The process development challenge for a new vaccine

/Home/Lyophilization Articles & Reports

The second critical step in the process was to make a vaccine for rapid evaluation in the clinic (assessed by its ability to generate antibodies against the HPV virus), recognizing that the method used to produce the vaccine would be crucial and would set the stage for all subsequent phases of the developmental program. To prepare the vaccine for initial clinical trials, a strategy was adopted that would be similar to one conceptually planned for eventual full-scale manufacturing once the vaccine was licensed. A crucial starting point was the development of a cell-based expression system that was stable over many generations and produced large amounts of high-quality VLPs. The decision to use Saccharomyces cerevisiae as the expression host for intracellular VLPs was excellent in this regard because the yeast can readily be grown in fermentors of several thousand liters and reproducibly forms intracellular VLPs during its growth cycle. The major reason that this particular host cell was chosen was because of the extensive experience at the Merck Research Laboratories using S. cerevisiae to make a successful hepatitis B vaccine (Recombivax). It was possible to finalize choice of this yeast strain based on productivity, reproducibility and the specific fermentation conditions at an early stage of the program, thus allowing process development to focus on scale-up, purification, formulation and the analytical methods required for product characterization and potency measurements.
Once the selection of the expression system to be used in vaccine manufacture was made, a master seed bank was established from the engineered yeast strain; this bank was sufficient in quantity for use throughout the duration of the program. A unique VLP was made for each of the four HPV types included in the vaccine, requiring technology development for the fermentation process, cell harvesting, cell disruption, cell debris removal, chromatography-based purification and absorption to the adjuvant of choice for each VLP to make the final formulated product. The VLPs are stable for several years under refrigerated conditions and the aluminum-based adjuvant was chosen to be the same as the Recombivax hepatitis B vaccine. Analytical methods based on antibody binding to the functional epitopes of the VLPs were developed to quantify the potency of the vaccine (in vitro using an enzyme-linked immunoassay) and that were sufficiently precise to be able to monitor VLP stability when stored as a refrigerated final product for several years, and also separately quantify each VLP representing the specific HPV types contained in the four-component vaccine. The challenge is to choose an antibody that recognizes the functional epitope for the HPV strain in question and which is not subject to interference from the other three HPV types present in the vaccine. Other key tests were also developed to search for impurities, such as highly sensitive assays to detect trace amounts of the undesired yeast cell proteins.
As mentioned earlier, a major challenge in commercial vaccine manufacturing is to ensure that the process is scalable so that it is possible to build a factory for tens of millions of doses, and that the vaccine made at this scale behaves the same way in the clinic as vaccine made at a much smaller scale earlier in the program. In addition, the analytics need to be sufficiently well developed so that the precise definition of potency at a particular point in the development cycle will not change as the program progresses. Specifically, it is essential to be able to link the clinical performance, comparing the vaccine made at an early stage of development to that used for final stage development and testing in the clinic. In the case of the HPV vaccine program, the vaccine used in the phase 1 clinical trials was manufactured in a pilot plant (a pilot plant uses the same type of equipment as the final factory) using a less-developed version of the production process and at a smaller scale of operation (300 liters). The bulk preparation of phase 3 vaccine was made at full-scale manufacturing, which is within a factor of 10 of pilot-scale manufacturing.
So, what essential components are needed for the successful development of a large-scale manufacturing process such as for the HPV vaccine described here? First, facilities are required to produce representative clinical supplies at the pilot scale. Second, a manufacturing facility needs to be designed and constructed. These facilities historically have been built by the company that develops the vaccine. Third, vaccine supplies need to be made within this manufacturing facility before licensure, and usually representative doses need to be tested in the clinic as part of the required program to gain approval of the vaccine for licensure. Fourth, sophisticated analytical characterization is required so that a rationale can be developed for making small changes in the process or changes in scale of production and bridging without the need for confirmation in the clinic. Fifth, a clinical research team needs to be able to coordinate large and complicated clinical trials as well as the development of serological assays that are used as a surrogate for protection. Sixth, the regulatory expectations for filing throughout the world need to be fully understood early in the process development effort. To carry out these diverse and complex development tasks, a committed team of approximately ten or more people is required to lead and coordinate such an inherently complicated project; this team would include representatives from the process development group, the analytical group, the clinical team, manufacturing, marketing, and from the internal regulatory organization. Patience and determination are needed, often for 10 years or more, to complete this cycle. After all this effort, the end product is 0.5 ml of safe and efficacious vaccine in a vial or syringe containing purified VLPs of four different HPV types at a precise and reproducible dose for each along with adjuvant.
Rotavirus. The vaccine being developed for rotavirus is a live attenuated virus vaccine that contains five different human-bovine virus reassortants6, 7, which have been adapted to large-scale production using VERO cells (Fig. 1). Rotavirus is a member of the Reovirus genus in the Reoviridia family of viruses. The final reassorted strain was selected from each of the five final types by taking advantage of the natural capability of the rotavirus genes to reassort in cell culture.
Figure 1. Rotavirus reassortant to generate oral live virus vaccine.

RotaTeq is a polyvalent vaccine consisting of five human-bovine reassortants: four G serotypes (G1, G2, G3, G4) representing 80% of the G strains circulating worldwide, and one P serotype representing >75% of the P strains circulating worldwide.
The general outline described for HPV would apply in terms of the approach for developing a vaccine but the technology-related issues are very different. The process used for rotavirus vaccine requires master-seed development for each of the five rotavirus reassortants as well as for the VERO cell bank. There are five different processes involved, with a separate process for each reassortant. The VERO cells are grown to confluence on a surface and then infected with rotavirus. The product is a live virus (initially developed at the Children's Hospital of Philadelphia) that needs to be formulated in a manner that maintains good potency in the refrigerator for each of the five assortants. The route of administration is oral. Potency measurements are based on quantification of attenuated live virus using PCR technology, which is used to quantify each of the individual five reassortants in the final formulated vaccine. The clinical trials required for this vaccine to prove both safety and efficacy have been very large and expensive, requiring more than 100,000 of vaccine doses made by a process representative of final-scale manufacturing (Fig. 2).
Figure 2. Overview of the development of a vaccine against rotavirus (RotaTeq).

G1-4, P1, rotavirus serotypes. RotaShield, a vaccine for rotavirus developed by Wyeth.
Varicella zoster. In the last example, the vaccine under development for postherpetic neuralgia or shingles (caused by reactivation of latent varicella zoster virus infections) is based upon Merck Research Laboratories' existing live attenuated chicken pox vaccine. The shingles vaccine is being developed for older adults, whereas the chicken pox vaccine is for young children. For increased immunogenicity in older individuals, the dose of the vaccine administered is much greater than that used to immunize children against chicken pox. Given that the requisite dose of vaccine is higher for use in adults for prevention of shingles, and that varicella virus is inherently difficult to grow to high titers in culture, significant obstacles had to be overcome to manufacture sufficient quantities of vaccine virus. Further, as the virus is unstable at normal temperatures, a unique technical challenge was to develop a method to preserve its stability using lyophilization in the presence of specially developed preservatives to permit an acceptable shelf life.
Again, the manufacturer must ensure that each dose of the vaccine is safe and effective. In the case of the varicella postherpetic neuralgia vaccine, formulation required knowledge of process capabilities, precision of measurement and stability assumptions. An adequate dose range must be evaluated in the clinic. For safety, the key is to obtain data representative of a vial soon after filling in order to reflect the highest dose. For efficacy determination, the key is to evaluate a dose representative of a vial held until the end of the claimed shelf life (i.e., when the dose will be lower as a result of loss of infectious virus upon storage). This example illustrates integration bet