Specific exosomes as novel media component to enhance manufacturing cell lines.

Lead Research Organisation: University College London
Department Name: Biochemical Engineering

Abstract

Characterisation and quantification of exosomes of manufacturing cell lines with a prefered phenotype (growth / productivity). Analysing the function of those exosomes by applying them in a quantitative manner on cells with contray phenotypes (slow to fast growth, low to high productivity and vice versa).
Generating cell lines which stably expresses exosomes loaded with a defined set of RNA/Protein and applying those exosomes to CHO cells to validate exosome triggered effects on phenotype.
The standard procedure in cell line development to select cells with the required attributes for manufacturing takes up to 6 months from DNA to clonal cell line. Reducing this period of time and improving product quantity as well as quality will save laboratory costs and help to reduce the overall project timelines and risks of failure. During the last decade an increasing number of publications propose that exosomes are able to influence the characteristics of up taking cells. Considering this the exosomes could be employed to shift the averaged behaviour of cell populations towards preferred characteristics like growth or productivity. To investigate potential effects, exosomes of cell lines with different phenotypes will be characterised, quantified and applied to cell lines with contrary phenotypes. The development of specific exosomes which drive cell phenotypes towards preferred behaviour enables the design of a novel tool box to enhance the cell line development on the molecular level in real time. Furthermore it extends our knowledge how to produce and purify exosomes on manufacturing scale which can have major impact for the exosome usage as a therapeutic format.
First, fundamental baseline understanding will be created by isolating from CHO pools cells that are either a) rapid growing or b) slow growing. These suspension-adapted cells will be propagated in shake flasks for 2 weeks and then resulting exosome product concentrated using tangiential flow filtration using techniques already available in the UCL Department of Biochemical Engineering. Exosomes from rapid growing cells will then be added to slow growing cells in culture (and vice versa) to determine whether exosomal signalling can direct cell behaviour. A matrix of experimental conditions that assesses timing, dosage and number of exosome doses will explore the effect strength over time in culture. A second set of experiments assessing CHO cells that have a) high productivity versus b) low productivity will be undertaken, whereby exosome product isolated from high producers will be used as a cell culture supplement for low producing cells (and vice versa). A range of measurements will be used to characterise exosomes, including nanoparticle number, size, concentration, RNA, DNA, protein.
Generating CHO cells that stably express reporter exosomes (generated using a commercial kit) where exosomes are loaded with a defined set of RNAs/proteins that elicit known responses will provide validation that exosomes are correctly targeting and regulating cell behaviour. Ambr15 will be used in fed-batch mode to validate the preliminary studies. During the second half of the project, a similar approach to that listed above will be undertaken to assess the effect of cell state-specific exosomes on adherent HEK-293 cells.

Planned Impact

The benefits of the proposed EPSRC Centre for Doctoral Training (CDT) aligned to the EPSRC Centre for Innovative Manufacture of Emergent Macromolecular Therapies will be significant. The CDT will address an acute skills storage of trained manpower, needed to take this industry forward for the benefit of the UK, and ultimately to improve the levels of healthcare provision nationally. This is a radical new opportunity for the industry which suffers from a lack of joined up thinking and hence tends to operate in discrete silos of expertise. The integrated approach offered by the CDT would pay high dividends. UK companies will benefit from access to highly skilled doctorates who will each have benefited from a wide and interdisciplinary research approach created by the CDT. Macromolecular medicines are complex and labile so that bioprocess development times and costs tend to be high due to unforeseen issues that occur during scale-up of the manufacturing process. Currently there is little scope to alter a manufacturing process because the effect of changes cannot be readily predicted. This is compounded by lack of individuals skilled in the methods needed. Our transformative CDT research training agenda will allow for the first time engineers and scientists to create the methods and approaches needed for UK companies to genuinely understand and control directly, for the first time, the quality of output during manufacture, in spite of biological variability. By creating and then testing manufacturing models and methods for whole bioprocesses using the resources of a national EPSRC Centre we shall gain fundamental engineering insights crucial for the more effective direction of acquisitions of experimental data and also the improved design and operation of whole bioprocesses. Manufacturing efficiencies will be raised and waste reduced. Such a vision is consistent with recent efforts by the regulatory authorities, and in particular the Quality by Design (QbD) initiative of the International Committee on Harmonisation (ICH), to develop science-based regulatory submissions for approval to manufacture new biological products. The CDT will create a network to provide a conduit for effective knowledge exchange from the very best academic groups in the UK. A key metric of success will be retention of CDT graduates within the industry where they will be effective in the application of Centre concepts with industrial practice and the adoption of the methods created. Potential patients will benefit as the innovations created by the CDT research will significantly aid reduction in development times of macromolecular medicines, which is particularly crucial for those addressing previously unmet clinical needs and the treatment of severe conditions such as arthritis, cardiovascular disease, viral infections and cancers. By providing industry the capabilities and tools to achieve changes to manufacturing processes we shall open up possibilities for major improvements to processes during production and hence reduce costs to the NHS. The capacity to treat conditions such as rheumatoid arthritis much more effectively in ageing populations is vital but it still poses a problem with respect to stretched NHS budgets. A significantly greater number of drugs will be capable of meeting NICE's thresholds and thus benefit extended patient populations. The UK economy will benefit because the academic research and training offered by the CDT will complement the country's strength in bioscience discovery. Collaboration between bioprocess engineers, process modellers manufacturing experts, regulators and physical scientists will ensure effective knowledge and skills transfer between the science and engineering base and UK industry and the regulating agencies. This will strengthen the UK position in the global healthcare market and attract further R&D investment from global business which recognises the UK as a good place to conduct these activities.

Publications

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Carrillo Sanchez B (2022) GFP-tagging of extracellular vesicles for rapid process development. in Biotechnology journal

 
Description Extracellular vesicles (EVs) secreted by cells are emerging as a promising therapeutic modality. Genetically encoded proteins expressed by cells have been designed to be destined to the lumen of said EVs. This provides a key analytic tool for characterization and evaluating the recovery of EVs from different process steps during isolation from cell culture. Additionally, a method of loading protein cargo of into EVs is in the works as part of the project funded by this award.
Exploitation Route Other researchers could employ a similar technique during development of their processes for isolation and recovery of extracellular vesicles. The method that has been developed could be adopted universally for any type of mammalian cell line expressing the fluorescent protein.
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology