Understanding recombinant therapeutic protein production at single cell resolution

The optimisation of manufacturing processes using CHO cells has been achieved through improvements in cell line development and the increased knowledge of biological characteristics associated with desirable bioprocess phenotypes such as sustained growth, productivity and product quality. Advances in next generation sequencing (NGS) technologies have paved the way for a greater understanding of CHO cell biology, accompanied by the publication of the Chinese hamster genome and subsequent analysis of omics data. Although beneficial, a critical limitation of many of these studies is the use of bulk methods to generate the data, therefore capturing average information across a population of cells. Recently emerged single cell sequencing methods permit the acquisition of omics data from thousands of individual cells in a single experiment and the ability to delineate intra-population heterogeneity. The overall aim of this work is to explore the utility of single cell sequencing approaches in different CHO cell populations to gain a deeper understanding of the underlying biology. A key novel aspect of the work outlined in this thesis is the first application of single cell ATAC sequencing (scATAC-seq) to study chromatin accessibility in CHO cell populations. Optimisation of an experimental workflow enabled efficient nuclei isolation and the generation of high-quality sequencing data in a non-transfected CHO K1-GS cell line. The resulting scATAC-seq data was used to identify genomic regions with favourable characteristics for potential transgene integration. Regions were compared to a previous publication using bulk ATAC-seq and the method was used to create a refined list of integration sites with homogeneous accessibility across the population. The identification of heterogeneous regions demonstrated the utility of scATAC-seq to provide additional insight and the ability to filter integration sites detected in only a proportion of the total cells in the population. Further application of scATAC-seq was used to perform a more comprehensive analysis and compare epigenetic changes associated with temperature shift in CHO cells at single cell resolution. Thousands of differentially accessible regions were found between temperature shifted and non-temperature shifted cells and specific transcription factors involved in the regulation of key biological processes were determined. Integration of scATAC-seq with single cell RNA sequencing (scRNA-seq) allowed additional insight into the regulatory mechanisms involved in production instability in a mAb-producing CHO cell line over long-term cultivation. A subpopulation of cells in an early passage was found to have comparable chromatin accessibility and gene expression to a later passage with significantly reduced mAb production – indicating early instability in the cell line. Key biological processes associated with the loss of mAb productivity were revealed and used to highlight potential routes for precise genetic engineering strategies. The identification of intra-population heterogeneity was permitted through the use of single cell sequencing and would not have been possible using traditional bulk sequencing techniques. Moreover, scRNA-seq was also used to study transcriptional changes in a CHO cell line with inducible mAb expression and generate a small list of genes linked directly to the burden of mAb production. Overall, single cell sequencing is a powerful tool for profiling chromatin accessibility and gene expression in individual cells and can provide an additional layer of understanding into CHO cell biology. This research demonstrates the utility of these methods and provides a benchmark for future research in biopharmaceutical manufacturing through the implementation of an optimised experimental approach and development of bioinformatic workflows for data analysis.