IC10 Vaccines manufacture

Question 1

Does vaccine production depend on aseptic production or final sterilization?

Answer 1

Aseptic production because biological compounds such as viruses and proteins are sensitive to high temp

*Sterilization methods require high temp (even chemical sterilization with “cold system” require 40-60dc)

Question 2

What are the 3 main components in the manufacturing procedure of vaccines?

Answer 2

1. Bioprocessing (Upstream, midstream, downstream)
2. Formulation, filling, capping and sealing
3. Finishing and packaging (labelling, QC, packaging)

Question 3

Explain upstream processing (bioprocessing)

Answer 3

Generation of antigen
- can be virus generated by primary cells, bacteria grown in fermenters, or recombinant proteins generated by bacteria, yeast or cell culture

Question 4

Explain midstream processing (bioprocessing)
What are the methods involved?

Answer 4

Removal of cell and cell debris typically via filtration

Methods involved:
1. Cake/alluvial filtration
2. Tangential flow filtration
3. Centrifugation (separation by weight)

Question 5

Explain the advantages and disadvantages of cake/alluvial filtration

Answer 5

Advantage:
- high filtering efficiency because filtration is against flow of the substance, filter becomes thicker as particles build up, improve efficiency

Disadvantage:
- backpressure will soon stop filtration, require maintenance

Question 6

Tangential flow filtration involves filter on the side, such that filtration is not against the flow of the substance. This causes efficiency to be low. How might we increase efficiency?

Answer 6

Efficiency dependent on the area
- Hence can use longer pipe, so theres more filtering area along the sides
- Large filtering area makes up for low filtering efficiency

Question 7

Explain the downstream separation and purification process (bioprocessing)
What are the methods involved?

Answer 7

Antigen is separated from impurities, antigen is released from substrate (via cell lysis)

Methods:
- Chromatography
- Ultrafiltration
- Precipitation
- Enzyme digest

Question 8

In bioprocessing of viral virus, what might be a concern in the downstream separation and purification step?

Answer 8

Carryover nucleic acid from lysed cell can be found in sample (because nucleic acid was able to pass through the membranes.

Benzonase endonuclease enzyme is added to degrade the nucleic acids, use together with chromatographic separation

Finally, a 0.22um sterilizing filtration is used (aseptic filtration)

Question 9

Why might concentration be an important step in the midstream process?

Answer 9

Yield and efficiency of filtration depend on concentration

If we skip concentration step, may lose product during filtration

Question 10

In bioprocessing of viral vector vaccines, explain the need for ultrafiltration after nuclease treatment in the downstream purification process.

Answer 10

Nuclease treatment - e.g., Benzonase to eliminate viral vector encapsidated nucleic acid impurities

Sterile FIltration using 0.22um filter to filter out the impurities

Question 11

Why might downstream process (e.g., VLP vaccines and mRNA vaccines) include an inactivation step?

Answer 11

Inactivation of baculovirus particles via formalin (formaldehyde 10%)
*Inactivate the virus involved in developing the vaccine

Question 12

Why must purification via TFF be done quickly for mRNA vaccines?

Answer 12

Possible lipid capsid degradation/damage may occur with TFF

Question 13

Why can’t formulated vaccine be filter sterilized?

Answer 13

Excipients are included in the formulation.

Adjuvant and purified antigens should be filtered and sterilized separately before being aseptically blended together

Question 14

What are the main concerns of formulated vaccine?

Answer 14

Thermal stability - e.g., lyophilization
Enzyme stability

Question 15

What are the advantages of microneedle?

Answer 15

Less painful than syringe
Controlled release - prevent pain at site of injection by releasing slowly
Avoid issue with disposal of needle

Question 16

What are the vial components for capping and sealing?

Answer 16

Glass vial - transparent for visual inspection

Rubber stopper - venting points to allow venting during lyophilization
-ve:
- trap moisture
- need lubrication to prevent break down
- break down into debris (possible source of contamination)

Aluminium cap + Stopper flange
- Aluminium cap must be crimped around the upper edge of the stoppered vial to ensure closure (prevent air flow, prevent leaking, prevent contamination, ensure sterility in vial)
- Usually centre-hole type: for reconstitution, pierce syringe through rubber stopper, shake product, take syringe out

Question 17

How is the quality of vaccines controlled?

Answer 17

1. Registration file (contain info on pdn process, materials, testing)
2. QC testing (parameters within specifications)
3. GMP inspection (compliance)
4. Pharmacovigilance (post approval clinical monitoring)

Question 18

What inspections must be done before and after approval (every 2-3y)?

Answer 18

GMP inspections - verification of compliance with legislation and registration file

• Compliance with guidelines
• Traceability of each batch
• Manufacturing, testing, quality of materials, QC methods => all properly conducted as described in registration file
• Results within acceptance limits
• GMP audits - run by authority, can have internal independent inspectors, external manufacturer inspection etc.

Question 19

What information must be described in the registration file for review and approval?

Answer 19

(contain info on pdn process, materials, testing)

Biological materials used screened for contaminants

Process, QC tests

Containers and equipments evaluated for possible leaching into vaccine

Impurity removal capacity of the process

Assessment to confirm safety of product

Residuals below acceptable safety limits

Question 20

What is QC testing? What parameters are tested?

Answer 20

Test to ensure every batch manufactured complies with a predefined set of specifications (via fixed panel of methods and results)

Parameters:
- General parameters: pH, appearance
- Identity
- Potency
- Purity/integrity
- Safety (sterility, endotoxins)

Others:
- Residual toxic compound (e.g., formaldehyde)
- Residual live virus (in inactivated vaccines)

Question 21

In the past, animal testing used (whereby vaccine product is compared to a positive control reference). However, initiatives are ongoing to implement in vitro testing. What are some methods and what are their concerns?

Answer 21

Grow organ on microchip
Use cells (natural or genetically engineered)

However, need to prove comparable to reference method

Question 22

What is allometric scaling?

Answer 22

PK data from nonclinical animal studies extrapolated and used to predict human drug exposure for a range of drug doses

More useful for small molecules only

Question 23

Potency can be assessed using in vitro immunoassay, whereby antigen content can be measured using ELISA. What is a downside of this method?

Answer 23

It only measures and quantifies the antigen content

Degraded antigen may still yield positive result (hence doesnt really test potency)

Question 24

Immunoassay may not be optimal as it only measures and quantifies and antigen content. What other methods can be used to monitor size, purity, integrity of antigen?

Answer 24

SDS-PAGE: Estimate purity of protein samples
- SDS is an anionic detergent, used to denature proteins
- Separation of protein dependent on differences in relative MW of polypeptides
- High resolution electrophoretic separation of proteins based on MW
- PAGE can give high resolution of protein folding, able to identify pattern of fragment

SEC-HPLC: size exclusion chromatography
- Porous particles in column, separate molecules by size

Question 25

What is used to determine potency of polysaccharide vaccines?

Answer 25

• Polysaccharide content
• Polysaccharide size
• Purity
• Degree of adsorption (for vaccines that adsorb on aluminium)

Question 26

What is used to determine potency of live viral vaccines?

Answer 26

To verify the viral titer of a testing virus

Cells are infected with various dilutions of vaccine, cell death quantified
- cell death tells us that the cells have been infected (potency)

Potency expressed as PFU/mL or TCID50
PFU: number of infective particle units
TCID50: median tissue culture infectious dose