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Development and strategies of cell-culture technology for

influenza vaccine

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 lineii) Manipulation of virus strainsiii) Optimization of infection conditioniv) Optimization of culture condition and cell survival rate

Conclusion

Introduction

Influenza virus belong to Orthomyxoviridae family of RNA viruses can causeepidemic 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-channelM2, 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 ofembryonated hen’s eggs.

Vaccine production in Eggs is labor intensive, low yield and unpurified vaccinescan cause fever and pathogenicity. To overcome this problems, cell-culture systemswere established to produce vaccines.

Advantages of cell cultured influenza vaccines over egg-based manufacture arestandard preparation technology, faster and easier scale-up for production andhigher initial purity.

Development of cell lines

Suitable cells lines for the replication of influenza viruses are Hamsterkidney cells (BHK21), Human embryonic retinal cells (PER.C6), African greenmonkey kidney (Vero) cells and Madin-Darby canine kidney (MDCK) cells wereused.

Only MDCK and PER.C6 cells used for large scale production in bioreactorsystems.

MDCK cells which transfected with human siat7e gene resulting 10 timesover expression viral protein Hemeglutinin (HA) in shake flasks with higher cellconcentration (7x 105 cells/ml).

MDCK cells are anchorage dependent, requires surface adhesion andexternal substrates (serum) to proliferate. This problems can be solved byusing micro-carrier and scale up the production in suspension culture inbioreactors.

PER.C6 cells are anchorage independent and produce 20 times more HAprotein with 1.0x107cells/ml cells density in absence of serum.

Bioreactor for vaccines production

Batch Culture systems:-Oxygen is added and nutrientsare not added after cultivation.

Fed Batch systems:-Oxygen and nutrients aremonitored and added duringcultivation.

Perfusion systems:-Nutrient and waste productsare monitored, controlled andremoved.

Micro-carrier:-For adherent cells and havinghighest surface-to-volume ratiofor high scale production.

Reasons for low viral yield in cell-culture

The influenza viruses extensively manipulate host-cell function to supporttheir replication through many different ways.

The host cells can cause significant changes after influenza virus infectionsuch as cellular mRNA and protein synthesis, morphology, metabolic changesand apoptosis.

Low titer of cell cultured influenza caused by low cells density and celldeath.

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 replicationrate,

Many cause in bioreactor such as mechanical agitation, nutrient limitationaccumulation of inhibiting factors, temperature sensitivity and replicationcycle.

Some extracellular inhibitors like trypsin , ammoniun and cloride inhibitesthe viral replication.

Strategies for improvement cell-cultured virus yields

Manipulation of cell line:-

The host cells able to become susceptible and propagate influenza virus andhaving high growth rate and cells density.

The new cell lines must proliferate at high rate in suspension cultures aswell as in media which is free of animal derived components and should releasehigh virus titers.

Manipulation of virus strains:-

Screen the replicated virus strain which are suitable for vaccine productionand new viral strain can be produced by reverse genetics techniques.

Isolation of high-yield producing strains from the mixed infection and thenew strains developed to increase the specific replication rate of the influenzavirus.

The new strain have been specifically adapted to grow in cell culture andthey are selected again because of their ability to grow on specific high yieldcell lines.

Strategies for improvement cell-cultured virus yields

Optimization of infection condition:-

The trypsin and serine proteases were added into medium resulting cleavegethe precursor protein of hemagglutinin into active hemagglutinin. Only cleavedhemagglutinin can leads to adsorption of the influenza virus on cells surface.

The volume of growth medium is reduced prior to infection and to allow theadsorption of virus to the cells in a minimum of culture volume and better virus tocell ratio.

Optimization of culture condition and cell survival rate:-

Physical and chemical culture condition for large scale production such asinoculums condition, medium component, cell metabolites, inhibitingfactors, 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 liquidoxygen vectors.

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.