Inactivated Polio

Vaccine (IPV)
By: Paul Brogee, Julia Manalil, Vikshit Girish Patel, Leila
Emami Taba, and Arpit Dharambhai Thumar
February 10, 2016
ChE 660
M. Aucoin

Poliovirus

1,2,3

Family:
Genus: Enterovirus

Picornaviridae

Genome:
+
single-stranded
RNA
Capsid: Composed of 60 heterotetramers
(VP1-4), arranged in icosahedral symmetry,
approximately 30 nm in diameter.
Envelope: None
Serotypes:
3
(PV1,
Entry: Via CD155 receptor
Transmission:
Reservoir:

PV2,

PV3)

Fecal-Oral
Humans

Figure 1: Radial depth rendering

of poliovirus capsid.

Poliomyelitis

Poliovirus typically infects the pharynx and GItract, resulting in asymptomatic or minor illness
(e.g., sore throat, fever)
Aseptic meningitis and paralytic poliomyelitis
occur when poliovirus spreads to motor
neurons and the central nervous system (CNS).
Cases are then divided into spinal, bulbar, and
spinobulbar classes based on localization of
infection.
Paralytic poliomyelitis results in death in 15-30%
of adult cases, and 2-5% in juveniles. Death is
caused by asphyxiation due to paralysis of the
respiratory system.

Figure
2:
Distribution
of
resultant
presentations due to poliovirus infection in
humans.

Epidemiology

4,5,6

Table 1: Cases of poliomyelitis in

2015

As of 2015, wild-type poliovirus is endemic only to

Afghanistan and Pakistan. Six other countries continue to
report instances of circulating-vaccine-derived-poliovirus
(cVDPV) infection, stemming from oral polio vaccine.

Country

Wild-type
Cases

cVDPV
Cases

Afghanista
n

19

The last reported cases of poliomyelitis caused by PV2 and

PV3 were in 1999 and 2012 respectively. PV2 was declared
eradicated in 2015 by the Global Polio Eradication Initiative.
All wild-type poliovirus infections in 2015 were due to the
PV1 serotype.

Guinea

Lao

Madagasca
r

10

Myanmar

Nigeria

Pakistan

53

Ukraine

Inactivated Polio Vaccine vs.

Oral Polio Vaccine 4,7

There are two forms of the polio vaccine: Inactivated Polio Vaccine (IPV) developed by Jonas Salk in
1952 and Oral Polio Vaccine (OPV) developed by Albert Sabin
Inactivated Polio Vaccine

Oral Polio Vaccine

Contains inactivated (killed) poliovirus.

Contains attenuated (live) poliovirus

Administered via subcutaneous or

intramuscular injection.

Administered orally.

Trivalent (PV1, PV2, PV3)

Contains 2-phenoxyethanol for preservative
effects.
Immunogenicity:
90% after 2 doses

Trivalent (PV1, PV2, PV3)

No preservatives.
Immunogenicity:
50% after 1 does
95% after 3 doses

IPV Components and

Characteristics 8,9

In Canada, there are two manufacturers of IPV, Sanofi Pasteur and GlaxoSmithKline.
Trivalent IPV is a clear, colourless liquid vaccine. It is distributed in single doses as prefilled sterile
syringes, or in sterile vials for 5 and 10 dose presentations.
Each 0.5ml dose contains the following components:
3 serotypes of inactivated poliovirus:
Type 1 (Mahoney) - 40 D-antigen units
Type 2 (MEF1) - 8 D-antigen units
Type 3 (Saukett) - 32 D-antigen units
M-199 media
2-phenoxyethanol (0.5%)
Trace amounts of:
Formalin (<0.02%)
Neomycin (<5ng)
Streptomycin (<200ng)
Polymyxin B (<25ng)
Residual calf bovine serum (<50ng)

IPV Production Platform: Cell

Lines 10,11
Two primate cell lines are used for viral production of Canadian
vaccines: Vero cells and MRC-5 cells. (Table 2)
Table 2: Summary of Vero and MRC-5 Cell Lines
Cell
Line

MRC-5

Vero

Source

Human
male lung
fibroblast
cells

African green
monkey
kidney
epithelial
cells

Media

CMRL 1969
medium w/
bovine calf
serum

EMEM
medium w/
bovine calf
serum

Sanofi

Td Polio

Imovax Polio

Figure 3: Confocal image of

Vero cells stained with DAPI
(DNA stain) and CytoPainter
(F-actin stain).

Step 1: Cell Culture

8,12,13

1. Cells (Vero or MRC-5) are adhered to

DEAE dextran microcarriers (CYTODEX)
and are cultured in the appropriate
media
(EMEM
or
CMRL
1969,
respectively) supplemented with bovine
calf serum.
2. The culture is grown in large scale
bioreactors (1000 L) for several days to
obtain a confluent culture.
3. Optimal culturing conditions are:
37 C, pH 7.4, 10% dissolved oxygen,
with a stirring speed of 30 RPM.
Figure 4: Cross-sectional illustration of
bioreactor for culture of Vero/MRC-5 cells

Step 2: Virus Production

1. Serum-containing media is removed and
replaced with M 199 media.
2. A strain of poliovirus (Mahoney Type 1, MEF1 Type 2, or Saukett Type 3) is used to infect
the culture. The virus is allowed to adsorb to
the cells for half an hour, while maintaining a
stirring speed of 30 RPM
Due to the biosafety risk associated with working with live
poliovirus, high containment measures (Biosafety Level 3) are
required
2. Viral production is conducted at 37C, pH 7.4, 10% dissolved
oxygen, with a stirring speed of 40 rpm for 72 hours
3. Stirring is stopped and the virus is harvested by withdrawing the
culture medium constituting the viral suspension

12,13,14

Step 3: Clarification

12,14,15

Once the resulting viral suspension has been harvested, the slurry is thinned, and excess
cellular debris is removed.
1. Harvested viral suspensions are filtered through a 75 m stainless steel sieve to remove
any microcarriers, followed by 0.45 and 0.22 m combination filtration. Sieves are pretreated with Celite, to improve filter performance.
2. Filtration units operate at set point for both inlet and outlet pressure, as well as retentate
cross flow rate, temperature, concentration and filter load.
3. Following each batch, units are flushed with 3-4 mL of fresh M199 medium to maintain
filter efficacy.

Step 4: Concentration

12,14,15,16

1. The product is concentrated ~1000x using tangential

flow filtration (TFF) via two consecutive ultrafiltration
(UF) steps.
2. For steady operation, the transmembrane pressure
difference in each filter is set to a maximum of 0.6
bar.
3. The observed recovery is a function of the filter
media, system feed flow, residence time, and the
quality of the starting material.
Figure 5: Tangential flow membrane
filter (TFF)

Step 5: Purification

12,13,14,15

1. Size Exclusion Chromatography (SEC) is performed using a column, ~ 80 cm in height,

containing separately sterilized CL-6B matrix.
2. A column, 22 cm in height, containing positively charged DEAE sepharose fast flow
matrix is utilized for Ion Exchange Chromatography (IEX).
3. For both SEC and IEX, the virus is eluted using an AKTAexplorer system (GE Healthcare)
with a 40 mM phosphate buffer (pH 7.0).
4. Critical parameters for chromatography are:
a. Bed height and linear flow rates
b. Column loading (mL product/L resin)
c. Temperature
d. pH and conductivity
e. Gradient height

Step 6: Inactivation

8,12,14,17

Thus far, the poliovirus has remained in a virulent state. To be

safely implemented as a vaccine, it must be chemically
inactivated. The inactivation process kills the virus while
retaining antigenic surface markers to elicit an immune
response. This process proceeds as follows:
1. The viral suspension is filtered using a 0.22 m cellulose
ester membrane.
2. Formaldehyde is added to a final concentration of 0.025%
and incubated at 37 C, with steady stirring for several
days.
3. On the 6th day, the viral suspension is filtered using a 0.22
m cellulose ester membrane and the inactivation is
continued at 37 C
4. After 12 days, it is homogenized and stored at 4 C

Figure 6: Formaldehyde

Step 7: Preparation of Final

Dosage 8,12,14

1. After purification and inactivation, monovalent suspensions from each of the three
poliovirus serotypes are mixed to produce a trivalent solution. The mixture is a 5:1:4 ratio
of PV1, PV2, and PV3, respectively.
2. The final trivalent mixture is homogenized and filtered using a 0.22 m cellulose ester
membrane.
3. After passing quality assurance, it is diluted and filled in vials for a final concentration of
(per 0.5 ml dose):
a. Type 1 (Mahoney) - 40 D-antigen units
b. Type 2 (MEF1) - 8 D-antigen units
c. Type 3 (Saukett) - 32 D-antigen units

Step 8: Packaging &

Distribution 18

Vaccines are classified for packaging on the basis of their thermostability and presentation.
IPV is designated as Class A Packing which requires temperatures to not exceed +8 C.
Temperature monitoring devices are included in all vaccine shipments to document whether
temperature limits have been exceeded.
The storage volume of the vaccine varies according to the type of vaccine, the number of
doses per vial, and the dimensions of the vial and secondary packaging.
Labeling is done for Primary Packaging (first level of container for the vaccine), Secondary
packaging (intermediate packaging that contains the primary packages) and Tertiary
packaging (the shipping container that contains the secondary packages)
Standard shipping and arrival procedures depend on the country of destination.

Approaches to Achieve an
Affordable IPV 13

One proof of concept study aimed to make IPV production more affordable by using the OPV Sabin strains but
processing them similar to IPV (sIPV). This was in an effort to reduce the biosafety risk of manufacturing wild polio,
ultimately lowering the cost of manufacturing and allowing it to be a process that can be conducted in middle- to lowincome countries. Below is a summary of the recovery of virus at each step in the manufacturing process. Low yields
for Sabin type 2 were observed, bringing into question whether this would be a competitive method compared to the
current methods. Nevertheless, studies are continuing down this avenue and products worthy of clinical trials are
being tested.

Approaches to Achieve an
Affordable IPV 19

Reduce Number of IPV Doses - Studies show new evidence that if 1 or 2 doses are sufficiently
immunogenic, the number of doses in an IPV schedule could be reduced. Research is being conducted
on whether a single dose of IPV can prime the immune system against poliovirus.
Intradermal Administration - Intradermal injection provides improved immune response as
compared to intramuscular injection, and thus requires a smaller quantity of vaccine. It may also be
administered by a non-medically trained person.
Adjuvants- IPV production cost can be reduced by the use of adjuvant to enhance immunogenicity. A
number of research groups have evaluated traditional adjuvants such as aluminium,
oligodeoxynucleotides and Vitamin D3 as being useful in reducing the magnitude of required
antigens.
Optimization of Production Processes The development of IPV from safer poliovirus strains and
noninfectious methods of production have become a priority. In addition, producers in developing
countries can take advantage of specific optimization techniques for current IPV production and can
further reduce costs.

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