Biotherapeutic protein production in mammalian cell systems is becoming commonplace as drug targets increase in complexity. Proper protein folding, assembly, and post-translational modifications are vital to a fully bioreactive end-product, bringing mammalian cell expression systems that closely mimic human processing to the forefront.
Suspension cell lines derived from CHO and HEK 293 cells are commonly used for mammalian protein production. These cell lines have many desirable traits including high expression levels, scalability (density and volume), and especially in the case of CHO cells, a history of regulatory approval.
Clinical biotherapeutics are frequently generated using stable transfectants for batch-to-batch consistency and low cost at extremely large scale. Many advances spanning the last decade such as improved cell lines, expression vectors, culture medium, and delivery methods have led to the adoption of transient transfection methods for mammalian protein expression.
In many drug discovery applications, it is beneficial to screen protein constructs quickly using transient transfection methods, allowing for the evaluation of various target molecules or protein isoforms simultaneously. In many instances, transient transfections are performed in parallel while more resource-intensive stable cell lines are under development.
Recent advances in transfection technologies, which include the TransIT-PRO® Transfection Kit by Mirus Bio, have allowed researchers to obtain high protein titers in suspension CHO and 293 derived cells in a simple and reproducible manner. Using Mirus Bio's technology, transfection complexes are prepared in serum-free media by adding plasmid DNA, TransIT-PRO Transfection Reagent, and PRO Boost Reagent.
The PRO Boost Reagent is an optional component and enhances gene expression in certain growth media formulations. After incubating complexes for 10–30 minutes, they can be added directly to cells in normal growth media. Transfection using the TransIT-PRO Transfection Kit eliminates the need for a culture medium change post-transfection and is suitable for both transient and stable transfection. For secreted antibody constructs, optimal titers are typically obtained 5–7 days post-transfection in batch fermentation.
Several parameters should be considered during the optimization of transient transfection protocols including cell density at the time of transfection, DNA concentration, reagent-to-DNA ratio, and cell culture medium.