“Therapeutic plasmid DNA vector” is promising technology for the future of gene therapy. Though still in the clinical trial stage, DNA vaccines have great potential of being successful as effective vaccination technology with negligible side effects.
One of the most important considerations in designing a successful DNA vaccine production process is the selection of an efficient cell line for the specific plasmid. Every plasmid, or protein for that matter, is unique and optimum yield can be achieved by selection of the appropriate cell line. Plasmids show different growth characteristics with respect to their sequences (inverted or direct repeats), length (> 15 KB), and modifications like methylation or structural characters. A single cell line is not fully capable of producing all kinds of plasmids efficiently. Different cell lines are available which are genetically tailored to overcome these differences and ensure efficient growth.
E. coli strains are widely used for DNA vaccine products and have proven to be a successful source of DNA plasmids. The DH10B cell line has been a highly successful strain for plasmid production with high growth and high yield in standard conditions. However, there can be certain challenges in the plasmid sequence for which DH10B may not be the best host strain. Today, availability of different cell lines makes process improvement and development easier and more efficient for different products, including plasmid DNA. Based on the specific plasmid characteristics, cell line optimization should be performed to maximize the potential of the process in regards to plasmid yield, growth, etc. Some DNA vectors contain viral protein sequences and long direct or inverted repeats as part of the construct. These kinds of sequences can pose the challenge of instability of the plasmid when grown in standard conditions. Many times DH10B fails to handle unstable plasmids, leading to poor or failed growth. There are many commercially available E. coli cell lines. The Stbl2 and Stbl3 cell lines from Life Technologies are modified to grow unstable plasmids that contain repeats and viral sequences. The Sure2 cell line by Stratagene has been developed to transform difficult to clone plasmids. Also from Stratagene, the XL-10 Gold cell line is recommended for transforming large plasmids. The NEB-10 Beta cell lines from New England Biolabs are tailored to transform and grow plasmids as large as 24 kb. There are cell lines available which are designed specially to produce single stranded DNA; for example, the DH12S cells from Life Technologies. Plasmids with structural challenges like hairpin structures in the DNA sequence could be grown using the GT-115 cell line from Invivogen.
An array of cell lines is available for other prokaryotic host systems and eukaryotic host systems like yeast cell lines and CHO cells. Cell line optimization is a promising tool not only for the development of DNA products but also for other biological products. Process development and improvement by optimum cell line selection results in high product yield at the upstream level. Better product yield at the upstream level leads to increased productivity at the end of downstream processing. With many cell lines to choose from, cell line optimization of plasmid DNA provides an excellent opportunity to make upstream processing more specific for products with more productivity.
– Neha Tiwari, PhD.
Processs Development Manager, VGXI