Efficient gene transfer into cells relevant for protein production purposes has been a major bottleneck. Viral gene transfer
systems usually have the advantage of high transduction efficiencies compared to non-viral methods. However, these methods
suffer from several limitations such as the time-consuming and laborious production of vectors, elevated laboratory costs
due to the high level of safety requirements, limitation of insert size, and possible immunogenic reaction in clinical trials.4
Non-viral gene transfer methods include calcium phosphate, lipofection reagents, electroporation, and ballistic gene transfer.5 Whereas electroporation and ballistic techniques usually lead to high cell mortality, calcium phosphate and lipofection
often result in low transfection efficiencies especially in suspension cells. The electroporation-based Nucleofector technology
is a valid alternative and has been proven to be efficient even in suspension cells relevant for protein production (Figure
Figure 2. Efficient Gene Transfer Using Nucleofection.
Stable protein production in mammalian cells
Cells used for the production of therapeutic proteins must comply with various requirements to ensure approval for the protein
as a drug. One of those requirements is to use a thoroughly defined clone. It must be guaranteed that the cells used for protein
production are derived from a single clone with a specifically-defined integration site. The generation of a stable clone
often requires six months or more due to selection procedures and adaptation to serum-free conditions (Figure 3A). A variety
of systems for selecting transfected cells exists, including resistance to antibiotics such as neomycin, hygromycin and puromycin,
dihydrofolate reductase (DHFR) and glutamine synthetase (GS) systems.
The aim of selection is to identify high producing clones, but this is a tedious and labor-intensive exercise. Several methods
for isolating clones are used, the most popular one being cloning by limiting dilution using multiwell plates. Once a stable
clone is selected, it must be adapted to serum-free suspension culture so that it can be used in efficient large-scale production.
This adaptation process is again time consuming and bears the risk that the clone will lose its desired high producing properties.
By applying novel transfection technologies such as the Nucleofector technology, suspension cells can be transfected directly
in a serum-free environment. This saves significant time and reduces the risk on the way to find the right clone (Figure 3B).
Figure 3. A. The generation of a stable clone requires six months or more due to selection procedures and adaptation to serum-free
conditions (SFM). B. If serum-free adapted suspension cells can be transfected, much time can be saved in the later process.
Transient Protein Production in Mammalian Cells