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Feature Articles : Mar 1, 2012 (Vol. 32, No. 5)

Modernizing Vaccine Production

  • Hannah Waters

Emerging bird and swine flu variants during the past decade haven’t turned into the epidemics some feared, but they have still sent a firm message to governments and drugmakers: Current manufacturing procedures are not ready to produce enough vaccine fast enough to counter such threats. As a result, the U.S. government has invested heavily in vaccine production technology. Beyond filling a health need, it’s building up an arsenal of production and manufacturing systems to ensure that flu vaccines can be delivered efficiently in the face of biological threats. And vaccines for other pathogens are hitching a ride on the funding tailcoats, modernizing technology across the entire field. At the “World Vaccine Congress” next month, vaccine-makers will gather to discuss their innovations.

“The pandemic threat that started eight years ago with avian flu is what really spearheaded this focal point to try to find a cell substrate that can handle high generation,” says Deborah Mosca, vp of product management at PaxVax. “We think novel cell substrates can respond much faster in the three-to-six month time frame with millions and billions of doses. And you can’t get there with eggs.”

Modern flu vaccine production methods haven’t changed much since the 1950s, and are still egg-based. Vaccine manufacturers inject thousands upon millions of chicken eggs with virus and let them incubate for 9–12 days. Then the eggs are cracked, and the virus is purified and prepared into a vaccine.

The process is slow, and occasionally entire batches of contaminated eggs have to be thrown out—hence the push toward cell-based production methods. “What makes cell culture attractive are the concerns about a bird flu,” says Tim Hahn, senior vp of manufacturing at NovaVax. “With a bird flu, the birds are infected, the eggs are infected, and eggs are used to make the vaccine.”

At NovaVax, researchers are harvesting virus-like particles (VLPs) grown inside insect cells to use as vaccine antigens. VLPs contain proteins that mimic viral structure but lack genetic material. Thus the immune system will form antibodies to the viral proteins, but there is no danger of replication and infection.

To generate the particles, the antigen sequence for pandemic or seasonal flu, two main disease areas at NovaVax, is encoded into a baculovirus, which then infects Sf9 insect cells grown in culture. Influenza spreads by budding new viruses off the cell membrane, and the researchers “keep that natural mechanism in the DNA coding,” says Hahn, so that the VLPs simply bud off the insect cell membrane. They’re then density-separated and purified, ready to generate an immune response.

The insect-based method isn’t cheap, however, so NovaVax has turned to other aspects of manufacturing to save money. It grows its cultures inside disposable bags housed in stainless steel incubators, reducing cleaning costs between batches. Its purification system also has disposable parts for the same reason. “It saves on infrastructure, saves on time, and gives us a better turnout between batches,” reports Hahn.

NovaVax’ seasonal flu vaccine will begin Phase II trials early this year, and it plans to begin human trials for a pandemic flu vaccine in the second quarter.

It’s taken a long time to get to the point where companies could even consider testing cell culture-based vaccines in people because regulatory authorities were unsure of their safety. NovaVax had some paving laid before it because GlaxoSmithKline has already broken in the FDA with its Cervarix vaccine against human papillomavirus, which is VLP-produced using an insect-baculovirus system.

“There has been tremendous resistance for introducing new cell types because of the unknowns,” says Mosca. “But when there’s been a need, there has been push and the regulatory authorities have acquiesced.”

One way to baby-step the regulatory dance is to use protocols already widely accepted, and tie in a new innovation—and this is exactly what PaxVax is doing. The antigen for its pandemic flu vaccine is encoded on an adenovirus vector, which has been used in military vaccinations for decades. “We had the benefit of all the experience from the military,” says Mosca. “Ten million recruits have taken it over the years with no significant adverse events.”

However, its real innovation is pioneering the use of a human cell line, A549, for use in vaccine production. Several human cell lines are already used to make vaccines, but one benefit of A549 is that it is immortal and thus can be used to continually produce vaccine proteins without having to restart cell cultures from scratch every 100 generations.

Aiming for Purity

A big challenge in vaccine production is not only designing an antigen that is safe and effective, but purifying the protein enough that it’s safe to inject into people. In 2010, pig viral DNA was found in a rotavirus vaccine—a remnant from the pig serum used in the cell culture medium. The scare was innocuous; nonetheless vaccine-makers have been trying to eliminate unnecessary animal products from manufacturing to make more pure vaccines.

PaxVax has taken steps in this direction by adapting its A549 human cell line to grow without calf serum in the culture medium, a common ingredient for human and other organismal cell lines. By slowly decreasing the serum concentration in the culture medium over time and selecting the cells that survived, the strain no longer requires calf serum, an ingredient that is expensive, potentially contaminating, and in limited supply worldwide.

“If there’s a pandemic and you need serum for vaccine, there may not be enough serum to feed your cells to make it,” says Mosca. “A serum-free suspension culture allows you to respond quickly.”

PaxVax has pushed the cell line even further, however. Most human cells are grown in plates, which are more labor-intensive because of continuous scraping and monitoring and take up a lot more space—another downside of egg-based manufacture. But A549 can be grown in suspension culture. “The actual facility and the equipment is much smaller for getting the same amount of doses,” says Mosca.

This also saves money on testing vaccine for purity and efficacy. “One of the things that people don’t realize is that the most difficult and expensive part of making a vaccine is not making the vaccine itself, it’s testing what you made,” says Alan Shaw, president and CEO of VaxInnate. If a single batch of vaccine contains many doses, all produced from the same suspension culture, can be purified and tested at once, “you just saved 90% of the cost,” says Mosca.

VaxInnate also grows its cell culture in suspension, but the researchers haven’t had to work as hard because they generate their vaccine antigens in Escherichia coli, an organism with incredibly well-characterized methods.

“Our purification system is pretty standard, using standard biophysical and biochemical purification processes,” says Shaw. But another reason its purification system is so simple, he adds, is that the company is making a basic “biopharmaceutical protein, which is a big contrast to the standard type of flu vaccines.”

The protein purified from this process is standard influenza hemagglutinin, commonly used to make vaccines. But to make the antigen more immunogenic, VaxInnate pairs it with flagellin, the protein that forms the bacterial motor flagellum.

The human immune system immediately recognizes the flagellin protein as nonhuman, generating a robust antibody response. “We just make this bifunctional molecule that’s efficiently made in bacteria, and humans respond to it very briskly,” says Shaw. “So if you’re in a hurry, this is a great way to do it.”

Cell Culture: Is It Cost-Effective?

Despite the benefits of using cell culture—making more vaccine more quickly in the face of a pandemic—it’s not necessarily a cost-effective process. Any new technology takes substantial research and development costs, for starters. But additionally, the vast testing and assays required by regulatory agencies to prove safety aren’t small change.

For example, Pharmathene has developed assays to characterize the protein, demonstrate vaccine stability, show consistent manufacturing processes, measure immunopotency and toxin neutralization—the list goes on.

“To go through the development and qualification of these assays is a lot of work,” says Tom Fuerst, CSO. “And then you have to establish system suitability criteria around them to make sure that they’re precise and accurate.”

Pharmathene also uses E. coli to culture its vaccines, but its focus is on anthrax. An anthrax vaccine has been licensed since the 1980s, using an attenuated strain. But the current method involves a “crude preparation,” says Fuerst, that is “attenuated with formaldehyde, which is now considered a carcinogen by the NIH”—certainly not an ideal situation.

Pharmathene’s vaccine antigen is a highly conserved recombinant protein from the anthrax bacteria grown instead in E. coli, for which “the cost of manufacturing is very low and production levels are exceptionally high,” says Fuerst.

One of the strengths of its vaccine is that it can be stabilized in an intermediate form, and Pharmathene aims for at least a three-year storage time. This method would allow it to mobilize quickly if another anthrax outbreak occurred.

“In event that there is an emergency, and the demand for an anthrax vaccine is very high, we are able to produce stable production intermediates that then can be polished very quickly and formulated into a vaccine,” says Fuerst.

All the companies mentioned are making vaccines for some diseases that already have vaccines available. Even if they are arguably making better versions of these vaccines, is all the time and money invested worth it?

“By and large what people find is that the cell culture system is cleaner, but a lot more expensive,” says Shaw. “The seasonal flu vaccine is a commodity product…and with cell culture, what you wind up with is a more expensive commodity product that’s not terribly economically interesting.”

But the U.S. government has made its investment clear. Later this year, a $1 billion, 430,000 square foot cell culture-based vaccine-making facility in Holly Springs, North Carolina, is slated for production.

“I don’t know if Novartis is going to break even; it will be more expensive to make than the standard flu vaccines by a factor of two or three I think,” Shaw says. “But enough money has been invested in the cell culture system, at least in the U.S., that they have to use it.”

A big investment, sure; but it is estimated to be able to produce 150 million doses of flu vaccine within six months of a pandemic declaration. This is a far cry from the circumstances surrounding H1N1’s discovery in 2009, when vaccine production didn’t begin until six month after the virus emerged, and even then it was only at a rate of 10 million doses per week.