Analysis of the total and subcellular proteome of antibody-producing cells to improve recombinant protein expression in CHO cells

Abstract

Recombinant protein therapeutics represent one of the most important classes of pharmaceuticals today as they revolutionized the treatment of severe illnesses e.g., cancer, autoimmune diseases and infertility. The majority of these pharmaceuticals are produced by Chinese hamster ovary (CHO) cells, first isolated in the 1960s from ovary tissue of the Chinese hamster (Cricetulus griseus) and since then developed into various cell lines for protein production purposes. Their three decades long usage allowed thorough media optimization, streamlining of the cell line generation process and numerous cell engineering studies. However, the ongoing success of recombinant proteins in diagnostics and therapy increases global demand beyond production capacity. Additionally, novel recombinant proteins increasingly consist of non-natural fusion proteins or bi-specific, monoclonal antibodies whose expression can be challenging for production cell lines. Therefore, further optimization of production cell lines aiming for higher protein product yield is needed to meet the global demand. This thesis aimed at comparing the proteomes of CHO cell lines to plasma-cell derived cell lines, which were optimized by evolution to secrete high amounts of antibodies, in order to characterize cell phenotype-specific traits, which can be used in rational cell engineering to increase recombinant antibody production in CHO cells. The proteomes and secretomes of two CHO cell lines and two murine myeloma cell lines, all secreting an Immunoglobulin gamma antibody were characterized using state of the art bottom-up shotgun LC-MS technology and compared with each other using bioinformatic tools to determine differentially regulated pathways affecting protein secretion. Additionally, subcellular protein distribution across ten subcellular compartments was analyzed using protein correlation profiling in combination with several bioinformatic workflows of which one first is introduced within this thesis to systematically compare subcellular protein distribution between different cell lines. Differentially expressed and distributed proteins mainly were enriched in vesicle-mediated transport, translation and transcription associated proteins. Additionally, plasma cell-derived cell lines showed enrichment of ER and Golgi-associated proteins, whereas CHO cells were characterized by elevated secretion of proteins associated with extracellular matrix. Based on their function and correlation with transcriptome data from the cell lines, a subset of these proteins was selected for cell engineering studies in CHO cells, where the overexpression of plasma cell transcription factors increased recombinant protein titer up to 50%. Moreover, transient knock-down of extracellular matrix-associated proteins in a CHO-DG44 cell line increased recombinant protein titer by 20%, validating the approach for rational CHO cell engineering towards a plasma cell-like phenotype described in this thesis as well as qualifying the reported data to be searched for novel protein targets in future engineering studies.