Adjuvants are included in inactivated vaccines to boost the immune response in the body.

Aluminum salts, added to vaccines in the late 1920s, are the most widely used adjuvant. Current alum-based adjuvants include aluminum hydroxide, aluminum phosphate and amorphous aluminum hydroxyphosphate sulfate (AAHS).

Since 1997, oil-in-water emulsions have been approved for use in vaccines available in Europe, and since 2009 in the US (MF59™ by Novartis > Seqiris, AS03 and AS04 by GlaxoSmithKline).

Merck's proprietary alum-based adjuvant AAHS was first marketed in the USA in 1997, used as an adjuvant in Comvax® (Haemophilus influenzae type b conjugate and hepatitis B vaccine)—that same vaccine was approved in Europe in 2004, under the brand name Procomvax®.

Each 0.5 mL dose contained ~225 mcg of AAHS, which was initially labeled as "aluminum hydroxide" in documentation for the FDA and the EMA—introduced without any prelicensure safety evaluation, under the false premise that it was equivalent to aluminum hydroxide. This mislabeling was a significant misrepresentation, given that AAHS is a proprietary, chemically distinct adjuvant with different safety and immunological properties. It obscured the novel nature of the adjuvant and delayed rigorous independent safety assessments—it was a blatant example of compromised scientific and ethical standards for which Merck paid no penalty.

AAHS is currently included in Merck's vaccines Gardasil®, Recombivax-HB®* (in US), HBVaxPro®* (in Europe), Vaqta® and Vaxelis®.
*given to newborns at birth!

Calcium phosphate; a naturally biodegradable mineral salt used as an adjuvant briefly in Europe in vaccines by Institut Pasteur. Though still approved by the WHO as a vaccine adjuvant, it is not currently used in any approved vaccines.

From the late 1960s, peanut oils, notably Adjuvant 65 by Merck, were used in unapproved formulas.

From 1997, proprietary oil-in-water emulsion MF59 by Chiron-Biocine (later Novartis > Seqiris) was approved for use in influenza vaccine in Italy, and later in Europe. It was approved in the US in the quadrivalent flu shot for adults over 65 years old, from 2016.

Download these key articles on vaccine adjuvants:
Zhao, T., Cai, Y., Jiang, Y. et al. Vaccine adjuvants: mechanisms and platforms. Sig Transduct Target Ther 8, 283 (2023). https://doi.org/10.1038/s41392-023-01557-7

Facciolà A, Visalli G, Laganà A, Di Pietro A. An Overview of Vaccine Adjuvants: Current Evidence and Future Perspectives. Vaccines. 2022; 10(5):819. https://doi.org/10.3390/vaccines10050819

Di Pasquale A, Preiss S, Tavares Da Silva F, Garçon N. Vaccine Adjuvants: from 1920 to 2015 and Beyond. Vaccines (Basel). 2015 Apr 16;3(2):320-43. doi: 10.3390/vaccines3020320. PMID: 26343190; PMCID: PMC4494348.

See the list of adjuvants available in US vaccines.

This is an essential component for viral and toxoid antigen-based vaccines to inactivate the antigen from its virulent properties.

The most common inactivating agents used in vaccines are:

Formaldehyde
Glutaraldehyde
Beta-Propiolactone
Phenol

https://www.vaccinesafety.edu/components-excipients/

This ingredient is used primarily in vaccines containing viral antigens to prevent contamination.

Some antimicrobials used in vaccines include:

Gentamicin Sulfate
Kanamycin
Neomycin
Neomycin Sulfate
Polymyxin
Polymyxin B
Polymyxin B Sulfate

https://www.vaccinesafety.edu/components-excipients/

Buffers are used in vaccines to help ensure chemical stability and maintain a pH balance similar to the body, ensuring the vaccine solution remains stable and effective, also allowing for a longer shelf life. 

Some buffers used in vaccines include:

• Calcium carbonate
• Citric acid monohydrate
• Monobasic/dibasic potassium phosphate
• Phosphate buffer
• Potassium chloride
• Sodium borate
• Sodium biocarbonate
• Sodium citrate
• Sodium citrate dihydrate
• Tromethamine/Tromethamine hydrochloride

Tromethamine, also known as Tris, is a buffer used in a few vaccines (RSV: Abrysvo®, mResvia®) including the Pfizer-BioNTech and Moderna COVID-19 mRNA vaccines. Though this is ingredient is also referred to as a "stabilizer"—see vaccine excipient list below.

https://www.vaccinesafety.edu/components-excipients/

These ingredients are important in maintaining the integrity and uniform dispersion of antigenic materials in the vaccine formulation, as well as preventing degradation during storage and transport.

Commonly used stabilizers in vaccines include:

• Sugars: lactose, sucrose
• Sugar alcohol: sorbitol
• Aromatic ether alcohol: 2-Phenoxyethanol (though mainly used as a preservative)
• Amino acids: glycine, monosodium glutamate
• Proteins: human or bovine (cow) serum albumin
• Hydrolyzed gelatine which is partially hydrolyzed collagen, usually of bovine or porcine (pig) origin
• Polysorbates

Polysorbate 80 and polysorbate 20 (also called Tween 80 and Tween 20 respectively) are examples of nonionic surfactants and emulsifiers used in vaccines to help blend fat-like substances with water, making sure the active parts of the vaccine are evenly distributed.

https://www.vaccinesafety.edu/components-excipients/

Preservatives are added to some vaccines to prevent bacterial and fungal contamination, both in single and multi-dose vials.

While multi-dose vials are now rarely used in most high-income countries—except for some influenza vaccines—preservatives like thimerosal, phenoxyethanol, and phenol have been used extensively. Today, 2-Phenoxyethanol is currently the most commonly used preservative in vaccines worldwide.

Thimerosal was historically the most widely used vaccine preservative since the 1930s, especially in multi-dose vials, and remains in several routine childhood vaccines used in Asia, Africa, and South America.

Benzethonium chloride is used as a preservative in the anthrax vaccine, Cyfendus® and BioThrax®.

https://www.vaccinesafety.edu/components-excipients/

Medium nutrient is used in vaccine production to sustain the growth of microorganisms.

Some examples:

Amino Acid
Bovine Casein
Bovine Extract
Calcium Chloride
Dextrose
Dimethyl-beta-cyclodextrin
Ferric (III) Nitrate
Histidine
Hydrocortisone
Lactalbumin Hydrolysate
L-cystine
L-histidine
L-tyrosine
Magnesium Sulfate
Peptone, Soy
Salts, Inorganic
Sodium Pyruvate
Vitamins (not specified)
Yeast

https://www.vaccinesafety.edu/components-excipients/

Manufacturing residues in vaccines are trace amounts of substances used during the production process that are not intended as active ingredients in the final vaccine. They may be residues of fetal cell culture material, like MRC-5 cells. These residuals are present in extremely small quantities, often measured in parts per million (ppm) or parts per billion (ppb), and sometimes in micrograms (µg) or nanograms (ng) per dose.

Common types of manufacturing residues include:

• Formaldehyde: Used to inactivate viruses or detoxify toxins. Residual formaldehyde is found in some vaccines.

• Antibiotics: Such as neomycin, polymyxin, or gentamicin, used to prevent bacterial contamination during manufacturing. Only trace amounts may remain in the final product.

• Egg proteins: Present in vaccines grown in eggs (e.g., some influenza vaccines), found in trace amounts.

• Yeast proteins: Used in recombinant vaccines, with only minute quantities remaining.

• DNA or proteins from cell cultures: Residuals from the cells used to grow viruses or express proteins.

• Other chemicals: Such as inactivating agents, coupling agents, or preservatives that might be used at various stages.

• Gelatin, human or bovine albumin: Used as stabilizers, can be present in trace amounts.

• Human fetal cell substrates: For example, MRC-5 cells are a line of human diploid fibroblast cells originally developed from the lung tissue of a 14-week-old male fetus in 1966. Used as the medium in which some viruses are grown during the manufacturing process for certain vaccines, such as those against hepatitis A, varicella (chickenpox), and rabies. The virus is then harvested and purified, and the final vaccine does not contain the original MRC-5 cells, though trace residual DNA fragments may remain.The WHO and US FDA guidelines recommend that 10 ng/dose and 200 base pairs be the limits of content and size of residual DNA in the final product dose. "Residual DNA (rDNA) is defined as the sum total of deoxyribonucleic acid (DNA) and fragments present in biological samples derived from recombinant host cells during expression, sometimes it is also referred to as residual host cell DNA. The potential risks associated with rDNA
  are infectivity (through virus such as HIV), oncogenicity (through oncogenes such as Ras), immunogenicity (through CpG rich sequences from bacteria), and mutagenesis (through transposons, retrotransposons, and DNA recombination)."
  Wang, X., Morgan, D.M., Wang, G. and Mozier, N.M. (2012), Residual DNA analysis in biologics development: Review of measurement and quantitation technologies and future directions. Biotechnol. Bioeng., 109: 307-317. https://doi.org/10.1002/bit.23343Other fetal cell lines used in vaccine development include the HEK293 (Human embryonic kidney cells) and WI-38 (Wistar Institute).Brumbaugh J, Aguado BA, Lysaght T, Goldstein LSB. Human fetal tissue is critical for biomedical research. Stem Cell Reports. 2023 Dec 12;18(12):2300-2312. doi: 10.1016/j.stemcr.2023.10.008. Epub 2023 Nov 16. PMID: 37977142; PMCID: PMC10724055.

Traditional amounts and measurement:

• Trace levels: Residuals are typically present at levels requiring measurement in ppm or ppb, which translates to micrograms (µg) or nanograms (ng) per dose.

• Regulatory approach: Safety assessments often use a worst-case estimate for the amount per dose. If a residual is present at or below the established safety threshold (such as the Threshold of Toxicological Concern, TTC), no further toxicological assessment is needed3.

• Examples:

  • Formaldehyde: The amount (in synthetic form) in a vaccine dose is 50–70 times less than what is found in its natural form in a newborn’s body.

  • Aluminium salts: Used as adjuvants, present in amounts like 0.8 mg per dose in DTPa vaccines (via injection), which is much less than dietary intake (via ingestion). Obviously aluminum absorption via injection versus via ingestion does not involve the same assimilation and excretion process, as injection bypasses the body's natural defense pathways and poses more strain on the kidneys and liver.

  • Antibiotics and egg proteins: Present in trace amounts, often below levels that would trigger allergic reactions except in highly sensitive individuals. Again, remember that these trace amounts are injected, not ingested!

"Polymers are used in various stages of vaccine production, including: syringes, stoppers, flexible tubing, flexboy bags, and final or intermediate containers for liquid storage. The use of polymers can result sometimes in the presence of unexpected extractable/leachables in the product. Furthermore, vaccine manufacturing processes occasionally result in the presence of residuals or contaminants in vaccines, for example: coupling agents used to link multivalent antigens, inactivating agents, antifoaming agents, preservatives, inducing agents used to induce expression of recombinant antigen, or antibiotics.

For these reasons, there is a potential need for common safety assessment processes when residuals or contaminants are detected. Of note; currently, there are no specific regulatory guidance (FDA, EMA, PMDA, WHO) for the safety assessment of vaccine residuals or contaminants. Although some guidelines exist, they are in support of more conventional pharmaceutical products rather than biologicals."

Below is the list of manufacturing residue as reported by the Johns Hopkins Vaccine Safety project.

https://www.vaccinesafety.edu/components-excipients/