viral vaccine production basics and manufacturing basics involved in development in research. Cell lines and characteristics of cell substrates and mode of operation useful for increased cell density. Basics of vaccine types and their features.

1. Viral vaccine and production

2. CONTENT
1. What is a vaccine ?
2. Classical vaccines & engineered vaccines
3. Replicating versus inactivated vaccines
4. Purpose of vaccination
5. Crucial aspect defining the process choice
6. Host cell characteristics & growth requirements
7. Advantages & disadvantages of insect cells
8. Virus/antigen stability
9. Vaccine production
10. Vaccine production in bioreactors
11. Process intensification in bioreactors
12. Manufacturing basics

3. 1. WHAT IS A VACCINE ?
• Biological preparation providing Active Acquired Immunity
• Goal of vaccination: to stimulate the body to develop a specialized, protective, immune
response in the absence of disease.
• Immune responses target viral proteins which can be delivered as :
i. Weakened (attenuated) viruses that replicate in the host without causing disease.
ii. Inactivated (killed) virions.
iii. Purified protein products.
iv. Nucleic acids to direct synthesis of desired proteins.

4. 2. CLASSICAL VACCINES & ENGINEERED
VACCINES
CLASSICAL VACCINES ENGINEERED VACCINES
1. Attenuated viruses usually obtained by repeated passage
in animals or cultured cells.
1. provide a safe source of antigen, representative of a
particular pathogen.
2. Unpredictable results and inadequately attenuated
viruses.
2.1 Virulent viruses weakened by deletion/modifications of
specific genes
2.2 Genes for capsid or envelope proteins can be
transferred to a safer virus.
2.3 genes of virulent virus moved into bacteria, yeast, or
cultured cells to produce large quantities of a particular viral
protein.
2.4 administer DNA (cloned genes) directly into the
recipient.
3. possibility of virus reverting to virulence. 3. use of adjuvants

5. 3. REPLICATING VERSUS INACTIVATED
VACCINES
LIVING VACCINE INACTIVATED VACCINE
1. Fewer, smaller doses and adjuvant
unnecessary
1. Do not replicate in recipient
2. Cheap to produce 2. Lower cost to develop
3. Longer protection and less chance of
hypersensitivity.
3. Unlikely to cause disease through
residual virulence
4. Stable on storage

6. 4.Purpose of vaccination
• Stimulate the production of neutralizing antibodies for the purpose of blocking infection
• Neutralizing antibodies might -
i. prevent a virus from binding to a receptor
ii. prevent membrane fusion
iii. prevent un-coating of the viral genome.

7. 5.Crucial aspect defining the process choice
5.1 VACCINE DEMAND:
i. Spread of a virus and its mutation rate.
ii. Time period Ex: seasonal influenza a time period as short as 5–6 months.
iii. Polio or measles require a more or less constant supply of the same vaccine strain for
worldwide application.
iv. Vaccines against dengue or yellow fever are only needed in certain regions of the world.

8. 5.2 VACCINE TYPE
i. Live attenuated viruses, inactivated viruses, virus subunits, viral vectors or recombinant
virus-like particles/proteins
ii. Viruses with a high mutation rate are unsuitable candidates for live attenuated vaccine
type as reversion may occur during vaccine production.
iii. Attenuated virus strains in cell culture gives lower process yield
iv. Wild-type live viruses require biosafety level 3
v. Recombinant vaccines may require higher antigen concentrations per dose

11. 5.3 VIRUS/ANTIGEN REQUIREMENTS FOR
GROWTH/EXPRESSION
i. Chemically defined protein-free media
ii. Hepatitis A (strain HM175) propagated in MRC-5 human diploid cells
iii. Recombinant hepatitis B surface antigen (HBsAg) produced in yeast cells grown in a
complex medium of extract of yeast, soy peptone, dextrose, amino acids, and mineral
salts
iv. Measles virus propagated in chick embryo cell culture

12. 6. HOST CELL CHARACTERISTICS & GROWTH
REQUIREMENTS :
i. Right cell line & optimal process parameter conditions e.g., temperature, pH value,
dissolved oxygen concentration, medium composition, etc.
ii. Human/higher animal cell substrates for whole virus replication.
iii. Insect cell substrates for recombinant antigen and virus-like particle (VLP) production.
iv. Continuous cell lines preferred over primary cells : Vero, MDCK, MRC-5, WI-38,
HEK293, PER.C6, AGE1.CR ,EB66
v. Media development- serum free

13. 7. ADVANTAGES & DISADVANTAGES OF INSECT CELLS :
i. Suspension cultures using serum-free media
ii. Insect cell-baculovirus expression system
iii. Difficultly to express membrane proteins , secreted glycoproteins at appropriate levels
or complex glycan structures
8. VIRUS/ANTIGEN STABILITY :
i. Hollow-fiber-based perfusion systems enables selective separation and further
processing of the product-containing medium.
ii. To avoid virus/antigen degradation in upstream processing, which is mainly caused by
the release of cellular proteases after cell lysis low thermo-stabilities.

14. 9. VACCINE PRODUCTION
Roller bottles
i. A practical and low-cost option for cell culture at laboratory scale and for large-scale
manufacturing of products
ii. Automated handling of up to 1000 RBs per batch (IDT Biologika)

16. 10. VACCINE PRODUCTION IN BIOREACTORS
DISCONTINUOUS BATCH CULTIVATION MODE
i. Low instrumental and operational intervention
ii. Good virus yield coefficients and high nutrient consumption
iii. Fed-batch or perfusion systems aims on higher cell concentrations & increased
volumetric virus yields

17. 11. PROCESS INTENSIFICATION IN BIOREACTORS
i. Microcarriers
ii. Fixed-bed systems or packed-bed systems
iii. Shake flasks , wave bioreactors or STRs
iv. Fed-batch mode
v. Perfusion systems (external hollow-fiber)
vi. Continuous bioreactor
vii. Use of ‘Single-use bioreactors’ in vaccine production

18. 12. MANUFACTURING BASICS
GENERATION of the
pathogen recombinant
protein derived from the
pathogen
RELEASE the antigen
from the substrate and
isolate it from the bulk of
the environment
PURIFICATION of the antigen
unit operations of column
chromatography &
ultrafiltration inactivation of
isolated virus.
FORMULATION of the
vaccine
include an adjuvant
QUALITY CONTROL (QC)
testing safety, potency,
purity, sterility, and other
assays specific to the
product
STORAGE addition of
stabilizers or lyophilization
, very low temperatures