Mammalian cell culture is and will remain the principle vehicle for protein production in upstream manufacturing, according to Florian Wurm, Ph.D., professor of biochemistry at the University of Lausanne and founder of ExcellGene (www.excellgene.com). Dr. Wurm reported that 70% of the $20-billion to $30-billion annual bioproducts inventory is generated with mammalian cells, and the gap separating them from other approaches (yeast, bacteria, baculovirus, transgenic plants, and animals) is only growing wider. “We anticipate that mammalian cells will continue to dominate bioproduction for the next 10 years as new technologies carry the process forward.”
Dr. Wurm spoke at a two-day symposium sponsored by Sartorius (www.sartorius.com) in which representatives from the academic and private sectors discussed their approaches to producing more and cleaner proteins, confronting both the upstream and downstream ends of the equation. Better promoters, enhancers, and other genetic elements have contributed to increased upstream production of proteins.
However, Dr. Wurm stated that the role of these elements on the DNA level has been overemphasized, and that the principle source of increased productivity comes from better cell growth and other process-related improvements that were obtained through media modifications, productivity enhancers, and media feeds. Indeed, this strategy has resulted in a 25-fold increase in cell density in cultures from 1986–2007.
“The conventional rationale for obtaining high producers is based on examination of transfected cell lines by low-throughput screening to identify the top producing clones,” Dr. Wurm continued. Subsequently, low-throughput process development strategies were pursued. Biotech firms assumed that highly delicate cells would not survive more robust technologies for growth and thus implemented highly expensive, fully controllable bioreactors. Time-consuming protocols were applied that could take up to 18 months.
ExcellGene has the ability to rapidly scale-up production. It has developed a disposable design, simple bioreactor system as an alternative to a classical stirred bioreactor that does not require oxygen probes and pH electrodes. These small Tubespin™ bioreactors are miniature editions of the much larger 100-L bioreactors. Hundreds or thousands of these reactors can be run in parallel, and vast amounts of data can be collected, allowing optimization over a wide range of culture conditions in a matter of weeks.
“This protocol can deliver better productivity and growth of CHO cells in a simpler and more efficient format than classical bioreactors, while exceeding the latter in performance,” Dr. Wurm asserted.
The heart of ExcellGene’s technology is an orbitally shaken bioreactor. The cylindrical bioreactors, ranging in volume from 1–100 L, are mounted on a circular moving platform. Orbital shaking allows for efficient mixing of suspension cultures, while oxygenating cells through the headspace. The vessel contains a disposable, sterile bag with appropriate connection tubes for seeding, feeding, gas supply, and harvesting of the culture. Compared to classical stirred tank bioreactors, orbitally shaken bioreactors are much simpler and less costly to operate, reported Dr. Wurm.
Published data from his lab established that oxygen can be transferred to cells more efficiently with ExcellGene’s technology than in other systems, an outcome reflected in the growth and productivity from these reactors. ExcellGene disposable reactors at the 100-L scale are equivalent and frequently better than those of fully instrumented, nondisposable, glass, or stainless steel stirred bioreactors, he said.
“Biology plays a dominant role in cell performance,” Dr. Wurm added, believing that the karyotypic makeup of the transfected cell lines needs to be carefully monitored during expansion. The insertion of foreign DNA into these cells is an unpredictable phenomenon. CHO cells have a highly variable and unstable karyotype, given that they are permanently transformed cells that have been cultured in vitro for decades.
Using FISH, Dr. Wurm was able to show the regions that carry the human DNA within the hamster karyotype. These fluorescent spotlights on the chromosomes represent the genetic region that codes for the protein of interest. By periodically monitoring the karyotype of the cells during the scale-up process, it is possible to guarantee the presence of the target marker, assuring that the cell will in fact produce the desired protein.
“There are virtually no diploid cell lines used in bioprocessing today,” Dr. Wurm continued, “and it’s a good thing we didn’t follow recommendations to use them that were current 20 years ago, as it would have set back the industry tremendously.”
Dr. Wurm observed that even using antiquated technologies, “we have seen extremely rapid progress in volumetric yields from batch processes.”
He said the industry is on the cusp of a massive run up in production driven by the demands of the marketplace. This will be realized through development strategies that closely match large-scale systems with resulting easier, shorter run times and much higher cell densities. Fewer reactors will be required as the cells are better adapted and can experience split ratios as high as 1–100, which would have been impossible 20 years ago.
There is good news and bad news on the upstream processing front, Dr. Wurm said. “On the one hand, we are not going to need 200,000-L systems, as process volumes will decline in the next decade. Volumetric yields will rise dramatically, easily exceeding the 10-g/L range. Transient gene expression will dramatically increase the number of proteins processed and recovered.
“The bad news is that the upstream-downstream gap is widening at an alarming rate. New technologies will have to be found and some old technologies will have to be reinvented. Simpler is more foolproof.”
“Current downstream process designs don’t match the upstream process improvements,” observed Alahari Arunakumari, Ph.D., senior director of process development at Medarex (www.medarex.com). “To meet these demands, we have formulated a comprehensive strategy for integrated process development, rather than attacking the problem piecemeal.”