Be that as it may, CHO cells are still the dominant stable producer lines in the pharmaceutical industry. Yet because R&D material is principally generated in HEK293-based cells, so as to minimize the (real and perceived) post-translational modification dissimilarities between transiently and stably expressed genes, there has been a call to develop a viable large-scale CHO transfection platform as an alternative. Regulatory submissions, too, would be simplified by using a common host to generate both preclinical and clinical matter.
Yves Durocher, Ph.D., research officer at the National Research Council Canada, has been heeding the call. His lab has developed a simple and robust transfection process that allows CHO cells to compete with the ease of use and expression levels of 293 cells.
The automation-compatible process utilizes serum-free medium (facilitating the recovery of secreted proteins), does not require medium exchange prior to or following transfection, and makes use of suspension cells, allowing for scalability.
“We are basically using the same approach, same protocols, and the same vectors to transfect CHO cells,” he said. As such they typically express every new target in both 293 cells and CHO cells, and test lines in parallel for the best expression and product quality/activity.
In some cases there have been differences in glycans or other post-translational modifications between the two hosts, which can be critical for many biological functions. This can be the result of rodents expressing different enzymes and in different proportions than humans, or by the fact that ovary metabolism may differ from that of the kidney.
“The more hosts we have, I think, the better,” commented Dr. Durocher. “It provides you with other options for different targets.”
But all else being equal, Dr. Durocher thinks that companies that already have stable CHO expression platforms for manufacturing biologics would rather use a large-scale CHO transient transfection system than use different platforms for research and production.
For most of the small percentage of proteins that can’t be made in the cells as they are now, “you can still stay with CHO cells and just engineer them to provide better biologics,” he said. “But of course you also have the opportunity to switch hosts and start from scratch in developing a new platform.”
Transfection Is Infection with DNA
Of course, there are certain circumstances when CHO cells just won’t do—like, for example, for the making of enveloped VLP vaccines. “Intrinsically you can’t perform the viral clearance that’s required from products in rodent cell lines,” explained Richard Schwartz, Ph.D., chief of the vaccine production program lab at the NIAID’s Vaccine Research Center.
“If you make a protein or product in CHO, you are required to show about 13 logs of viral clearance in your production process. So you generally have to do low pH viral or detergent inactivation and there’s a viral filtration.
“Low pH will basically destroy the VLP immunogenicity—it completely changes the envelope structure and it would destroy the actual activity of the VLP. And you can’t do a viral filtration, because VLPs are essentially the size of a virus and so would be removed by filtration.”
Add to this that enveloped VLPs would literally kill the host cells as they are produced by carrying the lipid membrane to form the VLP, and “this lipid membrane damage causes greatly decreased cell growth if not cell death, so you’ll never be able to passage the stably transfected cells enough to make master cell banks or to expand them into your final bioreactor,” he said. So what to do?
Dr. Schwartz has been using a transient HEK293 expression platform for early-phase clinical trials of a nonreplicating VLP vaccine for Chikungunya, a virus endemic to parts of Africa and Asia that can cause debilitating symptoms. The host cells can be expanded prior to transfection. “I don’t care about the cells at my last step of the process,” Dr. Schwartz confessed. “All I care about is the product.”
Transient transfections are notoriously difficult to scale up. Commercial media is highly inhibitory to these transfections, and so you have to do a large-scale centrifugation or media exchange of some sort—which becomes a problem at multiliter quantities.
Dr. Schwartz’ lab is currently scaling up production into 50 liter disposable bioreactors, as well as looking at inducible promoters, fed-batch processes, and other improvements for the next generation of VLP production.
Fortunately, the dosages of viral vaccines are so much smaller than those of therapeutic monoclonal antibodies—in the range of 20–100 micrograms, versus perhaps grams of mAb. So even with the productivity they’re currently achieving—about 100 mg/liter in 1 liter flasks—it’s easy to make sufficient doses for early-phase trials.