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A look at vaccine history, markets, manufacturing, and overcoming the scale-up dilemma.
As the world population expands, the need for human vaccines (to protect ourselves) and veterinary vaccines (to protect our pets and livestock food sources) has dramatically increased. Despite the demand over the past few decades, the industry has fallen behind in delivering new commercialization solutions to answer the needs of the population. Advances in cell-culture techniques have long been a key driver for enabling new vaccine solutions, and now, emerging process innovations and efficiency breakthroughs are solving traditional bottlenecks in vaccine development and production.
Jose Castillo, PhD
This article will review the history of and present advancements in vaccine production through mammalian cell cultivation for both weakened or killed forms of viruses (attenuated life viral vaccines or killed viral vaccines) and surface proteins of viruses or bacteria (recombinant sub-unit vaccines).
Vaccines, or biological preparations that improve immunity to a particular disease, have been around for centuries and still remain vital in the fight to ward off diseases. The term vaccine derives from the work of Edward Jenner (1749–1823), an English physician and scientist considered to be the father of immunology. His seminal work in 1796 used cow pox, or variola vaccinia (from the Latin vacca, meaning cow), to inoculate humans and protect them from smallpox.
The injectable polio vaccine was the first biopharmaceutical mass-produced using cell-culture techniques. It was made possible through the research efforts of Drs. Franklin Enders, Thomas Wellers, and Frederick Robbins, who were awarded the 1954 Nobel Prize in Physiology and Medicine for their discovery of a method for growing the virus in monkey kidney-cell cultures (Vero cells).
Vaccines typically contain an agent that resembles a disease-causing microorganism, and are often made from either weakened or killed forms of the microbe, toxins from the microbe, or a surface protein from the microbe. When they are manufactured to initiate immune system responses in humans and animals that help combat invading enemies, they act as an effective first line of defense against the spread of disease.
The global human vaccine industry is estimated to be worth $25 billion, with around 600 products currently on the market (1). Revenue is predicted to reach about 10% annual growth rate (CAGR) (2).
The human vaccine market is mainly in the hands of five large corporations that manufacture vaccines for the world's markets. There are small players as well, primarily in developing countries. Some countries, such as India, have a local biotechnology industry developing world-class vaccine manufacturing plants and expertise. Overall, the needs in Brazil, Russia, India, and China (the BRIC) are especially high, as they represent 50% of the world's population and fall behind the rest of the world in terms of manufacturing infrastructure.
On the other side of the equation, the global veterinary vaccine industry is estimated to be worth $5.24 billion, with approximately 2400 products on the market (3). Revenue for this industry is predicted to reach about 5.7% CAGR. The structure of this market is quite different when compared with that of human vaccines. Here, there are also several large, global suppliers. Unlike with human vaccine development, however, there is not a single species to treat. There is, instead, a vast variety of animal species, each with several diseases and specific needs. Consequently, vaccines from some companies are manufactured exclusively in roller bottles, because many batches of different vaccines are produced in parallel and processes using bioreactors have been slow to develop.
To understand how viral vaccines are manufactured today, we can refer to the pioneering work from the past two centuries, when both cells and viruses were studied in glass Petri dishes (German biologist Julius Richard Petri, 19th century) and glass Roux bottles (German biologist Wilhem Roux, 19th century).
When capacity was needed to produce larger quantities of viruses, the most obvious way to proceed was to multiply the number of these devices. As time went on, Petri dishes became tissue culture flasks and then cell factories or cell stacks. The Roux bottles then turned into roller bottles. Until recently, these were still made of glass; today they are made of plastics—mainly polystyrene or polyethylene terephthalate.
The use of such devices is quite straightforward. As scaling-up is strictly linear, no process development is needed. Increased capacity is obtained by a multiplication of the number of devices and a linear multiplication of footprint, operators, and operations. For some time, this approach was quite successful and used to produce both human and animal vaccines.
For other vaccines, like the polio vaccine (human), or the foot-and-mouth disease vaccine (animal), the need was so great that in the mid-1970s it appeared the multiplication of roller bottles was no longer a viable near-term option.
To meet the changing needs, large companies like GlaxoSmithKline, Merial, Intervet, Merck, and Sanofi invested in a different approach, combining large-scale, stirred-tank bioreactors, already proven for bacterial and suspension cell growth, with oligosaccharide-based microbeads to provide the adherent cells with a substrate to adhere to. This approach allowed a dramatic decrease in both the footprint and the number of operations by increasing the ratio surface per volume. It was a very clever and effective shift for the time.
However, a different set of ongoing challenges has emerged in the present time, mainly related to manufacturing scale. The most crucial challenge has to do with speed: a sufficiently high mixing speed is required for proper mixing distribution, but the mixing speed, however, must not be too fast to avoid negative effects on the process.
Microbeads must be in suspension to create a homogeneous distribution of surface into the volume of the bioreactor. In addition, microbeads and cells need to physically meet for the attachment process to occur. The high mixing speed enables this contact, but the microbeads are fragile (made of dextran) and have the mechanical properties of hydrogel. Moreover, the beads and cells need to come together for a sufficient amount of time to attach, therefore, mixing speed must be optimized to enable this time-sensitive attachment.
Because the scale-up of mixing is nonlinear to a large extent, it is easy to predict the difficulty in finding a new process that also offers control. In addition to this difficulty, the multiplication factor of cells on microcarriers lies typically in the range of five- to 15-fold, which makes several "preculture" steps necessary before cells can be seeded into the final-scale bioreactor. Typically, a 1000-L bioreactor will be seeded with cells from a 200-L bioreactor, which was seeded with cells from a 25-L bioreactor to meet scale. In practice, it means that engineers have to develop a process at 1000 L, a process at 200-L and a process at 25-L. For each volume, the cells need to be detached from the microcarriers in one bioreactor to seed the next bioreactor, which presents a challenge. In fact, many companies decided to give up on realizing a new process because of the lack of consistency and reliability in the outcome.
To increase capacity, companies using today's roller bottles have two options: increase the number of roller bottles or cell stacks, or implement stirred-bioreactors at large volume with microcarriers. Each has its drawbacks. Increasing the number of bottles or stacks means that additional cleanroom space and experienced labor (more capital) are required, which also brings an increase in contamination risks, as well as compromised consistency. Implementing stirred reactors at a large volume with microcarriers presents process development challenges, requires additional intermediate bioreactors, increases operating and manufacturing costs, and extends time horizons for results.
In the face of the cost and risk deterrents, it became clear that there were new opportunities to bring efficiencies and increase return on investments by taking a different approach to the challenge.An alternative approach is presented below, which bypasses some of the historical difficulties of scale-up. This approach uses three criteria for cell growth/viral production technology:
Figure 1: Scale-up comparison of Integrity iCELLis bioreactor with roller bottles.
This approach offers a bioreactor system with a simplified, minimal seed train requirement in which biomass would be multiplied by up to 500 times. Consequently, the Integrity iCELLis bioreactor can be inoculated at a very low cell density, and no preceding bioreactor is needed for seeding. In addition, the biomass is immobilized to avoid mechanical constrains at very high cell density and avoid additional perfusion tools. Because the system is compact, there is no need to increase the size of infrastructure to implement the capacity increase, enabling commercial-scale manufacturing in a typical laboratory environment
Innovation in process design is vital in meeting the world's growing vaccine demand. A technology such as the iCELLis bioreactor provides mass scale-up for vaccine production and enables commercial-scale manufacturing results. In the present day, speed to market and streamlined development are more crucial than ever before. As long as markets remain unsatisfied with the traditional pace of progress, innovation will continue to open the doors to the next levels of bioprocess advancements and breakthroughs.
Jose Castillo, PhD, is director of cell-culture technologies at ATMI LifeSciences, firstname.lastname@example.org.
1. Research and Markets, Human Vaccines: A Global Strategic Business Report (San Jose, CA, January 2011).
2. A. Hiller, "Vaccines Continue to Bolster Pharma Market," Dec. 2, 2010, www.pharmpro.com/articles/2010/12/busines-Vaccines-Continue-to-Bolster-Pharma-Market/, accessed Jan. 7, 2013.
3. ATMI, In-house market research: desk searches (companies and Institutions websites) and meetings with NOAH and ANSES (2012).