This document discusses strategies for improving cell culture technology for influenza vaccine production. It outlines the development of cell lines like MDCK and PER.C6 that are used for large-scale vaccine production. Reasons for low viral yields in cell culture include low cell density, cell death, and strains not growing well. Strategies to improve yields include manipulating the cell line, virus strain, infection conditions, and culture conditions to optimize cell survival and virus replication. The goal is to establish robust and scalable cell culture processes for purer and faster influenza vaccine production compared to egg-based methods.

1. ZXCzC
Development and strategies of
cell-culture technology for
influenza vaccine

2. CONTENTS
 Introduction
 Development of cell lines
 Bioreactor for vaccine production
 Reasons for low yield cell-culture
 Strategies for improvement of cell-culture virus yield
i) Manipulation of cell line
ii) Manipulation of virus strains
iii) Optimization of infection condition
iv) Optimization of culture condition and cell survival rate
 Conclusion

3. Introduction
 Influenza virus belong to Orthomyxoviridae family of RNA viruses can cause
epidemic respiratory illness in humans and animals.
 They are grouped into type A, B and C on the basis of their Nucleoprotein (NP)
and Matrix protein (M1).
 The viral proteins are Hemagglutinin (HA), Nuraminidase (NA), Ion-channel
M2, Polymerase protein (PA) and NS1 are act as vaccine antigens.
 Three types of vaccines are Virus-like particles (VLPs), Recombinant virus-
vectored and virosome vaccines are commercially produced in allantoic cavity of
embryonated hen’s eggs.
 Vaccine production in Eggs is labor intensive, low yield and unpurified vaccines
can cause fever and pathogenicity. To overcome this problems, cell-culture systems
were established to produce vaccines.
 Advantages of cell cultured influenza vaccines over egg-based manufacture are
standard preparation technology, faster and easier scale-up for production and
higher initial purity.

4. Development of cell lines
 Suitable cells lines for the replication of influenza viruses are Hamster
kidney cells (BHK21), Human embryonic retinal cells (PER.C6), African green
monkey kidney (Vero) cells and Madin-Darby canine kidney (MDCK) cells were
used.
 Only MDCK and PER.C6 cells used for large scale production in bioreactor
systems.
 MDCK cells which transfected with human siat7e gene resulting 10 times
over expression viral protein Hemeglutinin (HA) in shake flasks with higher cell
concentration (7x 105 cells/ml).
 MDCK cells are anchorage dependent, requires surface adhesion and
external substrates (serum) to proliferate. This problems can be solved by
using micro-carrier and scale up the production in suspension culture in
bioreactors.
 PER.C6 cells are anchorage independent and produce 20 times more HA
protein with 1.0x107cells/ml cells density in absence of serum.

5. Bioreactor for vaccines production
 Batch Culture systems:-
Oxygen is added and nutrients
are not added after cultivation.
 Fed Batch systems:-
Oxygen and nutrients are
monitored and added during
cultivation.
 Perfusion systems:-
Nutrient and waste products
are monitored, controlled and
removed.
 Micro-carrier:-
For adherent cells and having
highest surface-to-volume ratio
for high scale production.

6. Reasons for low viral yield in cell-culture
 The influenza viruses extensively manipulate host-cell function to support
their replication through many different ways.
 The host cells can cause significant changes after influenza virus infection
such as cellular mRNA and protein synthesis, morphology, metabolic changes
and apoptosis.
 Low titer of cell cultured influenza caused by low cells density and cell
death.
 Cells culture grown limited due to cells contact inhibition with micro carrier.
May be the viral strain could not grow well in cell culture, low virus replication
rate,
 Many cause in bioreactor such as mechanical agitation, nutrient limitation
accumulation of inhibiting factors, temperature sensitivity and replication
cycle.
 Some extracellular inhibitors like trypsin , ammoniun and cloride inhibites
the viral replication.

7. Strategies for improvement cell-cultured virus yields
 Manipulation of cell line:-
 The host cells able to become susceptible and propagate influenza virus and
having high growth rate and cells density.
 The new cell lines must proliferate at high rate in suspension cultures as
well as in media which is free of animal derived components and should release
high virus titers.
 Manipulation of virus strains:-
 Screen the replicated virus strain which are suitable for vaccine production
and new viral strain can be produced by reverse genetics techniques.
 Isolation of high-yield producing strains from the mixed infection and the
new strains developed to increase the specific replication rate of the influenza
virus.
 The new strain have been specifically adapted to grow in cell culture and
they are selected again because of their ability to grow on specific high yield
cell lines.

8. Strategies for improvement cell-cultured virus yields
 Optimization of infection condition:-
 The trypsin and serine proteases were added into medium resulting cleavege
the precursor protein of hemagglutinin into active hemagglutinin. Only cleaved
hemagglutinin can leads to adsorption of the influenza virus on cells surface.
 The volume of growth medium is reduced prior to infection and to allow the
adsorption of virus to the cells in a minimum of culture volume and better virus to
cell ratio.
 Optimization of culture condition and cell survival rate:-
 Physical and chemical culture condition for large scale production such as
inoculums condition, medium component, cell metabolites, inhibiting
factors, oxygen supply and initial cell culture medium.
 Controlling glucose concentration and production of lactic acid and ammonia.
 In bioreactor, mechanical agitation, non-ionic surfactant, aeration and liquid
oxygen vectors.

9. Conclusion
 Virus production in animal cells is the hub of industrial scale production.
There is a tremendous need for process development and optimization, also
including downstream process.