Protein-specific signal peptides for mammalian vector engineering

Abstract

ABSTRACTExpression of recombinant proteins in mammalian cell factories relies on synthetic assemblies of genetic parts to optimally control flux through the product biosynthetic pathway. In comparison to other genetic part-types, there is a relative paucity of characterized signal peptide components, particularly for mammalian cell contexts. In this study, we describe a toolkit of signal peptide elements, created using bioinformatics-led and synthetic design approaches, that can be utilized to enhance production of biopharmaceutical proteins in Chinese Hamster Ovary cell factories. We demonstrate, for the first time in a mammalian cell context, that machine learning can be used to predict how discrete signal peptide elements will perform when utilized to drive ER translocation of specific single chain protein products. For more complex molecular formats, such as multichain monoclonal antibodies, we describe how a combination ofin silicoand targeted design rule-basedin vitrotesting can be employed to rapidly identify product-specific signal peptide solutions from minimal screening spaces. The utility of this technology is validated by deriving vector designs that increase product titers ≥ 1.8x, compared to standard industry systems, for a range of products, including a difficult-to-express monoclonal antibody. The availability of a vastly expanded toolbox of characterized signal peptide parts, combined with streamlinedin silico/in vitrotesting processes, will permit efficient expression vector re-design to maximize titers of both simple and complex protein products.GRAPHICAL ABSTRACT