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Vaccine Manufacturing


The vast majority of the more than 5 billion doses of vaccines manufactured worldwide each year are given to perfectly healthy people. It is this fact that drives the requirements for vaccines to be among the most rigorously designed, monitored, and compliant products manufactured today. The ability to manufacture these vaccines safely and consistently is built on four competencies:

  • 1

    The manufacturing process that defines how the product is made.

  • 2

    The compliance of the organization to successfully complete that process.

  • 3

    The testing of the product and supporting operations.

  • 4

    The regulatory authorization to release and distribute the product.

This chapter examines how each of these components is established during the development of a new vaccine and how the field of vaccine manufacturing is responding to emerging challenges for increased capacity (e.g., coronaviruses vaccines, pandemic influenza vaccines), increased safety assurance (e.g., barrier isolator filling), and increasing complexities of manufacture (e.g., conjugate vaccines, multivalent vaccines, new adjuvant types). All of this must be accomplished while consistently delivering more than 5 billion doses annually at the relatively lower cost of similar therapeutic products. (As many of these more than 5 billion doses of vaccine product are multivalent vaccines such as DTP-HepB-HIB, MMR, influenza, pneumococcal, the total number of monovalent doses is likely more than 20 billion per year.)

In the United States, vaccines are regulated as biological drug products. The U.S. Food and Drug Administration’s (FDA) Center for Biologics Evaluation and Research (CBER) has the authority for the regulation of vaccines under Section 351 of the Public Health Service Act and specific sections of the Federal Food, Drug and Cosmetic Act. , Section 351 of the Public Health Service Act gives the federal government the authority to license biological products and the establishments where they are produced. Vaccines undergo a rigorous review of manufacturing, testing, nonclinical, and clinical data to ensure safety, efficacy, purity, and potency. Vaccines approved for marketing may also be required to undergo additional studies to further evaluate the vaccine and often to address specific questions about the vaccine’s safety, effectiveness, or possible side effects.

In the European Union, vaccines are regulated by the European Medicines Agency (EMA) through the EMA’s Committee on Medicinal Products for Human Use Vaccine Working Party that has oversight for human vaccines. Vaccines are licensed through a centralized procedure that allows for simultaneous licensure within all countries within the European Union. Human vaccines manufacturing is regulated under a Good Manufacturing Practices (GMP) Directive 200/94/EEC, Annex 16, and Annex 2.

Harmonization of licensing and regulating procedures for vaccines worldwide has obvious benefits in rapidly delivering safe and effective vaccines to the market. Impediments to harmonization include lack of standardized regulatory procedures and mutual recognition of licenses and inspections between countries and worldwide regulatory agencies. Harmonization of regulation continues to progress as joint FDA-EMA establishment inspection programs have become a reality and adherence to International Conference on Harmonization (ICH) guidance is expected. The World Health Organization, through a prequalification program, provides for means of accelerating access to vaccines world wide by leveraging the qualification of a country’s National Regulatory Authority against global quality standards, confirmation that vaccines are consistently being manufactured per GMP, and product testing and complaint monitoring. This initiative significantly expands access to vaccines by minimizing duplicative efforts, sharing of information, and utilizing harmonized product monitoring and maintenance (WHO Expert Committee on Biological Standardization, February 2012).

New vaccines are subjected to a well-defined regulatory process for approval. The approval process consists of four principal elements:

  • Preparation of preclinical materials for proof-of-concept testing in animal models; manufacture of clinical materials according to current GMP; and toxicology analysis in an appropriate animal system.

  • Submission of an investigational new drug (IND) application for submission to FDA for review (or equivalent process for other countries).

  • Testing for safety and effectiveness through clinical and further nonclinical studies (Phase I to Phase III clinical studies).

  • Submission of all clinical, nonclinical, and manufacturing data to the FDA and EMA in the form of a Biologics License Application (BLA) for final review and licensure.

This chapter outlines the basics of manufacturing a vaccine and a description of some examples of currently licensed products. It then discusses the development of new vaccines and vaccine analytics. The next section examines the great challenges in the field to deliver a product held to an ever-increasing standard of safety while providing sufficient doses at reasonable costs for an ever-increasing number of disease targets, including the development of platforms for developing new vaccines more quickly with similar equipment and technology. The chapter closes with considerations for low- to middle-income countries.

MANUFACTURING BASICS

The manufacture of vaccines is composed of several basic steps that result in the finished product. Table 6.1 summarizes these steps with examples for pathogens that have a licensed vaccine in the United States. The first step is the generation of the antigen or the generation of a coding sequence for the antigen used to induce an immune response. This step includes the generation of the pathogen itself (for subsequent inactivation or isolation of a subunit), the generation of a recombinant protein derived from the pathogen or the generation of a genetic sequence (mRNA or DNA) coding for the antigen. Viruses are grown in cells, which can be either primary cells, such as chicken fibroblasts (e.g., yellow fever vaccine), or continuous cell lines, such as MRC-5 (e.g., hepatitis A vaccine). Bacterial pathogens are grown in bioreactors using medium developed to optimize the yield of the antigen while maintaining its integrity. Recombinant proteins and virus-like particles (VLPs) can be manufactured in bacteria, yeast, cell culture, or in cell-free processes. The viral and bacterial seed cultures and the cell lines used for viral production are carefully controlled, stored, characterized, and, often, protected. The first step in manufacture is the establishment of a “master cell bank.” This is a collection of vialed cells that form the starting material for all future production. It is extensively characterized for performance and the absence of any adventitious agents. From this bank, working cell banks are prepared that are used as the routine starting culture for production lots. The final vaccine is a direct function of its starting materials, and a change in this bank can be as complicated as initiating a new product development altogether. Likewise, master virus banks are prepared and used to make working virus banks. The working virus banks are used to infect individual lots of cell culture for propagation of virus for the vaccine drug substance.

TABLE 6.1
Examples of Licensed Vaccine Manufacturing Processes
Disease Trade Name Generic Name Cell Culture/Fermentation Isolation Purification Formulation Preservative
Anthrax BioThrax Anthrax Vaccine Adsorbed Chemically defined protein-free media growing a microaerophilic culture of avirulent, nonencapsulated Bacillus anthracis ND Sterile filtrate of culture medium Aluminum hydroxide Benzethonium and formaldehyde
Haemophilus influenzae ActHIB Haemophilus b Conjugate Vaccine (Tetanus Toxoid Conjugate) Grown of Haemophilus influenzae type b strain 1482 grown in a semisynthetic medium Centrifugation Phenol extraction and alcohol precipitation; Hib polysaccharide conjugated to tetanus toxoid Lyophilized None
Hepatitis A Havrix Hepatitis A Vaccine, Inactivated Hepatitis A (strain HM175) propagated in MRC-5 human diploid cells Cells lysed to form a suspension Purification by ultrafiltration and gel permeation chromatography followed by formalin inactivation Adsorbed onto aluminum hydroxide 2-Phenoxy-ethanol
Hepatitis B Recombivax HB Hepatitis B Vaccine (recombinant) Recombinant hepatitis B surface antigen (HBsAg) produced in yeast cells grown in a complex medium of extract of yeast, soy peptone, dextrose, amino acids, and mineral salts Released from yeast by cell disruption Series of chemical and physical methods (ND) followed by treatment with formaldehyde Coprecipitation of HBsAg with amorphous aluminum hydroxyphosphate sulfate None
Influenza Fluzone Inactivated Influenza Virus Vaccine Propagation on embryonated chicken eggs Low-speed centrifugation and filtration Purification/concentration on linear sucrose density gradient using continuous flow centrifugation followed by additional purification by chemical means Phosphate-buffered saline with gelatin as stabilizer Thimerosal in some package configurations
Japanese encephalitis JE-VAX Japanese Encephalitis Virus Vaccine Inactivated Intracerebral inoculation of mice Harvest of brain tissue/homogenization Centrifugation, supernatant collection followed by formaldehyde inactivation; further purification by ultracentrifugation through 40% sucrose Lyophilized Thimerosal
Measles, mumps, rubella, and varicella ProQuad Measles, Mumps, Rubella and Varicella (Oka/Merck) Virus Vaccine Live Measles virus propagated in chick embryo cell culture; mumps virus in chick embryo cell culture; rubella virus propagated in WI-38 human diploid lung fibroblasts; varicella virus propagated on MRC-5 cells ND ND Lyophilized None
Meningococcal Menactra Meningococcal (groups A, C, Y, and W-135) Polysaccharide Diphtheria Toxoid Conjugate Vaccine Meningococcal strains are cultured individually on Mueller-Hinton agar and grown in Watson–Scherp media; Corynebacterium diphtheriae grown on modified Mueller and Miller medium Extraction of polysaccharide from cell Polysaccharide purified by centrifugation, detergent precipitation, alcohol precipitation, solvent extraction, and diafiltration; diphtheria purified by ammonium sulfate fractionation and diafiltration; conjugate purified by serial diafiltration Sodium phosphate–buffered isotonic sodium chloride None
Pneumococcal Prevnar Pneumococcal 13-valent Conjugate Vaccine (Diphtheria CRM197 Protein) Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7 F, 9 V, 14, 18 C, 19A, 19 F, and 23 F individually grown on soy peptone broth; C. diphtheriae strain containing CRM197 grown in casamino acids and yeast extract–based medium Polysaccharides isolated by centrifugation; CRM197 ND Polysaccharides purified by precipitation, ultrafiltration, and column chromatography; CRM197 purified by ultrafiltration, ammonium sulfate precipitation, and ion-exchange chromatography; conjugation done by reductive amination and the conjugate purified by ultrafiltration and column chromatography Aluminum hydroxide suspension None
Polio IPOL Poliovirus Vaccine Inactivated Types 1, 2, and 3 poliovirus individually grown in Vero cells on microcarriers using Eagle MEM modified medium supplemented with newborn calf serum Clarification (method ND) and concentration Purification by three chromatography steps: anion exchange, gel filtration, and anion exchange; inactivation by formalin Medium M-199 2-Phenoxy-ethanol
Rabies RabAvert Rabies Vaccine Rabies virus grown in primary culture of chicken fibroblasts in synthetic cell culture medium with the addition of human albumin, polygeline, and antibiotics Inactivated with β-propiolactone Purification by zonal centrifugation in a sucrose density gradient Stabilized with buffered polygeline and potassium glutamate; lyophilized None
Streptococcus pneumoniae Pneumovax Pneumococcal vaccine polyvalent ND ND ND Isotonic saline Phenol
Typhoid fever Vivotif Typhoid Vaccine Live Oral Ty21a Fermentation using medium containing a digest of yeast extract, an acid digest of casein, dextrose, and galactose Centrifugation ND Enteric-coated capsule containing lyophilized product None
Yellow fever YF-Vax Yellow Fever Vaccine Strain 17D-204 of yellow fever is cultured on living avian leukosis virus-free chicken embryos Homogenization Centrifugation Lyophilized product containing gelatin and sorbitol as stabilizer None
Data from vaccine package inserts.
ND, not disclosed.

The next step is to release the antigen from the substrate and isolate it from the bulk of the environment used in its growth. This can be isolation of free virus, VLPs, polysaccharides, or secreted proteins from cells or cells containing the antigen from the spent medium. The next step is purification of the antigen. For vaccines that are composed of recombinant proteins, this step may involve many unit operations of column chromatography, enzymatic treatment, and ultrafiltration. 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.

Formulation consists of combining all components that constitute the final vaccine and uniformly mixing them in a single vessel ( Fig. 6.1 ). The sequence of additions, rates of additions, mixing conditions, and many other factors are carefully controlled to ensure the final formulation is executed precisely as designed for consistency, stability, and efficacy. Operations are conducted in a highly controlled environment with employees wearing special protective clothing to avoid product contamination. Viable and nonviable control monitoring of the environment and critical surfaces is conducted during operations. Quality control (QC) testing at this stage usually consists of safety, potency, purity, sterility, and other assays specific to the product.

Fig. 6.1, Automated vaccine formulation vessels.

Once formulated, the product is then filled into vials or syringes. During this phase, individual, scrupulously cleaned, depyrogenated, single-dose or multidose containers are filled with vaccine and sealed with sterile stoppers or plungers. If the vaccine is to be lyophilized, the vial stoppers are inserted only partially to allow moisture to escape during the lyophilization process, and the vials are moved to a lyophilization chamber. All vials receive outer caps over the stopper for container closure integrity. To preclude the introduction of extraneous viable and nonviable contamination, all filling operations must take place in a highly controlled environment where people, equipment, and components are introduced into the critical area in a controlled manner. After filling, all containers are inspected using semiautomated or automated equipment designed to detect any minute cosmetic and physical defects. As with the formulation phase of the vaccine manufacturing operation, extensive control and monitoring of the environment and critical surfaces are conducted during operations. QC testing at this stage also consists of safety, potency, purity, sterility, and other assays that may be specific to the product.

Vaccine efficacy can be adversely affected by improper distribution and storage conditions. The sensitivity of vaccines to adverse environmental conditions, particularly temperature extremes, varies depending on their composition. Live attenuated vaccines tend to be more susceptible than inactivated vaccines and toxoids. Vaccines are formulated such that the potency at the end of shelf-life remains above the effective dose demonstrated in human clinical trials. As the product may degrade over the 2–3 years of shelf life, the release target potency may be significantly above the specified end-of-shelf-life specification. This “overformulation” can represent a significant production yield loss and cost-of-goods increase for the final product in order to support the necessary lead times to deliver and store the vaccines, especially if they are used as a rotating stockpile to protect against supply interruption or an emergency use that does not materialize. The addition of stabilizers or lyophilization, when feasible, tends to improve the thermal resistance of vaccines. Storage at very low temperatures within the manufacturing supply chain may be used to reduce potency loss during storage and distribution.

Although recommended storage conditions for many vaccines have been detailed, the vaccine manufacturers are responsible for developing data before and after licensing that demonstrate the stability of their vaccines under recommended storage conditions for the claimed shelf life. Generally, these programs provide data in excess of the claimed shelf life (up to 3 years) to support the development of new products intended for clinical use, as well as routine support of currently marketed products, expiration date extension, and supporting distribution conditions. , Accelerated studies conducted at elevated temperatures are commonly applied to better understand the impact of transient temperature excursions on the vaccine. Manufacturers are required to assure that products under their control are maintained under appropriate conditions so that the identity, strength, quality, and purity of the products are not affected.

Currently, only a limited number of vaccines are required by federal regulation to have specified shipping temperatures. Although most vaccine manufacturers use insulated containers and other precautions for the brief (usually 24–72 hours) shipping time, occasional, unanticipated temperature excursions may occur that could have a detrimental impact on the shipped product. Before accepting any vaccine shipment, users should look for any evidence of improper transportation conditions, including excessive transport time and possible adverse ambient temperature conditions.

EXAMPLES OF VACCINE PRODUCTION

Inactivated Virus (Influenza)

Influenza virus vaccine for intramuscular use is a sterile suspension prepared from influenza viruses propagated in chicken embryos. This vaccine is the primary method for preventing influenza and its more severe complications.

Typically, influenza vaccine contains two strains of influenza A viruses (H1N1 and H3N2) and a single influenza B virus. An additional strain of the influenza B virus was added, with the first four-antigen-containing-vaccine licensed in 2012. The two type A viruses are identified by their subtypes of hemagglutinin and neuraminidase. The hemagglutinin and neuraminidase glycoproteins of influenza A virus comprise the major surface proteins and the principal immunizing antigens of the virus. These proteins are inserted into the viral envelopes as spike-line projections in a ratio of approximately 4:1.

The trivalent and quadrivalent subunit vaccine are the predominant influenza vaccine used today. These vaccines are produced from viral strains that are identified early each year by the World Health Organization, the Centers for Disease Control and Prevention (CDC), and CBER. For U.S.-licensed manufacturers, the viral strains are normally acquired from CBER or CDC. European strains are typically provided by the National Institute for Biological Standards and Control, and Southern Hemisphere strains by the Therapeutic Goods Administration of Australia. These viral strains are used to prepare master and working virus banks at each manufacturer, which virus banks are ultimately used as the inoculums for vaccine production.

The substrate commonly used by producers of influenza vaccine is the 11-day-old embryonated chicken egg. A monovalent virus (suspension) is received from CBER or the CDC. The monovalent virus suspension is passed in eggs. The inoculated eggs are incubated for a specific time and temperature regimen under controlled relative humidity and then harvested. In the European Union, the number of passages from the original sample is limited. The harvested allantoic fluids, which contain the live virus, are tested for infectivity, titer, specificity, and sterility. These fluids are then stored wet frozen at extremely low temperatures to maintain the stability of the monovalent seed virus (MSV). This MSV is also certified by CBER.

Once the MSV is introduced into the egg by automated inoculators, the virus is grown at incubated temperatures, and then the allantoic fluid is harvested and purified by high-speed centrifugation on a sucrose gradient or by chromatography. The purified virus is often split using a detergent before final filtration. The virus is inactivated using formaldehyde before or after the primary purification step, depending on the manufacturer. This is repeated for three or four strains of virus, and the individually tested and released inactivated viral concentrates are combined and diluted to final vaccine strength. Fig. 6.2 outlines the overall process.

Fig. 6.2, Egg-based influenza vaccine manufacturing process flow. CBER, Center for Biologics Evaluation and Research (of the U.S. Food and Drug Administration); QA, quality assurance; QC, quality control.

The inactivated virus vaccine described above is used for most of the flu vaccine produced and sold today. In recent years, the inactivated influenza vaccine produced on mammalian cell culture has been approved in several countries. The process replaces the egg-based virus expansion with a certified cell line; the downstream processes are similar but focused on removing the host cell protein and DNA to below designated thresholds. A recombinant influenza vaccine produced in insect cells infected with a recombinant baculovirus to express the hemagglutinin protein has also been approved in the United States.

Recombinant Protein (Hepatitis B)

In July 1986, a recombinant hepatitis B vaccine was licensed in the United States. This vaccine built on the knowledge that heat-inactivated serum containing hepatitis B virus (HBV) and hepatitis B surface antigen (HBsAg) was not infectious, but was immunogenic and partially protective against subsequent exposure to HBV. HBsAg was the component that conferred protection to HBV on immunization. To produce this vaccine, the gene coding for HBsAg, or “S” gene, was inserted into an expression vector that was capable of directing the synthesis of large quantities of HBsAg in recombinant yeasts (e.g., Saccharomyces cerevisiae , Hansenula Polymorpha ). The HBsAg particles expressed by and purified from the yeast cells have been demonstrated to be equivalent to the HBsAg derived from the plasma of the blood of hepatitis B chronic carriers. , ,

The recombinant S. cerevisiae cells expressing HBsAg are grown in stirred tank fermenters. The medium used in this process is a complex fermentation medium that consists of an extract of yeast, soy peptone, dextrose, amino acids, and mineral salts. In-process testing is conducted on the fermentation product to determine the percentage of host cells with the expression construct. At the end of the fermentation process, the HBsAg is harvested by lysing the yeast cell, extracted from the cell extract by various techniques and then purified 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 sterile filtered, and then coprecipitated with alum (potassium aluminum sulfate) to form bulk vaccine adjuvanted with amorphous aluminum hydroxyphosphate sulfate. The vaccine contains no detectable yeast DNA but may contain not more than 1% yeast protein. , In a second recombinant hepatitis B vaccine, the surface antigen expressed in S. cerevisiae cells is purified by several physiochemical steps and formulated as a suspension of the antigen absorbed on aluminum hydroxide. The procedures used in its manufacturing result in a product that contains no more than 5% yeast protein. No substances of human origin are used in its manufacture. Vaccines against hepatitis B prepared from recombinant yeast cultures are noninfectious and are free of association with human blood and blood products.

Each lot of hepatitis B vaccine is tested for safety, in mice and guinea pigs, and for sterility. QC product testing for purity and identity includes numerous chemical, biochemical, and physical assays on the final product to assure thorough characterization and lot-to-lot consistency. Quantitative immunoassays using monoclonal antibodies can be used to measure the presence of high levels of key epitopes on the yeast-derived HBsAg. A mouse potency assay is also used to measure the immunogenicity of hepatitis B vaccines. The effective dose capable of seroconverting 50% of the mice (ED 50 ) is calculated.

Hepatitis B vaccines are sterile suspensions for intramuscular injection. The vaccine is supplied in four formulations: pediatric, adolescent/high-risk infant, adult, and dialysis.

All formulations contain approximately 0.5 mg of aluminum (provided as amorphous aluminum hydroxyphosphate sulfate) per milliliter of vaccine. Table 6.2 summarizes the QC testing requirements for the release of recombinant hepatitis B vaccine.

TABLE 6.2
Testing Requirements for the Release of Recombinant Hepatitis B Vaccine
Type of Test Stage of Production
Plasmid retention Fermentation production
Purity and identity Bulk-adsorbed product or nonadsorbed bulk product
Sterility Final bulk product
Sterility Final container
General safety Final container
Pyrogen Final container
Purity Final container
Potency Final container

Most vaccines are still released by CBER on a lot-by-lot basis, but for several extensively characterized vaccines, such as hepatitis B and human papillomavirus (HPV) vaccines, which are manufactured using recombinant DNA processes, this requirement has been eliminated. Their manufacturing process includes significant purification, and they are extensively characterized by their analytical methods. In addition, hepatitis B vaccine had to demonstrate a “track record” of continued safety, purity, and potency to qualify for this exemption. , ,

Conjugate Vaccine ( Haemophilus influenzae Type B)

The production of Haemophilus influenzae type b (Hib) conjugate includes the separate production of capsular polysaccharide from Hib and a carrier protein such as tetanus protein from Clostridium tetani (i.e., purified tetanus toxoid), CRM protein from Corynebacterium diphtheriae , or outer membrane protein complex of Neisseria meningitidis .

The capsular polysaccharide is produced in industrial bioreactors using approved seeds of Hib. A crude intermediate is recovered from fermentation supernatant, using a cationic detergent. The resulting material is harvested by continuous-flow centrifugation. The paste is then resuspended in buffer, and the polysaccharide is selectively dissociated from disrupted paste by increasing the ionic strength. The polysaccharide is then further purified by phenol extraction, ultrafiltration, and ethanol precipitation. The final material is precipitated with alcohol, dried under vacuum, and stored at −35°C for further processing.

Tetanus protein is prepared in bioreactors using approved seeds of C. tetani . The crude toxin is recovered from the culture supernatant by continuous-flow centrifugation and diafiltration. Crude toxin is then purified by a combination of fractional ammonium sulfate precipitation and ultrafiltration. The resulting purified toxin is detoxified using formaldehyde, concentrated by ultrafiltration, and stored at between 2°C and 8°C for further processing.

The industrial conjugation process was initially developed using tetanus toxoid by a team headed by J.B. Robbins at the National Institute of Allergy and Infectious Diseases (NIAID), Bethesda, Maryland.

Conjugate preparation is a two-step process that involves: (a) activation of the Hib capsular polysaccharide and (b) 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.

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