Suarez Heredia, Ricardo; (2024) A combined in-vitro and in-silico investigation on the design and optimisation of nutrient supplementation strategies for mammalian cell cultures. Doctoral thesis (Ph.D), UCL (University College London).

Text Suarez Heredia_Thesis.pdf - Other Access restricted to UCL open access staff until 1 February 2029. Download (29MB)

Abstract

The production of recombinant proteins remains a fast-growing market, expected to surpass $125 billion by 2020. Further improvements in manufacturing capacity and bioprocess yields are required to meet the increasing global demand for biologics. Fed batch cultures have been the dominant mode of production over the last 20 years due to increased productivity compared to batch cultures and relative simplicity compared to continuous cultures. Process intensification strategies for fed-batch cultures have been more lately implemented primarily through manipulation of: (1) feed media composition (e.g. adaptive media de-coupling upon culture stage), (2) mode of operation (e.g. concentrated fed batch with cell retention) and (3) feeding schedules (e.g. dynamic feeding regimes, feed format). The development of sophisticated, highly customised cell culture media has arguably contributed the most to the high cell densities and titres attainable today. Nevertheless, due to the high associated costs, media development has been carried out primarily by the industrial sector, leading to a considerable amount of knowledge and know-how being either semi-empirical or kept in-house as intellectual property (IP). In order to unlock further improvement potential and establishment of new processing modes, an in depth understanding of cellular metabolism and its response to nutrient and co-factor perturbations through cellular and process level analyses is required. The overall aim of this work was to investigate the basal metabolic requirements for growth and productivity along the cell response to variations in media composition, mode of operation and feeding schedule during bioprocessing conditions. Firstly, this works investigated the essential metabolic requirements of mammalian cells for growth, proliferation and heterologous protein production through a bibliome based medium library construction (MedLib, University College London) and experimental approaches (top-down and bottom-up). A detailed formulation of a fully chemically defined medium (UCBE-CHO, University College London) is presented and benchmarked against commercially available media in batch and fed batch cultures for three different CHO cell lines (IgG4 producing GS-CHO K1, parental GS-CHO K1 and native CHO-S). Moreover, medium library was further expanded into considering unconventional nutrients associated to metabolic pathways with a subsequent design of un-coupled nutrient supplementation strategies for feed and perfusion media. A series of batch, fed-batch and semi-perfusion GS-CHO cell culture experiments with introduced nutrient perturbations across 15 nutrient groups were performed and characterised in terms of growth and productivity. Overall, nutrient groups of particular interest for bioprocessing applications were highlighted for implementation into uncoupled dynamic feeding strategies for enhanced mammalian cell cultures. Subsequently, a series of fed-batch and concentrated fed-batch experiments were conducted to investigate the impact of different modes of operation, different feeding schedules (e.g., fixed vs variable volume additions) and feed formats (e.g. basal vs concentrated feed) on the performance and characteristics of GS-CHO cell cultures. Of particular interest was the performance observed during concentrated fed-batch (CFB) cultures which led up to a 5-fold volumetric increase in viable cell count (>70x106 cells mL-1) and 11-fold increase in antibody titre (>2 g L-1 day-1) compared to control fed-batch cultures of the same duration. Intensification of mammalian cell cultures through concentrated fed-batch cultures exploited the well-establishment of fed batch cultivation mode and perfusion technology while overcoming the challenges of continuous perfusion cultures such as volumetric footprint, diluted harvest stream and genetic instability of cell lines. To address the multi-parametric nature of these hybrid processes and a priori knowledge during early stages of their development, a digital framework for the rapid process design and optimisation of intensified cell cultures (CFB and continuous perfusion) was developed integrating evolutionary algorithms (Genetic Algorithms and Multigene Genetic Programming) and high-throughput automated micro-bioreactor systems (ambr15®). This adaptive methodology supported the improvement of intensified viable cell counts (>80x106 cells mL-1) and volumetric productivities (>2.5 g L-1 day-1) along with identification of key process variables through symbolic data analysis. Finally, along the in vitro observations, further investigation through a conjunctive heuristic framework and multivariate analysis (MVA) on the historical cell culture datasets allowed the identification and investigation of relevant process variables and nutrient compositions for the systematic design, development and optimisation of balanced media and feeding strategies for improved and intensified cell growth and productivity in mammalian cell cultures.

Download activity - last month

Export as JSONCSVXML

Download activity - last 12 months

Export as JSONXMLCSV

Downloads by country - last 12 months

Export as CSVXMLJSON

Archive Staff Only

View Item