Vaccine Production: Main Steps and Considerations

1.1

Bacterial Antigen Vaccines

The production of a traditional bacterial antigen vaccine provides a good foundation for understanding other vaccine types. For example, the production of tetanus toxoid vaccine starts with growth of the organism Clostridium tetani . A specific culture of the organism is obtained, expanded, and frozen to create a master seed for all future production. This master seed is typically further expanded to make working seeds, which are then used to start individual batches of product for release for use. The frozen working seeds are recovered on solid agar, then liquid culture allowing several days to a week between transfers for the bacteria to adapt to media and grow. The temperature and culture conditions are closely controlled; the transfers are executed in controlled environments to prevent culture contamination from the production environment. Ultimately, the culture has sufficient viable cell density to inoculate the production bioreactor. After the organism expands in the bioreactor, the culture is harvested and cells are removed via centrifugation and/or filtration, allowing the secreted toxin to be recovered. The toxin is treated with a chemical agent such as formaldehyde, which causes the toxin molecules to cross-link eliminating the toxicity, but retaining the protein structure needed to elicit a protective immune response. The resulting molecule is called a toxoid. The toxoid is purified by a variety of methods which may include precipitation (addition of a salt to cause the toxoid or impurities to selectively precipitate and to be removed from the solution), ultrafiltration (separation of the toxoid from impurities based on size differences), and/or chromatography (separation of toxoid from impurities based on differences of charge and/or size). The toxoid is tested for purity, lack of toxicity, and potency prior to formulation into the final vaccine. Tetanus toxoid may be mixed with an adjuvant to increase the immune response. Traditionally, tetanus toxoid is adjuvated with aluminum salts (aluminum hydroxide, aluminum phosphate, etc.). It can be administered as a monovalent vaccine or mixed with diphtheria and/or pertussis toxoids, as well as other antigens, in a combination vaccine. The tetanus toxoid is generally stable in this form without the additional of stabilizers or special processing (lyophilization), and hence represents a fairly simple, but not trivial, manufacturing process example. Diphtheria and pertussis toxoid vaccines are made in a similar fashion.

There are a number of bacterial-based antigen vaccines which follow similar production approaches but do not require the “toxoiding.” In some cases the antigen of interest is secreted as in the aforementioned tetanus example, in other cases, the antigen needs to be extracted from the cell paste following the bioreactor harvest (polysaccharide-based vaccine processes for Haemophilus influenzae type B; meningitis types A, C, W135, and Y; and pneumococcal vaccines, recover the polysaccharide from the cell wall). In some cases the purified product is not stable and needs to be lyophilized. Lyophilization, also known as freeze-drying, is a process that allows the removal of water at low temperatures to maintain potency during the manufacturing process and providing greater stability of the final drug product during storage and distribution to the end user. Table 5.1 shows examples of a variety of vaccines, the cultivation and purification approaches, and the stabilization requirements.

1.2

Live Virus Vaccine

Perhaps the most effective means of developing a robust and protective response, often with a low vaccine dose, is through the use of a live virus vaccine (LVV). The viruses used in production are altered from wild-type viruses to weaken, or “attenuate” them such that a robust protective response is obtained without severe disease. In some cases the virus may not replicate in the human host (cowpox used to protect from smallpox) or be altered genetically such that it does not replicate. Similarly, the live virus may be innocuous but used as a viral vector vaccine to deliver other antigens (an approach being tested for Ebola vaccine).

The production of viral vaccines adds a complexity to the bacterial antigen production processes in that viruses need a living organism to amplify and so in order to make virus, you must first expand a cell culture system for the viral expansion. Many traditional viral vaccines are grown in fertile chicken eggs such that the target virus is injected into the egg and then infects the embryo; after several days in controlled temperature and humidity conditions, the virus is harvested from the chicken embryo (yellow fever vaccine) or allantoic fluid (live-attenuated influenza vaccine) and is further purified and processed to make the final vaccine. Although the chicken embryo-based production has been a reliable method for making many vaccines today and for many decades, it is at significant biosecurity effort (vaccination, quarantine practices, limited access, extensive testing of flocks) that the chicken flocks be protected from disease that would reduce availability of eggs (avian influenza) or that could infect the manufacturing process. These LVVs use eggs that are certified to be free of avian viruses with extensive testing of the flocks and monitoring of bird health.

Many LVVs use an immortalized cell line which has been thoroughly tested and certified to be free of adventitious agents that would have a deleterious effect on the manufacturing process or vaccine safety. These cell lines, similar to the master seeds for the bacterial products, are specific for each product and are frozen into master and working cell banks allowing long-term availability and viability of the cells and manufacturing processes they support. Many cell lines require an attached surface to multiply and to be viable through the manufacturing process (eg, Vero cells, MRC-5 cells); this requires special equipment and processing to support the virus expansion. The most popular options for this production are roller bottles (bottles slowly turning to allow nutrients to wash over growing cells, while controlling temperature and dissolved gas concentration), flat plate reactors (which have multiple parallel plates for cell culture attachment and growth, pumping nutrients through the device), or microcarriers (small beads in suspension in a bioreactor allowing a surface for growth and bioreactor mixing for nutrient replacement). In each case, as the cells expand and need to be transferred to a large-scale device, they must be detached, typically with addition of a enzyme like trypsin, then reattached to the new surface (by removal/dilution of trypsin and addition of other nutrients). With these processes being done in a sterile environment, the equipment costs and complexity is high, often requiring robotics and clean room operations to reduce risk of failure. Once the expansion of the culture is complete, which could take several weeks, the culture is infected and the viral production is generally fairly fast (several days). When infection is complete, the virus may be collected from the culture media (if secreted) or purified from the disrupted cells. Unit operations in this case are similar to those described in bacterial antigen production.

Because a virus needs a living cell to expand, once the cells are removed, the virus may have limited stability. The processing times are strictly controlled to limit degradation of potency and often the material is frozen to −20 or −70°C to preserve potency between manufacturing steps. Most LVVs are ultimately freeze-dried (MMR, varicella) or may be delivered frozen (live attenuated influenza vaccine). Some need to be frozen until use even after lyophilization to prolong shelf life. There are exceptions like rotavirus vaccine which is stable at 2–8°C for 2 years.

1.3

Inactivated Virus Vaccines

For many viral diseases, exposure to the viral proteins, without an active infection, can produce protection against the disease. In these cases, one would produce the viral antigen similar to the processes described for LVVs, but the virus is inactivated by chemical means to render it noninfectious. The inactivation may take place before or after purification. The best examples of inactivated virus vaccines include inactivated influenza vaccine, largely grown on chicken embryos but also in cell culture, where the virus is inactivated with formaldehyde or BPL (β-propiolactone); inactivated poliovirus vaccine, grown in Vero cells on microcarriers in large bioreactors, inactivated with formaldehyde; and hepatitis A vaccine, grown in MRC-5 cells on flat plate reactors and inactivated with formaldehyde.

1.4

Recombinant Vaccines

Advances in genetic engineering have allowed the production of several vaccine antigens without use of the native infectious organism. In this case, a yeast culture, such as Saccharomyces cerevisiae can be altered to produce a vaccine antigen such as the hepatitis B surface antigen (HBsAg), which protects against hepatitis B infection. In this case the process resembles the bacterial antigen process. At the end of the fermentation process, the HBsAg is harvested by lysing the yeast cells. It is separated by hydrophobic interaction and size-exclusion chromatography. The resulting HBsAg is assembled into 22-nm diameter lipoprotein particles. The HBsAg is purified to greater than 99% for protein by a series of physical and chemical methods. The purified protein is treated in phosphate buffer with formaldehyde, sterile filtered, and then coprecipitated with alum (potassium aluminum sulfate) to form bulk vaccine adjuvated with amorphous aluminum hydroxyphosphate sulfate. The vaccine contains no detectable yeast DNA but may contain not more than 1% yeast protein. Similar approaches are used to make human papillomavirus (HPV) vaccines.

1.5

Conjugate Vaccines

The production of Haemophilus type b conjugate includes the separate production of capsular polysaccharide from H. influenzae type b and a carrier protein such as tetanus protein from C. tetani (ie, purified tetanus toxoid), CRM protein from Corynebacterium diphtheriae , or outer membrane protein complex of Neisseria meningitidis . The production of polysaccharide and tetanus toxoid was described earlier.

The industrial conjugation process was initially developed using tetanus toxoid by the J.B. Robbins team at the National Institute of Allergy and Infectious Diseases (NIAID), Bethesda, Maryland. Conjugate preparation is a two-step process that involves: activation of the Hib capsular polysaccharide and conjugation of activated polysaccharide to tetanus protein through a spacer.

Activation includes chemical fragmentation of the native polysaccharide to a specified molecular weight target and covalent linkage of adipic acid dihydrazide. The activated polysaccharide is then covalently linked to the purified tetanus protein by carbodiimide-mediated condensation using 1-ethyl-3(3-dimethylaminopropyl)carbodiimide. Purification of the conjugated material is performed to obtain high molecular weight conjugate molecules devoid of chemical residues and free protein and polysaccharide.

Conjugate bulk is then diluted in an appropriate buffer, filled into unit-dose and/or multidose vials, and lyophilized.

1.6

Vaccine Formulation and Filling

The focus of this chapter to this point has been the production of drug substance or active ingredient of the vaccine. The drug substance is further processed through formulation and filling, labeling and packaging to become drug product ready for use by the patient.

The formulation of the vaccine is designed to maximize the stability of the vaccine while delivering it in a format that allows efficient distribution and preferred clinical delivery of the product. The formulated vaccine may include an adjuvant to enhance the immune response, stabilizers to prolong shelf life, and/or preservatives to allow multidose vials to be delivered.

After formulation, the product is filled into vials or syringes under strictly controlled conditions to prevent introduction of any viable or nonviable contamination, and sealed to ensure container closure integrity during shelf life. Filled vials may be lyophilized in order to increase stability; in this case, the vials are fitted with special stoppers that are partially inserted during drying to allow moisture to escape, and fully inserted and capped after drying. Quality control (QC) testing at this stage usually consists of safety, potency, purity, sterility, and other assays specific to the product.