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Suma Ray, PhD, is a process development scientist, viral clearance and cell line development, Global Purification Technologies Group at Sartorius Stedim Biotech
Anurag S. Rathore is a professor in the Department of Chemical Engineering at the Indian Institute of Technology Delhi and a member of BioPharm International's Editorial Advisory Board, Tel. +91.9650770650, firstname.lastname@example.org.
Challenges of vaccine development include regulatory, technical, and manufacturing hurdles in translating a vaccine candidate into a commercial product.
Investment in research and development largely by the pharmaceutical industry has resulted in a broad range of vaccines targeting more than 25 infectious diseases. During the past 30 years, advances in biotechnology, genetic technology, and information technology have resulted in acceleration in the pace of vaccine development. As a result, the vaccine industry has recently introduced a number of new vaccines, such as those against cervical cancer, meningococcal infection, potentially pandemic influenza, pneumococcal diseases, rotavirus diarrhea, and varicella zoster.
Anurag S. Rathore
The manufacturing processes for vaccines have also come a long way, from using heat-inactivated cells to using recombinant DNA technology-driven antigen production. For example, inactivated polio vaccine as developed by Salk has been changed and improved and is currently being produced in a Vero cell line. Additionally, there are a few other examples of expression systems that are specifically developed for use in vaccine production. These include the PER.C6 cell line, the associated AdVac/Virosome technology (Crucell/DSM), and the avian-derived cell lines from Vivalis and ProBioGen. Another expression technology with potential benefits is the Pfenex Expression Technology from Dow, which has been used to generate high levels of vaccine antigens.
What sets manufacturing of vaccines apart from that of other biopharmaceuticals is the risk and safety considerations related to working with pathogens and pathogenic antigens. While removal of host cell-related contaminants (e.g., host cell proteins, DNA) has to be demonstrated just as for other biotech therapeutics, removal or inactivation of adventitious viruses remains a significant challenge (1). Key questions for manufacturers remain. How does one ensure that the starting materials and final product are consistently safe and of high quality? Who can manufacture the amount of vaccine needed for clinical trials and, eventually, commercial production?
This 27th article in the "Elements of Biopharmaceutical Production" series discusses the key technical, manufacturing, and regulatory considerations that need to be taken into account by the manufacturers today to make safe and efficacious vaccine products—whether manufactured in-house or outsourced.
Producing a safe and effective vaccine requires about 12–15 years of research and is estimated to cost between $100 million and $1 billion, depending on the type of vaccine being developed. It is estimated that 60% of vaccine production costs are fixed, meaning that vaccine products require a sizable market to be profitable (2). As a result, many potentially vaccine-preventable diseases, such as those primarily affecting the developing world, are left without large-scale research interest from multinational vaccine manufacturers. Attempting to fill this void have been networks led by private philanthropies, governments, and public-private partnerships (1, 2). Despite the challenges mentioned above, vaccine production has considerable lure for manufacturers. The global vaccine market is expected to increase by more than 100%, from $24 billion in 2009 to $56 billion in 2016 (1). Multinational vaccine companies historically have conducted much of the innovation, research, and development in the field of vaccine production. Many factors seem to discourage vaccine research and development, including liability concerns and price limits due to bulk purchasing. The vaccine industry is at present dominated by major players such as Merck, Sanofi-Aventis/Pasteur, GlaxoSmithKline, Novartis, and Pfizer (3). As illustrated in Figure 1, the number of biotech products including those that are in the pipeline indicates that there are more vaccines than recombinant proteins and monoclonal antibody products (4). The global vaccine market has experienced robust growth over the past few years, with economically emerging countries, such as India and China contributing effectively towards the industry's development. The governments in developing markets are spending significant amounts of money and resources for vaccine delivery and distribution. This spending, in turn, has led to the entry of more foreign vaccine players in these countries.
Figure 1: Distribution of products in pipelines of major biopharma companies (adapted from reference 4).
Process development is the technological foundation that underlies the manufacture of new vaccines and is central to successful commercialization. The key issue related to technology is the evolution and translation from a procedure used for making vaccines in a basic-research laboratory to a process that can be scaled up and run reproducibly in a manufacturing environment to make tens of millions of doses per year. Vaccine manufacture is broadly similar to other biotech manufacturing operations, but with some important differences. Purity is a particular challenge for vaccines, because some vaccine impurities have an immunomodulatory effect and can act like an adjuvant to the vaccine. Therefore, manufacturers have to scale up their production without losing the potency of the mixture of the vaccine and the impurity. Several new vaccine manufacturing platforms offer advantages including more effective vaccines, less costly vaccines, or more rapidly produced vaccines. At the same time, these manufacturing platforms raise new scientific and regulatory concerns that might potentially affect the vaccine's safety or efficacy.
For example, novel cell substrates raise new questions about potential adventitious viral agents. Vaccines also differ from conventional drugs in that some vaccines are generally stockpiled and are not used continuously. Therefore, vaccine manufacturing needs to be flexible, rather than continuous. Meanwhile, cell banks required for vaccine manufacture may become depleted with time and a new working cell bank may be needed, but the cells may not grow and perform as they did 10 years ago. Growing expertise in cell-culture technology and bioprocessing is helping manufacturers to overcome these problems.
Therefore, it should be possible to put many new vaccines into robust large-scale production during the next few years. The challenges of vaccine development are not limited to identification of suitable antigens, adjuvants, and delivery methods, but also include regulatory, technical, and manufacturing hurdles in translating a vaccine candidate into a commercial product.
A variety of technologies have been used to develop successful vaccines. These technologies include live attenuated bacteria and viruses such as Bacillus Calmette-Guérin and measles, mumps, and rubella (MMR); inactivated bacteria and viruses such as whole cell pertussis; proteins such as diptheria and tetanus toxiods; polysaccharides such as PneumoVax (Merck & Co.); conjugated polysaccharides such as Prevnar (Pfizer), and virus-like particles (VLPs). This variety can be a challenge to the vaccine manufacturer with respect to standardization of facilities and equipment. Also, while manufacturing processes and practices have significantly advanced with time, certain critical vaccines are still manufactured by traditional methods because of lack of suitable technology or lack of incentive for developing improved technology. For example, influenza vaccines are manufactured by methods fundamentally unchanged over several decades. This has made the manufacturers susceptible to disruptions in the supply of suitable eggs and to virus strains that do not propagate in the allantoic fluids. Although demand for this vaccine has increased over time, the motivation to improve the process is neutralized by the seasonal nature of influenza and the variable risk associated with the antigenic drift and antigenic shift associated with the pathogen. At the other end of the spectrum is Nesseria meningitis serogroup B vaccine, wherein the natural antigenic elements of the bacteria elicit an autoimmune response. Many newer versions of this vaccine are presently undergoing advanced clinical trials (4).
Compliance with the principles and guidelines of cGMP is a statutory requirement that applies to all pharmaceutical products including vaccines. In recent years, a number of novel tools and trends have been pursued in pharmaceutical manufacturing. These include novel ways of managing the manufacturing operations, such as operational excellence initiatives including lean manufacturing, total quality management, quality risk management, Six Sigma, and process and analytical technology.
Vaccines are complex and diverse biomolecules ranging from recombinant subunit antigens to live organisms. Therefore, their manufacturing has traditionally been relatively inefficient and technically challenging. As a result, a range of purification technologies and specialized development approaches are required to manufacture sufficient quantities of these products. To date, production of viruses and VLPs is primarily performed by density-gradient centrifugation, a labor-intensive process that is not readily scalable. An alternative strategy is to apply column chromatography. However, the problem with conventional porous chromatography resins is low binding efficiency due to the large size of viruses and low flow rate. With the adoption of membranes and monolithic columns, purification of recombinant vaccines is easier because of low steric restriction to the active binding channels (5). These formats also offer higher flow rates, resulting in smaller columns and shorter cycle times.
With advances in technology, vaccine manufacturers have access to a greater range of choices. For instance, traditional vaccines, which are often made through inactivation or crude fractionation of an infectious agent, are now being supplemented by vaccines based on pure proteins, engineered virus particles, DNA, or even cells. The latter may offer significant advantages in terms of safety and the ability to generate the required immune response. However, there are challenges in implementing such technologies as they require more sophisticated and expensive manufacturing technology, which could limit their broad availability and implementation (6).
Development of safe, effective and affordable vaccines requires that along with the use of suitable manufacturing approaches there is effective testing of manufacturing intermediates and an application of modern characterization approaches (see Table I). The difficulty of characterizing complex biological products such as vaccines makes it especially challenging to ensure that they can be manufactured in a consistent, reproducible, and commercially viable manner with assurance of safety, quality, and efficacy. The risk of manufacturing inconsistencies is especially high for novel products, because traditional testing technologies might not be able to identify subtle and unanticipated variability (7).
Table I: Advantages and disadvantages of approaches used in vaccine characterization. CD is circular dichroism, and FTIR is Fourier transform infrared spectroscopy.
Analytical testing of vaccines, just as any other pharmaceutical product, provides evidence that the vaccine and its intermediates meet the specifications defined within the license application. Safety, efficacy, and potency tests associated with a licensed vaccine are maintained within the approved filing and published in 21 CFR Part 610 in the US. In addition, several pharmacopeias (e.g., the Indian Pharmacopeia, British Pharmacopeia, US Pharmacopeia, and European Pharmacopeia) publish monographs for vaccines to provide standardized requirements for commercialization. Most countries require that vaccines be tested for both safety and efficacy by the manufacturer and a national testing laboratory (e.g., the FDA Center for Biologics Evaluation and Research in the US) before release and distribution.
Because of their simple composition (i.e., a few well defined immunogenic molecules plus adjuvant), component vaccines are the most amenable to analytical characterization. Live or killed/attenuated vaccines usually are a complex mixture of immunogens since they are directly derived from organisms such as killed or attenuated virus, intact bacteria, or multiple bacterial components. For such vaccines, the biological matrix is rather complex, allowing more characterization to be focused on the adjuvant. Technological advances such as proteomics are likely to permit the characterization of the biological components of such vaccines going forward. In the case of live and attenuated vaccine material, for example Bacillus Calmette-Guerin vaccine and oral polio vaccine, the efficacy of each vaccine batch is related to the number of live particles determined either by counting or by titration, that is, entirely in vitro. In vivo testing is only carried out for a new seed strain. Unlike live vaccines that are quantified by in vitro titration, an in vivo potency test is required for each batch of inactivated vaccines, although some exceptions do remain (8).
Classical methods for characterization of vaccines
These methods rely on the study of physical-chemical parameters such as differential scanning calorimetry (DSC), thermo-gravimetric analysis (TGA), pH, protein content determination, elemental composition, and studying the effects of stress such as freeze-thaw and agitation (9). TGA and DSC can be used to assess a change in the denaturing point of the protein or polynucleotide present. Appearance and pH can be used to monitor changes in composition or the impact of stress as observed by clumping or discoloration. Particle size analysis and particle size distribution can provide further insight into clumping and exposure to stressful conditions which can significantly affect the safety and efficacy of the vaccine. However, these methods are unable to determine small changes or relate the change in any of the measured parameters to an effect on potency and safety of the vaccine.
Advanced approaches to vaccine characterization
Lately, mass spectrophotometer (MS) based approaches have been applied towards product characterization as well as for routine monitoring during commercial manufacturing. These techniques include inductively coupled plasma MS (ICP–MS), gas chromatography MS (GC–MS), high performance liquid chromatography MS (HPLC–MS) and electrospray ionization time of flight MS (ESI–QToF–MS). ICP–MS can be used to characterize a vaccine preparation by measuring concentrations of heavy metals which may have been introduced unintentionally. HPLC–MS and GC–MS can measure the molecular weight of the vaccine components. To determine the exact location of any changes in molecular structure, a MS–MS instrument can be used because the fragmentation events for these types of molecules occur in a predictable manner that allow the interpretation of even complex spectra. ESI–MS has significantly increased the range of the size of molecules whose mass can be accurately measured and therefore provides a means for characterization of component based vaccines. In addition, newer MS-based instruments such as Q–TOF and Q–Trap have further improved sensitivity to where MS based measurements can provide detailed understanding of structural changes in an immunogen which can be correlated to the potency of an antigen.
The fact that analytical characterization plays a very crucial role in maintaining the potency and efficacy of a vaccine during purification and processing is evident from the Hepatitis B vaccine characterization reported by Seo et al. (10). They performed N-terminus sequencing of both monomers and dimers formed by complete and partial reduction, respectively, of the S-HBVsAg particles under reducing SDS–PAGE condition. They demonstrated that each polypeptide within a S-HBVsAg particle has an authentic sequence of N-terminus. Furthermore, a denaturation plot showed that the S-HBVsAg vaccine particles were extremely stable, especially in solutions with high acidity. Such information is not only important for formulation purposes, but also provides insight into appropriate conditions to be applied during downstream processing.
Another application highlighting use of advanced tools for characterization of vaccine products was used by researchers at FDA (11). Using NMR as a microscope to study polysaccharides at the molecular and atomic levels, the researchers probed the individual atoms and their locations in relationship to each other. This information helped determine the molecular shapes of these molecules, which in turn provided valuable insights into how polysaccharides interact with antibodies and proteins. Tools such as laser light scattering and circular dichroism were suggested to characterize the overall size and shape of polysaccharides, which, together with NMR, enable FDA and the pharmaceutical industry to ensure that polysaccharide vaccines meet regulatory requirements for safety and effectiveness.
It is clear that the relevance of an analytical method with respect to its usefulness in characterizing an antigen depends on the type of material (see Table II). A set of methods need to be employed to gain insight into the quality of a vaccine. Production of well-characterized vaccines paves the path for reduced animal testing while providing safe and potent vaccines to patients and is a path that must be pursued.
Table II: Diverse methods suitable for vaccine characterization listed based on the type of vaccine under evaluation. ELISA is enzyme-linked immunosorbent assay, NMR is nuclear magnetic resonance, MS is mass spectroscopy, and HPLC is high-performance liquid chromatography.
Despite the advances in vaccine manufacturing across the globe, regulatory hurdles still stand in the way of companies seeking to take a candidate product to the clinic and eventually to market. Identifying a suitable vaccine candidate, appropriate antigens, adjuvants, and delivery methods are just the beginning of vaccine development (12). Vaccines are usually injected into healthy people, hence the emphasis on having well characterized, safe vaccines (13). Additionally, because process development provides a technological foundation for manufacturing, analytical methods and assay development for characterization and potency determination must be included (12).
Major regulatory considerations in development and manufacturing of vaccines include use of adequately characterized, homogenous starting material of defined origin and acceptable quality including cells and production seeds; adequate validation of the production process to demonstrate that the conditions are reproducible for different production lots; demonstration of consistency of production to the satisfaction of the regulatory authority; and adequate pre- and postmarketing surveillance of the behavior of the product in the target population to demonstrate safety and efficacy.
Clinical testing plays a key role in establishing the safety and efficacy of the product. Vaccine-specific issues include determining correlations 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 (14). Because vaccines are generally given to healthy individuals, particularly children and infants, large-scale efficacy and immunogenicity studies to prove safety and efficacy are required before an approval is granted (13).
Risks versus benefits of vaccines are closely examined during the review process. Special considerations may apply. For example, it may be easier for a National Regulatory Authority (NRA) of a developing country to approve a product if the product has already been approved by a regulatory authority in a developed country. All NRAs need to also follow a process of continuous improvement with respect to their approval process. Tests that are outdated because of developments in technology and advances in understanding should be removed. On the other hand, expectations related to compliance with GMPs need to be maintained to ensure product consistency and safety. Further, novel analytical technologies for product characterization need to be put in practice. Newer regulatory initiatives such as quality by design (QbD) and process analytical technology (PAT) need to be implemented for vaccine manufacturing as well (15, 16).
The World Health Organization (WHO) has developed a major role in collaborating with and facilitating knowledge-sharing among the NRAs. WHO's prototype GMP guidelines have been adopted by more than 100 countries. WHO prequalification or recommendation is essential for many international tenders of UN agencies and other such large buyers. Other major regulatory agencies also have mechanisms of supporting product licensure in countries outside their jurisdiction. The European Medicines Agency (EMA) has a provision under Article 58 of giving scientific opinion on products with sponsors or manufacturers in Europe that will not be marketed in Europe (17). The process is as rigorous as the usual Marketing Authorization. Many times, experts in the endemic countries are included in the group that gives the scientific opinion. In the US, a similar kind of procedure is the FDA Global Disease Approach for vaccines (18).
For many vaccine products, the need is greatest in poor and developing countries. In such cases, besides the safety and efficacy of the products, affordability becomes another criterion of significance. NRAs of the endemic country can initiate a joint review of applications for which one or more regulatory authorities work with the local NRA to provide a comprehensive review process. Health Canada has participated in such a file review with the Drugs Controller General (India) to enable timely registration and approval of a certain vaccine (19).
Timeliness for approval may be an issue for the cases of pandemic vaccines. The prevailing strains of the flu virus change often and manufacturers have to use the prevailing strains for the season based on WHO recommendations. Selection of the strain for the development of a vaccine in such cases needs extensive research to study the prevailing wild type and the feasibility of that strain for use in a vaccine. Hence, influenza vaccines have a registration procedure which includes rapid reviews of annual strain updates so that the vaccine is available before the flu season starts. When the pandemic influenza vaccine of H1N1 was the need of the hour, many manufacturers and regulators worked together to develop, approve, and bring it to the market in a short time (20).
Today's vaccine manufacturers, whether manufacturing in-house or as a contract service, have to strive to reduce overall timelines for development and production and manage with limited resources. Meeting global demand while conforming to very strict quality and regulatory controls is an ongoing challenge requiring proper planning during the development of a vaccine. Key regulatory issues need to be thought of and a presubmission check list should be developed. Manufacturers should be ready for inspections and interactions with the health authorities. They should plan the registration strategies for various countries by gathering adequate information regarding the regulations and risks.
The vaccine industry is changing dramatically. Vaccine manufacturing technologies are changing, creating a push to produce newer products. Companies are working rapidly with new combinations where they can. There is tremendous competition at the technical and manufacturing levels. Market uncertainties are high, especially for those vaccines that will be largely used in public-sector programs. Government pricing policy poses another obstacle to vaccine development. Furthermore, for manufacturers in developing countries, challenges for vaccine development also include cost and lack of access to technology. Thus, the vaccine industry globally is looking for ways forward to counter these challenges. Various global initiatives based on push and pull strategies are being implemented. One type of initiative is a public private partnership, involving consortia between governments, industry, the international health community, and funding agencies. The best example of such an approach is the recent development and launch of a meningococal conjugate vaccine A in sub-Saharan Africa at an innovative pricing of less than 50 cents a dose (see Figure 2). The timely launch of this vaccine has been reported to have had a significant health impact in the meningitis belt of sub-Saharan Africa. Such consortium approaches need to be encouraged to target key challenges in vaccine development to encourage innovation and improve the chances of success.
Figure 2: Development of Meningitis vaccine through a consortium of government and industrial partners.
ANURAG S. RATHORE* is a biotech CMC consultant and a faculty member in the department of chemical engineering, Indian Institute of Technology, New Dehli, as well as a member of BioPharm International's editorial advisory board; SURESH JADHAV is an executive director in the Serum Institute of India, Pune; MAHESH BHALGHAT is a vice-president at Biological E, Hyderabad; SHIRAZ KANDAWALLA is a senior manager at Sanofi Pasteur, Mumbai; SUMA RAY is a process development scientist, viral clearance and cell line development, Global Purification Technologies Group at Sartorium Stedim, Bangalore; and ASHOK KUMAR PATRA is a group leader at Panacea Biotech, New Delhi. *To whom correspondence should be addressed, email@example.com.
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