, 2023PDF download

Development and Delivery of a Hands-On Short Course in Adeno-Associated Virus Manufacturing to Support Growing Workforce Needs in Gene Therapy.

The manufacturing of gene therapy products is a rapidly growing industry bolstered by the tremendous potential of these therapies to provide lifesaving treatment for rare and complex genetic diseases. The industry's steep rise has resulted in a high demand for skilled staff required to manufacture gene therapy products of the expected high quality. To address this skill shortage, more opportunities for education and training in all aspects of gene therapy manufacturing are needed. The Biomanufacturing Training and Education Center (BTEC) at the North Carolina State University (NC State) has developed and delivered (and continues to deliver) a 4-day, hands-on course titled Hands-on cGMP Biomanufacturing of Vectors for Gene Therapy. The course, which consists of 60% hands-on laboratory activities and 40% lectures, aims to provide a comprehensive understanding of the gene therapy production process, from vial thaw through the final formulation step, and analytical testing. This article discusses the design of the course, the backgrounds of the nearly 80 students who have participated in the seven offerings held since March 2019, and feedback from the course participants.

Validation of a GMP stem cell gene therapy manufacturing process for mucopolysaccharidosis type II (MPS II) in preparation for an approved phase I/II clinical trial

Validation of a GMP stem cell gene therapy manufacturing process for mucopolysaccharidosis type II (MPS II) in preparation for an approved phase I/II clinical trial

Evaluating the efficacy of hydrogen peroxide vapor against a retroviral vector in a gene therapy manufacturing facility.

An evaluation of the efficacy of 35% hydrogen peroxide vapor (HPV) against a novel Simian Immunodeficiency retrovirus (SIV) was conducted in a 624m3 GMP grade C manufacturing facility. SIV biological indicators, with an average titre of 7.11×108 TU/mL [equivalent to 1.3×108 Reverse transcriptase (RT) nanounits when analysed by F-PERT], were produced on-site using virus presented within an anticipated worst-case support matrix based on the manufacturers commercially confidential virus support media. SIV biological indicators were distributed around the manufacturing facility, including locations identified as key contamination control points by the manufacturer, and exposed to a HPV cycle. Commercially available biological indicators of 6 log10Geobacillus stearothermophilus were co-located with the SIV Biological Indicator (BIs). HPV was found to be efficacious in eliminating the SIV and commercial G. stearothermophilus BIs and offers a simple and robust method for biodecontamination of gene therapy facilities.

Machine learning and metabolic modelling assisted implementation of a novel process analytical technology in cell and gene therapy manufacturing

Process analytical technology (PAT) has demonstrated huge potential to enable the development of improved biopharmaceutical manufacturing processes by ensuring the reliable provision of quality products. However, the complexities associated with the manufacture of advanced therapy medicinal products have resulted in a slow adoption of PAT tools into industrial bioprocessing operations, particularly in the manufacture of cell and gene therapy products. Here we describe the applicability of a novel refractometry-based PAT system (Ranger system), which was used to monitor the metabolic activity of HEK293T cell cultures during lentiviral vector (LVV) production processes in real time. The PAT system was able to rapidly identify a relationship between bioreactor pH and culture metabolic activity and this was used to devise a pH operating strategy that resulted in a 1.8-fold increase in metabolic activity compared to an unoptimised bioprocess in a minimal number of bioreactor experiments; this was achieved using both pre-programmed and autonomous pH control strategies. The increased metabolic activity of the cultures, achieved via the implementation of the PAT technology, was not associated with increased LVV production. We employed a metabolic modelling strategy to elucidate the relationship between these bioprocess level events and HEK293T cell metabolism. The modelling showed that culturing of HEK293T cells in a low pH (pH 6.40) environment directly impacted the intracellular maintenance of pH and the intracellular availability of oxygen. We provide evidence that the elevated metabolic activity was a response to cope with the stress associated with low pH to maintain the favourable intracellular conditions, rather than being indicative of a superior active state of the HEK293T cell culture resulting in enhanced LVV production. Forecasting strategies were used to construct data models which identified that the novel PAT system not only had a direct relationship with process pH but also with oxygen availability; the interaction and interdependencies between these two parameters had a direct effect on the responses observed at the bioprocess level. We present data which indicate that process control and intervention using this novel refractometry-based PAT system has the potential to facilitate the fine tuning and rapid optimisation of the production environment and enable adaptive process control for enhanced process performance and robustness.

Regulatory requirements and product attributes for cGMP viral vector production

Navigating the constantly evolving regulatory landscape in cell and gene therapy manufacturing can pose critical challenges for developers and manufacturers. Failure to meet requirements can significantly raise costs, motivating the implementation of robust, reproducible, and compliant processes.