9

Question 1

Upstream process

Answer 1

Appropriate choice of cell substrate, growth media and fermentation or cell culture conditions that reproducibly produce the antigen in large quantities

Question 2

Downstream process

Answer 2

Vaccine purification designed to remove the host cell impurities, process additives to yield a bulk vaccine

Question 3

2 ways to process viruses

Answer 3

1. Viral removal/clearance
2. Viral inactivation

Question 4

Viral clearance

Answer 4

method to remove viruses from the product

Question 5

Viral inactivation

Answer 5

method to stop viruses from infecting the product

Question 6

Multistage approach to viral clearance

Answer 6

1) Chromatography
- separation based on resin and buffers
2) Viral inactivation
- disruption of lipid membrane or denaturation of viruses
3) Viral filtration
- nano/ultra filtration to remove parvovirus (15-24nm)

Question 7

Methods of viral inactivation

Answer 7

• Solvent/detergent inactivation (chemical)
• Heat inactivation (pasteurisation)
• Ultraviolet (UV) irradiation
• Low pH inactivation (acid) —> most commonly used

Question 8

Low pH inactivation

Answer 8

• Protein A capture step binds proteins while viruses and other contaminants are washed through the column.
• Acid inactivation performed to denature any remaining virus particles.
• Brought to pH range where proteins are still stable but enveloped viruses are not.
• Hold at low pH + mixing to ensure potential virus is exposed to acid treatment.
• Bring pH back up to 5-7 to make protein stable proceed with subsequent processes.

Question 9

Considerations of viral inactivation

Answer 9

1) Acid/base added too quickly can denature the protein due to the concentrated pool
2) Acid/base added to slowly can denature the protein due to overexposure to low pH
3) Hold time not long enough can result in:
- insufficient evidence to qualify log reduction of virus
- validated at a narrow range of hold time
4) Hold time too long can result in denaturing of protein due to overexposure to low pH

Question 10

Viral filtration

Answer 10

• Viral particle removal by size
• Molecular weight cut off
• Final steps in purification

Question 11

Viral filtration

Answer 11

• Viral particle removal by size
• Molecular weight cut off
• Final steps in purification