April 1, 2010
A recent private grant of US$10 billion for human vaccine applications illustrates the revival of interest in vaccine science (1). The 2009 response by vaccine manufacturers to the H1N1 pandemic revealed the convergence of three technological developments. First is a revolution in technology: Vaccines are being developed for diverse and unprecedented applications through a number of entirely new approaches. Second is the recent adoption of cultured cell-based production for a growing number of vaccines, such as influenza. And third is the rapid acceptance of single-use technology in bioproduction overall.
The Revolution in Vaccine Technology
Because of remarkable technological innovations in both prophylactic and therapeutic vaccine science, it’s important to establish the exact scope of this topic. From novel and unique vaccine types (e.g., DNA plasmid vaccines) to their unprecedented indications (such as cancer and diabetes treatment) to innovative means of delivery (e.g., inhaled nanoparticles) to entirely new production approaches (such as in genetically engineered plants), it has become a challenge just to stay on top of this field (2). Here, I address the use of higher-animal cell culture such as Madin-Darby canine kidney (MDCK) cells in production of a vaccine biological component (such as a viral vector) using disposable systems (such as single-use bioreactors).
Keep in mind that although much has been made of the recent move to human H1N1 flu vaccine production in cultured cell and disposable systems, these technologies also support a wide range of vaccine types against a number of pathologies in human and veterinary applications. This new world of vaccine design includes such diverse new approaches as recombinant vectors, virus-like particles (VLPs), and new adjuvants. Some related vaccine designs are not only facilitated by these newer manufacturing methods, they in fact depend on those advances.
Vaccine Production Through Cell Culture
The transition of vaccine production from primary animal tissues to cell culture began >50 years ago with the famous establishment of polio vaccine production in primary monkey kidney cells. A significant development occurred in 1987 with establishment of a World Health Organization guidance for using immortalized (continuous) cell lines for vaccine manufacturing (3). Since then, a large number of approved human and veterinary vaccines against a number of pathogens have been made using cell culture methods. For example, the ACAM2000 smallpox vaccine from Acambis, Inc. (www.acambis.com) is produced in Vero cell culture.
On the other hand, the transition to cell culture from flu vaccine production based on chicken eggs began only recently. Although it was heavily supported by >$1 billion in government funding in 2005–2006, it is not yet complete (4,5,6). In general, cell culture offers many advantages over animal tissue and egg-based manufacturing approaches, as listed in the “Features” box. A more specific concern is identification of the best from the many available cell-based production platforms, which range from human kidney cells cultured in serum-based media on microcarriers to immortalized duck embryonic stem cells in serum-free suspension culture.
FEATURES OF VACCINE PRODUCTION BY CELL CULTURE
Advantages
Eliminates expense of obtaining animal tissue or chicken eggs from biosecure flocks
Eliminates four- to six-month lead times for embryonated eggs
Eliminates time required to adapt new seed stocks to egg production
Facilitates use of nonadapted, wild-type strains in actual manufacturing
Eliminates risk of egg-adaptation- induced immunogenic changes in viruses
Generally supports greatly reduced high- volume production start-up times
Greatly supports rapid pandemic vaccine response
Frozen cell stocks greatly facilitate warm and surge capacity
Facilitates supplemental production when strain changes are necessary
Reduces extent/level of production facility classification (biosafety level)
Supports the linkage, automation, and closure of up- and downstream processes
Provides for a more reliable, expandable, and flexible process
Provides higher initial product purity in downstream operations
Reduces the potential of viable and nonviable contamination
Expands the population of potential vaccine recipients
Reported Concerns
Regulatory hurdles
Product comparability costs and challenges
New process development costs/delays
Multiple costs of implementing entirely new operations/equipment
Many factors must be considered in the choice of an appropriate cell line for any bioproduction application. Productivity is of course an important consideration, but other issues include ease and economy of use, scalability, reproducibility, safety, regulatory history and status, production platform compatibility, and availability of desired production media. Use in the production of a vaccine component can impose even a few more unique criteria, which is compounded by the state of remarkable growth in the field, so new and improved platforms are continually being presented.
Cells used in vaccine production may demand such added requirements as suitability for a broad range of viral strains or production of particles with unusual physicochemical characteristics. An illustration of vaccine-specific culture requirements occurred early in the development of Vero cell-based production of influenza virus. Despite excellent large-scale protein-free culture methods, it was well known that this system did not produce much active virus for use in vaccines. It was subsequently discovered that the simple addition of trypsin to the culture medium renewed the full activity of high-titer virus product (7).
Some viruses replicate best in cells that are historically anchorage- dependent, which can determine a requirement for such scalable culture technologies as multiple stacked-plate “factories,” roller bottles, or microcarrier beads. Cell attachment, uniformity of culture, and cell dissociation/separation approaches impose additional challenges in scale- up of these manufacturing processes. Fortunately, the cell culture support industry is responding with a number of materials that can facilitate this endeavor, such as microcarrier beads with divergent surface characteristics, specific densities, and other enabling features (Figure 1, LEFT). For maintaining animal-component-free status, standards-compliant cell dissociation reagents and culture media have been developed for such applications (Figure 1, RIGHT). Other vaccine-specific considerations include unique analytic and regulatory requirements and a sometimes heightened demand for “warm” or surge capacity.
A surprising number of cell lines are either currently being used or in late stages of development for production of human and animal vaccines. Cell lines currently popular in development of a human influenza vaccine alone include MDCK cells, Vero African green monkey kidney cells; the PERC.6 cell line from Crucell NV (www.crucell.com) derived from a single human retina-derived cell immortalized using recombinant DNA technology; Sigma Aldrich’s (www.sigmaaldrich.com) very new EBx stem cell line derived from chicken embryos; and a very interesting baculovirus-based platform that uses insect cells such as the Sf9 line.
The baculovirus vector system is based on a recombinant insect virus replicated in cultures of insect cells. This has proven to be a very powerful technology in vaccine development. At least three distinct types of influenza vaccine are using this system: surface- displayed antigens on the baculovirus itself (a recombinant vector vaccine), secreted free influenza proteins (a subunit vaccine), and self-assembling influenza protein virus-like particles (a VLP vaccine). For example, Novavax, Inc. describes a baculovirus-expressed VLP vaccine that includes the hemagglutinin (HA), neuraminidase (NA), M1, and M2 proteins (8).
Another significant factor in culture-based production is the type of media and supplements available for each platform. Modern serum-free media provide important advantages over the first generation, including supporting higher productivities and material definition as well as offering less lot-to-lot variability and potential for infectious contaminants than do serum-containing media. The number of considerations in selecting an appropriate bioproduction culture medium is surprising large and growing (9). Cell-based vaccine production invokes many of those demands, and as with cell line selection, the vaccine aspect can introduce a few new ones. An example of the continued vendor support here is found in the animal-component- free insect cell culture media and feed supplements that support production of all three flu vaccines described above while providing the safety and regulatory support of a material free of animal products.
Disposables-Based Production
Independent of vaccine interests, the biopharmaceutical industry has been rapidly adopting disposables into many process operations of cell-culture- based production systems. The list of benefits that even current hybrid implementations offer (listed in the “Disposables” box) is remarkable and still growing as reports are returned from recent placements. From upstream process material preparation through final product formulation, manufacturers not only have disposable technologies available, but they are increasingly presented with numerous solutions from several vendors (10). Systems available to support a growing number of manufacturing unit operations from a number of vendors begin upstream with disposable mixers and filters for buffer and culture media preparation, as shown in Photo 1, TOP (11).
Photo 1:
Vendors also offer single-use bioreactors that allow disposable-based vaccine production up to the 2,000-L scale (Photo 1, BOTTOM). Many such systems support full monitoring and control capability, with performance demonstrated either as seed or production reactors. Eliminating costly equipment such as centrifuges for clarification, single-use depth and diafiltration media allow disposable processing to begin directly from a bioreactor outlet. Single-use chromatography columns (and large- scale membrane chromatography systems) for use in vaccine purification through polishing have been developed (12).
Disposable production systems provide cost savings through a number of means including reduction in initial equipment capital, process footprint, and plant design costs, as well as the requirement for clean water and steam services. Efficiency is gained through greatly reduced process turnaround time, ease and safety in product changeover, and economy of site-to- site transfer or replication.
While providing many unique added benefits such as ease and economy of process reconfiguration, disposables also support generally required production capabilities such as existing sensing and monitoring systems, devices for aseptic connections, and cold chain logistics. When compared with monoclonal antibodies (MAbs), vaccines produced in cell culture can introduce processing steps and constraints that give manufacturers an additional set of challenges to consider (e.g., the common use of adherent culture). But a number of single-use values actually lend themselves to vaccine production. One is the ability to multiply wrap sterile liquid containment bags for ease of transfer between areas of divergent classification or biosafely levels.
Despite the many advantages afforded by disposable systems, a number of challenges have been reported, also listed in the “Disposables” box. Some are very real, such as the need to design novel process flow paths for disposable system implementation. However, they are usually more than compensated for by such factors mentioned above. The environmental impact of waste material and components generated in these operations has been specifically addressed (13). As in other industries, final answers to concerns such as the relative aggregate carbon footprint are troublesome due to such issues as model assumptions and implementation-specific considerations (14). However, in most cases it appears to be that when all factors are considered, the transition to disposables-based operations provides a net reduction in environmental impact (15). Open dialogue is ongoing in a number of forums (16).
New entrants to single-use manufacturing can benefit from increasing publication of application data and case-studies by disposable systems providers and — better yet — the industry’s early adopters (17). Such reports present actual results obtained in implementing commercially available systems in real-life operations and can reveal valuable implementation details, observations, and summaries of particular value for those just getting started. They frequently include examination of some considerations presented in the “Disposables” box and often provide detailed information on subtopics such as product contact leachables, practical flow rates, and disposable bioreactor oxygen transfer coefficients (KLa). Such reports are sometimes available as journal articles, but due to corporate legal requirements, valuable details are frequently presented only at meetings dedicated to the topic (18,19,20).
For example, at an ISPE meeting last year Miriam Monge of Biopharm Services Ltd. presented on the economics of disposable systems in bioproduction (21). Her model illuminated the cost-saving implications of specific technologies for defined process steps, considering such input as the scale and specific productivity of a given process and the percent of facility use. She provided details on the impact of such disposable systems on water service and CIP skid requirements, environmental impact relative to conventional systems, aggregate facility throughput, and overall cost of goods (COG, expressed in grams or, with vaccines, particles per liter of culture). Multiple scenario and sensitivity analyses with this model made it possible to analyze, for example, where hybrid solutions combining stainless steel and disposables would make the best sense — or beyond which scale the value of disposables becomes more limited. Monge presented a case study of a major vaccine manufacturer that had some debate regarding the optimal level of disposables integration for a given process. Her model provided clear, objective data on the impact of specific disposable technology choices.
Other sources of information on disposables implementation include patents and reports from government commissioned studies (22).
Although existing systems for single-use processing have been accepted in bioproduction for many operations, new developments in the technology and scale of application continue to be presented. Exciting advancements are occurring in connectivity, sampling, and monitoring of disposable containers (including bioreactors). Aseptic sampling ports and means of accommodating classical dissolved oxygen and pH probes have long been available for disposable containers (Figure 2, LEFT). But recently introduced sensing technologies for parameters from pH to pressure are either noninvasive or disposable, themselves.
One that has been very well received provides dissolved oxygen (DO) and temperature sensing with a single-use sheath preinserted in a disposable bioreactor bag port before irradiation sterilization (Figure 2). An optical reader provides accurate in situ measurement of DO using phase fluorometric detection. Concurrent temperature measurements are obtained through a stainless steel thermal window embedded in the sheath.
Excitement is growing for the use of near-infrared (NIR) technologies in many bioprocessing applications, from QC testing of raw materials to final product quantitation (23). NIR could even provide on-line information regarding substrate, biomass, product, and metabolite concentrations, which can improve monitoring or control. Real-time, in-process component monitoring is supported through development of on-line and at-line autosamplers and even disposable, noninvasive NIR porting.
DISPOSABLES-BASED PRODUCTION FEATURES
Reported Advantages
Reduces both overall capital investment and cost of goods
Reduces plant development and start-up time
Provides cost distribution gains: later instead of up-front, variable instead of fixed
Reduces capital equipment design, installation, and validation cost
Eliminates CIP/SIP water and steam capital costs
Eliminates use of harmful cleaning materials
Eliminates cost and downtime related to cleaning operations
Reduces overall carbon footprint (in most cases)
Reduces both production process and classified area footprint
Reduces production facility service requirements
Reduces manufacturing and quality labor activities/costs
Increases sterility assurance (irradiation over steam)
Reduces lot and product cross-contamination risk
Reduces risk of contaminations
Improves compliance values (reduces error potential)
Eases material transfer between diverse biosafety or classification levels
Eases CGMP “cold chain logistics” and “good storage practice”
Increases plant capacity, scheduling flexibility, and campaigning
Simplifies and accelerates product changeover and turnaround
Facilitates flexible approaches in process flow and layout
Supports an evolving “plug-and play” of operating modules
Increases ease in reconfiguring and extending production facilities
Supports inexpensive establishment of efficient surge capacity
Reduces process flow and equipment modification costs
Supports economical and rapid site-to-site facility transfer
Provides for general equipment/process “facility independence”
Provides demonstrated scalability within available system sizes
Processes and models in infancy: yet to reflect true potential
Supports existing process sensing, monitoring, modeling, and control
Supports growth in novel systems (e.g., connectivity, sensing, chromatragraphy)
Provides for developing freezing capability in many containment systems
Expands range of “closed system processing” across unit operations
Expands application of disposables in operations up- and downstream
Reported Concerns
Scalability limitations: 2,000 L is currently the largest available working volume
Processes and models in infancy: based on preliminary data, process “robustness” unproven
Materials inventory, storage, and repetitive purchases required
Can involve single or novel systems and vendor dependency (e.g., single- source)
Observed variability in vendor “maturity” and capabilities
Can present new and different vendor audit issues
Systems categorized as “generic,” “standard,” or “specialized”
Continual innovations in a young field can be a burden
Presents novel validation demands and engineering and process layout and flow designs
Developing industry and regulatory standards and definitions
Product containment failure concerns/contingencies
Can present contents transfer flow rate limitations
Connectivity issues: standardization and continued solutions desired
Questions regarding new materials: leachables and extractables
Concerns over particulate load and relevant compliance
Materials compatibility with products and processes must be established
Questions remain regarding both carbon footprint and the fate of used materials
An Optimistic Outlook
Growth and technological revolution in vaccine technology show no sign of abating. Concurrent with the adoption of disposable systems for many operations in bioproduction is the implementation of cell-based manufacturing approaches for a growing number of vaccines. Together, these innovations are changing the face of vaccine production — and some industry leaders predict closed, fully disposable vaccine production process trains for some manufacturing scales in the very near future.
1.) McNeil, DG. 2010. Gates Foundation to Double Spending on Vaccines. The New York Times www.nytimes.com/2010/01/30/world/30vaccine.html.
2.) Allary, C, M Parker, and S. Rhodes. 2009. Using Innovation to Drive Competitive Advantage. BioProcess Int. 7:10-17.
3.) 1987. Cells, Products, Safety, Backgrounds: Papers from the WHO Study Group on Biologicals. Dev. Biol. Standard. 68:1-90.
4.) WHO Initiative for Vaccine Research (IVR) Tables on the Clinical Trials of Pandemic Influenza Prototype Vaccines.
5.) Neuzil, KM, and RA. Bright. 2009. Influenza Vaccine Manufacture: Keeping Up with Change. J. Infect. Dis. 200:835-837.
6.) Reisinger, KS. 2009. Subunit Influenza Vaccines Produced from Cell Culture or in Embryonated Chicken Eggs: Comparison of Safety, Reactogenicity, and Immunogenicity. J. Infect. Dis. 200:849-857.
7.) Kistner, O. 1998. Development of a Mammalian Cell Line (Vero) Derived Candidate Influenza Virus Vaccine. Vaccine 16:960-968.
8.) Kang, SM. 2009.Influenza Virus-Like Particles As Pandemic VaccinesCurrent Topics in Microbiology and Immunology: Vaccines for Pandemic Influenza, Springer, Berlin:269-289.
9.) Decaria, P, A Smith, and WG. Whitford. 2009. Bioproduction Culture Media Functions. BioProcess Int. 7:44-55.
10.) McLeod, L. 2009. The Road to a Fully Disposable Protein Purification Process: Single-Use Systems Eliminate Time- Consuming, Non-Revenue-Generating Activities. BioProcess Int. 7:S4-S8.
11.) Glasser, V. 2009. Quest for Fully Disposable Process Stream. Gen. Eng. News 29.
12.) Wolff, MW. 2010. Capturing of Cell Culture-Derived Modified Vaccinia ankara Virus By Ion Exchange and Pseudo-Affinity Membrane Adsorbers. Biotechnol. Bioeng. 105:761-769.
13.) Rawlings, B, and H. Pora. 2009. A Prescriptive Approach to Management of Solid Waste from Single-Use Systems. BioProcess Int. 7:S40-S47.
14.) Searchinger, T. 2009. Fixing a Critical Climate Accounting Error. Science 326:527-528.
15.) Walton, D.. Using Contained Processes to Reduce the Carbon Emissions.
16.) Rawlings, B, and H. Pora. 2009. Environmental Impact of Single-Use and Reusable Bioprocess Systems. BioProcess Int. 7:S18-S26.
17.) Williamson, C, R Fitzgerald, and A. Shukla. 2009. Strategies for Implementing a BPC in Commercial Biologics Manufacturing. BioProcess Int. 7:S24-S33.
18.) 2009..
19.) Bhatia, R. 2009.Economic Analysis of Implementation and Use of Disposable Technology in cGMP FacilityBioProduction 2009, Barcelona.
20.) Eisenkraetzer, D. 2009.Disposable Systems As Platform Technology for R&D and Clinical SupplyBioProduction 2009, Barcelona.
21.) Monge, M. 2009.Biological Products Manufacturing Challenges: Now and in the Future ISPE Managing Knowledge Through Science and Risk Assessment, Strasbourg.
22.) Baird, A. 2009. Ensuring Biologics Advanced Development and Manufacturing Capability for the United States Government, A Summary of Key Findings and Conclusions, University of Pittsburgh Medical Center, Pittsburgh.
23.) Cervera, AE. 2009.Review Article: Process Sensing and Control. Application of Near-Infrared Spectroscopy for Monitoring and Control of Cell Culture and Fermentation Biotechnol. Proogr..
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