Recombinant antibody therapeutics take up the biggest market share of the biopharmaceutical industry. This places great demand on efficient recombinant protein production. Despite this, there is still a great deal of unpredictability about how certain sequences will express in mammalian cell lines. Protein expression and secretion are highly regulated processes and a plethora of molecular events throughout the pathways can cause âbottlenecksâ to secretion. These bottlenecks are still largely undetermined. For the biopharmaceutical industry, this can have huge implications towards cost and product quality. This thesis is an attempt to understand how molecular handling of certain sequences leads to vastly different expression titres. A panel of IgGs with a high degree of sequence identity was generated and expressed in CHO cells by transient electroporation transfection. Analysis of mRNA, intracellular protein and secreted protein revealed that translation of each antibody was taking place, but levels of secretion varied dramatically. A solubility fractionation assay and a bioimaging assay revealed poorly expressed antibodies were forming insoluble intracellular aggregates. A computational pipeline was developed to provide a unique method of quantifying these aggregates. Further to this, work was performed to gain understanding into whether the UPR response differed to antibody sequences that expressed at varied titres, before a number of cell-culture based strategies were used in an attempt to overcome aggregation and improve expression. Finally, computational biology methods were employed in order to increase understanding into sequence characteristics that lead to poor expression titres.
|Date of Award||1 Aug 2018|
- The University of Manchester
|Supervisor||Alan Dickson (Supervisor) & Eileithyia Swanton (Supervisor)|