The Quality and Safety of Vaccines: Formulation, Chemistry and Use of Adjuvants in Vaccines

Question 1

Biological sourced materials can include:

Answer 1

 Bioactives – toxins, fragments
 Bacterial polysaccharides, oligosaccharides
 Proteins, peptides, lipoproteins and glycoproteins, peptidoglycan, lectins
 Lipids, lipopolysaccharides e.g. Lipid A
 Chemically modified substances e.g. subunits, DNA/si-dsRNA

Question 2

Adjuvant

Answer 2

 This is a drug/immunological agent/formulation aid that modifies the effect of other
agents
 They’re a variety of particles (natural and/or synthetics) which when added to
vaccines, they stimulate the immune system to response to the target antigen but
don’t directly give immunity
 Adjuvants can act in many ways in presenting an antigen to the immune system
 Adjuvants of 0.1-0.9mg/dose often act as a depot for the antigen, presenting the
antigen to the body over a longer duration than IV administration – this maximises the immune response prior to bodily clearance

Question 3

adjuvants.2

Answer 3

 E. coli heat labile enterotoxin
 Cholera toxin
 Tetanus toxoid
 Diphtheria toxoid

Question 4

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Answer 4

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Question 5

Entrapment or absorption gent adjuvants

Answer 5

 Silica
 Latex
 Calcium phosphate  Starch
 Gelatine
 Virosomes – virus structured liposomes
 Bacterial flagellin – Helicobacter pyloris, E.coli, Salmonella
 Bacterial fimbriae and pili e.g. E. coli
 Haemophilus influenza (Hib)

Question 6

Formulation stabilisers

Answer 6

 Freeze-drying cryoprotectants e.g. such as sucrose and lactose
 Buffers e.g. amino acids such as glycine or Na glutamate
 Complexing and suspending agents e.g. proteins such as human serum albumin or
gelatin or EDTA
 Delivery acids e.g. gums and viscosifiers (e.g. gelatine)
 Preservatives e.g. BHA
 Co-solvent e.g. phenoxyethanol
 Emulsifiers e.g. Tween
 Relics e.g. foetal bovine serum remnant from cell culture, deactivation substrates

Question 7

Antibiotic stabilisers

Answer 7

 Multi-use vaccines need special formulation
 They contain antibiotics e.g. penicillin, sulpha drugs, cephalosporins and
thiomersal/thimerosal fungicides – used to prevent fungal and bacterial infection as
consecutive aliquots are removed from the same bulk container
 Examples of antibiotics used during vaccine manufacture include neomycin,
polymixin B, streptomycin and pentamicin
 Antibiotics are used in some vaccines e.g. flu production methods to reduce bacterial
growth in non-sterile eggs during ‘passaging’ processing steps
 These antibiotics can be found in final products at very low concentrations

Question 8

Chemical inactivation agents

Answer 8

 Chemical remnants e.g. cross-linkers are often found in vaccines
 Cross-linkers remove pathogenicity and act as a disinfectant
 Formaldehyde (methanal) remnant used to inactivate viruses e.g. polio virus and
bacterial diphtheria toxin etc.

Question 9

Other inactivations

Answer 9

toxin, virus, whole cell pathogen, subunit, conjugate inactivation
vaccines

Question 10

Purification of antigens

Answer 10

example of HBsAg (Hep B surface antigen)
A. Differential precipitation/solubilisation – ammonium sulphate, pH
B. Ultracentrifugation
C. Final purification

Question 11

Final purification by:

Answer 11

1. Ultrafiltration/nanofiltration (porous membrane molecular weight cut-off)
2. Gel permeation chromatography (GPC)
3. Hydrophobic interaction chromatography (HIC)
4. Affinity (ligand) chromatography
   D. Tested by enzyme-linked immunosorbent assay (ELISA) concentration in ELISA units per ml

Question 12

Enumeration – ELISA

Answer 12

 Enzyme-linked immunosorbent assay
 A colour indicating popular microtitre plate-type
analytic assay of a solution, which uses a solid-phase immobilised enzyme in an immune-assay to detect the presence of a vaccine antigen
 Used routinely for quality control

Question 13

Antigens in vaccines

Answer 13

 Homotypic vaccines – one type of component e.g. Hib, flu
 Heterotypic vaccines – many types of antigenic components e.g. in TB BCG jab
(various strains of M. bovis bacille Calmette-Guerin)
 Whole cell: live or killed
 Toxoid
 Pathogen components

Question 14

Valency – refers to the number of strains used and thus universality e.g. influenza vaccines

Answer 14

 Bivalent – 2 strains

 Quadrivalent – 4 strains

Question 15

Quality control

Answer 15

 Sterility – free of live microorganisms
 Chemistry standpoint – correct adjuvant, preservative (if needed) and pH
 Content – uniformity of content and potency (PCQ)
 Safety – overdosing would carry no effective risk and correct labelling of the product
 Efficacy – each antigen present meets regulatory requirements and satisfies key tests
and no component interference
 Virulence testing – via passaging in suitable model
 Toxicity – no harm-causing constituents
 Environment – no risk or harm of spread or dissemination
 Stability test – shelf life, optimal storage determination
 Chemical quality control – moisture content, headspace vacuum, pH

Question 16

Dosage issues

Answer 16

 Variable: dose, dissipation, response, maturity of the immune system, temperature
history, security
 Constancy: formulation injection properties e.g. viscosity, constant content (metered
dose per vial), actives and excipients, stability profile

Question 17

Delivery

Answer 17

the basic premise of effective vaccine delivery is based upon:
 Efficient encapsulation of the active
 Successful targeting of the active to a specific region of the body and pathogen
 Successful release of that active in situ (in position)

Question 18

Delivery routes

Answer 18

 Liquids
 Parenteral (needle and syringe common) liquids e.g. liposomes, micelle solubilisation, adsorbates on colloidal solids (e.g. alum)
 Suspension (oral/IV)
 Solution oral syrups

Question 19

Solids/semi-solids (oral/topical)

Answer 19

 Oral films, gels
 Solid dispersions and glasses (buccal, sub-lingual)
 Tablets
 Capsules
 Mucous membrane entry: aerosol inhalers –
pulmonary and nasal
 Implants, micro-needle technology
 Creams

Question 20

Principal routes of vaccine delivery

Answer 20

 IM
 ID
 SC
 IN – intranasal: aerosol, liposome
 Pulmonary – MDI, PDI, liposome
 Oral – buccal, sub-lingual
 Biodegradable implant – bioneedles
 Transdermal – needle-free
-  Patch
 -Liquid injection
-Powder injection
-Pre-filled reconstitution
- Vaginal, cervical, anal, penile

Question 21

The Quality and Safety of Vaccines: Production, Storage and Handling of
Vaccines – Manufacturing Response to Antigenic Shif

Answer 21

 Regulation guidelines on biological vaccine products
 The FDA, MHRA and EMEA define key criteria for vaccines
 Vaccines in Europe are covered by The Committee for Medicinal Products for
Human Use (CHMP) which describes the definitions of vaccines or vaccines
components (which are also known as biologicals)
 EMEA provides guidelines on biologicals containing biotechnology-derived proteins
and active substance in terms of quality issues
 Impact of quality changes in the manufacturing process of a biological medicinal
product must be ascertained
 Governance spills over into other committees for vaccines that include:
 The Paediatric Committee (PDCO)
 The Committee for Advanced Therapies (CAT)

Question 22

Vaccine production

Answer 22

 Must have requirements in vaccine production
 Clean rooms (bacterial load) and laminar flow, positive pressure conditions
 Sterile or virus segregation equipment (pathogen load)
 Segregation of personnel

Question 23

Growth

Answer 23

 Virus grown on primary cells – chicken eggs or cell lines
 Bacteria in bioreactors e.g. Hib
 Proteins or parts of the organism can be grown inside cells – yeast, bacteria, cell
cultures
 Inactivation – using agents and typically heat
 Purification – filtration, chromatography, centrifugation (rotates at high speed to
separate substances)
 Formulation = antigen + adjuvant + stabiliser + preservative

Question 24

Sterilisation of vaccines

Answer 24

 Z-value defines temperature
 D-value defines the total number decreases at Z-value
 F0 defines the total effective sterilisation time

Question 25

Sterilisation time/temperature – 121 0C (pressured steam) for 6 minutes (gives >10 log cycle reduction in numbers and is used for:

Answer 25

 Vegetative cells
 Spores
 Denaturation of enzymes
 Thermo-destruction of macromolecules

Question 26

Manufacturing needs of vaccines

Answer 26

 Safety reviewed by CHMP (by the EMEA) and biological panels
 Staff requirements
 Additional bio-specific training
 Immunological status or staff
 In-process controls
 cGMP
 Starting material suitability
 Preparation protocol
 Finished goods: PCQ (purity, consistency and quality)

Question 27

Storage

Answer 27

 Very careful control of ideal transport temperature is needed
 The cold chain (temperature-controlled supply chain – system or storage transport)
has a minimum and maximum
 There is a clear effect of temperature on potency
 Aim for 5 ± 3oC storage in cold chain (shelf-life months)
 Calibrate all refrigeration facilities – all calibration equipment must be accurate
within 1oC
 Cool box interim transfer systems – use bubble wrap insulants
 Immunoglobulins can be stored at room temperature for up to 1 week
 Vaccines are frequently sensitive to light, heat and freezing
 Sensitivity to sunlight, fluorescent lightings and UV
 Always store in the original pack until used

Question 28

Handling

Answer 28

 Needs inventory management
 Emergencies procedure
 All attempts to reduce wastage, which can account for up to 50% of all produced
vaccines
 Most vaccines are damaged at freezing temperatures producing agglomerates
(collected or formed into a mass) and precipitates