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Author: Brett Verstak, Director of Cell Line Development
In today’s rapidly evolving biopharmaceutical landscape, speed and efficiency in cell line development are more critical than ever. While gene integration methods such as transposase technologies have garnered significant attention, it is essential to not overlook the fundamental aspects of gene expression. Regardless of the method used to drive stable integration of the recombinant gene into the host chromosome, it is the quality of the gene expression cassette that truly drives high cellular productivity (Qp). This needs to be combined with a compatible host cell line that can support high levels of gene expression and a process that supports high cell density, cell viability and productivity. All three of these need to be in place to develop a robust process for bioproduction.
By focusing on refining vector components – such as promoters, signal peptides, and untranslated regions (UTRs) – and combining these with a fast-growing, robust cell line and a process for rapid screening and selection of high-quality, stable clones, we can derisk and accelerate cell line development activities to help bring innovative medicines to patients sooner.
One of the key factors affecting the productivity of a cell line is the expression vector. The vector carries multiple genetic elements that control the expression of the transgene(s) and allows for efficient selection of transfected cells. Developing a productive vector needs systematic consideration of each of these genetic elements, their role in gene expression and how they may interact with one another.
Promoters are the engines of gene expression and, typically, a strong constitutive promoter is selected to ensure robust RNA transcription of the gene of interest. The untranslated regions (UTRs) play a critical role in the transcribed RNA stability, processing and transport out of the nucleus, as well as the affinity for ribosomes. These are often overlooked sequences that can play a critical role in translation efficiency. Fine-tuning these regions allows you to capitalize on the choice of a strong promoter and ensure that more of the message transcribed results in a translated polypeptide.
Signal peptides guide the nascent polypeptide through the secretory pathway, and optimizing these sequences can improve protein folding and secretion efficiency, which is critical in maintaining cell health, productivity and product quality.
The selection marker’s design and regulation can significantly impact the speed and reliability of post-transfection selection, cloning, expansion, and characterization of clones. The position and orientation of the different gene expression cassettes can also impact expression levels, through both trans- and cis- mechanisms.
By carefully designing the gene cassette, we can also influence epigenetic factors that stabilize gene expression over time. This stability is crucial for maintaining high protein production levels throughout manufacturing and complying with regulatory guidance. Holistic optimization of vector architecture can result in less stress on the cells and eases the initial phases of cell line development, allowing for the more efficient selection of high-producing, stable clones.