Role of Cell Culture Technology

in new Vaccine Development

Asst. Prof. Dr.Kanokwan Poomputsa

School of Bioresources and Technology

King Mongkut’s University of Technology Thonburi

KMUTT
 Outline

• Introduction to cell culture

• Cell culture and vaccine production
– Cells as virus host
• JE vaccine, Influenza vaccine, Rabies vaccine
– Cells as protein factory
• Influenza vaccine (protein, VLP)
– Cells as antigen presenter
• Cancer vaccine (personalized vaccine)

KMUTT
 Cell Culture

Process by which animal cells are grown

under controlled laboratory conditions.

KMUTT
 Maintaining cell culture

Basic environmental requirements

• Controlled Temperature
• Appropriate medium that maintains the
correct pH and osmolarity
• Good substrate for cell attachment

KMUTT
Animal cell culture media is
complex

• Animal is made up of hundreds of different cell

types, each specialized to perform one or a few
functions.
• Different cell types synthesize many different
molecules needed, and these are secreted into
the extracellular fluid and are circulated by the
blood throughout the body.
• The mammalian cell medium is an attempt to
recreate this extracellular fluid.

KMUTT
Growth factors are present in cell media

KMUTT
 Culture Medium
Culture medium should provide;
– basic nutrition requirements;
– Amino acids, vitamins, minerals and
carbohydrates which cells used to build new
proteins and other components and provide
the energy necessary for metabolism
– Necessary growth factors and hormones
– 5-20% of animal blood such as calf serum
– Help to regulate and control the cell’s growth
rate and functional characteristics.
– Regulate the pH and osmolarity
– The medium control the pH range and buffers
the cells from abrupt changes in pH.

KMUTT
 Cell Culture
Removal of cells, tissue or organ from an animal
or plant and their subsequent placement into
an artificial environment conducive to growth.
• Organ Culture/tissue culture
A three dimensional culture of undisaggregated
tissue retaining some or all of the features of
the tissue in vivo.
• Cell Culture
Single cells, no longer organised as tissues.
Derived from dispersed cells taken from the
original tissue.

KMUTT
 Isolation of cells
There are 2 basic methods;
• Explant Cultures : small pieces of tissues are
attached to a glass or treated plastic culture vessel
and bathed in culture medium. After a few days,
individual cells will move from the tissue explant
out onto the culture vessel surface or substrate
where they will begin to divide and grow.

• Enzymatic Dissociation : addition of proteolytic

enzyme e.g. Trypsin or collagenases, to the tissue
fragment to dissolve the cement holding the cell
together. This create a suspension of single cells.

KMUTT
 Explant Enzymatic Dissociation

Human foreskin explants

in Culture dish (2 of 9
explants failed to grow)

1 week old
Poeciliopsis lucida
cells from embryo

KMUTT
 Cell culture
(Primary cell VS Continuous cell)

‰ When cells are surgically removed from an organism and

placed into a suitable culture environment, they will attach,
divide and grow.

‰ Most primary cell cultures have limited lifespan. After a

certain number of population, doubling cells undergo the
process of senescence and stop dividing, while generally
retaining viability.

‰ Some cells may become immortal, continue to divide

indefinitely, and are called continuous cell lines. They
have undergone a fundamental irreversible changes or
transformation.

KMUTT
 Continuous Cell Lines
An established or immortalized cell line has
acquired the ability to proliferate indefinitely
through;
– Spontaneously modification such as
random mutation by drug, radiation or
viruses.
– Deliberate modification such as artificial
expression of the telomerase gene.

KMUTT
 Continuous cell lines

• CHO (Chinese hamster ovary) cells

• Vero (Monkey kidney) cells
• MDCK (canine) cells
• BHK (Baby hamster kidney) cells
• NSO (mouse myeloma) cells
• HEK 293 (human embryonic kidney) cells
• PER.C6 (human retinoblasts) cells
• MRC-5 (fetal lung) cells

KMUTT
 Cell Culture Systems
• Monolayer Culture Systems
• Adherent cells or Anchorage-
Dependent cells
• cells attach to a glass or
treated plastic substrate
• Cell growth is limited by
available surface area on which
cells can grow

• Suspension Culture Systems

• Anchorage-Independent
- Cells grow floating free in
suspension

KMUTT
Monolayer Culture Systems

Dishes T-flaskes

Roller bottles Microcarrier

KMUTT
Suspension Culture Systems

Erlenmayer flasks Spinner vessels

KMUTT
 Types of Cells
• Epithelial-like :
• Cells that are attached to a
substrate and appear flattened
and polygonal in shape.

• Lymphoblast-like :
• Cells that do not attach
normally to a substrate but
remain in suspension with a
spherical shape.

• Fibroblast-like
• Cells that are attached to a
substrate and appear elongated
and bipolar, frequently forming
swirls in heavy cultures.

KMUTT
 Cell Culture and Vaccine
Manufacturing

• Control of product properties e.g. glycosylation

• Cost efficient manufacturing process –
especially for high volume products
• Speed in development, especially for early
phase

KMUTT
 Cell cultures can be scaled-up for
biomanufacturing using bioreactors

KMUTT
Cell Culture and Vaccine Production

•Viral vaccines
Cultivation of virus for vaccine production e.g.
polio, rabies, chicken pox, hepatitis B & measles

• Recombinant protein vaccines

Production of recombinant proteins from animal
cells using different expression system

KMUTT
 Viral Vaccine Production

• Cell-Culture-Based Vaccine Production

‰ virus is propagated in animal cells and
harvested
‰ downstream processing parameters for
purification
‰ filling, and packaging of the vaccine

cells
Infection
with seed virus Splitting, inactivation and purification

KMUTT
 Cells for virus production

Propagation of cells
Virus-
Infection

Seed Preculture
Virus Propagation
cells

Production
fermenter

Cell Viral Particles

 Vaccine Production Plant

Fermenter 4

Virus
Production

Fermenter 2 Fermenter 1
Fermenter 3 Cell propagation
Cell propagation Cell propagation
High Cell Density

KMUTT
 Purification

Virus-
Infection Virus Propagation

Seed Preculture
cells Harvest

Centrifuge
Ultrapurification (Cells/Debris)

Ultracentrifugation;
BPL-Inactivation/ Chromatography +
+ Virus splitting Ultra-/Diafiltration
Filter
(small particles)

KMUTT
 Japanese Encephalitis
Vaccines
• JE virus (JEV), a mosquito-borne flavivirus,
is the most common vaccine-preventable
cause of encephalitis in Asia
• Two JE vaccines are licensed in the United
States.
¾ An inactivated mouse brain–derived JE
vaccine (JE-VAX [JE-MB])
¾ An inactivated Vero cell culture-derived
vaccine (IXIARO [JE-VC])

KMUTT
 Japanese Encephalitis
Vaccines
• An inactivated mouse brain–derived JE vaccine [JE-MB])
• It has been licensed since 1992 to prevent JE in persons aged
≥1 year traveling to JE-endemic countries.
• Use of mouse brains as the substrate for virus growth has
raised concerns about the possibility of neurologic side effects
associated with the JE vaccine.
• Moderate to severe neurologic symptoms including
encephalitis, seizures, gait disturbances, and parkinsonism,
have been reported at a rate of 0.1-2 cases per 100,000
vaccinees with variation by country.
• Supplies of this vaccine are limited because production has
ceased.

KMUTT
 Japanese Encephalitis
Vaccines
• An inactivated Vero cell culture-derived vaccine (IXIARO
[JE-VC])

• It was licensed in March 2009 for use in persons aged ≥17

years.

• JE-VC does not contain gelatin or murine proteins, it might be

associated with fewer hypersensitivity or neurologic adverse
events than the mouse brain–derived vaccine.

KMUTT
 Japanese Encephalitis
Vaccines

MMWR Recommendations and Reports March 12, 2010 / Vol. 59 / No. RR-1 KMUTT
 Influenza Vaccines
• Influenza Vaccine Manufacturing Today
• Vast majority (90%) of licensed capacity is in egg-based
products
• Reliable process for seasonal production but the egg-
based process has significant limitations and lacks
flexibility (embryonated eggs require~6 months from
order to delivery)

Egg Lead Time Production Time

~6 Months ~6 Months

No Chickens 1x
No Eggs ~ 1x
No Vaccine
 Influenza Vaccines
• Cell Culture: An alternative to Egg-based
production
‰ No egg lead time involved, production can start
as soon as seed virus is available
‰ Cell culture is a practical alternative
• Industrializable – can be run routinely and cost
effectively
• Established processes can be run at practical scale
to provide sufficient vaccine for interpandemic and
pandemic needs

KMUTT
 Cell-based vaccine production:
critical points
• Cell line
™Virus production
™Regulatory path
™Industrialization capacity
• Growth in suspension or on microcarriers
• Synthetic or complex medium

• Vaccine manufacturing and formulation

™Whole virus, split, subunit vaccines
™Removal of DNA
™Adjuvants to enhance immunogenicity and dose
sparing

KMUTT
 Influenza Vaccines
• Cell lines
• PerC.6 (Crucell)
• EBx™: a stem cell line derived from chicken embryos
(Sigma-Aldrich Group)
• VERO: a kidney cell from the African green monkey
(GSK)
• MDCK : Madin-Darby canine kidney cells used by
Chiron

• Requirements
• Good cell-virus interaction
• Broadly and highly permissive for a wide variety of flu strains
• Restricted growth of non-flu human pathogens that may be
present in the viral seed
• Scalable, industrializable high yield, high volume production
• Cell growth in chemically defined medium (no animal-derived
components)

KMUTT
 Cell-based vaccine production:
regulations
• There must be documentation to support the complete
removal of the cells from the final product and
documentation to show that the cell line does not bring
any transforming agent (oncogenic transformation) into
the final product

• Documentation must show that no genetic material is

left from the cell line in the final product that can cause
tumors to be formed. All residual DNA must be removed
or inactivated so it cannot give rise to tumors in animal
models

• Documentation must show the removal and/or

inactivation of infectious and/or oncogenic agents from
the final product, regardless of whether they originated
in the media or the cell line.

KMUTT
 Influenza Vaccines
• EU approves Novartis's cell-based
flu vaccine
• Jun 13, 2007 (CIDRAP News) – The European
Union (EU) has approved Novartis's seasonal
influenza vaccine, Optaflu, the first flu vaccine
grown in cell culture rather than eggs.
• The vaccine has been approved for use in all 27
EU member states plus Iceland and Norway.

KMUTT
 Rabies Vaccine
• Rabies is a zoonotic disease caused by RNA viruses in the
Family Rhabdoviridae, Genus Lyssavirus.
• Virus is typically present in the saliva of clinically ill
mammals and is transmitted through a bite.
• After entering the central nervous system of the next host,
the virus causes an acute, progressive encephalomyelitis that
is almost always fatal.
• Cell-based rabies vaccines which are licensed in the United
States:
• human diploid cell vaccine, MRC-5 (HDCV, Imovax®
Rabies, sanofi pasteur),
• purified chick embryo cell vaccine, primary cultures of
chicken fibroblasts (PCECV, RabAvert®, Novartis
Vaccines and Diagnostics)

KMUTT
Cell Culture and Vaccine Production

•Viral vaccines
Cultivation of virus for vaccine production e.g.
polio, rabies, chicken pox, hepatitis B & measles

• Recombinant protein vaccines

Production of recombinant proteins from animal
cells using different expression system

KMUTT
 Established & Novel Expression
Established Systems
Systems
Slow SPEED Fast

yeast E. coli
transgenic plant mammalian cells baculovirus
$$$$ COST $

mammalian cells yeast E. coli

transgenic plant baculovirus
Unlikely FDA Approval Likely

transgenic plant baculovirus yeast E. coli mammalian cells

Promising New Platforms

Cell Nucleic
free acid

duckweed crustacean
 Animal Cell Expression System

A. Transient transfection
B. Stable transfection
C. Viral vector-mediated protein expression (e.g. adenovirus, baculovirus)

KMUTT
 Influenza Virus Vaccine
based on the Baculovirus-Insect Cell Expression
System

• FluBlok®
• First recombinant influenza vaccine
• First cell-based influenza vaccine in U.S.
• FDA licensure in 2010
– No additional safety or efficacy studies required

• The pandemic solution

• Only pandemic vaccine that can be quickly
manufactured and/or transferred to and
manufactured in other countries

KMUTT
 HA (Hemagglutinin)

• Coat of the influenza virus

• Antibodies against HA protect against influenza
• Changes in HA require annual update of vaccine

KMUTT
 Recombinant HA protein
from Baculovirus-Insect Cell Expression System
Baculovirus with
influenza gene Days post infection

Recombinant Influenza HA protein

Produced in infected Insect Cells

Purification of recombinant HA Formulate

protein With PBS
To Vaccine

KMUTT
 Safety & Immunogenicity of FluBlok
Potential Benefits
• rHA antigens are produced in insect cells – protein based
vaccine with low endotoxin content
• highly purified and does not contain egg protein or other
contaminants from eggs
• Selection or adaptation of influenza virus strains that
produce at high levels in eggs is not required
• FluBlok can be prepared within 2 months
• FluBlok does not require large amounts of embryonated
chicken eggs
• Manufacturing of FluBlok does not require biocontainment
facilities
• Manufacture of rHA does not include formalin inactivation or
organic extraction procedures

KMUTT
 Pandemic Preparedness
Development Timeline recombinant HA1 vaccine

KMUTT
 Virus-Like Particle
(VLP)-based Influenza Vaccines
• What are VLPs?
• Made of recombinantly-expressed viral proteins that
• spontaneously assemble into 3-D structures similar to
parent virus
• By electron microscopy, VLPs mimic the parent virus
• Immune response is similar to that which would be
seen if exposed to the parent virus
• Do not contain genetic material
• Cannot replicate

KMUTT
How are Influenza VLPs Constructed?

KMUTT
Potential Immunologic Advantages
of Influenza VLPs

KMUTT
 Possible Limitations of Vaccine
Production using cell culture

• Not all infectious agents can be grown in culture

• Animal/human cell culture is expensive
• Yield of viruses from cultures can be low
• Safety precautions for culture of live agents

KMUTT
 Personalized Cancer Vaccine

• Use of material from a patient’s own tumors

to induce immune response
• Cells harvested from patients are fused with immune cells
and expanded in the special instrument--

KMUTT
Antigen-Presenting Cells and cancer
 DC-based cancer vaccine
• Dendritic cells (DC), a leukocyte population, represent unique
antigen-producing cells capable of sensitizing T cells to both
new and recall antigens.
• DCs are the most potent antigen-presenting cells (APS).
• Tumor antigens in different forms (DNA, RNA, proteins,
peptides, viruses, cell lysates) become immunogenic when
presented to T-lymphocytes by DCs.
• Immunization with ex vivo generated DC has proven feasible
and permits the enhancement as well as the dampening of
antigen-specific immune responses in man.
• Several DC-based clinical trials have demonstrated potent
immunological and some clinical responses.

KMUTT
 DC loading and activation

Ex vivo generated dendritic cells(DC) can be loaded with antigens and re-infuse to
the patients, or they can be used for ex vivo expansion of anti-tumor lymphocytes.
 DC loading and activation
• Ex vivo generated dendritic cells(DC) can be loaded with
antigens and re-infuse to the patients, or they can be used
for ex vivo expansion of anti-tumor lymphocytes.

scale up
of patient
Derived cells

KMUTT
 Ex vivo expansion of loaded APCs

AutovaxID

GMP and GTP regulations

 Personalized Cancer Vaccine

• More than 150 DC-based clinical studies for the

treatment of solid or hematological malignancies
have been reported in 2008 including;
• Melanoma (〉 40 published clinical studies)
• prostrate cancer (20)
• Renal cell carcinoma (16)
• Breast cancer (12)
• Multiple myeloma (9)
• Leukemia (9)
• Colorectal cancer (9)
• Glioma (9)

Nencioni, A. et.al. Critical Reviews in Oncology/Hematology 65(2008), 191-199

KMUTT
Role of Cell Culture Technology
in new Vaccine Development

• Host for viral vaccine production

• Factory for gene expression for
vaccine
• recombinant proteins
• VLP
• Personalized vaccine

KMUTT
 Thank you
คณะทรัพยากรชีวภาพและเทคโนโลยี
School of Bioresources and Technology
Bangkhuntien Campus
Established in 1993

www.bioresources.kmutt.ac.th