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Feature Articles : Apr 1, 2010 ( )
Cell Culture Moving Past Previous Technical Limits
Researchers Employ Insect and Duck Cells, Novel Media, and Next-Generation Automation Technologies
Spodoptera frugiperda (the fall armyworm caterpillar) and the duck are gradually replacing the chicken as the creature of choice for vaccine production. New cell-culture techniques and, particularly, recombinant technologies, are bringing faster production cycles and higher purity yields, according to researchers who will present their insights at the Bharatbook conference on “Vaccine Manufacturing” next month in London.
Protein Sciences is about to commercialize its FluBlok™ seasonal flu vaccine, which was developed through a baculovirus protein-expression technology that uses insect cells as host cells. The Spodoptera frugiperda insect cell line (expresSF+® cells) grows in suspension culture in the absence of serum. “We will have one of the first products made in insect cells licensed in the U.S.,” reports Clifton McPherson, Ph.D., director of quality control. GlaxoSmithKline was the first, using a different insect virus.
“We use a recombinant baculovirus in which a highly expressed gene is replaced with the hemagglutinin gene, and this recombinant baculovirus is used to infect expresSF+ cells,” Dr. McPherson explains. Using an insect cell line also reduces the potential of cross-over infections, as few viruses can infect both insects and humans.
Although chicken and egg technologies have been widely used for decades, they are expensive and have certain production issues. Namely, “the influenza virus is adapted to grow in eggs,” Dr. McPherson says. In contrast, “ours can be a perfect match for the virus in the wild.”
Protein Sciences’ technology is also scalable. The company is in the process of scaling up from 500 L batches using the same bioreactors used for mAbs. It’s also safe, he adds. “We never handle live virus. We start with RNA or with the gene sequence.”
The same technology used for FluBlok is also being used to develop a pandemic vaccine called PanBlok™ that is in Phase II trials in partnership with UMN Pharma and with support from BARDA in the U.S.
PanBlok is based on a purified recombinant hemagglutinin antigen from the H5N1 avian influenza. Protein Sciences is working to speed pandemic response with the goal of producing 50 million doses within six months of an outbreak. Protein Sciences’ technology allows for a rapid response, and the production volume is more assured because recombinant technology is not limited by egg production concerns.
Vivalis chose avian stem cells for its cell-culture line. There are two major advantages, according to Majid Mehtali, Ph.D., managing director and CSO. “The avian nature of the cells allows Vivalis’ EB66 cell line to be used as an alternative manufacturing platform for the production of all human and animal vaccines previously produced on chicken eggs. The use of duck instead of chicken cells prevents the risk of contamination by endogenous retroviral particles, which are common in chicken cells but not in duck cells.
“The use of embryonic stem cells is beneficial since such cells are the only ones that are naturally immortal and genetically stable due to their naturally high levels of telomerase activity, which stabilizes the chromosome structure and length of telomeres. Consequently, EB66 cells can be grown for hundreds of generations without major changes in their biological properties.”
A broad variety of viral vaccines can be produced in EB66, with production yields for influenza viruses that are comparable to those of mammalian cells, Dr. Mehtali reports. Because stem cells differentiate depending on the cell-culture environment, the procedure used to generate the EB66 cell line was designed for great stability and will only differentiate in harsh conditions.
“Additionally, a strategic collaboration with SAFC-Biosciences allowed the development of custom EB66 cell culture media, which allows efficient cell growth at high cell densities (more than 30 million cells/mL) and a short population doubling time of about 15 hours without any differentiation,” Dr. Mehtali adds.
Manufacturers report that the greatest challenges to the widespread adaptation of cell-culture production techniques in the vaccine industry are regulatory.
Vivalis undertook a stringent and extensive characterization of the sanitary status of the EB66 cell line and thoroughly documented its history and the procedures necessary for its establishment, according to Dr. Mehtali. That information was included in the biological master file that was submitted to the FDA in 2008. The company anticipates receiving an IND for a human vaccine sometime this year.
Much to its chagrin, Protein Sciences’ process is basically regulated as a vaccine and as a recombinant protein—a combination that is not yet commonplace for regulatory bodies. Consequently, both sides have a learning curve. “We’re in the final stages of licensing with the FDA,” Dr. McPherson notes.
Ferruccio Messi, Ph.D., founder of Cell Culture Technologies, has developed minimal culture environments that streamline the purification process for cell-culture based products. “They are protein- and peptide-free and consist of a mixture of water and molecules only,” he says, which simplifies purification.
A complex culture medium is, by its very nature, an undefined nutrient mixture, Dr. Messi stresses. “You can’t control your culture process if you don’t know what’s around the cell. Cells continuously interact with their environment, and if you don’t know what’s in that environment, there’s no way to accurately predict how cell metabolism will be affected.
“With our technology, we eliminate that problem.” Cell Culture Technologies’ minimal culture media are made exclusively of tissue culture, water and chemically defined small molecules. Consequently, users can develop a methodology that governs cell behavior, based upon understanding and maintaining the nutritional balance of the culture media.
“There’s also an advantage in upstream development by knowing what chemicals surround the cell.” That results in better control, which produces a more consistent outcome, lower costs, and speeds purification. “Product can be harvested in almost a purified form.”
In 2006 MedImmune was awarded a Department of Health and Human Services (HHS) contract to develop a cell culture system for seasonal and pandemic flu. The project involves both upstream and downstream work and, according to George W. Kemble, Ph.D., vp of R&D and GM, “we came up with a robust program.”
The vaccine is based on the Madin-Darby Canine Kidney (MDCK) cell line. When compared to several other popular, potential substrates for virus production, “MDCK cells supported high levels of vaccine virus production for multiple different live attenuated influenza vaccine (LAIV) subtypes in both serum-containing and serum-free media. These results suggest that MDCK cell-based production can be used as an alternative production platform to the currently used egg-based LAIV production system,” Dr. Kemble explains.
“Lab-scale bioreactors can produce 10,000 to 100,000 doses or more with this cell line,” he adds. Additionally, this cell line has a lower tumorigenic potential than many other similar cell lines used for vaccine development.
MedImmune has developed “a more robust, purification process,” Dr. Kemble says, because live viruses can’t tolerate the harsh conditions typical for inactivated viruses. This new process is at “late preclinical stage now. An IND has been submitted to CBER,” and MedImmune is talking with HHS about how to proceed. As it moves into clinical trials, “we’ll probably use a prototype pandemic virus strain.”
As cell-culture applications grow, so does the need for automation. In response, The Automation Partnership (TAP) introduced ambr™, an automated microbioreactor system, earlier this year. This system replicates the conditions in 5–10 L bioreactors within 10–15 mL vials. The system also provides stirred sparged culture with closed loop control of DO and pH along with automated sampling and feeding.
“Results from ambr’s beta testers have shown that the productivity values match those of larger bioreactors for fed-batch processes,” according to Richard Wales, Ph.D., systems development. Those early users also found a correlation in the amount of protein produced.
In many labs, “there’s a queue for bioreactors,” Dr. Wales says. “People tell us one FTE can manage four to eight bench-scale bioreactors at the 2 to 10 liter scale.” In contrast, that same FTE can manage 24 microbioreactors in the ambr system in only a quarter of the time. “There’s quite an overhead associated with larger bioreactors,” he explains, in terms of labor and cleaning, as well as materials consumption.
In contrast, ambr automates tedious tasks, including set-up, feeding, sampling, and maintenance. This saves a significant amount of time, as does the disposable nature of the bioreactors. ambr reportedly has applications throughout the biodevelopment process, but TAP is focusing initially on cell-line selection and characterization and early process development.
Another recent addition to cell-culture automation, TAP’s Sonata™ cultures and processes insect and mammalian cell lines in shake flasks. It can count cells, add and decant media, centrifuge and harvest cells, and provide refrigeration, allowing optimization to occur at any time, thereby increasing workflow, Dr. Wales says. With automation, “you get a much higher level of consistency.”
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