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Like the egg-based vaccine production process, producing a vaccine under cGMP conditions using mammalian cells can be a lengthy process, taking a minimum of six to 12 months.
The traditional method for developing a vaccine uses substrates isolated from chicken eggs or mammalian cells. This method has several drawbacks that could pose many problems. The first major issue is the time taken to develop a vaccine using this method, which could be longer than six months. The nature of the disease agent can present significant challenges to cell- and egg-based production depending on the species and strain of the agent. Moreover, there is a concern that people with allergies to eggs may also react negatively to an egg-based vaccine. Using modern plasmid DNA-based vaccines to combat infectious diseases is an alternative to traditional egg-based and mammalian-based vaccines. This article compares these two methods of developing and producing vaccines.
The use of vaccines to fight infectious diseases is not a new practice. Vaccines have been around for over two hundred years with one of the first documented being the vaccine for smallpox, which was developed and introduced in the 1790s. Over the years, science and medicine have made great strides in developing and producing numerous successful vaccines to combat various diseases. The smallpox vaccine developed by Jenner was prepared by taking scrapings of cowpox lesions directly from infected cattle. The two polio vaccines developed in 1952 by Jonas Salk and Albert Savin used a revolutionary new technique that allowed the propagation of polio virus in cultured monkey kidney cells.1 Today, effective vaccines for human diseases are produced by a variety of methods.
Traditional methods for developing and producing vaccines use substrates from either embryonated chicken eggs or mammalian cells. The primary techniques for vaccine production used today are shown in Table 1. The table shows that chicken embryos are primarily used for vaccines for viral diseases. The production of vaccines using fertilized eggs has over a fifty-year history. Though this method has been successful in the fight against numerous diseases including influenza, measles–mumps–rubella (MMR), and rabies, it has several drawbacks. A significant disadvantage is the time that it takes to develop a vaccine. Vaccines that are developed using chicken eggs can take upwards of six to 12 months for development, production, and release testing activities. This timeline would prove to be excessively long if a pandemic outbreak were to occur.5
Table 1. The primary techniques for vaccine production 4
Another concern regarding the production of vaccines in chicken eggs is that production capacity is limited to the number and availability of specific pathogen-free fertilized eggs and the finite capacity of current manufacturing facilities. For example, with the influenza vaccine, one egg is required for each dose of vaccine. Therefore, for a million doses, a million chicken eggs must be processed. This method is not ideal for rapid large-scale production scale-up requirements. The specific strain and species of the disease also can present challenges to the actual production of the vaccine in embryonated eggs. Certain virus strains may not replicate productively in an embryonic avian host, which can then require additional process optimization and production time. For example, the H5N1 strain of the avian influenza is generally deadly to chicken embryos and therefore, an alternative method must be employed for the production of the vaccine.6 In addition, even for avian flu strains that could be produced in chicken embryos, in the event of a pandemic, the availability of eggs would be severely impacted. Another disadvantage of producing vaccines in eggs is that people with allergies to eggs may react negatively to egg-based vaccines.
The technology of using mammalian cells for the production of vaccines has grown rapidly in recent years. In the first step of production, mammalian cell lines are used to produce a master cell bank (MCB), which is then characterized and used to grow the pathogen or antigen subunit under controlled cell culture or fermentation conditions. The subsequent cell culture or supernatant is then harvested, purified, and several virus inactivation steps take place. The use of bioreactors and optimization of fermentation conditions allows mammalian cell production of vaccines to be a more scalable process than the egg-based method of vaccine production. Like the egg-based vaccine production process, producing a vaccine under current good manufacturing practices (cGMP) conditions using mammalian cells can be a lengthy process, occurring over a minimum six- to 12-month time period. It takes longer because of the regulatory constraints for mammalian cell production. The MCB must undergo extensive testing and characterization prior to release for cGMP manufacturing. This testing can take up to four months to complete. Process validation for the viral inactivation steps is also an important part of mammalian cell production of vaccines and can also require months to complete. Also, the final product testing for vaccines produced using mammalian cell production often requires assays, which take more time for development and execution. These additional safety requirements are all related to the fact that mammalian cells can harbor various human pathogens or become infected during cell culture expansion.
Using modern plasmid DNA–based vaccines to combat infectious diseases is an alternative to traditional egg- and mammalian cell-based vaccines. Plasmid DNA vaccines are produced by transforming the engineered plasmid into a host strain of E. coli. The plasmid DNA with the vaccine gene insert is constructed using recombinant DNA technology. The gene fragments that are inserted into the plasmid DNA strand carry genes that specify one or more antigenic proteins. Antigenic proteins are normally made or expressed by the selected pathogen. The benefit of inserting the selected genes into the plasmid DNA is that genes that would enable the pathogen to reconstitute itself and trigger the disease are absent from the plasmid DNA. This ensures a safe vaccine that is unable to produce an infection.2–3 Unlike mammalian cells, E. coli can not produce viruses or mycoplasm and therefore, do not require virus inactivation processing steps and final product testing for human pathogens.
In most plasmid DNA vaccination platforms, the segment of DNA that encodes the protein antigen is incorporated into the plasmid DNA backbone. Upon delivery of the plasmid DNA vaccine to a patient, the plasmid is taken up by the targeted cells and can exist independently of chromosomal DNA and transcribe the gene encoding the antigen of interest. The production of small amounts of antigen can lead to the induction of both antibody and cellular responses within the patient or one specific response if certain adjuvants are used.3
The use of plasmid DNA vaccines has a number of potential advantages compared with the current egg- and mammalian cell-based vaccines. The first advantage is safety. Because only certain genes from the selected pathogen are inserted into the plasmid DNA vaccine, the risk of pathogen replication or spreading is essentially nonexistent. Plasmid DNA vaccines will be unable to cause infection in the patient because such vaccines lack the genes needed for the pathogen's replication.3 These vaccines also avoid the potential for allergic reactions of egg-based vaccines in humans.
Another advantage of plasmid DNA-based vaccines over the current egg and mammalian-cell based vaccines is the overall development and production timeline. The genes from the specific pathogen can be isolated and inserted into the plasmid DNA construct within a very short timeline. The plasmid DNA construct can be then transformed into an E. coli strain and a cGMP master cell bank can be produced within a week. The testing required for the release of a MCB for plasmid DNA is less extensive and the timeline is much shorter than the testing required to release a mammalian cell bank. The release testing for the MCB can be completed in as short as two weeks from the time the cell bank is produced.
The plasmid DNA production process is a readily scalable process which can be scaled up to meet worldwide manufacturing demands for vaccines. Recent process improvements to plasmid DNA production have significantly shortened the manufacturing timeline. New high-cell-density fermentation methods have dramatically increased plasmid DNA productivity and shortened the overall production timeline. These new methods are able to achieve overall plasmid yields of greater than 1 gram of purified plasmid DNA per liter of fermentation. Scaled-up downstream lysis and purification activities also have greatly reduced the overall production timeline. Large-scale purification techniques have incorporated the use of a single chromatography column, which results in higher overall yield and consistently pure plasmid DNA. It is possible to produce a de novo plasmid DNA vaccine through manufacture, filling, testing, and release in less than three months. Stability studies performed on numerous formulated plasmid DNA lots have shown that they are rock stable when properly formulated and can be stored for extended periods of time at controlled temperatures, which makes plasmid DNA a good candidate for vaccine use in fighting significant public health diseases, where delivery and stockpiling of vaccines is a serious concern. Lyophilization and formulation optimization are now being used to develop DNA vaccines, which will be stable without requiring cold storage.
Though a plasmid DNA vaccine has not yet been approved for commercial use in humans, there are numerous clinical trials currently being conducted using these vaccines. New technologies for plasmid DNA delivery such as electroporation are currently being used in clinical trials to enhance the immune response of the patient. Other methods of vaccine delivery include the "prime boost" method, in which the patient is first primed with the plasmid DNA vaccine and then a protein subunit vaccine is delivered to the patient in order to boost the antibody or cellular immune response.2 These new methods of plasmid DNA vaccine delivery have shown promising results in preclinical studies and clinical trials.
Early in the development of DNA vaccines, there were a number of concerns regarding the manufacturability, stability, and safety of such products. Safety concerns focused on two primary questions: would the introduced DNA remain at the site of injection, or could it migrate through the body to the reproduction organs and be incorporated into gamete cells (egg or sperm)? The second question was: could the introduced gene incorporate itself into the target cell's chromosome where it could potentially activate an oncogenic gene or inactivate a tumor suppressor? These two phenomena have been observed at a very low frequency with viral vectors; however, they do not appear to occur in the case of DNA vaccines. New constructs are still evaluated in good laboratory practices (GLP) controlled biodistribution and integration studies prior to use in clinical trials as a safety precaution.
Though plasmid DNA vaccines for human use are still in the stages of clinical development, the vaccines have a number of potential advantages over traditional vaccines. The plasmid DNA vectors can be designed and produced quickly. The overall plasmid DNA production, from cell bank production and testing, through fermentation, purification, filling, and release testing of final product can be completed in a very short timeframe. The process scalability and rapid production time ensures that plasmid DNA vaccines could be developed and produced to meet aggressive timelines if there were a pandemic outbreak of disease. The immune responses achieved with plasmid DNA and the use of delivery techniques, such as electroporation and the prime boost method have shown to have long-lasting results and provide disease protection.
While DNA vaccines have been approved for veterinary applications, some significant challenges remain in terms of optimizing the targeted immune responses in humans. Various strategies have shown great promise in resolving this last hurdle; scientists are now using novel adjuvants and electroporation to enhance immunogenicity and plasmid delivery to target cells. While the field of DNA vaccines is still in its infancy, and real challenges remain, the early concerns about safety and the ability to produce large quantities of DNA in a scalable, regulatory compliant manner have been resolved. The manufacturing process, which in the early days employed cesium gradients and animal-derived enzymes, such as RNAse, and a tremendous amount of labor to produce a few milligrams has evolved to the point where hundreds of grams of DNA can be produced within as short as 10 days. As science progresses forward in the treatment of diseases, it is certain that plasmid DNA will play a role in future vaccines for use in humans.
Richard B. Hancock is the executive vice president and chief operating officer at Althea Technologies, San Diego, CA, 858.882.0123, email@example.com. At the same company, Melissa M. Rosness is the director of contract management.
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2. David B, Ronald C. Genetic vaccines. Scientific America Inc. 1991 Jul.
3. Gareth M. Rapid-response vaccines— does DNA offer a solution? Nature Biotechnol. 2005 Sept.;23(9).
4. VFC: Approved vaccines and biologicals. Centers for Disease Control and Prevention. Available from: www.cdc.gov/vaccines/programs/vfc/parents/apprvd-vaccs.htm.
5. World Health Organization (WHO). WHO biosafety risk assessment and guidelines for the production and quality control of human influenza pandemic vaccines. Available from: www.who.int/biologicals/publications/ECBS%202005%20Annex%205%20Influenza.pdf.
6. World Health Organization. Development of a vaccine effective against avian influenza H5N1 infection in humans. Jan. 2004. Available from: www.who.int/csr/don/2004_01_20/en/.