Methods for cell line and protein engineering

Sammanfattning: Therapeutic proteins are becoming increasingly important. They are desirable, as they typically possess low adverse effects and higher specificity compared to the traditional, small molecule drugs. But they are also more complex and involve different intricate and expensive development and production processes. Through new technologies in protein and cell line development, more efficient and safer drugs can be readily available and at a lower cost. This thesis gives an overview of how protein therapeutics are developed and produced. It explores strategies to improve the efficacy and safety of protein drugs and how to improve production yields. In the present investigation, two papers present new methods for high-throughput cloning and site-directed mutagenesis using solid-phase immobilization of DNA fragments. These methods were designed to generate new drug candidates with swiftness and ease. Three papers show the development of a new cell line screening system that combines droplet microfluidics and the split-GFP reporter system. This combination allows for relative quantification of secreted recombinant proteins between individual cells and provides a tool for the selection of the best-producing clones for final production from a heterologous cell pool. The final paper explores the possibility to produce proteins at a higher cell density by examining how the metabolome and proteome of a perfusion bioreactor evolve as the cell density reaches exceptionally high levels. The consistent goal of all of these studies is to expedite the development and improve the production of therapeutic proteins, to assist the discovery of new drugs and to bring down production and development costs. Engineered proteins can be used to cure previously incurable diseases or give current medications a higher efficacy. Lower production and development costs can make the treatments available to more people.