Most cell-culture–based viral vaccine production has historically been performed using anchorage-dependent cultures: The first large-scale virus culture of animal cells was accomplished in the 1950s using an array of 5-L glass (Povitsky) flasks. Roller bottle use was originally developed at Johns Hopkins University for growing large quantities of adherent cells. In addition to increased surface area, the bottles exhibited other advantages over static cultures in preventing gradients from forming in cell culture media and in improving gas exchange with thinner layers of cell culture medium overlay. Disposable plastic roller bottles appeared on the market in the mid-1970s and soon became a mainstay for vaccine production.
Today's manufacturers can choose from a number of diverse formats supporting either attached or suspension cell cultures. Although many bioprocessors have long-since transitioned to using steam-in-place (SIP), stainless-steel, stirred-tank reactor systems, roller bottles remain in significant use today for legacy processes. They provide robust, well-characterized solutions for some new products as well, for which other platforms have not yet been demonstrated. But the modern bottle shapes, plumbing and closure configurations, culture surface treatments, and housing apparatus would hardly be recognizable to those early culturists (Figure 2).
The next wave of production modes have continued with the disposable theme. These include an engineered extension of the T-flask: stacked-array reactors such as the Thermo Scientific Nunc Cell Factory system from Thermo Fisher Scientific (Figure 2). The scalable system provides a more compact footprint than do traditional roller bottle systems, and it comes in several versions with one to 40 trays and a range of culture media transfer mechanisms, port dimensions and styles, and other equipment to assist in handling.Microcarriers
Interest in using well-developed and efficient stirred-tank bioreactor systems resulted in further developments for attachment-dependent culture platforms. New low-density matrices that can be easily suspended in bioreactors come as solid or microporous substrates composed of diverse rigid or pliable materials. New manufacturing formats and optimized procedures have emerged to complement them.
Like roller bottles, microcarriers have a long history of use for adherent cell culture. As the move from egg-based to cell-culture–based influenza vaccine production has accelerated, so too has the focus on overcoming some challenges associated with this platform. Mark Szczypka, (president of SoloHill Engineering, Inc.) told us, “Advancements in bioreactor applications — such as new solutions in microcarrier culture — are now providing powerful alternatives to the older methods of vaccine manufacturing.”
One such accomplishment comes in the ability to culture the large numbers of adherent cells required to seed bioreactors of >1,000-L capacity. Microcarrier-based expansion in bioreactor seed trains has been demonstrated as a viable solution to this challenge. Specialty microcarriers present such characterized and designatable features as cross-linked polystyrene copolymer composition, cationic-modified surfaces, and buoyant densities (Table 3).Table 3: Microcarriers in vaccine production
For example, SoloHill Engineering addressed recent demands in cell culture processes by developing ADCF products such Hillex II microcarriers (11). These specifically charged beads have demonstrated utility for supporting serial passage of many common cell lines as well as success in high-titer vaccine production with ADCF serum-free media (Figures 2 and 3, Table 1).
Other innovations supporting applications in vaccine manufacturing include microcarrier products with ultralow particulate counts, products amenable to gamma irradiation without loss of function, and distribution in convenient and compliant single-use bioprocess containers. Together, these developments support further advancement in vaccine manufacturing, such as the ability to provide single-use bioreactors (SUBs) preloaded with microcarriers for fully integrated systems.Disposables
Single-use technology has become an accepted component of bioproduction based on animal cells (Figure 4). Both off-the-shelf and custom-designed systems are now in regular use to some extent as part of nearly every production process at contract manufacturing organizations (CMOs) and biopharmaceutical companies, including vaccine manufacturing (12). Drivers for this rapid acceptance and widespread use in such a conservative industry have been well reviewed in recent years. Some of the most important to vaccine production include advantages in facility development and installation time, cost of goods, prevention of lot cross-contamination, rapid turn-around times, production scheduling flexibility, and surge capacity (13).
Disposable components being used in vaccine manufacture include systems for process fluid mixing and storage, bulk material and product storage, filtration, and chromatography, as well as distribution manifolds, sensors, connectors, and production-scale bioreactors (14). Their acceptance by the vaccine industry is evidenced by both the number of companies successfully using them and in published applications (Figure 5).
More than a dozen SUB designs suitable for vaccine production support lot sizes >100 L. Some styles come in the range of 2,000-L capacity. Distinctions among systems offered include agitation and gas-sparging mechanisms, overall dimensions and fluid containment approaches, and applications data and support available. The field remains in a state of rapid development, as evidenced by the recent introduction of an interesting new disposable system involving hollow-fiber bioreactors for virus production in vaccine manufacturing (15). They provide several fundamental advantages not found in other reactor systems (Figure 2). Combining single-use production systems with modular, portable, cleanroom technology promises low-cost, flexible, vaccine-manufacturing facilities that comply with good manufacturing practices (GMPs) to assist developing countries and provide all with swift pandemic response (16).
Driven by technological innovations and such regulatory initiatives as process analytical technology (PAT) and QBD, bioprocess design — including for vaccine manufacturing — is in a period of significant development (17). Increased process understanding, novel analytics, and risk-based methods — and such goals as establishing a life-cycle approach, design space, and critical process parameters — have as much bearing on vaccines as any other products. Just as for other biologics, new guidance and regulations on these topics should help clarify the application of such programs for vaccine manufacturers (18,19).
Virus stock generation techniques such as clone selection and plaque assays used to be performed manually using standard Petri dishes or multiwell plates. Today, a number of specialized procedures using advanced, automated, and even high-content techniques provide product and process developers with increased understanding of virology and bioprocesses. For example, videographic evidence revealed in 2010 that Vaccinia virus infections can demonstrate a “surfing” phenomenon (vigorous repulsion of superinfecting virions) with implications for both in vitro and in vivo infections (20).