There are two functionally different types of DNA transfection (with a gray area between). On the one hand, stable transfections allow newly introduced genetic material to be passed down as part of the fixed genome. On the other hand, transiently transfected cells carry transgenes episomally.
We rarely hear much about transfection—it’s more of a behind-the-scenes player. Yet whether and why to make stable lines vs. transiently transfect, as well as seeming nuances such as which host cell line to use, how to introduce the DNA, under what conditions to culture the cells—both short term and long, and even what kind of media to use, can lead to (or avoid) untold frustrations in the process of generating the cells for manufacturing or experimental purposes.
Such nuances were certainly on the mind of researchers who shared their transfection experiences at Informa’s “Cell Line Development and Engineering” meeting held last month.
Science doesn’t always proceed by “aha!” moments—sometimes it’s incremental advances over a myriad of seemingly small parameters cumulatively coming together that lead to big improvements. That is the case at UCB Pharma UK, where Bernie Sweeney, Ph.D., group leader for protein expression and purification, generates antibodies for early-stage research and preclinical development.
“Back in 2006, we were making about five grams over the year,” she said, comparing that to the 40–50 grams of protein they’re producing now from an equivalent number of transfections.
Over the past five years UCB has engineered a CHO host overexpressing both XBP-1 and ERO1-Lα, two key regulators of protein secretion and cellular oxidative stress, respectively. Use of this host has resulted in routines titers between 400–700 mg/L.
Supplying reagents for several ongoing projects at early-stage discovery through in vivo studies means that their system needs to be able to cope with both large- and small-scale production. At the moment its preferred method of transfection is electroporation using individual cuvettes, which for large-scale production requires “an awful lot of cuvettes,” Dr. Sweeney remarked.
Her group recently delivered a 13 g batch of purified IgG1 in 10 weeks, which would have taken 16–20 weeks using a bulk pool or stable cell-line process, she noted. While labor-intensive, the great benefit of using a transient expression system to generate gram-quantities is its ability to reduce timelines.
Transient transfection is generally seen as a short-term solution for making small amounts of a protein. Yet as several speakers argued, there are distinct advantages that transient transfection offers over its more laborious stable cousin.
Among the most obvious is the time it takes to generate a stable cell line—a major barrier for discovery and preclinical programs that require the screening of multiple different components in conjunction with one another.
And this is especially the case when those components are parts of a complex multimeric protein. Manuel Carrondo, Ph.D., professor of chemical and biochemical engineering at, and CEO of, Portugal’s Instituto de Biologia Experimental e Tecnológica (iBET) and his team developed a HEK293-based virus-producing packaging cell line that allowed for easy replacement of the gene(s) of interest by homologous recombination.
Once the high-producing line was established, it could be reused to generate transient lines with predictable high viral production without the need for further exhaustive clone screening. Compatible retroviral vectors could be swapped in and out in a matter of weeks, in a process called recombinase mediated cassette exchange (RCME).
Typically recombinant viruses and virus-like particles (VLPs) would be produced by co-infection. Jumping from the co-infection strategy to targeting where we know that there is a single insertion and where it is, allows for better management of both the stoichiometry of the unit and also the timing, Dr. Carrondo noted.
“For example, you need a given protein to arrive at a later stage to cover up or to link to the one that has to be there at an earlier stage. Those are thermodynamic and stoichiometric issues involved. By doing this with our recombinant mediated cassette exchange, you can test many more options for VLPs or multimeric proteins in a shorter time than through co-infection and screening.”
Production and assembly for the different multimeric proteins can change much faster, vastly improving the iterative process at the preclinical stage, he said.
E. coli and yeast are surely simpler systems, but animal cells are needed to produce complex proteins with post-translational modifications. Dr. Carrondo’s group recently set its sights on using this “tag and target” methodology for VLPs and multimeric proteins in insect cell lines.
Compared to mammalian cells they are generally more robust, metabolically easier to deal with, less expensive to maintain, and are also more genetically stable.