Vaccines are regulated by FDA as biological products. The general requirements are listed in national laws and international guidances. For the U.S., national requirements are codified in 21 CFR, the 200 and 600 sections, with additional recommendations available in FDA Points to Consider and Guidance documents (www.fda.gov). International guidances are available from the International Conference on Harmonization (ICH) (www.ich.org; see Appendix 2) and the World Health Organization. New methodologies are continually being developed and validated and will be included in USP as they become available. Reference standards are available from USP and FDA.

Manufacturers should have a general layout of manufacturing facilities, including diagrams that show the following: flow of raw materials and process inputs; movement of product, intermediates, waste streams, and personnel; and air flows and pressurization levels. These diagrams assist in minimizing the risk of potential product contamination from various sources. These sources can include cross-contamination from other products, contamination from different batches of the same product, and extraneous contamination from microorganisms and personnel. Evaluation of the flow diagrams can assist with strategies for development of engineering controls, personnel procedures, and monitoring systems to enable compliance with Good Manufacturing Practices (GMPs). Analysis of potential risks may also provide insights about what information should be recorded in batch documentation to facilitate consistent manufacture and also to facilitate failure investigations. Together, physical facilities, procedures, personnel, training, and quality systems make up the GMP environment in which a vaccine will be produced.

The manufacturing process includes process inputs such as raw materials and processing aids and unit operations comprising both the initial and downstream processing steps. A process flow map for the manufacturing process is useful and assists in validation of the manufacturing process. This map shows all unit operations, the inputs to each operation, and the outputs to subsequent manufacturing steps. Analytical testing done at relevant steps and the specifications required to proceed to the next stage of processing may be added to the map. A process map also supports a processing space to facilitate a rugged process, i.e., one based on suitable characterization studies to establish boundaries within which manufacturing can occur to promote unchanged safety and efficacy outcomes.

The process flow map should include all steps from making the seed/cell bank (described below) to formulation and filling of the final product. The validation strategy should include the steps that require validation, along with identification of the process space, associated critical process parameters (CPP), and critical quality attributes (CQA). The critical process parameters are those that directly affect core quality attributes needed to successfully manufacture a batch of product. Some manufacturers identify other processing parameters that are important for processing but do not affect critical quality attributes. These important but noncritical factors help identify the process development space, can contribute to the development of a rugged process, or can be useful when the company assesses processing deviations. The concepts of quality by design and exploration of the process space are relatively new to the biologics/vaccine industry but are becoming considerations for the overall development-planning process.

Manufacturing surveillance is the continual observation of how the process and the resulting product are performing. This section is not exhaustive; rather, the points raised here outline the types of considerations recommended for a manufacturer during development of a vaccine. Manufacturing surveillance includes the following:

Taken as a whole, these activities allow a manufacturer to assess the state of the process and product and to evaluate which, if any, operations need to be modified. These same systems also provide a surveillance matrix to evaluate changes. In any of these programs it is also valuable to develop additional characterization assays that are not used for process intermediate or product release purposes but may be used for further evaluation when additional information is needed or desired. These additional assays for characterization are often based on different underlying analytical procedures to provide different ways to evaluate materials.

Routine surveillance processes are increasingly implemented to attempt to detect changes in processes before any critical quality attributes are adversely affected. Not all vaccine processes can be characterized to the same extent or level (e.g., a live virus vaccine vs. a recombinant protein vaccine), and statistical tools are often used to determine alert or action levels in surveillance programs. Exceeding these levels requires the manufacturer to evaluate the situation but does not necessarily signal product failure.

GMP manufacturing entails facility design, process development, quality systems, and manufacturing surveillance. Together these systems help the manufacturer to control the production of a vaccine. As noted, many types of vaccine are marketed, and each has its unique features and therefore requires different plans for each of the steps mentioned in this section.

In the bacterial seed lot system, a master seed is subcultured to produce a working seed one passage beyond the master seed. An aliquot of the working seed is then expanded to produce a vaccine lot. The strain(s) used for the master seed lots are identified by historical records that include information about their origin. Information about the bacterial seed lot system should include source, passage history, and raw materials to which it was exposed, with specific emphasis on raw materials of ruminant origin. Seeds should be stored at an appropriate temperature in more than one location within a facility or at a distant site in order to decrease catastrophic risk.

Identity tests may include inoculation onto suitable biochemical media, Gram stains, genotype, and serological identification with suitable specific antisera. Special tests may be added, for example, to show culture viability but also lack of virulence.

Purity of the bacterial strains used for seed lots is verified by methods of suitable sensitivity to ensure that no adventitious agents are present. These purity tests often are performed in the presence of the seed under conditions where growth is inhibited by the presence or the absence of specific nutrients. Streaking can also be used to show that the cultured seed is a pure culture.

The derivation and passage history of viral seeds should be recorded in detail. Any manipulation of the viral phenotype (e.g., cold adaptation, development of temperature sensitivity, or attenuation of virulence) or intentional genetic manipulations (e.g., reassortment or recombination) should be documented.

These viral seeds are commonly differentiated into a master viral seed and working viral seeds or working viral stock. Viral seeds should be stored at cryogenic temperatures to promote stability and in more than one location within a facility or at a distant site to decrease catastrophic risk. Manufacturers should assess the following characteristics of the viral seed stock:

The master viral seed should be extensively characterized to demonstrate the stability of genotype and phenotype for a number of passages beyond the level used in production. Generally, during assessment of genetic stability, a master seed undergoes a minimum of five passes beyond the passage that will produce the final vaccine.

Tests should be performed for identity (e.g., sequencing the entire virus or a portion of it), adventitious agents, viral phenotype, genetic stability, and, if applicable, agents that might be present in the seed as a result of its passage history. Viral phenotype can be assessed further for tissue tropism, attenuation properties, and temperature sensitivity. Not all of these tests may be necessary for every viral seed strain.

In some cases the viral seeds may have a broad host range and therefore may require neutralization of the vaccine virus before they are tested for adventitious agent(s). If possible, testing for adventitious agents should be done without neutralization in order to avoid an antiserum that may inadvertently neutralize an adventitious agent present in the seed. Sometimes it is not possible to effectively neutralize a viral seed, and in such cases alternative strategies can be used. For example, the test can be performed in a cell substrate that does not permit replication by the vaccine virus. However, such a substitution of the substrate cell may compromise the test's sensitivity for detection of other adventitious agents. Therefore, the tests may be supplemented with use of polymerase chain reaction (PCR) assays.

Assessment of neurovirulence may be appropriate if the virus is known to be neurotropic. Manufacturers should consult with regulators about appropriate animal models, methods, and scoring systems for this assessment before they initiate such studies. For viruses that are neurovirulent or may revert to neurovirulence (e.g., polioviruses), it may be necessary to assess neurovirulence beyond the master seed.

If the master viral seed is well characterized, the working viral seed may not require extensive characterization. For example, it may not be necessary to repeat testing for all the relevant viruses from the derivation history.

For a vaccine produced via a biotechnology-engineered cell-expression system, a master seed lot or a master cell bank will be established during product development. The seed lot or cell banks should be homogenous, which is often accomplished by limiting dilutions. The seed lot or cell bank system should be characterized in a manner analogous to that used for the cell substrate discussed in the next section, and additional tests can be used to demonstrate the genetic stability of the expression system.

The nutrients consist of materials like proteins, sugars, inorganic trace elements, amino acids, and vitamins needed for bacterial growth. The protein component may be as simple as free casein (milk protein), or it can be as complex as extracts from bacterial, plant, or animal sources. Any fermentation nutrients of animal origin are sourced carefully and tested for adventitious agents. The composition of a medium is often customized to optimize product quality attributes. Medium components that are known to cause allergic reactions should be avoided.

The types and composition of media used for isolation and all subsequent culture of components of viral vaccines need to be recorded in detail. Chemically defined media without materials of animal origin are preferred. The medium should be tested for sterility and suitability for the cells used in product production. If materials of animal origin are used, they are assessed for freedom from adventitious agents. If human albumin is used in a U.S.-licensed vaccine, it must be licensed by FDA. The final product should be within specified limits of residual medium components such as serum, antibiotics, selection agents or reagents added for growth enhancement.

The requirement for media used for the fermentation and propagation of biotechnology-engineered cells is the same as that noted above for bacterial fermentation and cell culture growth.

Propagation of bacteria for bacterial vaccines is performed under specified conditions for the inoculum preparation and the fermentation phases. In-process monitoring and testing should be conducted for quality assurance. All controls and testing performed after production (e.g., purity, viability, antigen yield, and phenotypic identity) should be documented. The first step of drug-substance recovery is harvesting from the bioreactor. A variety of equipment is available, and the process equipment used depends on the nature of the process. Procedures should be established to ensure containment and prevention of contamination during harvesting and to monitor bioburden (including acceptance criteria) or sterility. The storage conditions and the stability time limit for the harvest material should be described. For most bacterial vaccines, an inactivation step is necessary. Personnel involved in bacterial inactivation should consider the following: how cell culture purity is verified after inactivation, whether culture purity should be defined before inactivation, choice of the inactivation agent, and validation of the procedure(s).

The manufacturing of viral vaccines using eukaryotic cell culture includes a two-phase production process. The first is the expansion of the cell cultures used as a substrate for viral replication. The second phase includes the initial virus infection and subsequent replication and virus production.

Bacterial fermentations are typically highly productive and yield large amounts of biomass. For bacterial subunit products or recombinant components expressed by bacteria, fermentation can produce very high concentrations of the desired active ingredient. Manufacturers should initiate culture purity testing before further processing.

Of special concern in the purification of recombinant-derived vaccine components is the issue of residual host cell components that could produce an adverse immunogenic response in patients. This response could be exacerbated by the presence of vaccine adjuvants.

The embryonated chicken egg is a highly productive growth substrate for certain viruses, such as those used to make vaccines for yellow fever and several influenza vaccines. In the case of influenza vaccines, vaccine virus is harvested from egg allantoic fluid. In the case of yellow fever vaccine, the vaccine virus is harvested from embryo tissues. Therefore, residual egg or embryo components are special considerations in vaccine purification.

Egg-based vaccine production, like all biomass expansions, requires care and quality control of the virus seed lots and egg substrates to avoid contamination with other organisms.

Intermediates are manufactured from starting materials by one or a combination of different processes (e.g., fermentation, cultivation, isolation, or synthesis). Subsequent steps of the procedure involve preparation, characterization, and purification, eventually resulting in the drug substance. Quality systems documents are adopted for production and all applicable information should be recorded in a controlled document (i.e., a batch record). When applicable, stability studies and release tests should be performed before proceeding to the next steps (see below).

The quality attributes of the intermediate are commonly tested in conjunction with further processing. Characterization beyond release testing should be considered. Characterization methods can use appropriately qualified procedures. Some tests are routinely performed before the intermediates are converted to the final bulk, depending on individual vaccines.

If intermediates need to be stored and/or subsequently shipped to a different location for further processing, the stability of these materials should be demonstrated. Stability tests can be a combination of both physicochemical analysis and biological assays.

Appropriately controlled amounts of all ingredients are blended to uniformity to produce the final bulk. The processing may include one or more steps such as buffer exchange and addition of diluents, bulking agent, stabilizing excipients, adjuvants, and preservatives. Final bulk may be prepared aseptically or processing may include a sterilization step.

The quality attributes of the final bulk should be tested. Appropriate testing should be performed with respect to identity, purity, potency, sterility (see Sterility Tests 71), and antimicrobial effectiveness (see Antimicrobial Effectiveness Testing 51). Tests demonstrating safety, if applicable, are performed. The list includes, for example, tests for the absence of adventitious agents, mycoplasma, and other microorganisms.

Testing is required for specific process-related and product-related impurities, depending on the vaccines being manufactured. In addition, tests are required for the bulking agent, stabilizing excipients, adjuvants, and/or preservatives, if used. All the testing should be done according to respective standard operating procedures (SOPs), and all tests should have specifications (or provisional specifications, where applicable).

If final bulks are stored and/or subsequently shipped to a different location for further processing, the stability of these materials should be demonstrated. Stability tests can be a combination of both physicochemical analysis and biological assays. Implementation of a stability program is required for formal stability studies, and the studies should be executed according to a protocol that contains detailed information about types of tests, including specifications, testing intervals, and data and analysis.

In addition to specific antigens, vaccines often include excipients and other additives that are intentionally added to the vaccine by the manufacturer for a specific purpose. These include adjuvants, antimicrobial preservatives, and stabilizers. Vaccines also contain manufacturing residuals, which are trace amounts of various components used during manufacturing. Thus, the combinations of these components comprise and define the complete vaccine product. Manufacturers must adhere to regulations governing permissible limits of such components, as indicated in the product's license.

Aluminum compounds have long been the most widely used adjuvants worldwide. Two methods traditionally have been used for combining aluminum adjuvant to antigen to form aluminum-adsorbed vaccines. The first involves the addition of the antigen solution to preformed aluminum precipitate. The second involves the addition of an antigen to aluminum in solution and the addition of a compound that will coprecipitate the aluminum salt and the antigen in situ. Solutions of aluminum potassium sulfate, known as alum or aluminum chloride, have been used together with phosphate salts as precipitating agents. A number of aluminum adjuvant formulations are used in vaccines.

Tests for aluminum are based on metal detection tests described in the general test chapter Aluminum 206. Regulations limit the amount of aluminum permitted in a dose of vaccine. The Code of Federal Regulations [21 CFR 610.15(a), Ingredients, preservatives, diluents, adjuvants] states that “the amount of aluminum in the recommended individual dose of a biological product shall not exceed:

The third criterion above aligns U.S. regulations with World Health Organization guidance for aluminum content in a single human dose of a vaccine product.

Note that adjuvants are not licensed by themselves; they do not constitute a product. Rather, a vaccine consisting of specific antigen(s) and an adjuvant are licensed together as a drug product.

The microbial preservatives currently used in vaccines are thimerosal, 2-phenoxyethanol, benzethonium chloride, and phenol. These agents must pass the appropriate antimicrobial effectiveness test, as described in Antimicrobial Effectiveness Testing 51. Antimicrobial test challenges should be conducted as part of the normal formal stability program, including at expiration date. Various tests for preservatives can be found in Antimicrobial Agents—Content 341.

When a protein is chosen as a stabilizer, two main safety concerns arise. One stems from the source of the protein: animal or human origin raises the possibility of the presence of an adventitious agent. The second concern is the possibility of an allergic reaction in persons sensitized to that protein. This should be evaluated as part of the clinical program during vaccine development. At present two proteins are used as stabilizers for vaccines: HSA and gelatin. FDA requires that any serum-derived albumin used in manufacturing be U.S.-licensed HSA. FDA guidance further recommends that a statement indicating the source and related risks appear in the “Warnings” section of the labeling for HSA-containing products.

Gelatin or processed gelatin also is used as a vaccine stabilizer. The gelatin source may be either bovine or porcine. Although the conditions of manufacturing gelatin are harsh (i.e., the product is subjected to extremes of heat and pH), there remains a concern with bovine sources about the presence of the transmissible spongiform encephalopathy (TSE) agent, because this agent is known to resist such conditions. Therefore, if gelatin added to a vaccine or used in manufacturing is from a bovine source, the material should have the appropriate documentation certifying that it comes from a country or region that is in compliance with TSE guidance for industry.

Viral vaccine manufacturing requires cell substrates to produce the viruses, which are then taken through purification processes. Generally, killed viral vaccines are more highly purified than are live attenuated ones. Depending on the method used to manufacture the vaccine, manufacturers work with FDA to develop prudent specifications for the final vaccine. Animal-derived host cells have been used extensively in vaccine manufacturing, particularly viral vaccines. For example, influenza and yellow fever vaccines are produced, respectively, in egg allantoic fluid and chicken embryos. Mumps, measles, and some rabies vaccines are produced in chick embryo cells. The labels of these products must state that residual chicken proteins may be present in the final vaccine, and the label may indicate how much is present. Further, the label also urges practitioner caution when vaccinating a person with known hypersensitivity to eggs.

Two U.S.-licensed hepatitis B vaccines are based on recombinant DNA-derived proteins expressed in yeast cultures. In both cases, the labels notify health care professionals that yeast protein may be present in the vaccine and recommend that suitable precautions should be exercised. In the case of live viral vaccines, considerations may be given to the reduction of cellular residual materials (e.g., host DNA, proteins).

The overall experimental plan for evaluating the stability profile of a given set of product or intermediate batches typically includes specific definition of the conditions under which the samples will be stored and why these conditions are relevant, the length of time the samples will be stored at each condition, when samples will be tested during this time course, and the analytical measurements at each time point. Additionally, these stability protocols include itemization of the analytical procedures to be used. For stability studies that occur early in product development, the studies may be conducted to confirm the suitability of the product formulation and/or storage conditions. Later in development, stability studies are typically conducted to provide data supporting product dating period or intermediate hold time, to provide more elaborate product characterization, and to evaluate manufacturing consistency. These latter studies define product end–expiry specifications that allow definitions of acceptable and unacceptable product. Unacceptable product is defined as product that is no longer acceptable for use in clinical studies or for commercial use (e.g., because of degradation or loss of potency). Stability studies should be conducted over a duration sufficient to determine the point of loss of acceptable potency or other relevant parameters.

Manufacturers should consider the rigor of the analytical method(s) used to evaluate the stability of complex products and improve their understanding of the parameters that are critical to immunogenicity (including stability-indicator parameters). Selection of the stability-indicator parameters varies with each vaccine's unique characteristics.

The primary parameter that reflects stability for most vaccines is the potency assay (see Potency Tests in Lot Release Testing, below). This assay can take many forms, depending on individual vaccines (e.g., an infectivity assay for a live virus vaccine or a measure of the proportion of conjugated polysaccharide for a polysaccharide–protein conjugate vaccine). The potency assay is generally the key analytical result predicting whether a vaccine remains suitable for use and whether it will produce the expected clinical response. Other analytical measurements can provide important supplemental data, particularly those that have a clear link to the potency of the product. Examples include degradation profile, dissociation of a carrier protein from conjugated vaccines, and dissociation of an adjuvant from an antigen complex. Additionally, other common assays typically are performed as part of the stability study and may address physical or chemical changes in the product that may or may not affect its potency (e.g., general safety, degree of aggregation, pH, moisture, container, preservative, and enclosure).

Vaccines must remain within potency specifications at the expiration date, provided that the product was stored under the normal conditions specified. Manufacturers should conduct stability studies to determine those storage conditions and that dating period to demonstrate that the product remains within the potency specifications. Manufacturers should conduct stability studies on a continuing basis. If a major manufacturing process changes, additional stability studies should be conducted to verify that there is no adverse impact on the stability profile. Under certain conditions such as process changes, accelerated stability studies could be conducted. An accelerated study involving temperatures both higher and lower than routine can evaluate the impact of temperature excursions on products. A similar evaluation should be done for product intermediates to establish how long a given intermediate can be held under defined conditions before it is processed further or discarded.

Provisions are made for the following labels:

The label should be affixed to the container in a manner that allows visual inspection of the contents for the full length or circumference of the container. If no package exists, the container bears all of the information required for the package label.

The full container label normally contains the following:

In addition to the information required on the container label, the package label should describe the following:

Detailed information about a vaccine appears in its prescribing information, commonly called the package insert. Increasingly, vaccines are distributed with patient package inserts written in lay language. Prescribing information (21 CFR 201.56 and 201.57) includes the following:

Manufacturers perform all appropriate tests for the licensed specifications for the product, according to 21 CFR 610.1 and 610.2. Samples of each licensed lot and protocols containing the manufacturers' test results are submitted to FDA. After FDA evaluates the protocol to ensure that the product specifications are met, and after satisfactory confirmatory testing, FDA approves the release of the lot if all tests meet the standards of safety, purity, and potency established for the particular vaccine product. After approval is granted, the manufacturer distributes and markets the product.

Guidelines are available regarding alternatives to lot release and a surveillance system. All of these variations are subject to the regulations in 21 CFR 610.2 that allow FDA to require that samples of any lot of licensed product (e.g., vaccine), together with the protocols showing results of applicable tests, be sent to FDA.

The tests common to all lots of all products include tests for potency, general safety, sterility, purity, identity, and constituent materials. The manufacturer completes these tests for conformity with standards applicable to each product. The results of all tests are considered, except when a test has been invalidated as a result of causes unrelated to the product (21 CFR 610.1).

Assay precision and reproducibility vary with the different methodologies that are used in potency assays, ranging from the high accuracy and precision of chemical tests at one end of the spectrum to bioassays at the other end. The general test chapter Design and Analysis of Biological Assays 111 provides guidance for bioassays and applies to vaccine potency assays. Other tests should be validated as described in the general information chapter Validation of Compendial Procedures 1225.

Formulations that combine several vaccines must be licensed as combinations (21 CFR 610.17). The potency of each vaccine in the combination is individually tested and must meet the specifications in the context of the final combined product; other appropriate quality tests apply as well. For vaccines that are physically combined in clinical locations just before administration to a patient, prescribing information should describe specific procedures to follow in those settings.

In general, quality control systems for vaccine manufacture are identical to those routinely employed for production of other pharmaceuticals. These include raw material testing and release, manufacturing, process-control documentation, and aseptic processing. Manufacturers formally assign responsibility to designated staff for maintaining the continued safety, purity, and potency of the product and for ensuring compliance with applicable product and establishment standards, along with compliance with current GMPs. Analysts use reference standards and validated methods to determine active ingredients, residuals, and impurities. Manufacturers determine product safety in a variety of ways that may include the use of experimental animals, procedures to demonstrate product sterility, and tests to ensure product potency. The complexity of the quality control systems for vaccines lies in the variety of methods used to produce and control production. Lot release testing proceeds according to 21 CFR 610.2 and involves evaluating lots for safety, purity, and potency before release. Manufacturers follow FDA and applicable international standards for testing and validation. The basic considerations for validation are included in Validation of Compendial Procedures 1225, in addition to guidance documents issued by FDA and the International Conference on Harmonization (ICH) (see Appendix 2).

Modification of test methods or manufacturing processes as licensed may be permitted if the regulatory authority can be assured that the modifications cause no reduction in safety, purity, potency, and effectiveness of the biological product. It may be necessary for the manufacturer to file the proposed changes prior to implementation (21 CFR 601.12 and 21 CFR 610.9).