Drug makers enamored with insect cell bioreactors cite its strengths for producing gene therapy, vaccines, and more.
This image shows some of the equipment used by Cronin, Pfizer Global R&D, for small-scale screening. The 24-well culture block (left) is used for 5 mL expression cultures of incest cells and the 250 mL Erlenmeyer flask (right) is sometimes used for 100 mL cultures. (Source: Ciarán Cronin, PhD)
Insects are one of the most biologically-diverse groups of animals on Earth. And although they appear primitive to the untrained eye, insects are remarkably complex organisms. Insects are multi-cellular, have specialized organ systems including a highly-evolved nervous system, and have been the subject of biological studies for decades. One prime example of the use of insects in basic research is the genetic model, Drosophila melanogaster, the fruit fly. This little critter has been used for decades and has contributed to our knowledge of genetic interactions, gene mapping, chromosome arrangement, and much more. Another example is the honey bee, which has been used to help us understand social behavior in animals and has also been bred to produce honey on an industrial scale; honey production will likely improve with the recent completion of the honey bee genome. A major contribution of the insect to the study of biology is the insect cell bioreactor—a system for producing therapeutic proteins.
“The system works by programming baculoviruses with the genes [of interest]. And when the baculovirus is infecting Sf9 insect cells, over the two to three days of infection is when the high levels of gene expression of a foreign gene or cloned gene you have introduced into the system is produced,” says Gale Smith, PhD, vice president of vaccine development, Novavax, Inc., Rockville, Md.
“Sf9 insect cells were originally isolated from the ovaries. So it is an ovarian cell line of a Lepidoptera, which is a butterfly and moth, and of Spodoptera frugiperda,” says Smith, who adds that the Sf9 cell line was cloned in the early 1980s and will be in culture indefinitely because they are immortalized cells.
According to Smith, the insect cell bioreactor has a lot of advantages. One advantage is that insects do not generally carry any human pathogens so they are safe to use for the production of therapeutic proteins. “The baculovirus that is used normally infect these Lepidoptera—butterflies and moth—and there are quite a few baculoviruses that are used for pest control,” says Smith. “[Baculoviruses] are extremely safe. They have never been shown to infect man or any other mammals. They are found on virtually all green leafy vegetables. So, for example, whenever you have a salad, you are consuming some baculoviruses.”
Another advantage of the insect cell bioreactor is that, in contrast to Escherichia coli and yeast cell bioreactors, insect cells can add post-translational modifications such as glycosylation, thus allowing this system to produce complex human proteins as well as vaccines.
Producing gene therapies
“I think the big success of [insect cell bioreactor] technology came through the use of the baculovirus system,” says Amine Kamen, PhD, head of Animal Cell Technology, Biotechnology Technology Institute, National Research Council, Montreal, Canada. “Since its discovery, it was the most rapid and straightforward system to get the protein from recombinant DNA.” According to Kamen, one of the major advantages of the insect cell system is its ability to produce high yields of recombinant protein at high cell density in serum-free culture medium.
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This graphic is a schematic representation for the production of recombinant adeno-associated virus from baculovirus-infected insect cells. (Source: Juan Alejandro Negrete Virgen, PhD)
Although Kamen does not use the insect cell bioreactor as much as he used to, his main use is in generating protein targets for drug screening, for which he is collaborating with big pharma companies. He is currently using baculovirus to produce adeno-associated virus (AAV) that will eventually be used as a gene therapy vector to deliver, for example, dystrophin to patients with Duchenne muscular dystrophy.
Another researcher using an insect cell bioreactor to produce recombinant adeno-associated viral vectors (rAAV) for gene therapy applications is Juan Alejandro Negrete Virgen, PhD, Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Md. “This system requires three different baculoviruses. One containing the genes expressing the capsid proteins of AAV, a second containing its replication genes, and the third containing the gene of interest,” says Negrete. All three viruses are used to simultaneously infect insect cells. “The beauty of this system is that insect cells and baculoviruses are utilized to produce human viruses for gene therapy applications; our technology was described for the first time in 2002.”
One application of this technology includes the gene therapy for Duchenne muscular dystrophy. To restore the functions of dystrophin, a protein found in human muscle cells, some exon skipping constructs have been evaluated in animal models. “After gene therapy with this recombinant AAV, around 80% of the muscle activity is restored. This pre-
clinical trial in animal models is still ongoing”, says Negrete.
In order to produce sufficient rAAV to complete pre-clinical animal testing, Negrete has scaled-up the manufacturing process up to the 250-liter scale. With the developed process, it is possible to produce approximately 1e17 particles. He is looking to stirrer tank vessels to reach several thousand liter cultures with minimal adjustments. “Our system has some big advantages because none of the mammalian genes are involved, the cells can grow in serum-free media in suspension, it is relatively easy to scale-up, and there is no difference in the quality of the product compared to the rAAV produced in mammalian cells” says Negrete. Two factors make scale-up of the insect cell bioreactor more cost-effective than scale-up of the mammalian cell system. First, because baculovirus is being used to carry the gene of interest, there is no need to perform expensive plasmid production and purification. And secondly, the insect cells can grow in suspension in serum-free conditions, so there is no need to purchase expensive serum.
Novavax uses the insect cell system to produce its influenza vaccine. “It is very easy and very rapid for engineering genes from influenza virus or any other source for producing the recombinant products/proteins,” says Smith. He adds that it takes a few days to produce the desired proteins and often the yields are as high or higher than what can be achieved in, for example, mammalian cells.
“We are currently producing at 100-liter scale for lots that are going to the clinic. And the yields are high enough,” says James Robinson, vice president of technical and quality operations at Novavax. “And one of these 100-liter batches produces as much flu as 70,000 eggs in the egg-based, flu vaccine production program.” Novavax has had a pandemic flu vaccine candidate in the clinic since the middle of 2007; it is currently in Phase 2.
The insect cell bioreactor is also safer for manufacturing than other systems. “The fact that we are not using a live flu virus, but we are using a nonpathogenic baculovirus to infect our cells means that we don’t have to operate in a contained manufacturing environment,” says Robinson. “If the bioreactor spills, there is no safety issue. Whereas if you spill a bioreactor filled with live flu virus, you have a huge environmental risk.
This image shows a Wave Bioreactor, which is used by Cronin to scale-up expression of recombinant protein in insect cell culture. (Source: Ciarán Cronin, PhD)
Producing drug targets
Another researcher using the insect cell bioreactor is Ciarán N. Cronin, PhD, head of the Parallel Protein Production Group, Pfizer Global R&D, La Jolla, Calif. Cronin has generally used Sf9 for protein production in insect cells for the following reasons:
• It is the most commonly-used insect cell line for baculovirus transfection and its shorter doubling time makes it more suitable to recombinant protein production scheduling than Sf21 cells.
• Also, Sf9 cells often show less endogenous target proteolysis than T.ni (High Five) insect cells.
Most of the proteins are being produced in Wave Bioreactors (GE Healthcare, Somerset, N.J.) from the five to 100-liter scale. The majority of the proteins are oncology targets produced for the purpose of structure-based drug design (SBDD) efforts or to perform high-throughput screening of compound libraries.
After these proteins are produced in the Wave Bioreactors, Cronin uses a generic protocol to purify them. This protocol, which works for about 90% of their proteins, includes the use of cleavable histidine (His) tags, i.e., the recombinant protein is tagged with histidine residues during the cloning steps. In general, the purification protocol starts with an IMAC column, followed by His tag removal, a second IMAC column, and then, in some cases, a final clean-up by gel filtration on HPLC (Dionex Corporation, Sunnyvale, Calif.).
“For crystallization, proteins need to be reasonably well–purified, in a mono-dispersed state, and preferentially a single species in terms of post-translational modification,” says Cronin. To determine the structure, the protein does not need to be biologically-active, although it should have ligand-binding capability for SBDD efforts. However, for compound-screening assays, the protein target must be biologically-functional with regard to ligand-binding or activity.
In summary, the insect cell bioreactor appears to have an unlimited number of applications for drug discovery and development. The insect bioreactor can produce protein targets for performing high-throughput screening of compound libraries, proteins for structure determinations, viral vectors for gene therapy, and viral proteins or particles to be used as vaccines. And based on this success, it appears that insect cell bioreactor will be used for drug discovery and development long into the future.
This article was published in Drug Discovery & Development magazine: Vol. 11, No. 4, April, 2008, pp. 26-28.
Filed Under: Drug Discovery