Vaccine Additives and Manufacturing Residuals in Vaccines Licensed in the United States

Preservatives

Preservatives are added to vaccine formulations to prevent the growth of bacteria or fungi that may inadvertently be introduced into the vaccine during use. In some cases, preservatives are used during the manufacturing process (e.g., in buffers and column washes) to prevent microbial growth. Improvements in manufacturing technology, however, have decreased this need for the addition of preservatives to control bioburden during the manufacturing process. The Code of Federal Regulations (CFR) requires that, with certain defined exceptions, or with the approval of the Center Director (discussed later), preservatives must be added to multidose vials of vaccines. In the past, tragic consequences followed the use of multidose vials that did not contain a preservative, and served, in part, as the impetus for this requirement (see Wilson for a discussion of incidents related to the lack of preservatives in vaccines). Specifically, 21 CFR §610.15(a) states; “Products in multiple-dose containers shall contain a preservative, except that a preservative need not be added to Yellow Fever Vaccine; Polio-virus Vaccine Live Oral; viral vaccines labeled for use with the jet injector; dried vaccines when the accompanying diluent contains a preservative; or to an Allergenic Product in 50% or more volume in volume (v/v) glycerin.”

Although the regulation does not specify a quantity, it does require that the preservative used “shall be sufficiently nontoxic so that the amount present in the recommended dose of the product will not be toxic to the recipient, and in the combination used it shall not denature the specific substances in the product to result in a decrease below the minimum acceptable potency within the dating period when stored at the recommended temperature.”

The CFR does not, however, provide a definition of a preservative. The definition (i.e., antimicrobial effectiveness) that has been used by the FDA for vaccines and other biologicals is found in the U.S. Pharmacopoeia (USP). This is a functional definition, wherein the final formulation of the vaccine, including the preservative, is challenged with specified quantities of the following organisms: Candida albicans , Aspergillus brasiliensis , Escherichia coli , Staphylococcus aureus , and Pseudomonas aeruginosa. The test sample (preservative-containing vaccine plus the microorganism) is incubated at 20–25°C, and the number of viable microorganisms is determined on days 7, 14, and 28. A preservative is deemed acceptable if the following are achieved:

• Bacteria: a reduction of not less than 1.0 log ₁₀ from the initial count at 7 days, and not less than a 3.0 log ₁₀ reduction from the initial count after 14 days, and no increase in the 14-day count at 28 days.

• Yeasts and molds: remain at or below the level of the initial inoculum on days 7, 14, and 28.

Note that the antimicrobial agent is not tested by itself; rather, it is the final vaccine formulation that is tested.

Preservatives cannot completely eliminate the risk of bacterial or fungal contamination of vaccines; moreover, they do not address any potential viral contamination. Although it occurs rarely, and not in the recent past, the scientific literature does contain reports ^, (see also Wilson ) of bacterial contamination of vaccines despite the presence of a preservative, emphasizing the need for meticulous attention to technique when withdrawing vaccines from multiuse vials. At present, the following preservatives are used in U.S.-licensed vaccines: phenol, benzethonium chloride plus formaldehyde, 2-phenoxyethanol, and thimerosal (termed thiomersal in some other countries). In 2011, the FDA amended the biologics regulations to permit exceptions or alternatives to the regulation for constituent materials (21 CFR §610.15), which includes preservatives and adjuvants. The following section was added to the regulation: “(d) The Director of the Center for Biologics Evaluation and Research (CBER) or the Director of the Center for Drug Evaluation and Research (CDER) may approve an exception or alternative to any requirement in this Section. Requests for such exceptions must be in writing.”

The amended regulation could, as an example, allow the use of particular vial adaptors to prevent contamination of products in multidose vials without the use of preservatives. As noted in the final rule, the Director of CBER or the Director of CDER “would not approve an exception or alternative when the data or conditions of use, including the indication and patient population, do not provide a sufficient scientific and regulatory basis for such an approval.” Of note, the three COVID-19 vaccines by Pfizer-BioNTech, Moderna, and Janssen that were granted Emergency Use Authorization by the FDA in late 2020 and early 2021, are available in multidose vials without preservatives. Labeling for each COVID-19 vaccine supplied in multidose vials includes detailed instructions for storage and in-use time after first puncture in their labeling.

As noted, a preservative “shall not denature the specific substances in the product to result in a decrease below the minimum acceptable potency within the dating period when stored at the recommended temperature.” Certain preservatives are not compatible with certain antigens; compatibility must be established. For example, it has been known for a number of years that thimerosal has a deleterious effect on the potency of inactivated poliovirus vaccine (IPV). ^, An alternative preservative is necessary for IPV. A preservative that is used in other products, 2-phenoxyethanol, has been found to be compatible with IPV vaccine formulations; it is used as a preservative in both of the currently U.S.-licensed IPV vaccines (IPOL [Sanofi Pasteur SA] and Poliovax [Sanofi Pasteur Ltd.; not currently marketed in the United States]).

Phenol is currently used in three U.S.-licensed vaccines: the polysaccharide vaccines Pneumovax 23 (a 23-valent pneumococcal polysaccharide vaccine manufactured by Merck Sharp & Dohme Corp.) and Typhim Vi ( Salmonella typhi capsular polysaccharide vaccine manufactured by Sanofi Pasteur SA), and ACAM2000 (the smallpox vaccine manufactured by Emergent Product Development Gaithersburg, Inc.); each of these vaccines contains 0.25% phenol as a preservative (phenol is contained in the diluent for ACAM2000). According to the Minimum Requirements of the National Institutes of Health (NIH), ^, phenolic compounds (such as phenol or the various creosols) are not permitted as preservatives in diphtheria- and tetanus toxoid–containing products. *

* At the time of the writing of these requirements for diphtheria and tetanus toxoids, U.S. vaccine regulation was the responsibility of the NIH.

This requirement is also reflected in other regulations or requirements, such as those of the World Health Organization (WHO). It has been reported that phenol affects diphtheria toxoid, “so that its immunizing power falls rapidly.” Benzethonium chloride with formaldehyde is currently used in only one U.S.-licensed vaccine, anthrax vaccine adsorbed (BioThrax; the preservative is 25 µg/mL benzethonium chloride and 100 µg/mL formaldehyde), manufactured by Emergent BioSolutions.

In recent years, considerable controversy has surrounded the use of thimerosal, an organomercurial, in vaccines. Although allergic responses to thimerosal have been described, a controversy, arising in the late 1990s, centered on the hypothesis that exposure to thimerosal, a derivative of ethyl mercury, may be causally linked to autism and other neurodevelopmental disorders in children. Although there were no clear or definitive data to support a link between thimerosal and neurodevelopmental disorders, the U.S. Public Health Service (PHS), first in July 1999 and again in June 2000, in an effort to reduce mercury exposure in children from all sources, recommended that thimerosal be removed from pediatric vaccines as expeditiously as possible. The July 1999 PHS statement was issued jointly with the American Academy of Pediatrics; the June 2000 PHS statement was issued jointly with the American Academy of Pediatricians, the American Academy of Family Physicians, and the Advisory Committee on Immunization Practices (ACIP). Letters from CBER of the FDA, in 1999 and again in 2000, to the various vaccine manufacturers noted that the removal of thimerosal from vaccines was merited and requested manufacturers’ timelines for thimerosal removal or submission of an explanation as to why thimerosal removal was not currently feasible.

In 2004, the Institute of Medicine (IOM)’s Immunization Safety Review Committee of the National Academy of Science’s IOM issued its final report, examining the hypothesis that, inter alia, thimerosal-containing vaccines are causally associated with autism. The committee concluded that the body of evidence favors rejection of a causal relationship between thimerosal-containing vaccines and autism, and that the hypotheses that were generated concerning a biological mechanism for such causality were theoretical only. The European Medicines Agency also noted, as a precautionary measure, “that, although there is no evidence of harm caused by the level of exposure from vaccines, it would be prudent to promote the general use of vaccines without thiomersal and other mercury-containing preservatives.” Of note, the WHO continues to recommend the use of vaccines containing thimerosal because the need for multidose preservative-containing vaccines and, thus, the benefit of using such vaccines outweighs the theoretical risk of toxicity. Additionally, the Global Advisory Committee on Vaccine Safety has stated that it remains of the view that there is no evidence supporting a causal association between neurobehavioral disorders and thimerosal-containing vaccines. A more comprehensive update on the thimerosal-autism hypothesis for vaccines may be found in Chapter 82.

At present, with the exception of inactivated influenza vaccines presented in a multidose presentation, all of the U.S.-licensed, routinely recommended pediatric vaccines (hepatitis B, diphtheria–tetanus toxoid–acellular pertussis [DTaP], Haemophilus influenzae type b, IPV, meningococcal conjugate, pneumococcal conjugate, human papillomavirus [HPV], hepatitis A, rotavirus, measles-mumps-rubella [MMR] and varicella) are thimerosal free or contain only trace amounts (<1 µg of mercury per dose) as a residual from the manufacturing process ( Table 8.1 ). Of the seven U.S.-licensed quadrivalent injectable influenza vaccines, five are approved for pediatric use: Fluzone Quadrivalent (Sanofi Pasteur Inc.), for use in infants 6 months of age and older; Afluria Quadrivalent (Seqirus Pty Ltd.), for use in infants 6 months of age and older; Fluarix Quadrivalent (GlaxoSmithKline Biologicals), for use infants 6 months of age and older; FluLaval Quadrivalent (ID Biomedical Corporation of Quebec), for use in infants 6 months of age and older; and Flucelvax Quadrivalent (Seqirus, Inc.), for use in children 2 years of age and older. These five inactivated influenza vaccines are available in thimerosal-free presentations (Fluzone Quadrivalent, Fluarix Quadrivalent, Afluria Quadrivalent, FluLaval Quadrivalent, and Flucelvax Quadrivalent). FluMist Quadrivalent (MedImmune, LLC) for use in children 2 years of age and older is a live, attenuated quadrivalent influenza vaccine and does not contain thimerosal.

Two Tdap (tetanus, diphtheria, and pertussis for adolescents and adults) vaccines (tetanus, diphtheria, and acellular pertussis vaccines, with the lowercase letters indicating a reduced antigen content for the diphtheria toxoid and one or more of the pertussis antigens) for use in adolescents and adults, Adacel (Sanofi Pasteur Ltd.) for use in persons 10–64 years of age, and Boostrix (GlaxoSmithKline Biologicals) for use in persons 10 years of age and older, are licensed in the United States. Neither product contains thimerosal. The quadrivalent (Groups A, C, Y, and W135) meningococcal conjugate vaccines, Menactra (Sanofi Pasteur Inc.), Menveo (GlaxoSmithKline Biologicals) and MenQuadfi (Sanofi Pasteur Inc.) do not contain any preservative. Similarly, the two meningococcal serogroup B vaccines BEXSERO (Novartis Vaccines and Diagnostics S.r.l.) and TRUMENBA (Wyeth Pharmaceuticals Inc.) and the HPV vaccine Gardasil 9 (Merck Sharp & Dohme Corp.) do not contain any preservative.

A diphtheria and tetanus toxoid vaccine (DT) for use in children younger than 7 years of age is available from Sanofi Pasteur Inc. It contains only a trace amount of thimerosal as a manufacturing residual.

Adjuvants

Adjuvants are materials that enhance and direct the immune response (see Chapter 6). Vaccine adjuvants are not licensed separately; rather, the adjuvant is a constituent of the licensed vaccine, and it is the vaccine formulation, in toto, that is tested in clinical trials and is licensed. An adjuvant cannot be added or removed, or its amount in a licensed vaccine changed, without submitting a supplement to the vaccine license and obtaining approval from the FDA. The various aluminum salts (aluminum hydroxide, aluminum phosphate, alum [potassium aluminum sulfate], or mixed aluminum salts) are the most commonly used adjuvants in U.S.-licensed vaccines. One vaccine, Cervarix (GlaxoSmithKline Biologicals), which contains AS04, an adjuvant system composed of an aluminum salt and monophosphoryl lipid A, a detoxified lipopolysaccharide (LPS), has been licensed. Of note, as of 2020, the manufacturer no longer markets Cervarix in the United States. In 2013, as part of the U.S. government’s national pandemic preparedness initiative, an H5N1 monovalent influenza vaccine, approved for use in persons 18 years of age and older, containing the unique adjuvant, AS03, manufactured by ID Biomedical Corporation of Quebec was licensed. The AS03 adjuvant is an emulsion composed of squalene, dl -α-tocopherol, and polysorbate 80. In 2015, an inactivated influenza vaccine, Fluad (Seqirus Vaccines Ltd.), which contains the squalene-based adjuvant MF59C.1, was licensed. Audenz (Seqirus Vaccines Ltd.), an H5N1 vaccine approved in 2020 also contains MF59C.1. In 2017, Shingrix and Heplisav-B were licensed with novel adjuvants. Shingrix (GlaxoSmithKline Biologicals) contains the adjuvant AS01 _B , which is composed of 3- O -desacyl-4’-monophosphoryl lipid A (MPL) from Salmonella minnesota and QS-21, a saponin purified from plant extract Quillaja saponaria Molina, combined in a liposomal formulation. Heplisav-B (Dynavax Technologies Corp) contains the CpG 1018 adjuvant, a 22-mer phosphorothioate linked oligodeoxynucleotide in a phosphate buffered saline.

Despite worldwide use of aluminum salts for more than 50 years, surprisingly little has been known about their mechanism of action as adjuvants (see, e.g., Chapter 6 and HogenEsch ). For many years, the prevailing thought was that the aluminum salts functioned as depots for the vaccine immunogens. More recently, it was shown that the aluminum salts also activate inflammasomes, clusters of proteins found inside certain cells. Inflammasomes respond to stresses such as infection or injury by releasing cytokines, which, in turn, stimulate an immune response.

Table 8.1 presents the specific aluminum salt (hydroxide, phosphate, sulfate, or mixed) and the quantity of aluminum that is contained in a number of commonly used vaccines. (The aluminum content that is listed for some vaccines noted in Table 8.1 represents the upper limit of the specification; the vaccine may routinely contain less aluminum.) By regulation (21 CFR §610.15[a]), the aluminum content of a vaccine cannot exceed 0.85 mg of aluminum per dose if the amount is assayed, or 1.14 mg/dose if determined by calculation based on the amount of the aluminum compound that is added. To harmonize with WHO recommendations, this regulation was amended in 1981 to permit up to 1.25 mg of aluminum per dose. However, the higher amount was permitted only “provided that data demonstrating that the amount of aluminum used is safe and necessary to produce the intended effect are submitted to and approved by the Director, Center for Biologics Evaluation and Research” (21 CFR §610.15[a]). The European Pharmacopoeia likewise restricts the aluminum content to 1.25 mg/dose. It should be noted that the above regulation for aluminum content refers to an individual dose of a biological product; thus, for example, for a hypothetical combination vaccine derived from licensed components, the aluminum content for the combination vaccine may still not exceed 0.85 mg.

Recently, the FDA amended the CFR requirements for constituent materials including adjuvants to permit, when justified, exceptions or alternatives such as the use of an increased aluminum content in a vaccine; this change may have a greater impact on certain therapeutic vaccines than on the preventive vaccines.

Concerns have been raised in recent years about the use of aluminum in vaccines and potential adverse outcomes that may be associated with its use at the levels that exist in individual vaccines and through the additive effects of multiple vaccinations. These concerns about the use of aluminum in vaccines prompted a workshop that was sponsored by the National Vaccine Program Office in May 2000. The general use of aluminum salts in vaccines and aluminum toxicokinetics were reviewed during the workshop. In their overall summary of the workshop, Eickhoff and Meyers noted that “based on 70 years of experience, the use of salts of aluminum as adjuvants in vaccines has proven safe and effective.” A more recent study by FDA scientists, using updated parameters for toxicokinetic assessments, including current recommended vaccines and aluminum excretion data, concluded that the risk from aluminum exposure from vaccines and the environment to infants was extremely low.

Stabilizers

Various stabilizers are added to vaccines to help protect them from adverse conditions such as the freeze-drying process (for those vaccines that are freeze-dried) or heat. For freeze-dried (lyophilized) preparations of vaccines, it is also necessary to add materials that provide a bulk matrix for the vaccine. The amount of an immunogen that is contained in a vaccine can be extremely small, on the order of tens of micrograms or less. If sufficient amounts of various materials were not added to the vaccine before lyophilization, the vaccine would not be readily observable and would most likely adhere to the vial wall. By way of illustration of vaccine immunogen masses, ActHIB (Sanofi Pasteur SA), a polysaccharide conjugate vaccine that is marketed in a freeze-dried presentation, contains approximately 10 µg of purified polysaccharide conjugated to 24 µg of tetanus toxoid. The immunogen mass for live viral vaccines is even less, on the order of nanograms (approximately 10 ³ –10 ⁵ viral particles per dose). Thus, there is a need to provide a matrix to contain these vaccines during freeze-drying.

The types of material that are added to vaccines as stabilizers include sugars (such as sucrose or lactose), amino acids (such as glycine or the monosodium salt of glutamic acid), and proteins (such as gelatin). Table 8.2 lists the stabilizers that are used for a number of common vaccines.

Added proteins may be of concern for two principal reasons. The first concern arises from the potential for animal- and human-derived protein to contain one or more adventitious agents. The second concern arises from the potential for these proteins to elicit an allergic reaction. The two animal- or human-derived proteins that are currently used as stabilizers in U.S.-licensed vaccines are human serum albumin (HSA) and gelatin. Additionally, FDA has recommended that the following statement appear in the Warnings section of the package insert for blood-derived HSA-containing products: “This product contains albumin, a derivative of human blood. Based on effective donor screening and product manufacturing processes, it carries an extremely remote risk for transmission of viral diseases. Although there is a theoretical risk for transmission of Creutzfeldt–Jakob disease (CJD), no cases of transmission of CJD or viral disease have ever been identified that were associated with the use of albumin.”

Gelatin or processed gelatin is also used as a stabilizer. Gelatin may be bovine or porcine derived. The concern for gelatin relates to allergic responses. Allergic responses to gelatin, although rare, have been described in the medical literature. It has been hypothesized that in Japan, use of partially hydrolyzed gelatin, which contained a small amount of high-molecular-weight gelatin, contributed to an increase in the incidence of allergic reactions. ^, Nakayama and Aizawa noted that a change to hydrolyzed modified porcine gelatin, together with discontinuation of the use of gelatin-containing DTaP vaccines, may have contributed to a decrease in the incidence of allergic reactions after administration of monovalent measles and mumps vaccine in Japan. A severe allergic reaction to gelatin is a contraindication to receiving gelatin-containing vaccines.

Various buffers (e.g., phosphate buffer) are also used in vaccines to maintain a particular pH range, and salts (e.g., NaCl) may be added to achieve isotonicity.