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Cell-culture technology and financial incentives give influenza vaccine makers a much-needed shot in the arm, but many downstream processing issues remain unaddressed.
If necessity is the mother of invention, then funding is its father. For decades, vaccine manufacturers have known that the process for making influenza vaccines using hens' eggs needed to change. But there was no existing market, no business incentive, no financial support. Improving vaccine production would require significant investments and any return on those investments would most likely be pocket change compared with the revenue of a blockbuster drug. Of course the egg-based method developed half a century ago was tedious, time-consuming, and heavily dependent on the health and biological timing of large flocks, but it was also time-tested, GMP-approved, and a consistent technique for producing whole viruses.
Growing influenza viruses in eggs also avoided the compliance work required for making postapproval changes. "Until a few years ago, nobody cared too much because vaccine makers had a perception that the old processes were grandfathered in. If they made any changes to these processes, they would have to get new regulatory approval. So the consensus was to let things be and just continue making them the same way they were made since the 1930s and 40s," says Vijay Singh, president of Wave Biotech (Somerset, NJ, www.wavebiotech.com).
Then, in 2004, a discovery of Serratia marcescens temporarily shut down one of only two US flu vaccine suppliers, and, for the first time, manufacturers could not meet demand. The need for an alternative process was clear, and there was a renewed interest in extending cell-culture technology to influenza vaccine development. "The disaster with Chiron changed everything," says Singh. "A lot of companies realized they had an obligation to bring their vaccine manufacture to current standards, and that meant getting away from the old 1940's processing. That is why we are seeing a new interest in vaccine production and cell culture."
As the deadly avian flu spread unpredictably across the Eastern hemisphere, it became very clear that any alternative production process must not only be safer, but also much, much faster. Public health organizations and government agencies finally gave vaccine manufacturers the support and some of the funding it needed to begin redesigning its processes.
Industry will have to address how cell-culture technology for producing influenza or pandemic vaccines may affect manufacturing operations such as vial filling. (BAXTER HEALTHCARE CORPORATION)
Vaccinologists are well aware of the shortcomings of egg-based production. The traditional method takes six to nine months from the time the World Health Organization identifies the vaccine strain and the Centers for Disease Control and Prevention grows that strain as the seed virus to the time when the virus is sent to manufacturers, grown, processed, and distributed to the market. "During this time, you are hoping that the strain of virus that is circulating does not change," say Rick Bright, PhD, vice-president of vaccine research, Novavax (Rockville, MD, www.novavax.com)
Producing a vaccine to protect against a pandemic would require a year-round supply of eggs, and a pandemic strain may emerge while egg supplies are lower than usual because of a previous epizootic in chickens. Avian strains have been difficult to grow in eggs, but even if they could, the time restrictions of egg-based production would be useless. "If a pandemic lasts two or three years, and it takes nine months for a first dose, but a whole year before you really have a large amount of vaccine produced, you are going to have a whole year where people are dying before you have the vaccine out there giving people their first inoculation. Then if you are going to require two inoculations a month apart, it's just hopeless," explains Bright.
As announced last month (see www.pandemicflu.gov), the first H5N1 strain already has mutated, and a second strain is currently circulating in Asia, Africa, and Europe. To combat a virus that has drifted, industry experts have pointed out that suitable cell substrates will need to grow high yields of various influenza virus strains. Cell-based vaccines also will have to induce the appropriate antibody responses as traditional egg-derived vaccines.
Although several vaccines have been produced in cell culture, influenza-type strains have been particularly difficult to grow. Vaccine developers are actively working toward finding a viable cell substrate and most of this work so far is still in clinical or preclinical trials. Manufacturers are keeping watch on these efforts, knowing that the production methodologies for the next generation of influenza or pandemic vaccines will depend on their understanding of how the antigens were developed. "It's a race to see who has the best approach, makes the best immune response, and can scale it up to make enough to save the world. That is what it's going to come down to," says Bright.
"Two factors will dictate the way a vaccine will need to be purified: the type of cell and the type of vaccine that you want to obtain," says Hélène Pora, vaccines application development director, Pall Life Sciences (Paris,France, www.pall.com. Among the platforms being studied are vero cells (e.g., Baxter), human cell line (e.g., Aventis Pasteur with Crucell's PER.C6 line), and mammalian kidney cells (e.g., GlaxoSmithKline). Few details about these investigations have been disclosed, and if any new technologies were approved, their processing would be proprietary. A few companies, however, are providing some information of interest to vaccine production managers.
Chiron, for example, is working on the Madin Darby Canine Kidney–University of South Dakota (MDCK-SD) cell line. MDCK cell lines have proven effective in producing the inactive polio vaccine. Alison Marquiss, Chiron's director of corporate communications (Emeryville, CA, www.chiron.com) says the company's current work is on the seasonal (trivalent) influenza vaccine but the company anticipates it will apply this technology to pandemic vaccines as well. According to Marquiss, "Our vaccines are the same whether produced in eggs or in cells, so a subunit vaccine produced in eggs would become a subunit vaccine produced in cells." In November 2005, the company participated in an FDA advisory committee meeting after which the committee recommended continued development of its flu cell-culture products. Chiron's development program in Europe is currently in Phase III.
Novavax is one company working with insect cells to grow virus-like particles (VLPs) in disposable bioreactor bags (see Figure 1). The bags contain Sf9 (Spodoptera frugiperda) insect cells which are infected with a modified baculovirus. As the cells grow in the bag, they produce the VLPs containing the hemagglutinin and the neuraminadase surface proteins of influenza virus. The company matches the sequence of virus and uses that sequence to make a recombinant vaccine in about 6 weeks and a finished product in 12 weeks. The 100-μm particles contain virus. There is no virus and no DNA. "In an egg base, once you grow the live virus you have to chemically inactivate the virus, and in doing so you are chopping off these surface proteins, in doing that you are destroying several critical components of those proteins that enhance the immune response. When the VLP vaccine is delivered, the body thinks it's seeing a virus, the immune response very closely mimics the immune response it would make to a live virus," explains Bright.
The purification method is a simple sucrose gradient procedure in which the particles are centrifuged through a sucrose gradient that separates the impurities from the particles based on size. The band in the sucrose gradient containing those particles is removed and the result is put back into a generic buffer solution, resulting in the vaccine. "It's a similar process for the human papillomavirus vaccine that is being made and in clinical trials," adds Bright. The company is finishing a preclinical trial and assembling an IND package to meet with the FDA in hopes of gaining approval to begin a clinical trial by this fall.
Dowpharma is in preclinical animal studies in its development of a fully contained platform for producing plant-derived VLPs of vaccine antigens. These VLPs are produced either in plants grown in highly controlled greenhouses or cells grown in a fermentor. The company system for the vaccine market. Its "Pfénex" expression technology is founded on modified strains of Pseudomonas fluorescens. The Pfénex system produces high levels of soluble, nonglycosylated protein. The bacteria and plant-cell methodologies are similar to that of mammalian cell culture or an Escherichia coli fermentation. The gene making the vaccine protein of interest is integrated into the host cell, which is then grown in a fermentor to provide sufficient yield and volume of product. Both the plant-based approach and Pfénex are being used to produce antigens, adjuvants, and VLPs.
Figure 1: Disposable "Wave Bioreactor" (Wave Biotech) bags, seated on temperature-controlled trays, rock back and forth to create wave agitation. The inflated, gamma-irradiated bag provides an internal sterile environment. The wave motion takes the air from the headspace and forces it to the liquid, providing the oxygen needed for cell growth. It also prevents cells from settling to the bottom, which avoids the formation of a dead-cell layer.
Dowpharma researchers report that vaccines comprising a protein or peptide antigen are generally easier to characterize than vaccines of inactivated virus. "From a quality standpoint the procedures are in place to analyze these and maintain a high quality standard," says Kurt Hoeprich, global commercial director, biopharmaceuticals, Dowpharma (San Diego,CA, www.pharma.dow.com). "Further, we are able to target protein expression in the plant or in Pfénex so that the antigen is easier to purify compared with other systems. Specifically, there are fewer non-antigen components to deal with when you start the purification." The company has adapted its production technologies so they integrate into accepted purification methodologies. "Although the antigen is grown in a different way, inside a plant cell or inside a bacterial cell, when we extract it and start to purify it, we use the same basic methods that the industry has used for a long time and are approved by regulatory agencies. We also use the same analytical tests to characterize the product as those used on products that are on the market today such as ones that come from eggs."
The elimination of allantoic fluid, other animal-derived proteins, particulates, and the preservatives usually associated with egg-based media will result in higher purity batches (typically 1000–2000 L) coming out of bioreactors and going into downstream purification processes. "With cell-based systems, there is a sense of having more control of the purification," says Shawn Knopp, PhD, project manager, Baxter BioPharma Solutions (Bloomington, IN, www.baxter.com).
Cells growing on "Hillex II" microcarriers (SoloHill Engineering). Many cells now being studied in vaccine research are anchorage dependent (i.e., they require a surface on which to grow). Microcarriers provide large surface areas in small volumes for this purpose. The two major types of microcarriers are dextan-based beads (Cytodex 1 and 3, GE Healthcare) and polystyrene-based beads (SoloHill Engineering, Ann Arbor, MI). SoloHill's 150-250 Âµm spherical beads are either inert or may have surface coatings, which decrease the amount of energy required for the cell to attach and grow. "The advantage of getting out of eggs is that you just don't have to handle hundreds of millions of eggs, you readily have microcarriers and cells. you can put a lot of surface area in a big bioreactor and make a lot of material very quickly," says William Hillegas, chief technical officer, SoloHill Engineering.
A cell-based system may not be free of difficulties, however. To meet supply demands, cells would have to be grown in suspension, most likely in large fermentors. "Using cells in a suspense system often leads to a purification challenge because cells are not as good at replicating the viruses. You a lot of non-intact virus particles or even just the building blocks. The solution is a wash with the building blocks of the viruses. Chicken eggs produce a great number of viruses in a much more concentrated form," says Richard Pearce, program director, purification technologies, Millipore (Billerica, MA, www.millipore.com). Therefore, a cell-based process may need to include very large fermentors because the virus titer may not be as high as the virus titer obtained from egg-based systems.
Although serum-free cell culture is a much cleaner feedstock (even if it is lipid-loaded), which makes the initial clarification easier, the volumes tend to be bigger from cell culture than they would be from eggs. "You get a very high concentration of virus growth within the egg. Within a stirred tank bioreactor, the virus concentration tends to be lower," says Pearce.
Chiron's cell-culture process involves storing proven cultures in liquid nitrogen at temperatures less than -180 Â°C. They are revived at the beginning of the production process.
Because cell-culture technology is still in its infancy and a company's vaccine development program is proprietary, no one is quite sure yet which downstream technologies will be most efficient and result in the best quality. Process systems for vaccines grown out of cell-culture may turn out to resemble those of egg-based systems. For example, for a process that involves growing virus in cell culture, the first step typically is standard clarification using depth filters to remove the cells (egg-based systems would use centrifugation), followed by a simple chromatography step (e.g., an anion exchange column for simple affinity purification), then typically a concentration by ultrafiltration. "That would be the same whether it was within an egg-based system or a cell-culture system," explains Pearce.
One difference may be that material coming out of chicken eggs may have a very high endotoxin load, compared with the lower endotoxin load of material coming out of cells. Another difference is that material coming out of cell culture would have host cell proteins and genomic DNA that would need to be removed.
Pearce notes the big differences between egg-based and cell-culture-based processes: the increasing adoption of disposable technology and more chromatographic purification to separate virus vaccine product from similar impurities such as misformed virus particles. "If you look at a lot of the old vaccine processes for flu, for example, there is no chromatography, it's just filters or ultracentrifugation."
As Pora observes, "The downstream purification process for cell-culture derived vaccines is proprietary, but the general tendency recently has been toward increasing the use of ultrafiltration and chromatography." Ultrafiltration, however, cannot distinguish between products and contaminants that are about the same size. In this case, a more-selective methodology such as chromatography, especially ion-exchange chromatography, should be implemented. "Sometimes hydrophobic interaction is used as well, both techniques are usually more productive and efficient than size chromatography."
"When trying to filter a vaccine, the size of the virus is a primary consideration" says Pora. In some cases it may be impossible to filter the virus. In addition, viruses have a tendency to aggregate, which will require close monitoring throughout the development cycle. Moreover, because viruses are charged molecules, they may tend to bind to the membrane. "Anything you can do to develop filters with very little adsorption capability and are open in structure to let the virus flow would be of interest to the marketplace. That is why, as a company, we've been working on the development of polyethersulfone membranes."
Processing experts also predict an increase in membrane-based chromatography, a technology that would help alleviate problems with separating out large molecules. Because membrane chromatography doesn't have the diffusive effects associated with microporous resin beads (used in the manufacture of conventional chromatographic media), one can obtain a much higher capacity for large molecules such as viruses and DNA. "We are seeing a lot of development and interest in membrane chromatography technology for vaccines as well as gene therapy because it provides increased capacity to as much as a 10-fold increase," says Pora.
Filtration scientists also predict that diafiltration or tangential flow filtration would continue to be used at the end of the process to concentrate the final product and to get it into the right formulation buffer before sterile filtration and vialing, "Coming off a chromatography column, the product might have a high salt level, and to reduce the salt level one would use diafiltration or tangential flow filtration. At this point, one of the challenges is that you're trying to do it with relatively low volumes, so you really don't want to be losing your product when you are at the end of the process and it's highly concentrated," says Pearce.
Processing managers also note that disposable technologies could prove cost efficient during a pandemic or surge in supply demand. "During a pandemic crisis, you can't just pop up a new facility overnight. A typical vaccine plant built with tank technology would cost between 20 and 30 million dollars for even a small facility. Whereas if a facility had disposables, it would typically take a few million dollars to build the same capacity," says Singh. Vaccine developers such as Novavax are already are using disposable bioreactors, for example, in their investigations into cell-based influenza vaccines.
Development experts also predict an increased implementation of barrier technology. "Barrier technology has improved. It is still used more for operator safety when you have hazardous entities that you are processing rather than being a standard alternative to traditional cleanroom technology. So I think the verdict is still out if that is going to be a standardized technology that could help us with vaccines. The drive to use barrier technology is going to be the same drive that, if it happens, would move the rest of the industry toward barrier technology in the general sense," says Knopp.
Knopp also predicts that disposable aseptic connections will continue to move the industry toward safer processing. "This technology helps you maintain a closed system, whereas before a lot of the aseptic connections you had to rely on the environment and the personnel to maintain the aseptic condition. These greatly reduce the risk of operator error and environmental contamination." Knopp also sees disposable tubing and closed-loop sampling devices being used more frequently than systems using hard piping, obviating the need for cleaning validation and breaking of the processing line to take samples. "Closed, disposable technology definitely reduces the risk of environmental contamination during aseptic processing and the need for cleaning validation."
Safety and capacity
Safety remains the biggest issue in vaccine production, and cell-based systems present their unique challenges. Specifically, it is still not clear how the genetic material of the cells could affect the antigen. Moreover, cultured cells are not as efficient as chicken eggs at producing whole viruses or well-formed subunits and split viruses. Current purification technology may or may not be able to resolve this issue. "There are two big challenges I see still unmet with cell culture purification. The first is how to separate the components of a badly formed virus from a whole virus. The second is how to separate possibly contaminating viruses from the cell culture," says Pearce.
The potential influence on pharmaceutical manufacturing is further complicated because there is still no consensus about whether capacity will be a major factor. Companies are actively building or acquiring facilities and seeking strategic partnerships or license agreements to help avoid a potential capacity shortage. Industry experts at Chiron, for example, predict "current production capacity of the world's vaccine manufacturers, which is determined by ongoing seasonal epidemic demand, is significantly less than would be required to vaccinate the world's population in the event of a pandemic."
"Traditional vaccine plants could never be built in time in the event of a pandemic," Singh points out. The traditional tank-based plant takes about two years to build from design to validation. The fear that we heard from a number of people is that the typical avian pandemic will only last eight or nine months, so the facility wouldn't be of much use anyway."
One proposed strategy is to commission biologicals plants for routine products, but design them so that they can be repurposed in an emergency to produce vaccines against pandemic strains. Traditional nondisposable facilities for biologics production are inflexible and may be difficult to reconfigure quickly and at reasonable cost. Disposable processing systems, including for buffer preparation and cell-culture, could make it easier to repurpose existing facilities in the event of a pandemic or to meet surge demand.
Another issue that has not been addressed is the risk of retroviruses. When mammalian cell is contaminated it will produce whole retroviruses or retroviral protein products. "When your product is a protein and you have a virus contaminant, you can use technologies that will separate viruses from proteins," says Pearce. The problem is much more complicated for viruses produced out of cell culture. "If a virus were to contaminate the cell, how can you separate a virus from a virus? It is very, very difficult. You also do have that risk with egg-based systems as well but you have obviously built safeguards into the flocks and into the raw materials coming in. That is an unmet need of cell cultured systems: How do you make sure you have the same viral safety of your product equivalent to proteins produced in a cell culture systems?"
Impact on fill and finish
Compared with the huge emphasis placed on producing the raw material, very little attention has been paid to the impact on fill and finish processing. "I think the question of capacity has been placed more on the active substance side and it is kind of a footnote now that we need to address fill finish," says Knopp.
For example, vaccines produced from cell-culture system could be produced in a completely contained system, as opposed to an open egg-based systems that rely on a clean environment, personnel training, and gowns to protect the product. "The biggest advantage between the two is that a closed system has the ability to meet a higher standard for CGMPs," says Knopp. "It is going a level beyond and saying we are going to process this in a way that dramatically reduces the potential for environmental or personnel contamination. You have a better potential to incorporate those types of technologies by virtue of the manufacturing process on the upstream side."
With the introduction of cell-based technologies, the burden of time to market could shift the focus to the fill-and-finish manufacturers as well as raise the debate of whether there is available capacity at this end. "I don't think there is an over or an under abundance of capacity industrywide, but the ability to meet a surge demand is not built in," says Knopp. "Having not only the ability for surge demand on the vaccine raw material side but also on the fill–finish side should be considered. I think you could see that vulnerability if we're not careful."