vaccine production Flashcards
Vaccination is active or passive immunisation
active
○ Host immune system encounter pathogen
Activated to produce immune response
what is passive immunisation
Administer specific Ab for short-term immunological protection of host
level of vaccination
- LEVEL 1: Instrument of indiv prevention, protection from possible adverse short/ long-term conseq of infection w/ pathogen
- LEVEL 2: herd immunity
- LEVEL 3: global prevention, avert export of pathogens to regions not endemic
*LEVEL 4: global eradication of disease, protect current and future generations
requirements of a good vaccine
○ 100% effective in all age grp
○ Long-term protection after single application (LIFELONG)
○ No side effects
○ Stable in diff conditions
○ Easily accessible, inexpensive
○ Protect > 1 disease at a time
stability of vaccine
§ Heat, light, transportation
□ Stored, transported, applied in special conditions
§ Since biological pdts (protein, lipids, carbohydrate, nucleic acid)
□ Freezing, temp (<0C) negative effect on substance. Affect potency
Aim 2-8C
cold chain transport temp
2-8*C
shake test
to see if vaccine is damaged/ suspect freezing
- Freeze and thaw one batch (positive control)
- Other batch without freezing (test
- Compare after shaking both
- Should have flakes , suspension
- See if sedimentation rate is same/ faster
=-That means your vaccine has been frozen, should not be used anymore - If slower:
Not affected, batch can be used
components inside vaccine
Immune antigens
Suspension fluids
preservatives
stabilisers
Surfactants/ emulsifiers
Adjuvants
Trace components
Immune antigens
API, viral. Bact antigens that directly stimulate immune system
But do not cause disease
Suspension fluids
Lq that contains chemicals used during production that kill/ weaken germ for use in vaccines
preservatives
Prevent bact, fungal growth
(only in multi-dose vials)
Ensure vaccine content and potency remains unchanged
Sterilisation not 100% safe, add preservatives to kill off possible contaminants
* Aseptic production
* No final sterilisation, due to proteins DNA, mRNA component
* Sensitive to temp
3 eg of preservatives
- Formaldehyde
- Kills, inactivate unwanted germs in vaccines
- 2-phenoxyethanol
- Antimicrobial preservatives used in cosmetics, TOP pharm formulations
- Narrow range of actions
- Mostly in combi (synergistic effects + less amts of preservatives)
- Easily eliminated, little toxicity
- Thimerosal
- Alternative to benzalkonium chloride/ other phenylmercuric preservative
- Bacteriostatic, fungistatic activity
○ Parenteral formulation at conc of 0.01%
○ Hypersensitivity reactions, not used
stabilisers
1) Keep vaccine stable after manufacturing (Maintain effectiveness during storage)
2) Stop chemical reactions from occuring IN THE VACCINE
3) Prevent components from SEPARATING
4) During transport, storage prevent STICKING to vaccine vial
** if lyophilised, no need stabiliser, will be reconstituted with NS water
4 eg of stabilisers
- Monosodium glutamate (MSG)
- Protect vaccine from heat, light, humidity, acidity while stored
- Gelatin
- Protect vaccines from heat while they are stored
- Sorbitol
- Stabilise proteins when in solution
- Buffers (monopotassium phosphate, sodium borate, sodium chloride)
- Adjust tonicity
- Maintain osmolarity (Na content in body)
- pH range for IM, IV
surfactants/ emulsifiers
Amphiphilic molecules with lipophilic part linked to hydrophilic part
Used as solubilisers or stabilisers of emulsions, also can be adjuvants
Surfactants/ emulsifiers 3 eg
- Sorbitan esters
- Lacithins
- Mannide oleates (Montanide)
- Esters of oleic acid (lipo) + mannitol (hydro)
- Lipopolysaccharides (LPS)
- Gram-ve bact
- Saponins, glycosides
- Quilaja saponaria tress
- Dimethyl dioctadecyl ammonium bromide (DDAB)
+ve charged organic salt
Adjuvants
Adjuvant: component to induce more potent immune response
* * reduce need for booster * Used for flu shots
Modern vacc, weak immunogens + need good antigen delivery system
2 ways adjuvant can work
- Vaccine delivery systems: conc and target antigen
a. Microparticles
b. Nanoparticles - Immunostimulatory adjuvant
a. Immunopotentiators activate innate immunity directly
i. Cytokines
b. Pattern recognition receptors
i. PRRs
3 eg of adjuvants
- Alum salts
- Aluminum phosphate, hydroxides
- Weak effect, slows down rate of release of antigens
- Incr duration of antigen interaction with immune system
- Complete Freund’s adjuvant (suspend in oil)
- Heat-killed mycobacteria in oil
- Primary agent resp for stimulating Ab production
- Severe SE = inflammations
- Oil-in-water// water-in-oil emulsions = TOXIC, DANGEROUS to use
other adjuvant emulsions
- water-in-oil emulsions
○ Prepared from non-metabolisable oils: incomplete Freund’s adjuvant
○ Very effective
○ SE: cutaneous reactivity - MF59 (Oil-in-water)
○ Submicron oil-in-water emulsion
○ Contains squalene (influenza vaccine) - Montanide (oil based adjuvant)
○ Family of oil based adjuvant
§ Natural, recombinant and synthetic antigens
○ Experimental vaccines: dog, rates, mice, cats
Trace components
Residual inactivating ingredients for virus/ bact
- formaldehyde
- residual cell culture material
Trace amt = no effect
Route of administration
○ ORAL
§ Drops to mouth – rotavirus (Rotarix, RotaTeq)
○ INTRANASAL
§ Intranasal vaccine intro each nostril using manufacturer filled nasal spray
□ Incr pt compliance but hard to formulate
§ Live attenuated influenza (FluMist) Vaccine
○ SUBCUTANEOUS
§ Admin into fatty tissues below dermis – above muscle tissue
○ INTRAMUSCULAR
§ Admin into muscle through skin and sc tissue
○ Jet-injectors
Vaccines manufacture steps
bioprocessing (upstream –> mid stream –> downstream separation & purification)
formulation, qc, filling (formulation –> filling –> cap, seap)
finishing, packaging (label –> qc –> packaging)
upstream
a) Upstream: antigen generated
□ Pathogen itself/ antigen from recombinant protein
□ 3 types of antigen:
-Virus generated by 1* cells/ continuous cell line
-Bacteria grown in fermenters
-Recombinant proteins (from bact, yeast, cell culture)
Midstream process + 3 clarification methods
□ Removal of cells and cell debris
□ Between upstream(fermentation) & downstream (pdt purification)
□ Filtration one after another, several in place
(CAKE/ alluvial)
(tangential flow)
(centrifugation)
(CAKE/ alluvial)
◊ Particles accumulate at first few layers
◊ This will thicken filter = more effective, prevent big particles psss through
◊ BUT incr back pressure of filter, until cannot filter anymore (need maintenance)
Tangential flow filtration TFF
◊ Pipe, have a flow
◊ Filter on side of pipe
◊ Big particles go with flow
◊ Particles go through holes at side of pipe
1) Disadv: poor efficiency
– Long pipe, force flow to go through the pipe
– filter more, more area
2) Adv: no clog
Centrifugation
◊ Separation by weight
◊ Bigger particles thrown out
◊Smaller particles go centre
Downstream
- Antigen is separated from impurities in downstream process
- Antigen released from substrate (cell lysis) if necessary
○ Chromatography
§ Column to retain certain target particles
○ Ultrafiltration
§ Retain certain particles, let others go through
○ Precipitation
§ depend on protein
○ Enzyme digest
FINAL sterile filtration (0.22um)
summary of bioprocessing of diff vaccine types
- clarification (remove)
- inactivation
- sterile filtration
viral virus (remove cell debris. benzonase/ endonuclease degrade nucleic acids)
virus-like particle vacc (inactivate by formalin. weak ion-exchange chromatography resins)
polysacc conjugate vacc (chromatography to get polysacc. couple with nontoxic/reactive carrier proteins. purification)
viral vector (cell lysis, clarification –> nuclease + UF, chromatography –> sterile filtration)
summary of bioprocessing of diff vaccine types
- clarification (remove)
- inactivation
- sterile filtration
viral virus (remove cell debris. benzonase/ endonuclease degrade nucleic acids)
virus-like particle vacc (inactivate by formalin. weak ion-exchange chromatography resins)
polysacc conjugate vacc (chromatography to get polysacc. couple with nontoxic/reactive carrier proteins. purification)
viral vector (cell lysis, clarification –> nuclease + UF, chromatography –> sterile filtration)
mRNA (purification by TFF, formulation (lipids), inactive virus particles via FORMALIN, TFF purification again in case of lipid degradation)
Formulation
- All components that constitute final vaccine are combined in formulation process
- Designed to maximise:
○ Stability
○ Efficient distribution
○ Preferred clinical delivery method
- Designed to maximise:
- May include (adjuvant, stabiliser, preservatives)
- Formulated vaccine CANNOT be filtered sterilised
○ Adjuvant, purified antigens are filtered and sterilised separately, aseptically blended
filling
capping and sealing
- sterile cotnainers filled, load into lyophiliser chamber.
apply vaccum and heat to evaporate water from FROZEN
stopper by hydraulic/ screw stop mechanism - Stopper flange
○ Close via crimping around upper edge of vial
○ Prevent leak, contamination
○ Automatic vial visual inspection
○ Usually centre-hole type
§ Tight around glass vial
QC of vaccines (controlled at multiple levels)
- Before approval
i. In depth review of registration file
ii. Contain all infor on production process/ materials/ testing- QC testing
i. Every single vaccine batch tested by manufacturer according to predefined set of specifications
ii. Approved by authorities - GMP (Good manufacturing process)inspections
i. Beore and after approval (every 2-3yrs)
ii. Verification of compliance w/ legislation and registration file - Pharmacovigilance
i. Post-approval clinical monitoring
ii. Pop larger sample to identify problem that wasn’t brought up in clinical trials - Additional QC testing by INDEPENDENT LAB
- QC testing
QC testing
○ General parameters (pH, appearance)
○ Identity (of antigen)
○ Potency (verify antigen content)
○ Purity/ integrity of antigen
○ Safety (sterility, endotoxins)
- Specific tests for:
○ Potency, purity, integrity
○ Type of antigen or vaccine - animal testing
potency testing by 3 ways
1) immuno assay (recombinant, inactivated vacc)
In vivo assay replaced by in vitro immuno-assay
antigen content measured by ELISA
2) potency live viral vaccines
cell death quantified from in vitro cell culture
TCID 50
3) Potency polysaccharide vaccines
polysacc content, size, purity
degree of adsorption
potency false +ve
○ In case of degraded antigen, immunoassay
○ Additional assays required to monitor size, purity, integrity of antigen (SDS-PAGE, SEC-HPLC)
