Engineering Conferences International
Not a member yet
9271 research outputs found
Sort by
Process development and scale-up for gene circuit engineered CAR-NK cell manufacturing
Get PDFAllogeneic Natural Killer (NK) cell therapy has shown promise in recent years for treating cancer in patients without inducing graft versus host disease and with potential for off-the-shelf administration. Senti Bio is using gene circuits to introduce logic-gating and regulated expression of payloads into next-generation CAR-NK cell therapies to broaden the therapeutic indications and improved efficacy in liquid and solid tumors. Key process development objectives for gene circuits include the ability to efficiently and stably transduce multi-gene constructs into primary NK cells while retaining cell expandability and anti-cancer function. Here, we describe a scalable GMP-ready manufacturing process for generating clinically relevant numbers of CAR-NK cells, and we demonstrate its potential applicability to our product pipeline.
To achieve a batch size target of \u3e10^11 NK cells, we aimed to develop a process to start with ~25*10^6 isolated NK cells, achieve \u3e40% CAR+ transduction, and obtain \u3e5,600-fold expansion over 21 days. Enrichment of adult apheresis material from 12 healthy donors via CD3 depletion and CD56 selection yielded an average of ~3*10^8 NK cells, which were cryopreserved for later use. Upon thaw, NK cells were activated using proprietary irradiated gene-modified feeder cells and expanded in a closed system 1L G-Rex chamber. Seven days later, NK cells were transduced with retroviral vectors using closed system procedures, resulting in up to 80% CAR+ population. Gene circuits were tested across multiple retroviral vector delivery systems, and successful constructs were developed into producer cell lines (HEK293) using various single cell cloning techniques with the goal of generating stable, high titer vector producer clones. Primary NK cell transduction efficiency was optimized by testing a range of MOI, comparing different vector addition and spinoculation vessels, and the effect of GMP-compatible transduction enhancers. Transduced NK cells were expanded further in multiple closed system G-Rex culture vessels for a total process time (initial NK thaw to CAR-NK harvest) of approximately 21 days. Different expansion methods were assessed including different irradiated modified cell lines and feeder-free NK expansion technologies achieving ~10,000-fold expansion in the 1L vessels. At cell harvest, the cell suspension was volume-reduced, harvested and formulated into cryopreservation medium using an automated cell processing system, yielding ~4*10^9 cells per liter of culture. Formulated cells were filled in vials and stored in liquid nitrogen vapor phase. Functional assessment was performed via both in vitro and in vivo studies, demonstrating significant CAR-specific cancer cell killing compared to non-transduced NK cells. We also evaluated multiple donors for transduction efficiency, growth characteristics, cancer cell killing specificity, scalability, immunomodulatory function, single cell transcriptomics and distribution and kinetics in vivo to determine desirable attributes for manufacturing. This CAR-NK manufacturing process is expected to be suitable for translation to GMP clinical manufacturing in support of Senti Bio’s internal allogeneic CAR-NK cell pipeline
hiPSC and hiPSC-cardiomyocytes are alternative EV biofactories for cardiac regeneration
Get PDFIn cardiac regenerative medicine, there is a growing interest in using EV as cell-mimetic therapeutics due to their potential superior efficacy and overall advantages over cell transplantation: i) absence of oncogenic risk, ii) low immunogenicity [1], iii) easier large-scale manufacturing and iv) consistent product profile. However, most studies have focused particularly on the potential of either mesenchymal or cardiac progenitor cell-derived EV to promote cardiac repair [2].
Here, we study the potential of human induced pluripotent stem cells (hiPSC) and hiPSC-derived cardiomyocytes (hiPSC-CM) as alternative cell factories for the production of a high yield of therapeutic EV for cardiac regeneration. Due to their high self-renewal ability and capacity to differentiate into functional cardiomyocytes, these cells can provide an unlimited source of EV for application in cardiac regeneration.
We generated and characterized EV derived from key stages of hiPSC-CM differentiation and maturation, i.e. from hiPSC (hiPSC-EV), cardiac progenitors (CPC-EV), immature (CMi-EV) and mature (CMm-EV) cardiomyocytes, with the goal of studying their potential role as therapeutics, and whether their yield and function was influenced by the state of their parent cell. Two hiPSC lines were differentiated into hiPSC-CM and cultured as 3D spheroids in a fatty acid supplemented medium to improve CM maturation [3,4]. EV isolation was performed based on density separation on an iodixanol discontinuous gradient, and EV were characterized in terms of particle size and particle size distribution, presence of EV-specific markers, and imaging through transmission electron microscopy. Functional studies were performed using human umbilical vein endothelial cells (HUVECs) to evaluate EV-uptake, migration and angiogenesis.
EV yield varied along CM differentiation stages, with a minimum for CPC, for both cell lines. Bioactivity assays with HUVECs showed that uptake of PKH26-labelled EV could be blocked by dynasore, an inhibitor of dynamin-2, a GTPase that plays a crucial role in clathrin and caveolin-dependent endocytosis. Increased migration was observed in HUVECs treated with hiPSC, CPC and CM-derived EV (92.25 ± 14.69% wound closure at 24h for hiPSC, 77.13 ± 13.64 % for CPC, 74.71 ± 19.86% for CMi, 69.2 ± 19.12% for CMm versus 45.65 ± 7.26% for control), but angiogenic properties were found only for hiPSC-EV (fold change of 11.2 ± 4.59 in total segment length vs. control, p\u3c0.001).
Current efforts towards the characterization of EV small RNA cargo aim at understanding the correlation between cargo composition and in vitro activity, to identify the optimal cell factory for scalable therapeutic EV production.
Funding: FCT PhD fellowship PD/BD/139078/2018; IC&TD Projects MetaCardio” (PTDC/BTM-SAL/32566/2017) and NETDIAMOND (SAICTPAC/0047/2015), and iNOVA4Health Research Unit (UIDB/04462/2020).
1. Zhu, X., et al. Comprehensive toxicity and immunogenicity studies reveal minimal effects in mice following sustained dosing of extracellular vesicles derived from HEK293T cells. Journal of Extracellular Vesicles 6, 1, 2017.
2. El Harane, et al. Acellular therapeutic approach for heart failure: in vitro production of extracellular vesicles from human cardiovascular progenitors. European Heart Journal 39, 20, 2018.
3. Correia, C., et al. Distinct carbon sources affect structural and functional maturation of cardiomyocytes derived from human pluripotent stem cells. Scientific reports 7, 1, 2017.
4. Correia, C., et al. 3D aggregate culture improves metabolic maturation of human pluripotent stem cell derived cardiomyocytes. Biotechnology and Bioengineering 115, 3, 2018
Erbi Biosystems - Cell culture development with a 2 mL continuous perfusion bioreactor
Get PDFWith five chimeric antigen receptor (CAR) T cell immunotherapy products currently approved by the FDA for the treatment of hematological cancers, there are increasing efforts to develop novel and better manufacturing technologies and processes for cell therapies to improve efficacy, reduce variability, and reduce cost. Knowledge in the field about how to optimize cell expansion for consistent and reproducible cell-based treatments is improving, but major challenges still exist in experiment reproducibility and robustness during process development. For autologous processes, donor variability and differences between healthy donor-derived versus patient-derived material can confound experiment results. Materials are also costly, including peripheral blood mononuclear cells (PBMCs), growth factors, viral vectors, and serum or chemically defined media. Therefore, process development experiments in cell therapy are typically done in milliliter sized, often static culture with minimal environmental control. While these systems can generate data quickly, the lack of control and monitoring can result in variabilities that may prove difficult to translate to larger culture systems. To address this gap, Erbi Biosystems has developed the Breez™ True Perfusion™ bioreactor. This fully closed sterile single-use perfusion bioreactor operates at a 2 mL working volume and can operate outside of a biosafety cabinet to replicate bench scale perfusion processes to industrially relevant cell densities in excess of 100e6 cells/mL. The advanced microfluidics, including bubble free mixing, on-line cell density monitoring, and automated dO/pH control allow the Breez™ to achieve excellent cell growth in a 2 mL scale, reducing labor and bench space required. By introducing a milliliter scale perfusion reactor, material from a single patient can be used for many bioreactor experiments, enabling process development under controlled conditions. Using the Erbi Breez™ bioreactor, we have demonstrated equivalent performance to a static 24-well G-rex for expansion of CAR-transduced T cells when supplying the same total volume of media, with similar phenotypes at the end of a 14-day culture. We then explore the advanced capability provided by the Breez™ automated perfusion system to perform in-place activation, transduction, and expansion. We show that with improved media exchange rates, cell expansion performance is improved, achieving nearly patient dose levels with more than 400-fold expansion from 0.6 million cells at the point of transduction to more than 200 million cells at 14 days post-transduction at consistently high cell viability of above 95%. This is in contrast to maximum viable cell density (VCD) in the range of 10e6 cells/mL for the 24-well G-rex, with a similar total media volume consumed to total cell number ratio. Initial transduction efficiency was comparable between the bioreactor (without transduction enhancer) and spinoculation on a retronectin-coated 24-well plate. Additionally, on-line data showing optical density, pH, and dissolved oxygen will be presented, providing insight into the metabolic state of cells during culture. These data show that the Breez™ platform is well suited for cell therapy process development and process characterization studies and having already demonstrated cell doses nearly sufficient for patient infusion, may be a promising future production platform
Protocol development to overcome bioprocess bottlenecks in the large-scale expansion of high quality hIPSC aggregates in vertical-wheel bioreactors
Get PDFHuman-induced pluripotent stem cells (hiPSCs) have generated a great deal of attention owing to their capacity for self-renewal and trilineage differentiation. hiPSCs are cultured as adherent colonies at small scale, which is sufficient to generate cells for experimental purposes but impractical to achieve large quantities for clinical applications. Bioreactor-based processes are the method of choice for efficient expansion and differentiation of cells. Current protocols for the expansion of hiPSCs, however, utilize horizontal impeller, paddle, or rocking wave mixing method bioreactors which require large static cell-culture starting populations and achieve only moderate cell fold increases within the bioreactor. We have recently demonstrated that the vertical-wheel bioreactor produces a unique fluid flow pattern that results in a homogeneous distribution of hydrodynamic forces, making it the opportune environment for systematic bioprocess optimization of hiPSC expansion.
Please click Additional Files below to see the full abstract
Manufacturing of gene-modified human mesenchymal stromal cells in microcarriers and agitated conditions
Get PDFHuman mesenchymal stromal cells (hMSCs) are one promising cell type whose properties have been extensively investigated over the last two decades. During this period, the safety and efficacy of these cells were evaluated in over 900 clinical trials worldwide. Due to their safety profile and their short life span following administration, hMSCs have attracted interest not only as cell therapy products but also as cellular agents to deliver genes with anti-cancer or pro-vascular properties, amongst others. The focus of this study was to develop a viral-based method to introduce genes of interest (GoI) into the hMSCs and subsequently expand the transduced cells in microcarriers and agitated conditions.
A 2nd generation lentiviral vector system (LV) was used to prepare the vector carrying either green fluorescent protein (GFP) or vascular endothelial growth factor (VEGF) genes to transduce the umbilical cord tissue human mesenchymal stem cells (UCT-hMSCs). The LV system consisted of a packaging, an envelope and a transgene plasmid. After quantifying both physical and infectious LV titres, the vector was used to transduce UCT-hMSCs at a multiplicity of infection (MOI) of 2. One additional passage in monolayer was performed before the cells were expanded in agitated conditions. The microcarrier-based suspension expansion was then carried out using spinner flasks. The working volume was set at 40 mL on day 0, and an intermittent attachment protocol was performed (25 minutes rested, 5 minutes agitated at 25 rpm during the first 8 hours). One day after seeding, the working volume was increased to 80 mL, and medium exchange was performed using 50% of the active volume every other day.
Compared to untransduced UCT-hMSCs, both GFP and VEGF transduced cells (UCT-hMSCs-GFP and UCT-hMSCs-VEGF, respectively) showed similar growth kinetics, reaching similar maximum cell concentrations. The metabolite analysis of the untransduced and gene-modified cells identified comparable glucose, lactate, and ammonia consumption/production patterns. From a characterization standpoint, the gene-modified cells showed over 90% expression of hMSCs-markers (CD73, CD90 and CD105) together with less than 10% expression for negative markers (HLA-DR, CD11b, CD19, CD34 and CD45). The immunophenotype obtained was comparable to the one observed for untransduced UCT-hMSCs. This study has demonstrated the feasibility of using LV vectors to transduce hMSCs, and once genetically modified, the cells were successfully expanded using microcarriers in spinners flasks
Scalable downstream purification of recombinant adeno-associated viral vectors
Get PDFScalable manufacturing technologies are essential for ensuring modern medicines can be produced to meet the needs of clinical trials, process development, and commercial manufacture. Recent advances in in vivo gene therapies have resulted in multiple regulatory approvals of rAAV vectors for gene transfer in humans. These vectors can be produced using transient transfection of mammalian cells, baculovirus infection of insect cells or produced via engineered stable producer cells. These production methods are performed in single-use bioreactors and utilize other scalable technologies as used in commercial monoclonal antibody manufacture. In this work, we evaluated the use of existing single-use filtration and separation technologies for downstream purification of an rAAV5 viral vector. rAAV5 vector was produced by transient transfection of HEK293 cells in the Pall iCELLis® Nano bioreactor. Bioreactor harvest lysis material was clarified using direct flow filtration with both depth and sterilizing grade filters. The product was concentrated 10x using 100kD OmegaTM flat-sheet tangential flow-filtration (TFF) before primary purification using affinity chromatography. The rAAV5 vector was then polished using Mustang® Q membrane chromatography to enrich for full capsids. A second TFF step was performed to concentrate and buffer exchange with flat sheet TFF with the same 100KD Omega membrane. Final sterile filtration was performed using Supor® EKV validated sterilizing grade filters. All downstream unit operations resulted in acceptable performance. Feasibility of a complete downstream process was established with a theoretical whole process yield of ~25%. This process results in a very low contaminant profile as host cell protein (HCP) and host cell DNA were reduced to near and below the assays’ limits of quantitation during purification. Of particular interest, Mustang Q polishing resulted in retention of only ~10% of total capsids, while recovering ~50% of full capsids enriching the ratio of full capsids to empty capsids by 4.5 fold
Directed evolution of novel AAV vectors for clinical gene therapy
Get PDFGene therapy – the delivery of genetic material to the cells of a patient for therapeutic benefit – has been increasingly successful in human clinical trials over the past decade, and there are numerous FDA-approved gene therapies. The most successful gene delivery vehicles, or vectors, are based on adeno-associated viruses (AAV); however, vectors based on natural versions of AAV face a number of delivery barriers that limit their efficacy and will thus preclude the extension of these successes to the majority of human diseases. Furthermore, efforts to overcome these barriers simply by increasing dose incur major manufacturing challenges and risk inflammatory responses within patients. Such delivery limitations arise since the parent viruses upon which these vectors are based were not evolved by nature for our convenience to use as human medicines. Unfortunately, due to the highly complex mechanisms of virus-host interactions, there is currently insufficient mechanistic knowledge to enable rational design to be sufficiently successful in creating new vectors. As an alternative, however, we developed the concept of using directed evolution to engineer highly optimized variants of AAV for a broad range of cell and tissue targets. Directed evolution involves the iterative genetic diversification of a biomolecule to create a gene pool and functional selection to isolate variants with optimal properties. Using this approach, we have engineered AAV variants with greatly improved delivery efficiency to multiple organs including the retina; lung, and muscle; targeted delivery to specific cell types; and the capacity to evade immune responses. Our novel AAV variants are currently used in 8 human clinical trials involving delivery to the retina, heart, and lung. Furthermore, we have been employing genomewide CRISPR/Cas9- based screening to identify genes whose overexpression in manufacturing cells results in increased AAV production. The integration of high throughput screening technologies to improve AAV efficacy and production can enable a broad range of basic and therapeutic gene delivery applications
Preliminary evaluation of PendoTECH® single use sensors post X-ray irradiation
Get PDFWith the ongoing growth of the biopharmaceutical, vaccine, and cell and gene therapy markets, and the increasing demand on existing sterilization facilities for materials required in manufacturing processes, X-ray irradiation for sterilization has caught the eye of the industry. As an innovative industry leader, PendoTECH has evaluated the compatibility of its various Single Use Sensors with X-ray irradiation for sterilization. This poster details a preliminary study performed on PendoTECH Single Use Pressure, UV/Turbidity, and Temperature sensors. A small sample size of sensors was X-ray irradiated at 41.5 to 44.6 kGy, and then checked for physical integrity (via leak testing) and accuracy to validate the performance of the sensors. All pressure, UV/turbidity, and temperature sensors were demonstrated to be reliable, and were within specification post X-ray irradiation. Although this is a preliminary study, these results suggests that PendoTECH sensors are compatible with X-ray irradiation and they are expected to pass the full qualification currently in progress.
Please click Additional Files below to see the full abstract
A new single-use disk stack separator for pilot scale CHO cell-based antibody productions
Get PDFSingle-use systems are well-established in modern antibody productions. This is especially true for upstream processing (cell production, fermentation and cell harvesting), where complete single-use lines have become reality [1]. However, it has proven rather difficult to convert centrifugation for cell harvesting to single-use technologies, even though Sartorius and PneumaticScale Angelus have had solutions available for some time with their KSep (fluidized bed technology) and Carr Centritech UniFuge (conventional separation) systems. Previously, there were no single-use disk stack separators on the market. However, such a system, the kytero® 500, has been developed by GEA and was launched in summer 2021.
This poster summarizes the results of tests of the kytero® 500 with CHO suspension cells. The biomass to be separated was from fed-batch cultivations of immunoglobulin G (IgG) producing ExpiCHO-S cells in a 200 L BIOSTAT STR from Sartorius. The cells were cultivated with Gibco’s chemically defined media (Efficient-ProTM Medium and Efficient-ProTM Feed 2). Separation of the biomass from the culture broth was performed at a living cell density of 17 x 106 cells mL-1 and a viability of 98.9%. To study the performance of this new centrifuge over as wide a range as possible, various separation parameters, including separator speed, feed flow, and biomass concentration in concentrate, were tested.
Regardless of the separator settings, no solid particles were detected in the centrate, which was also confirmed by turbidity measurements. Nephelometric Turbidity Unit-based clarification efficiencies ranged from 96.6% to 97.5%. In addition, no increase in DNA or Host Cell Protein concentration in the IgG-containing centrate phase and no loss of IgG were detected. The biomass was concentrated to a maximum of 30-fold, yielding 99.3% of the liquid phase. The kytero® 500 is characterized by its ease of use and compact design. The highly efficient cell separation delivered by the kytero® 500 may result in a smaller depth filter area in product purification processes and thus a reduction in process costs.
1. BioPlan Associates, 17th annual report and survey of biopharmaceutical manufacturing capacity and production: A study of biotherapeutic developers and contract manufacturing organizations, 2020