Bioprocessing and engineering characterisation of T-cell therapy manufacture in an ambr® 250 bioreactor

The use of engineered CAR-T cells in clinical trials has been growing over the last years. The recent approval of Kymriah® (Novartis) and Yescarta® (KitePharma) made CAR-T cell treatments available to a broader public. However, despite the recent successes and significant improvements, there are different aspects that need to be further assessed in order to develop a reproducible, cost-effective manufacturing process for the production of personalized T-cell therapies. This requires an approach, which generates sufficient quantities of patient-specific cells at the appropriate quality required for clinical application, overcoming the challenge imposed by significantly different starting material. Please click Additional Files below to see the full abstract

A cost/quality analysis of primary human T-Cells in different expansion systems

Recent developments in cell and gene therapy, in particular the emergence of Chimeric Antigen Receptor (CAR) T-cell immunotherapies, have demonstrated a new therapeutic modality to combat life-threatening diseases. Kymriah® (Novartis) and Yescarta® (Gilead) are the first CAR-T therapies that have been approved by both FDA and EMA. They are autologous therapies, where T-cells are isolated from the patient and engineered in order to express a specific CAR which recognizes and targets cancer cells. The clinical success of these products is driving the need for a consistent and cost effective manufacturing process to meet the lot-sizes required for commercial production. Current T-cell manufacturing and production capabilities are greatly lagging and will not be able to meet the predicted surge in demand (£6.8 bn by 2028, CAGR of 46%) unless new technologies and processes are developed. These technologies need to be highly regulated and consistently achieve high yield, without compromising the quality of the cells, irrespective of donor. A number of systems have been used for CAR-T cell expansion, namely flasks, bags (static or dynamic), G-Rex® (WolfonWilson) and CliniMACS Prodigy® (Miltenyi Biotec). All these technologies have many advantages, but they generally lack in scalability, suffer from manufacturing bottlenecks or incur in high suppliers costs and manual intervention. The aim of this work is to understand the relation between reduction of production costs and cell growth quality. We compared the growth of human primary T-cells from multiple healthy donors in the aforementioned culture platforms, characterizing cell growth and phenotype. Cells were expanded for 7 days in each of the platforms with the aim of comparing the different systems with respect to scalability, productivity and efficiency. The phenotype composition of the final product was determined by flow cytometry. Please click Additional Files below to see the full abstract

Process development and manufacture of primary human T-cells in scalable, automated stirred-tank bioreactors

Engineered Chimeric Antigen Receptor (CAR) T-cell products have recently gained FDA and EMA approval and have demonstrated significant clinical efficacy against non-Hodgkin lymphoma and pediatric B-cell acute lymphoblastic leukemia. Despite the significant clinical and commercial progress these products represent, the high costs associated with patient-specific cell therapy manufacture needs to be addressed. The work presented here focuses on the growth of human primary T-cells and CAR-T cells across a range of commercially available expansion platforms, including stirred tank bioreactors, which although routinely employed for the production of biologics, are not commonly used for the manufacture of T-cells. Initial experimental studies were carried out in an automated ambr® 250 single use bioreactor system which has demonstrated significant success for suspension and adherent mammalian cell culture applications. Building on previous work undertaken in the group which developed a new bioreactor vessel for microcarrier culture, both the new and existing bioreactor vessels were characterized with respect to cell yield, fold expansion, viability, metabolite profile, T-cell subpopulations and kLa. The comparison between the two vessels was performed based on power per unit volume, kLa and stirring speed, ranging from 100 to 200 rpm, using at least 3 different donors per condition. Please click Additional Files below to see the full abstract

Development and testing of advanced methods for the screening of Enhanced-Oil-Recovery techniques

Enhanced Oil Recovery (EOR) techniques must undergo preliminary laboratory and pilot testing before implementation to field-wide scale, and the whole evaluation process requires heavy investments. Hence forecasting EOR potential is a key decision-making element. A critical difference amongst EOR techniques resides in the oil-displacement mechanism upon which they are based. The effectiveness of these mechanisms depends on oil and reservoir properties. As such, similar EOR techniques are typically successful in fields sharing similar features. Here we implement and test a screening method aimed at estimating the optimal EOR technique for a target reservoir. Our approach relies on the information content tied to an exhaustive set of EOR field experiences. The basic screening criterion is the analogy with known reservoir settings in terms of oil and formation properties. Analogy is assessed by grouping fields into clusters: we rely on a Bayesian hierarchical clustering algorithm, whose main advantage is that the number of clusters is not set a priori but stems from data statistics. As a test bed, we perform a blind test of our screening approach by considering 2 fields operated by eni. Our predictions for analogy assessment are in agreement with the EOR techniques applied or planned in these fields

Scalable manufacturing of gene-modified human mesenchymal stromal cells with microcarriers in spinner flasks

Due to their immunomodulatory properties and in vitro differentiation ability, human mesenchymal stromal cells (hMSCs) have been investigated in more than 1000 clinical trials over the last decade. Multiple studies that have explored the development of gene-modified hMSC-based products are now reaching early stages of clinical trial programmes. From an engineering perspective, the challenge lies in developing manufacturing methods capable of producing sufficient doses of ex vivo gene-modified hMSCs for clinical applications. This work demonstrates, for the first time, a scalable manufacturing process using a microcarrier-bioreactor system for the expansion of gene-modified hMSCs. Upon isolation, umbilical cord tissue mesenchymal stromal cells (UCT-hMSCs) were transduced using a lentiviral vector (LV) with green fluorescent protein (GFP) or vascular endothelial growth factor (VEGF) transgenes. The cells were then seeded in 100 mL spinner flasks using Spherecol microcarriers and expanded for seven days. After six days in culture, both non-transduced and transduced cell populations attained comparable maximum cell concentrations (≈1.8 × 105 cell/mL). Analysis of the culture supernatant identified that glucose was fully depleted after day five across the cell populations. Lactate concentrations observed throughout the culture reached a maximum of 7.5 mM on day seven. Immunophenotype analysis revealed that the transduction followed by an expansion step was not responsible for the downregulation of the cell surface receptors used to identify hMSCs. The levels of CD73, CD90, and CD105 expressing cells were above 90% for the non-transduced and transduced cells. In addition, the expression of negative markers (CD11b, CD19, CD34, CD45, and HLA-DR) was also shown to be below 5%, which is aligned with the criteria established for hMSCs by the International Society for Cell and Gene Therapy (ISCT). This work provides a foundation for the scalable manufacturing of gene-modified hMSCs which will overcome a significant translational and commercial bottleneck. KEY POINTS: • hMSCs were successfully transduced by lentiviral vectors carrying two different transgenes: GFP and VEGF • Transduced hMSCs were successfully expanded on microcarriers using spinner flasks during a period of 7 days • The genetic modification step did not cause any detrimental impact on the hMSC immunophenotype characteristics

Characterization of a single-use stirred-tank bioreactor vessel for microcarrier-based adherent cell culture processes using experimental and computational fluid dynamics studies

Renewed interest in microcarrier-based processes for the large-scale culture of adherent cells for vaccine and cell therapy applications drives the need for effective, high-throughput, single-use, process development tools that can be translated successfully into industrial-scale systems. The automated ambr250® platform is one such technology, operating at a volume between 100 – 250mL and which is both high-throughput and single-use. The ambr250 has demonstrated significant success for suspension-based mammalian cell culture applications. However, no studies have been reported investigating microcarrier-based processes for the culture of adherent cells. With any cell culture process, the fluid dynamics characteristics of the bioreactor must be sufficiently well understood to enable successful scale-up to larger scale bioreactors. With microcarriers, there is an additional challenge as the fluid dynamics must take into account the presence of the particulate solid phase. A critical aspect for cell cultivation on microcarriers is the minimum agitator speed required to achieve complete microcarrier suspension, NJS. Under these conditions, the surface area of the attached cells is available for transfer of nutrients (including oxygen) to the cells and metabolites from them, whilst higher speeds hardly increase these transport processes and may lead to damaging fluid dynamic stresses being generated1. This suspension condition can be studied experimentally if equipment is specially modified to make easy visual observation of the two-phase flow in the bioreactor which during actual culture is very difficult. Therefore, it is extremely beneficial to both measure NJS and then to compare the measured values with predictions based on computational fluid dynamics (CFD) in order to validate the latter. Once validated, CFD modelling is a very useful tool for analysing flow patterns, mixing time, mean and local specific energy dissipation rates and other parameters important for scale up in order to optimise the overall bioreactor geometry. In addition to the above fluid dynamic aspects, cell culture studies was also performed in parallel to analyse the cell growth at and around the minimum speed for microcarrrier suspension, NJS and the results were compared to the culture performance in well-characterised traditional spinner flask bioreactors2.The CFD and experimental results with the single-use ambr250 bioreactor will be discussed in detail along with their scale-up implications. References 1. Nienow, A. W., Coopman, K., Heathman, T. R. J., Rafiq, Q. A. and C. J. Hewitt (2016). “Bioreactor Engineering Fundamentals for Stem Cell Manufacturing”. In: “Stem Cell Manufacturing”, (Eds. J.M.S. Cabral, C.L. de Silva, L. G. Chase and M. M. Diogo), Elsevier Science, Cambridge, USA; Chapter 3, pp 43 – 76. 2. Rafiq, Q. A., Brosnan, K. M., Coopman, K., Nienow, A. W. and Hewitt, C.J. (2013) Culture of Human Mesenchymal Stem Cells on Microcarriers in a 5 L Stirred-Tank Bioreactor. (with Q. A. Rafiq, K. M. Brosnan, K

Characterization of a single-use stirred-tank bioreactor vessel for microcarrier-based adherent cell culture processes using experimental and computational fluid dynamics studies

Renewed interest in microcarrier-based processes for the large-scale culture of adherent cells for vaccine and cell therapy applications drives the need for effective, high-throughput, single-use, process development tools that can be translated successfully into industrial-scale systems. The automated ambr250® platform is one such technology, operating at a volume between 100 – 250mL and which is both high-throughput and single-use. The ambr250 has demonstrated significant success for suspension-based mammalian cell culture applications. However, no studies have been reported investigating microcarrier-based processes for the culture of adherent cells. With any cell culture process, the fluid dynamics characteristics of the bioreactor must be sufficiently well understood to enable successful scale-up to larger scale bioreactors. With microcarriers, there is an additional challenge as the fluid dynamics must take into account the presence of the particulate solid phase. A critical aspect for cell cultivation on microcarriers is the minimum agitator speed required to achieve complete microcarrier suspension, NJS. Under these conditions, the surface area of the attached cells is available for transfer of nutrients (including oxygen) to the cells and metabolites from them, whilst higher speeds hardly increase these transport processes and may lead to damaging fluid dynamic stresses being generated1. This suspension condition can be studied experimentally if equipment is specially modified to make easy visual observation of the two-phase flow in the bioreactor which during actual culture is very difficult. Therefore, it is extremely beneficial to both measure NJS and then to compare the measured values with predictions based on computational fluid dynamics (CFD) in order to validate the latter. Once validated, CFD modelling is a very useful tool for analysing flow patterns, mixing time, mean and local specific energy dissipation rates and other parameters important for scale up in order to optimise the overall bioreactor geometry. In addition to the above fluid dynamic aspects, cell culture studies was also performed in parallel to analyse the cell growth at and around the minimum speed for microcarrrier suspension, NJS and the results were compared to the culture performance in well-characterised traditional spinner flask bioreactors2.The CFD and experimental results with the single-use ambr250 bioreactor will be discussed in detail along with their scale-up implications. References 1. Nienow, A. W., Coopman, K., Heathman, T. R. J., Rafiq, Q. A. and C. J. Hewitt (2016). “Bioreactor Engineering Fundamentals for Stem Cell Manufacturing”. In: “Stem Cell Manufacturing”, (Eds. J.M.S. Cabral, C.L. de Silva, L. G. Chase and M. M. Diogo), Elsevier Science, Cambridge, USA; Chapter 3, pp 43 – 76. 2. Rafiq, Q. A., Brosnan, K. M., Coopman, K., Nienow, A. W. and Hewitt, C.J. (2013) Culture of Human Mesenchymal Stem Cells on Microcarriers in a 5 L Stirred-Tank Bioreactor. (with Q. A. Rafiq, K. M. Brosnan, K

Experimental and Computational Fluid Dynamics study of microcarrier suspension during the cultivation of Mesenchymal Stem Cells in an ambr250 bioreactor

The ambr250 unit is a fully automated disposable 100-250 ml bioreactor for R&D that has been developed by TAP Biosystems, now part of Sartorius-Stedim, widely used for scale down and scale up modelling studies. Recently, mesenchymal stem cells (MSCs) have become strong candidates for cell-based therapies based on in vitro growth on microcarriers in stirred bioreactors. However, to fully realize the MSCs potential, a number of key processing issues need to be addressed because of the huge number of cells that are required. Thus, the fluid dynamics characteristics of the stirred ambr250 bioreactor must be sufficiently well understood to enable scale-up to larger bioreactors to be efficiently accomplished particularly because of the special issues arising from the presence of the particulate solid phase. One of the most critical aspects for MSC cultivation on microcarriers is the minimum agitator speed required to achieve complete microcarriers suspension, NJS. Under these conditions, the surface area of all the attached cells is available for transfer of nutrients (including oxygen) to the cells and metabolites from them, whilst higher speeds hardly increase these transport processes and may lead to damaging fluid dynamic stresses being generated1. This suspension condition can be studied experimentally if equipment is specially modified to make easy visual observation of the two-phase flow in the bioreactor but during actual growth that is very difficult. Therefore, it is extremely beneficial to both measure NJS and compare the measured values with predictions based on computational fluid dynamics (CFD) for validation. Once validated, then CFD is a very useful tool for analyzing flow patterns, mixing time, mean and local specific energy dissipation rates and other parameters important for scale up. In this work we examined the fluid dynamics of the two-phase particle-liquid system with an experimental analysis and a CFD simulation using a lattice-Boltzmann base software and particle tracking of an ambr250 vessel at different stirring conditions. Cell culture was also performed in parallel to analyse the cell growth at and around NJS and the results were compared to the performance in a spinner flask bioreactor. The CFD and experimental results will be discussed in detail along with their scale-up implications. References 1) Nienow, A. W., Coopman, K., Heathman, T. R. J., Rafiq, Q. A. and C. J. Hewitt (2016). “Bioreactor Engineering Fundamentals for Stem Cell Manufacturing”. In: “Stem Cell Manufacturing”, (Eds. J.M.S. Cabral, C.L. de Silva, L. G. Chase and M. M. Diogo), Elsevier Science, Cambridge, USA; Chapter 3, pp 43 – 76

Italy

This report is part of the project The Factbook on the Illicit Trade in Tobacco Products. It focuses on Italy, where the illicit trade in tobacco seems to have grown in recent years. This fact, combined with the geographical location of the country and the consolidated presence of organised crime, makes Italy an interesting country to explore in terms of ITTP flows in the Mediterranean basin and towards North European countries

Experimental and computational fluid dynamics studies of adherent cells on microcarriers in an ambr® 250 bioreactor

Interest for microcarrier-based processes for the large-scale culture of adherent cells has recently grow, due to possible application in vaccine and cell therapy. This opportunity drives the need for effective, high-throughput, single-use, process development tools that can be translated successfully into industrial-scale systems. The automated ambr® 250 platform is one such technology, operating at a volume between 100 – 250mL, both high-throughput and single-use. The ambr250 has demonstrated significant success for suspension-based mammalian cell culture applications. However, additional investigations need to be performed on microcarrier-based processes for the culture of adherent cells. The fluid dynamics characteristics of the bioreactor must be sufficiently well understood to enable successful scale-up to larger scale bioreactors. Physical parameters such as fluid velocity, power number and shear stress are important for any cell culture. With microcarriers, there is an additional challenge as the fluid dynamics must take into account the presence of the particulate solid phase. A critical aspect for cell cultivation on microcarriers is the minimum agitator speed required to achieve complete microcarrier suspension, NJS. Under these conditions, the surface area of the attached cells is available for transfer of nutrients (including oxygen) to the cells and metabolites from them, whilst higher speeds hardly increase these transport processes and may lead to damaging fluid dynamic stresses being generated. It is also extremely beneficial to predict the flow dynamics of the stirred tank based on computational fluid dynamics (CFD). Once validated, CFD modelling is a very useful tool for analysing flow patterns, mixing time, mean and local specific energy dissipation rates, shear stress, and other parameters important for scale up in order to optimise the overall bioreactor geometry. In addition to the above fluid dynamic aspects, cell culture studies was also performed in parallel to analyse the cell growth at and around the minimum speed for microcarrier suspension, NJS. The CFD and experimental results with the single-use ambr250 bioreactor will be discussed in detail during the final presentation