Therapeutic antibodies are central to modern drug development, but growing mammalian cells for protein expression is complex. Credit: Shutterstock
“Bacteria just grow,” says Rodrigo Ribeiro, molecular biologist and regional marketing manager at Eppendorf North America. “I used to work with E. coli for protein expression, but when you’re working with mammalian cells, it’s much more complex.”
While bacterial cells may be faster to grow, the proteins expressed from these cells don’t always include the post-translational modifications required to produce biologically active protein-based drugs. Many researchers use mammalian cells to produce therapeutic proteins — their output is more likely to resemble endogenous human proteins and therefore be functional and clinically relevant.
Cell lines commonly used for monoclonal antibody expression include Chinese hamster ovary (CHO) and human embryonic kidney 293 (HEK293) cells. But even if researchers have worked with these wild-type cell lines in plates, they will have to learn to work with adapted versions of these cells that are cultivated as suspension cultures.
For researchers looking to grow mammalian cells for protein expression, what are the key lessons?
Contamination
“With mammalian cells, we have to watch out for contamination constantly,” says Ribeiro.
Mammalian cell cultures are more prone to contamination than bacterial cultures, and because their growth cycle is longer, the consequences are more severe. The presence of fungi, yeast, mycoplasma, bacteria or viruses in the medium affects the consistency and quality of protein expression, not to mention wasting time and money. Careful handling and proper lab technique reduces the risk of contamination, as does using easy-to-clean incubators, which are key contamination sites.
It’s important to regularly test for contamination and quarantine any new cells arriving from other labs. Cells should be thoroughly tested before being introduced to the main work area.
Reproducibility
Standardization is another important consideration for protein expression from mammalian cells. To reproducibly express monoclonal antibodies, conditions need to be consistent between batches. “How accurate your sensors are, how effectively you control CO2 or the temperature or the pH — all affect reproducibility,” says Ribeiro.
Suspension cell cultures need to be consistently shaken while they grow. Ribeiro recommends easy-to-clean shakers with CO2 control, but notes many scientists use an open-air shaker inside a regular CO2 incubator to grow cells, which can cause problems.
“The shaker generates vibration and heat inside the incubator, affecting cell growth and ultimately protein quality,” he says. “It also becomes a potential source of contamination since we’re not able to clean the shaker and incubator properly.”
Scaling up
Ramping up the scale of mammalian cell culture is less straightforward than in bacteria. If the amount of therapeutic protein you can produce becomes a bottleneck, it might be time for a bioreactor. These have several benefits besides scale.
Even a small benchtop bioreactor will be a step up from flasks and shakers. It can be challenging to master the controls of a bioreactor, but with a little optimization you'll be able to tightly monitor culture parameters. “You gain many features and can better control growth conditions,” Ribeiro notes.
With a growing list of regulatory approvals sparking sustained interest in producing monoclonal antibodies, Eppendorf has been supporting researchers who are working with both bacterial and mammalian cultures.
Moving to mammalian cell cultures comes with a learning curve, but by keeping on top of contamination, focusing on reproducibility, and being prepared to scale up, any research team should be ready to explore the exciting possibilities in this space.