An energy-based modelling tool for culture medium design and biomanufacturing optimization

Demand for high-value biologics, a rapidly growing pipeline, and pressure from competition, time-to-market and regulators, necessitate novel biomanufacturing approaches, including Quality by Design (QbD) principles and Process Analytical Technologies (PAT), to facilitate accelerated, efficient and effective process development platforms that ensure consistent product quality and reduced lot-to-lot variability. Herein, QbD and PAT principles were incorporated within an innovative in vitro-in silico integrated framework for upstream process development (UPD). The central component of the UPD framework is a mathematical model that predicts dynamic nutrient uptake and average intracellular ATP content, based on biochemical reaction networks, to quantify and characterize energy metabolism and its adaptive response, metabolic shifts, to maintain ATP homeostasis. The accuracy and flexibility of the model depends on critical cell type/product/clone-specific parameters, which are experimentally estimated. The integrated in vitro-in silico platform and the model’s predictive capacity reduced burden, time and expense of experimentation resulting in optimal medium design compared to commercially available culture media (80% amino acid reduction) and a fed-batch feeding strategy that increased productivity by 129%. The framework represents a flexible and efficient tool that transforms, improves and accelerates conventional process development in biomanufacturing with wide applications, including stem cell-based therapies

Key environmental stress biomarker candidates for the optimisation of chemotherapy treatment of leukaemia

The impact of fluctuations of environmental parameters such as oxygen and starvation on the evolution of leukaemia is analysed in the current review. These fluctuations may occur within a specific patient (in different organs) or across patients (individual cases of hypoglycaemia and hyperglycaemia). They can be experienced as stress stimuli by the cancerous population, leading to an alteration of cellular growth kinetics, metabolism and further resistance to chemotherapy. Therefore, it is of high importance to elucidate key mechanisms that affect the evolution of leukaemia under stress. Potential stress response mechanisms are discussed in this review. Moreover, appropriate cell biomarker candidates related to the environmental stress response and/or further resistance to chemotherapy are proposed. Quantification of these biomarkers can enable the combination of macroscopic kinetics with microscopic information, which is specific to individual patients and leads to the construction of detailed mathematical models for the optimisation of chemotherapy. Due to their nature, these models will be more accurate and precise (in comparison to available macroscopic/black box models) in the prediction of responses of individual patients to treatment, as they will incorporate microscopic genetic and/or metabolic information which is patient-specific.peer-reviewe

Patients with gastrointestinal irritability after TGN1412-induced cytokine 2 storm displayed selective expansion of gut-homing αβ and γδ T-cells

This is an accepted manuscript of an article published by Springer in Cancer Immunology, Immunotherapy on 13/10/2020, available online at: https://doi.org/10.1007/s00262-020-02723-4 The accepted version of the publication may differ from the final published versionFollowing infusion of the anti-CD28 superagonist monoclonal antibody TGN1412, three of six previously healthy, young male recipients developed gastrointestinal irritability associated with increased expression of ‘gut-homing’ integrin β7 on peripheral blood αβT-cells. This subset of patients with intestinal symptoms also displayed a striking and persistent expansion of putative Vδ2+ 7 γδT-cells in the circulation which declined over a two-year period following drug infusion, concordant with subsiding gut symptoms. These data demonstrate that TGN1412-induced gastrointestinal symptoms were associated with dysregulation of the ‘gut-homing’ pool of blood αβ and γδT11 cells, induced directly by the antibody and/or arising from the subsequent cytokine storm

Biology of dendritic cells in health and in acute myeloid leukaemia

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Differentiation of murine embryonic cells towards the haematopoietic cell lineage using the HepG2 conditioned medium and encapsulation in a rotating wall vessel bioreactor

Embryonic stem cells (ESCs) are known for their unique property to be maintained almost indefinitely in an undifferentiated, proliferating state with the potential to give rise to all types of cells. Current established protocols for the culture and differentiation of ESCs are cumbersome and inefficient involving three stages: a) maintenance or expansion of undifferentiated ESCs, b) spontaneous differentiation through formation of embryoid bodies (EBs), and c) dissociation of the EBs and replating leading to the terminal differentiation to the desired lineages. One of the major challenges in the use of ESCs for cell therapy is controlling their differentiation pathways. Optimal culture conditions and requirement as well as precise differentiation mechanisms and cellular interactions within EBs are still not well characterised resulting in sub-optimal control of homogenous differentiation especially due to the formation of all three germ layers. Attempts on developing an efficient culture protocol have been widely reported in order to overcome the limitations. Recent research approaches have shown that treatment with conditioned medium derived from HepG2, a human hepatocarcinoma cell line enhances the formation of multipotent mesodermal progenitors from ESCs. This promotes a greater control of ESC differentiation in a lineage-specific fashion possibly resulting in efficient haematopoietic differentiation. In this study, we have developed an integrated, single step bioprocess for ESCs hematopoietic differentiation that: a) uses HepG2-conditioned medium (HepG2-CM), that stimulates mesoderm formation, b) facilitates three dimensional (3D) culture through encapsulation of undifferentiated ESCs in hydrogels, c) bypasses EB formation, and d) involves culture in a rotating wall vessel bioreactor that does not require passaging of the cells and is scalable and automatable. In conclusion, this thesis reports the development of a novel culture system for the efficient single-step haematopoietic differentiation of ESC resulting in a reproducible, scalable, high-intensity culture system of mESCs for ex-vivo blood manufacture.EThOS - Electronic Theses Online ServiceMinistry of Higher Education Malaysia, Richard Thomas Leukaemia Fund and Northwick Park Hospital Leukaemia Research Trust FundGBUnited Kingdo

Differentiation of murine embryonic cells towards the haematopoietic cell lineage using the HepG2 conditioned medium and encapsulation in a rotating wall vessel bioreactor

Embryonic stem cells (ESCs) are known for their unique property to be maintained almost indefinitely in an undifferentiated, proliferating state with the potential to give rise to all types of cells. Current established protocols for the culture and differentiation of ESCs are cumbersome and inefficient involving three stages: a) maintenance or expansion of undifferentiated ESCs, b) spontaneous differentiation through formation of embryoid bodies (EBs), and c) dissociation of the EBs and replating leading to the terminal differentiation to the desired lineages. One of the major challenges in the use of ESCs for cell therapy is controlling their differentiation pathways. Optimal culture conditions and requirement as well as precise differentiation mechanisms and cellular interactions within EBs are still not well characterised resulting in sub-optimal control of homogenous differentiation especially due to the formation of all three germ layers. Attempts on developing an efficient culture protocol have been widely reported in order to overcome the limitations. Recent research approaches have shown that treatment with conditioned medium derived from HepG2, a human hepatocarcinoma cell line enhances the formation of multipotent mesodermal progenitors from ESCs. This promotes a greater control of ESC differentiation in a lineage-specific fashion possibly resulting in efficient haematopoietic differentiation. In this study, we have developed an integrated, single step bioprocess for ESCs hematopoietic differentiation that: a) uses HepG2-conditioned medium (HepG2-CM), that stimulates mesoderm formation, b) facilitates three dimensional (3D) culture through encapsulation of undifferentiated ESCs in hydrogels, c) bypasses EB formation, and d) involves culture in a rotating wall vessel bioreactor that does not require passaging of the cells and is scalable and automatable. In conclusion, this thesis reports the development of a novel culture system for the efficient single-step haematopoietic differentiation of ESC resulting in a reproducible, scalable, high-intensity culture system of mESCs for ex-vivo blood manufacture.EThOS - Electronic Theses Online ServiceMinistry of Higher Education Malaysia, Richard Thomas Leukaemia Fund and Northwick Park Hospital Leukaemia Research Trust FundGBUnited Kingdo