Vaccine

1. FMD Vaccine Manufacture:
Controls and Constraints
Tim & Claudia Doel
Hanoi, October 2015
Global Foot and Mouth Disease Research Alliance
Hanoi, Vietnam
20-22 October, 2015

2. Compliance
 Most vaccine manufacturers must comply
with national or international standards of
manufacture (GMP) and submit a product
dossier to Regulators before it can be sold.
 Additionally, there may be mandatory
requirements in pharmacopoeias of which
the European Pharmacopoeia Monograph
on FMD Vaccines is particularly important.
Hanoi, October 2015
GMP
Certificate
For Premises Located at 1
Penrose Way, Glasgow,
Scotland. Dated 05 October.
2015

3. Compliance Costs!!!
• Under full GMP, every employee will commit
some time to GMP and QC/QA staff may
represent 20% or more of the total
headcount.
• Perversely, Compliance requirements can
inhibit potential product improvements
because of the time and costs required to
conduct and gain approval for a revised
product dossier.
Hanoi, October 2015
GMP
Certificate
For Premises Located at 1
Penrose Way, Glasgow,
Scotland. Dated 05 October.
2015

4. New Product Registration
 Changes to media, inactivation method,
downstream processing, adjuvant/delivery systems
etc require major work including safety and efficacy
of dose, overdose and repeated dose in the target
species, including specific claims relating to
pregnant animals, juveniles etc.
 A few Regulators have argued that even a change of
virus strain should prompt such studies with
particular reference to efficacy!
Hanoi, October 2015

5. Media
 Most production media will be based on MEM,
GMEM, DMEM etc and supplemented with a
protein hydrolysate, antibiotics, some
miscellaneous additives and bovine serum.
 Because of TSEs in particular,
media components of
animal origin attract special
attention from Regulators.
Hanoi, October 2015

6. Media
 Bovine serum is expensive. In Europe, it must be
sourced from a minimal risk TSE country (notably
NZ) using the supplier(s) registered in the dossier.
Each batch must be tested for freedom from
general and specified adventitious agents before
being gamma irradiated.
 It is necessary to prove the serum is of bovine
origin – Adulteration in transit of high value NZ
serum with human plasma (ex-blood banks) was
reported many years ago in the UK.
Hanoi, October 2015

7. Cells
• Cell production requires a 2-3 day growth cycle and
the conditions used are also optimal for bacterial
contaminants. In Europe, strong antibiotic cover
using Penicillin and Streptomycin is not permitted.
• After 3 days, cell numbers in the range 2 - 6 million
per ml can be achieved. Many years ago, titres of up
to 9 million cells per ml were achieved but there
seemed to be no additional virus productivity
above 3 million cells per ml.
Hanoi, October 2015

8. Cells
• While all BHK cells originated from Glasgow and,
later, Pirbright in the case of the suspension line,
virus growth in different lineages varies
significantly (panel A).
• In the 1970s, Clarke derived a range of cell lines of
which the AC9 lineage is reputed to give high virus
yields (panel B).
Hanoi, October 2015

9. Hanoi, October 2015
0
20
40
60
80
100
120
140
160
O BFS 1860 C Noville Asia1 Iran 1/73
Pirbright
Brescia
Lelystad
Lindholm
Wellcome Spain
PANAFTOSA
CF
Titre
0
20
40
60
80
100
120
140
160
O BFS 1860 C Noville Asia1 Iran 1/73
AB8
AC9
AE11
AF8
CD11
CD12
Clarke and Spier, 1979
Panel A
Panel B

10. Virus
• Growth of established, ‘classical’ strains is generally
straightforward.
• New vaccine strains are not always so easy. To increase the
chances of obtaining a high yielding vaccine strain with
good stability and able to confer relevant protection, a
comprehensive selection of field isolates will be the
minimum requirement.
• Regardless, a new vaccine virus will need full, lengthy and
costly testing to ensure freedom from adventitious agents!
Hanoi, October 2015

11. Virus
• In the case of an emergency situation, a position
paper by the European Medicines Evaluation Agency
does allow ‘fast-tracking’ using risk assessment to
rule out at least a substantial number of potentially
contaminating viruses.
• Fortunately, FMDV is resistant to organic solvents
such as ether and chloroform whereas many
potential contaminants are not and can be excluded
from the testing process.
Hanoi, October 2015

12. Hanoi, October 2015
Source:
Position
Paper on
FMD
Vaccines,
2004
EMEA
Committee
for
Medicinal
Products
for
Veterinary
Use.

13. Virus – Matching the Vaccine Strain with
the Field Strain
• How closely should the vaccine strain match
with the prevalent field strain?
• Should the serological relationship between
the viruses be the deciding factor?
• What about characteristics such as stability
and immunodominance (whatever that
means!)?
Hanoi, October 2015

14. Hanoi, October 2015
Vaccination Points (Months)
0 6 12
Estimated
Percentage
Protection
99
95
70
Adapted from Pay, 1994
r = 1.0
1
r = 0.4
1
r = 0.1
1
Adapted from Pay, 1994
1

15. Virus – Matching the Vaccine Strain with
the Field Strain
• While the relationship between the two
viruses is clearly important, it is worthwhile
reflecting that many of the popular and
effective current vaccine strains have been
used successfully for decades (eg A24 Cruzeiro,
O Campos, Asia1 Shamir, O Manisa, A22 Iraq).
• Whereas, there has been a tendency for new
strains to come and go quite quickly (Iran
96/99/2001).
Hanoi, October 2015

16. Hanoi, October 2015
146S Analysis
A critical test which will be used for yield analysis as well as
vaccine payload decisions
 Our experience is that it is quite common
to find deficiencies in the method
including the calibration of the
spectrophotometer.
 This doesn’t really matter as long as the
deficiencies are constant over a period of
years. Payload and yield trends will still be
(internally) meaningful.
 Naturally, it matters greatly if the method
is variable from day to day.
 Extreme caution is needed if a customer
decides to compare the payloads claimed
by different manufacturers.

17. Hanoi, October 2015 Doel and Mowat, 1985

18. Hanoi, October 2015
Inactivation
A critical stage of FMD Vaccine Manufacture.
 Many manufacturers favour a
two dose, two tank BEI
process to ensure full
inactivation.
 In fact, the European
Pharmacopoeia only requires
that the inactivation kinetics
are linear and there must be
less than 1 infectious particle
per 10,000 litres bulk virus at
the end* of the inactivation
process .
Log
10
Virus
Titre
Hours of Inactivation
For the first dose of BEI, complete
inactivation is generally required by 67%
of the inactivation period. Eg 16 hours
assuming the first cycle is 24 hours.

19. Hanoi, October 2015
Inactivation
A critical stage of FMD Vaccine Manufacture.
 Failure to get a statistically acceptable regression line means
one thing for the antigen batch!!!

20. Virus Before AEI* After AEI* % Loss 146S
A24 Cruzeiro 1.7 1.8 0
A27 3.5 3 14.3
A27 2.3 2.2 4.4
O Campos 3.2 (216) 1.3 (190) 59.4
O Campos 0.8 0.1 87.5
O 9489 1.8 0.2 88.9
O 9489 1.9 0.3 84.2
Instability of 146S During Inactivation with AEI
*Production samples. Same viruses grown and inactivated at Pirbright did
not show significant breakdown. High instability of the Colombian O
preparations was never explained, but was not due to pH.
Gomez and Doel, 1984
Hanoi, October 2015

21. Downstream Processing
Many manufacturers concentrate and partially
purify their bulk antigens.
• Historically, one of the aims was to reduce allergic
responses of animals to non-viral proteins.
• Semi-purified concentrates also opened the door for
antigen banks as well as giving significant commercial
and manufacturing flexibility for some companies.
• Increasingly, however, there is a major perceived benefit
in removing NS proteins in order to minimise NS
antibody responses.
Hanoi, October 2015

22. Downstream Processing
How is it done?
• Three basic methods used individually or in
combination.
1. Ultrafiltration. Primarily a method of concentration but
will remove molecules up to 1 million daltons depending on
the filtration cartridge.
2. Gel Permeation Chromatography. Used only by one
manufacturer to our knowledge.
3. PEG Precipitation. An old but very widely used and
proven method. By cycles of different PEG concentrations,
it is possible to get very high levels of purification.
Hanoi, October 2015

23. Downstream Processing
Hanoi, October 2015
1. In some countries,
manufacturers are required
to purify antigens such that
NS antibodies are not
induced.
2. Certainly, it will reduce the
possibility of adverse
reactions in livestock.
3. Concentrates can be stored
almost indefinitely in freezers
providing commercial and
manufacturing flexibility and
strategy.
1. Most probably, only a modest
level of purification is necessary.
Garland showed many years ago
that even crude vaccines did not
induce anti-3D (VIAA) except after
three doses.
2. High levels of purification can
significantly reduce yields. This
can mean only 50 or 60% of the
original culture yield is recovered.
3. It is absolutely vital to quickly
purify and store the antigens if
proteolysis of the VP1 is to be
avoided/minimised.

24. Antibody Titres of Vaccinated Guinea Pigs
Vaccine
Virus
Control Trypsin Treatment
Cells Mice Cells Mice
OBFS 2.4 6.4 1.5 1.1
A24 2.6 6.5 1.8 6.1
C 2 5.9 1.7 6
Asia1 2.4 4.9 2.1 4.4
SAT1 2 3.4 1.7 2.4
SAT2 2.4 0.9 1.5 0
SAT3 2.1 3.9 2 3.6
Proteolytic Cleavage of the VP1 of O and SAT2 Reduces
Neutralising Antibody Titres by over 90%
Doel and Collen, 1982
Hanoi, October 2015

25. ‘Innocuity’ Testing
Hanoi, October 2015
•Another critical test is the demonstration that
the inactivated antigen contains no infectious
virus.
•The European Pharmacopoeia requires that it is
necessary to inoculate and passage not less than
200 dose equivalents of antigen in a sensitive
cell line and demonstrate absence of CPE in all of
the passages.
•This test is far more sensitive than animal
testing.

26. Vaccine Design
Hanoi, October 2015
• A fundamental complication is that FMD vaccines are
expected to cover all scenarios. This invites the question
of whether formulations should/could be developed to
suit the ages and species of animals as well as livestock
systems, with particular reference to pigs.
• 1. With short-lived animals, the vaccine needs to induce
rapid immunity, often in the face of maternal antibodies,
and protective immunity is required for a relatively short
time. Duration of immunity is less important and immune
memory probably irrelevant.
• 2. With longer-lived animals, immune memory and
duration of immunity are essential.

27. Vaccine Design
Hanoi, October 2015
Possible Approaches:
•Higher payload primary dose vaccines designed to
establish a good primary immune response and,
possibly, overwhelm the effect of maternal antibodies.
•Secondary dose vaccines with lower payloads to
ensure boosting and development of immune
memory.
•All of this would require extensive animal studies + a
new product dossier + full cooperation between the
vaccine supplier and customer to ensure product was
used correctly in the appropriate animals.

28. Vaccine Design
Hanoi, October 2015
•Manufacturers will use their 146S assays to routinely
determine the payloads of the antigen to be used in
the vaccine.
•A perception that very high payloads are desirable
for general vaccine efficacy is not supported either by
experimental work (Rweyemamu) or from a
fundamental immunological perspective.

29. Hanoi, October 2015
Effect of Antigen Payload on Antibody Titre
7 14 21 28 60 90 120
0.5
1
1.5
2
2.5
3
3.5
Days Post Vaccination
Log
Neutralising
Antibody
Titre
330 ug
55 ug
9.2 ug
1.5 ug
0.25 ug
0.04 ug
0.007 ug
ug 146S
Rweyemamu et al, 1984

30. Vaccine Design
Hanoi, October 2015
• With some serotypes, there are perceived benefits in using a
mixture of strains:
• When a new field strain emerges and is recommended for
use, it is prudent to use a mixture of the current and new
vaccine strains for at least a year until it is clear that the
current strain is no longer as relevant and the new strain
remains appropriate.
• In fact, it is not uncommon to use a mixture of related
vaccine strains to broaden the serological response and
cover more effectively the population of field viruses.
Don’t throw the
baby out with
the bathwater

31. Hanoi, October 2015
0
0.5
1
1.5
2
2.5
3
1 2 3-4 5-6 7-9 10 or More
O Lausanne (homologous)
O Manisa
O Campos
Dubourget et al, 1987
Repeated Vaccination Doesn’t Fundamentally Change the Heterologous r1
Relationship Between a Vaccine Strain and Other Related Strains.
VN
Titre
Number of Vaccinations

32. Thank You for Your Attention
Hanoi, October 2015