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An evaluation of the technologies needed to develop a safe, effective, and economically efficient vaccine. This article is part of a special section on vaccines.
The biotechnology era has experienced significant changes in the number of companies involved in vaccine manufacturing as well as in the production systems they use. Nevertheless, challenges in this area are multiple. In the current vaccine-manufacturing environment, time to market and cost effectiveness are key issues that need to be addressed in addition to smooth R&D and clinical studies. Furthermore, scale up and safety are important for maintaining a successful manufacturing process.
As a result, state-of-the-art technologies to simplify vaccine development and manufacturing are becoming ever-more crucial. In this article, the authors discuss these challenges and evaluate the technologies and process parameters that need to be considered when developing a safe, effective, and economically efficient vaccine. The article primarily deals with prophylactic vaccines.
Vaccines are a group of biologics considered to be the lifeline of the human race. It has been said about vaccines that, "With the exception of safe water, no other modality, not even antibiotics, has had such a major effect on mortality reduction..." (1).
A good vaccine is one that elicits an appropriate immune response for the particular pathogen, which could either be a cell-mediated response to tuberculosis or an antibody response to a bacterial or viral infections. It should be safe to use in a variety of patients and the vaccine itself should not cause disease or induce adverse effects. A vaccine should offer long-term protection (ideally lifelong) with one dose and retain its immunogenicity despite adverse storage conditions before administration. Furthermore, it must be inexpensive to make and buy (2).
The development and manufacturing of vaccines must follow four ground rules (3):
The largest market for vaccines is found in the developing world, where doses need to be less expensive than those sold in the developed world in order to have any chance of getting to the patients who need them (4). Companies, therefore, need to weigh the risks of killed or weakened whole organisms against the costs of recombinant vaccine production for products meant to help a large percentage of the economically weak population.
As part of the "Elements of Biopharmaceutical Production" series (see sidebar), this article presents an overview of the various challenges encountered during process development for the manufacture of prophylactic vaccines.
Elements of Biopharmaceutical Production
Process development for vaccines can pose unique obstacles for manufacturers. Because most vaccines are new products, there is no history or experience to rely on with regard to how subjects will respond to the drug. Furthermore, promising preclinical results in animal models are generally not duplicated when the therapy is tested in humans. Often, it is challenging to develop robust manufacturing processes and to validate quality control assays for these products because there is a need for a specific biological assay for each product (5).
One important difference between the production of vaccines and other biopharmaceuticals is the risk-safety consideration related to working with pathogens and pathogenic antigens. As with all biomolecules purified from crude biological material, the removal of contaminants (e.g., derivatives from host cell such as DNA, protein, or leachables), must be documented. However, the removal or inactivation of adventitious viruses remains a unique challenge (3).
The process of vaccine development begins with a good understanding of the underlying biology involved.
Egg-based versus cell-culture based
There has been much debate regarding whether the egg-based method for manufacturing a viral vaccine is more effective than the animal cell-culture based method. A third method using virus-like particles (VLPs) is also in use. This method is similar to the recombinant-vaccine manufacturing process in terms of cloning and expressing recombinant proteins.
Although the egg-based viral vaccine manufacturing process is an old and familiar technology that has been practiced for more than 60 years, it has its own complex issues. Egg-based viral vaccine manufacturing, for example requires a large number of specific pathogen free (SPF) eggs because the virus needs to be propagated inside the fertilized chicken eggs. However, typcially, one can only produce one or two doses of vaccine per egg. Here, the nutrition status of the poultry is also important because changes in their diet can dramatically affect the virus yield from the eggs. The process is labor intensive and highly susceptible to bacterial contamination. In addition, individuals who are allergic to eggs might have allergic reactions to such vaccines.
On the contrary, animal cell-culture driven viral vaccine processes are being encouraged by regulatory authorities, particularly because the process reduces the lead time from laboratory to market. Furthermore, during an emergency, chances of scaling up production capacity of an egg-based vaccine is dismal considering the reliance on SPF eggs, compared with cell culture, which can be propagated multiple times from frozen cellbanks. In addition, the footprint for cell-culture-based production is considerably smaller, and processing takes place in closed systems, thereby reducing chances of contamination.
However, an important issue with cell-culture processes is the tumorigenic and oncogenic potential of the cells in which the virus is propagated. Although highly tumorigenic cell substrates have never been used in vaccine manufacturing, it is important to keep in mind that some Madine-Darby Canine Kidney epithelial cell (MDCK) sublines are highly tumorigenic. Such cell substrates can pose significant regulatory challenges (e.g., if the cell line harbors oncogenic or tumorigenic viruses, that virus could integrate into the recipient's genome and cause a tumor). Other cell-culture concerns include the oncogenic potential of virus producing cells due to the presence of contaminating DNA in the product. Additionally, the use of animal-derived components in the cell-culture media—whether serum-sourced from calf or trypsin-sourced from porcine—could be potential sources of animal viruses. The International Conference on Harmonization (ICH) Q5A guideline on the quality of biotechnology products, therefore, recommends using serum-free, chemically defined media for cell culture and genetically engineered trypsin to ensure the absence of any animal-derived component in the raw materials used in manufacturing.
Use of VLPs in vaccine development
During the past few years, the use of VLPs for the manufacturing of vaccines is becoming more popular. For example, the H1N1 influenza vaccine developed jointly by Novovax and Cadila Pharmaceuticals uses VLPs. The advantages of a VLP-based vaccine platform is that it uses a recombinant-vaccine technology with a baculovirus expression system and, as a result, one does not need to handle a live pathogenic virus. This process rules out the need for a containment facility. There are no safety concerns and the process is easily scalable to large quantities and more economical in terms of facility, materials, labor, and utility costs (6). When using recombinant technology, one can select the exact genetic match for hemagglutinin, neuraminidase, and matrix proteins of the circulating virus strain. The entire process of manufacturing, from cloning to development and release, takes about 12 weeks (6). Other VLP-derived vaccines include Merck's Gardasil, which protects against human papillomavirus types 6, 11, 16, and 18.
Current technologies for vaccine manufacturing can help ensure quality and reduce time. Some of these technologies include analytical methods such as surface plasmon resonance, upstream technologies such as microcarrier beads for adherent cell lines, high-throughput screening methods, and downstream technologies such as membrane chromatography and cross-flow filtration. Figure 1 outlines the schematic flow for complete vaccine-process development.
Figure 1: Process-development scheme for vaccine manufacturing.
Upstream process development
The productivity of large-scale cell culture can be increased either by scaling up to larger volumes with cell densities of 2–3 x 106 /mL, or by intensifying the process in smaller volumes but with higher cell densities (up to 2 x 108 cells/mL). When intensifying cell densities, more frequent media changes are needed and perfusion is eventually applied (7).
Many alternative technologies are also available. Cross-linked dextran beads (i.e., microcarriers), for instance, provide an extended surface and a stable environment for optimal cell growth. Microcarrier culture of anchorage-dependent or entrapped cells reduces volume and thus belongs to the latter of the options cited above (8). The technique, in general, has many advantages for the commercial manufacturer. It can be operated in batch or perfusion modes during cell culture and it is well-suited to efficient process development and smooth scale-up. Washing and changing culture media just before viral infection is easier because there is no virus inside and one does not have to follow additional precautions as required while handling a virus. The reactors can also be modified to grow other organisms.
Downstream process development
As evident from Figure 1, the downstream processing and purification steps occupy a major portion of the vaccine-manufacturing process. As a result, the use of robust and economical steps to develop and optimize parameters for purification is beneficial. For example, in general, downstream steps are more expensive to carry out than upstream steps because of the use of chromatography columns and membranes. The onus for generating a product that is absolutely free of pathogens and other contaminants such as host cell protein (HCP), host cell DNA, or endotoxin, relies on downstream steps. If this cost escalates, the manufacturer could run into business risks.
The current industry trend for purification of biological therapeutics, including vaccines, involves the use of membrane chromatography to purify viruses as well as for polishing applications. Membrane chromatography offers many benefits compared with density gradient ultracentrifugation, including the removal of HCPs, contaminating DNA and endotoxins. Membrane chromatography compared with centrifugation offers many advantages (e.g., processing time is faster, virus yield is higher, cleaning validation is not needed, the carbon footprint is smaller) (see Table I). This method can therefore bring greater effectiveness to the key stages of bioprocessing.
Table I: Comparison between the use of membrane chromatography and density gradient ultracentrifugation for virus purification.
The implementation of membrane chromatography for virus purification, in place of density gradient ultracentrifugation, is gaining prominence in many areas, even though the latter remains the gold standard among traditional vaccine manufacturers.
Membrane chromatography has been successfully used in multiple applications for virus purification. In a recent study, human and equine influenza A virus in cell-culture supernatant (i.e., serum-free and serum-containing cultivation) was directly adsorbed onto Sartobind Q 75 anion-exchanger, and eluted out by displacement with sodium chloride (up to 1.5 M, pH 7.0), which resulted in average yields of 86% based on hemagglutinin activity (9). In another instance a virulent wild type NIA3 strain of Pseudorabies, grown in porcine kidney epithelial (PK15) cell monolayers was purified using Sartobind S cation exchanger membranes (10).
In another study, Sartobind D membrane adsorber was used in a larger scale downstream process, for the purification of rotavirus VLPs to a clinical grade at 46% global recovery yield, and with nearly 100% removal of host bulk DNA together with approximately 98% of HCPs (11). Sartobind D was also used for purification of recombinant baculoviruses that are widely used as vectors for the production of recombinant proteins in insect cells (12).
Another robust technology is cross-flow filtration (using tangential-flow filters), which is best suited for higher solid contents, more viscous feed solutions, or in cases where concentration, recovery, and purification of cells or target species is desired. This technology is largely used for concentrating and washing feed streams before chromatography (3).
For vaccine manufacturing, particularly virus purification, fully scalable macrovoid-free hollow-fiber technology applied to ultrafiltration and microfiltration offers great advantages in the virus-purification process due to its open porous structure. The open-flow path design of hollow fibers gently processes cell suspensions and other particulate feed streams and reduces shear forces, thereby maintaining the integrity of the virus. Hence, recovery rates of the target virus and overall process economics are improved (13).
Figure 2: A generic process for egg-based vaccine manufacturing using platform technologies. SPF is specific pathogen free. UF is ultrafiltration. DF is diafiltration.
One way to eliminate bottlenecks, especially in downstream-process development, is to use purification platforms. The biopharmaceutical manufacturing industry has communicated timesavings of 3–8 months using a fast-track development approach when technology platforms are applied in all key aspects of development. This includes upstream as well as downstream processing, such as cell-line development, cell culture, downstream processing, analytical concepts, and filling (3). Major considerations when designing a manufacturing platform include the infrastructure, resources and manufacturing capacity available, the degree of scalability and productivity desired, the time available for producing the first dose and the dosing regime. Figures 2 and 3 illustrate some platform processes for vaccine manufacturing in eggs and in animal cell culture. A complete platform for downstream processing as noted in one study includes three steps: depth filtration, ultra/diafiltration (UF/DF), and membrane adsorption for purification of recombinant baculoviruses. Global recovery yields reached 40% at purities over 98% (12).
Figure 3: A generic process for animal cell culture based vaccine manufacturing using platform technologies. HCP is host cell protein.
Another important parameter to consider when manufacturing a vaccine is its thermostability. All vaccines lose potency over time, and the rate of potency loss is temperature dependent. Hence, there is a need to develop, monitor, and maintain cold-chain systems to ensure that the potency of vaccines is maintained under refrigerated conditions (mostly between 2–8 °C) until the point of use. The World Health Organizaion (WHO) recommends that reconstituted vaccines be kept cold and that any unused vaccine from a multidose vial be discarded after 6 h (14). This is primarily due to the instability of reconstituted vaccines and also due to the chances of bacterial contamination because live vaccines do not contain preservatives (15). New approaches to develop thermostable vaccine formulations that are resistant to damage caused by freezing or overheating are necessary to eliminate dependence on a cold chain. Such approaches could have great economic benefits in terms of reducing vaccine wastage and preventing adverse health consequences of administering damaged vaccines to recipients. Furthermore, such approaches would improve the effectiveness of vaccines and enable delivery of the vaccine to remote populations (15).
In addition to ensuring vaccine stability, optimizing the vaccine supply chain through quality management in vaccine manufacturing plays a key role in safe and effective vaccine manufacturing. In this context, performing a risk analysis for the manufacturing-process parameters that affect product quality, as well as an assessment of each validation step, including cleaning protocols, monitoring of the air filters, and assurance of sterility of the source materials, are also crucial to a quality management process (16).
Vaccines are generally (although not always) prophylactic biomolecules, therey making their development and commercialization complex. A set of basic regulatory criteria from the WHO applies to vaccine manufacture, regardless of the technology used to produce the products. Licensure of a new vaccine is based on the demonstration of safety and effectiveness, and the ability to manufacture in a consistent manner. The manufacturer facilitates the development and evaluation of new vaccines by anticipating and addressing the regulatory issues involved. General regulatory issues that are applicable to other biologicals, such as detection of adventitious agents and improved test methods that are reliable and sensitive, are valid for vaccines as well. Additional vaccine-specific issues include determining correlates of protection necessary for evaluating efficacy, improving assays for potency, or finding animal models that can be used for the evaluation of efficacy when human clinical trials are not feasible or unethical (17).
Although the use of animal-cell culture for manufacturing viral vaccines is the current practice, regulatory challenges tied to this protocol are extensive. The WHO requires additional reports to ensure safety of the population receiving vaccines generated out of cell culture. Required tests include confirming tumorigenicity, checking for extraneous agents of the cell substrate, residual HCP and residual DNA, equivalence of cell culture and egg-based vaccines (i.e., antigenic characterization by cross-reactivity of specific antisera and animal protection studies).
Generally speaking, single-use technologies reduce bottlenecks in manufacturing environments. Implementation of single-use technologies in vaccine manufacturing should be the gold standard. Single-use steps eliminate the need for cleaning and validation, which automatically eliminates any kind of cross-contamination, while maintaining the aseptic path. Apart from eliminating cleaning and validation, the reduced set-up time associated with these systems is crucial to meeting the time-sensitive demands of vaccine manufacturing. Single-use process steps can be easily assembled and are quickly configurable, thereby reducing downtime and capital investment in facility and equipment.
Aseptic processing has always received attention from regulators in the biopharmaceutical sector because of the high risk of microbial contamination that can affect patient health, particularly when the molecule is a prophylactic material. Traditionally, aseptic processing is done in critical or controlled areas, depending on the risks associated with certain steps in the manufacturing process (16).
Interestingly, discarding a device, without having to prove that it has been sufficiently cleaned, is one step that both regulatory authorities as well as manufacturers seem happy to embrace (18). This approach is especially beneficial for vaccines, because vaccinating large, healthy populations, carries much greater risks than treating relatively small groups of people with conventional drugs.
For manufacturers attempting to minimize the risks of vaccine-batch contamination, single-use technologies provide an important avenue for enhanced safety. Single-use systems that the supplier presterilizes and bundles together can further simplify set-up. With fewer opportunities for operator error, single-use technologies can improve safety and production economics. Some single-use products, such as bags, also save space, because they lie flat and can be stacked before use. Unlike permanent storage tanks, single-use bags are typically ordered on an as-needed basis to avoid excess unused equipment (18).
Although single-use technologies can play a prominent role in egg-based vaccine manufacturing, in the areas of filtration, storage, and at connection points, their use multiplies in cell-based applications. More specifically, single-use technologies can be used for media preparation, clarification, and cell harvesting in upstream processes as well as in buffer preparation, capture, and polishing chromatography steps, purification and filling in downstream processes. Single-use bioreactors are an example of the increasingly growing role of single-use technologies in upstream processing (18). A recent study demonstrated the successful development of a complete single-use downstream process by implementing depth filtration, UF/DF, and membrane chromatography for the purification of recombinant baculoviruses (12).
Finally, environmental concerns have been cited as key motivators for moving toward single-use systems in biomanufacturing (19). This belief stems from the observation that although single-use systems require significant energy to produce them and generate plastic waste, the amount of energy and water consumed in the production of water-for-injection and steam used in clean-in-place/steam-in-place operations can more than offset the waste issue. When waste-to-energy plants are considered for disposal of the plastics, the environmental benefits of single-use technologies are further enhanced (20). By one estimate, the commutes of the plant employees account for more than 50% of the total carbon emissions associated with biomanufacturing (19).
The above observations demonstrate that single-use technologies make it possible to develop and scale up processes quickly and facilitate manufacturing by reducing the cleaning-validation burden.
Despite the advances in vaccine manufacturing across the globe, regulatory, technical, and manufacturing hurdles still stand in the way of companies seeking to take a candidate product to the clinic and eventually to market. The identification of suitable vaccine candidates is only one of many hurdles involved in the translation of a vaccine candidate from the bench to the clinic (21). Identifying suitable antigens, adjuvants, and delivery methods are just the beginning of vaccine development (22). Public demand for safe and effective vaccines continues. In addition, regulatory requirements have led to an emphasis on well characterized, safe vaccines (21).
The need for well defined, single-use platform processes to reduce complexities, ensure safety, and maintain timelines to market, therefore, is only growing. Additionally, because process development provides a technological foundation for manufacturing, analytical methods and assay development for characterization and potency determination must be part of a company's approach. Such an approach helps to lay the groundwork for successful commercialization (22).
SUMA RAY is a senior process development scientist at Sartroius Stedim India Private, Ltd., firstname.lastname@example.org.
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The author would like to acknowledge colleagues from Sartorius Stedim Biotech, Mahesh Prashad and Frank Meyeroltmanns for sharing their knowledge and designing the generic schemes for platform processes in vaccine manufacturing. The author appreciates the support of Dr. Uwe Gottschalk, vice-president of purification technologies at Sartorius Stedim Biotech. The author would also like to acknowledge Anurag S. Rathore, PhD, a faculty member at the Indian Institute of Delhi, India, for his editorial assistance with this paper. Rathore is a member of BioPharm International's Editorial Advisory Board and the author of the journal's series on the Elements of Biopharmaceutical Production.