Iabs Conference 3RS and Consistency Testing in Vaccine Lot Release Testing

IABS CONFERENCE
3RS AND CONSISTENCY TESTING IN VACCINE LOT

RELEASE TESTING
VACCINE LOT RELEASE TESTING
Egmond aan Zee, The Netherlands

September 16 18, 2015
Welcome from the Organisers

We are delighted to welcome you all to the Hotel Zuiderduin in Egmond aan Zee for what will
be a stimulating and successful three days. Many people think that a radical change to the
process of vaccine lot release testing is long overdue. The stepwise approach of replacing
each animal test by a non-animal method is painfully slow and ultimately unsatisfactory when
these new tests are incorporated into an existing framework that emphasises the testing of
the final lot rather than the consistency of production. Over the next few days, we will hear
about and discuss many new non-animal methods since these are still badly needed to
replace several animal tests that are scientifically, economically and ethically inadequate
whether they are used for final lot testing or as in-process controls. But we will also discuss
the concept of the consistency approach and see whether we can at last move from theory to
examples of practice, and, most importantly, to define what will be needed for more general
acceptance and use of the approach.
We have an exceptional list of speakers and panellists and we hope that everyone will
participate openly and honestly in our efforts to deal with this important issue. We look
forward to your arrival and to an excellent meeting.
The Organisers
Coenraad Hendriksen,
Catrina Stirling,
Ian Ragan,
Intravacc, The Netherlands
Zoetis, Uk
NC3Rs, UK
IABS CONFERENCE
'3Rs ALTERNATIVES AND CONSISTENCY TESTING IN VACCINE LOT RELEASE
TESTING'
EGMOND AAN ZEE (THE NETHERLANDS), SEPTEMBER 16 - 18, 2015
WHERE INNOVATION IN VACCINE QUALITY CONTROL MEETS ANIMAL WELFARE.
Vaccine development and quality control include models based on studies in laboratory
animals, substantial numbers of which are used. Increasingly, however, regulations on
animal experimentation and testing now urge the use of non-animal methods, reduction of
animal numbers in tests still being performed and refinement of animal procedures and
animal husbandry practices; the principle which is generally known as the 3Rs.
This need for humane science aligns with a broad wish to make vaccine development and
quality control more science based, more economic and less time consuming.
Substantial progress in 3Rs implementation in vaccine development and quality control has
been achieved; several non-animal models are being validated and a new testing strategy
which integrates analytical tools, in vitro assays and quality systems; the consistency
approach, is now under study.
The IABS conference will be the focal point for well recognised experts presenting the latest
developments on the most important 3Rs topics in vaccine development and quality control.
The conference will be THE platform for exchange of information on the 3Rs and testing
strategies with representatives from industry, academia, guideline bodies and regulatory
authorities. There will be a mix of presentations and interactive sessions offering an excellent
opportunity for all those who are interested in state of the art vaccine quality control in the
context of improving animal welfare.
Congress Committees
Organising Committee
Coenraad Hendriksen,
Institute for Translational Vaccinology (Intravacc), Bilthoven (NL)
Catrina Stirling,
Zoetis, Tadworth. Surrey, UK
Ian Ragan,
National Centre for the Replacement, Refinement & Reduction of Animals
in Research (NC3Rs), London, UK
Scientific Committee
Local Organising Committee
Juan Arciniega (FDA)
Arnoud Akkermans (RIVM)
Lukas Bruckner (IVI)
Johan van der Gun (BBio)
Karl-Heinz Buchheit (EDQM)
Marieke Hoonakker (Intravacc)
David Dusek (USDA)
Gideon Kersten (Intravacc)
Marlies Halder (EURL-ECVAM)
Fabrizio de Mattia (MSD-AH)
Richard Isbrucker (Health Canada)
Bernard Metz (Intravacc)
Suresh Jadav (SII)

Carmen Jungbaeck (PEI)
Congress secretariat
Laurent Mallet (Sanofi Pasteur)
Marieke Schimmel (Intravacc)
Eddy Rommel (Rommel Consulting Partners)
Abbigail Charlet (IABS)
Thea Sesardic (NIBSC)
Rebecca Sheets (IABS)
About the Region
Egmond aan Zee (www.vvvhartvannoordholland.nl)

Over 1000 years ago, the town began as a fishing village, but since the beginning of the last
century, it has attracted tourists. Both its fishing and tourism history are still evident in the
streets and the Jan van Speyk lighthouse on the main boulevard can be seen from all parts
of the village. The village offers the holidaymaker plenty of entertainment with promenades,
restaurants and a shopping area with something for everyone. In summer there is every
week an event village with many entertainment that wonderful on the Pompplein from there
you can enjoy an outdoor cafeThe shops are open all year 7 days a week and accessible for
everyone. The wide beach offers numerous possibilities for relaxation: beach chairs,
sunbeds, windbreaks and beach pavilions. There are of course hiking, swimming, sailing and
surfing, and it is possible to take part in, for example kite surfing or canoeing which will result
in an unforgettable beach experience.
About Alkmaar (www.vvvalkmaar.info)

Although Alkmaar is and continues to be a Dutch cheese city, the title covers only part of
what is has to offer. For years the city has been entitled to call itself one of the best shopping
cities in the Netherlands. As you wander through the streets with their impressive
monuments you will be amazed at the beauty of the old town. This beautiful city is just fifteen
minutes from the beach.
About Amsterdam (www.amsterdam.info)

There is a broad spectrum of recreational and cultural sights in Amsterdam that range from
fascinating old buildings like the Oude Kerk, to oddities such as the Hash Marihuana
Museum. The main Amsterdam tourism attractions are its museums. Everyone knows the
Rijksmuseum, Van Gogh Museum and Stedelijk Museum, but there is much, much more.
The city has over fifty museums which attract many millions of visitors every year.
Temperature
The avarage temperature in September is 14.50C. It can be chilly in the evening and some
rain may be expected.
General Information
Congress Venue
All conference activities, including registration, take place at
Hotel Zuiderduin,
Egmond aan Zee,
in the Netherlands.
Hotel Zuiderduin
Zeeweg 52
1931VL
Egmond aan Zee

The Netherlands
T. + 31727502000
info@zuiderduin.nl
www.zuiderduin.nl
How to get to Hotel Zuiderduin from Amsterdam (Schiphol) airport
BY PLANE + TRAIN + BUS/TAXI

Amsterdam Schiphol railway station is situated directly below the airport. You can pick up a
free luggage trolley from the platform. Via Schiphol Plaza, you can walk straight to the
departure or arrival hall.
The ticket machines are located near the platforms at Schiphol Plaza. Tickets are also
available from the Ticket- and Service desks, which are situated close to the red/whitechecked cube at Schiphol Plaza. Staff at the desk will also be able to provide you with train
departure information and general information on travelling by train in Holland.
One-way ticket full-fare for 2nd class from Amsterdam Schiphol railway station to Alkmaar
railway station (with 1 transfer in Amsterdam Sloterdijk) is 9.10. Train departs every 15
minutes and the travel time is approximately 50 minutes. From Schiphol take the train to
Amsterdam Sloterdijk, and then change train to Alkmaar.
From railway station Alkmaar you can take bus line 165 to Egmond aan Zee, bus-stop
Zeeweg (right next to the hotel). Tickets are available at the driver, one-way ticket 4,00
(coins are needed) travel time is 20 minutes. You can also take a taxi from railway station
Alkmaar to Hotel Zuiderduin. The taxi stand is in front of the train station. One way taxi ride
27,75 and the travel time is 20 minutes
For those traveling by train: * dont forget to validate your ticket

* be aware of pickpockets
BY PLANE + TAXI
From Amsterdam Schiphol Airport, take a taxi in front of the airport directly to Hotel
Zuiderduin in Egmond aan Zee or make a reservation at Taxi Zwart in advance (see next
page). You can use their 'Schipholservice' , via their website.
One way taxi ride 88.40 travel time - 45 minutes
Taxibedrijf P.L. Zwart B.V.

De Hoefsmid 37
1851 PZ Heiloo
T: 072 533 13 13/ I: http://www.taxizwart.nl/ E: centrale@taxizwart.nl
(no responsibility is taken for the correctness of the information on prices)
Congress Bureau
During the conference a special conference desk will be set up in the lobby of the conference
center, next to the Lamoraalzaal. It will be open for registration from 10am on Wednesday,
16 September to Friday 13pm, 18 September.
Map of conference venue/hotel
Plattegronden invoegen
Coffee breaks
Coffee/tea will be available free of charge in lobby of the conference center during the coffee
breaks from 16 18 September.
Lunches
Wednesday, 16 September and Friday, 18 September: Buffet lunches will be served

in the restaurant
Thursday, 17 September : walking lunch will be served in the conference lobby
Insurance
The organisers can accept no liability for personal injuries or loss or damage of property
belonging to the Congress participants, either during or as a result of the event.
Internet access
Free of charge Wi_Fi connection will be available in all meeting rooms of Hotel Zuiderduin as
well as in all guest rooms.
No-smoking
Smoking in the conference area and hotel lobby is not allowed.
Cell Phones
Participants are kindly requested to turn off or switch their cell phones to silent mode in the
meeting room during the sessions.
Hotel Zuiderduin & Facilities

All congress participants stay at Hotel Zuiderduin, Zeeweg 52, Egmond aan Zee. For
address details see : Congress venue
Check inn > 15.00pm
Check out < 12.00pm
Hotel activities
Restaurant
Bar
Bistro
Pub
Swimming pool/Sauna
10
Bowling
Squash
Fitness room
Bike rental
11
Registration
Registration/Information
Location: next to the Lamoraalzaal in the conference lobby
The registration/Information desk is open on Wednesday from 10.30am 6.00pm; on
Thursday from 8am 6 pm and on Friday from 8.30am 1pm.
The full participation includes:
Admission to the conference activities
The Get-Together Party on Wednesday evening
The Congress Dinner on Thursday evening
Two nights at Hotel Zuiderduin (16 18 September)
Breakfast (17/18 September), Lunches (16/17/18 September), Dinner (16 September,

conference dinner 17 September)
Drinks in the bar (apart from the welcome drink in the Pub) and any other extra
activity/entities will be credited to your own account.
Social Events
No special programme has been prepared for accompanying persons during the conference.
However, the staff of the Zuiderduin hotel will be happy to advise you on special visits to
Alkmaar or other places of interest.
12
CONFERENCE PROGRAMME
Wednesday, September 16, 2015
Registration will be open from 10:30. Coffee will be available
12.00 13.00
Lunch (buffet) in restaurant
13.00 - 13.30
Opening of conference (Lamoraalzaal)

Chairs : Coenraad Hendriksen (Intravacc) / Marlies Halder
(EURL-ECVAM)
13.00 13.05
John Petricciani, chairman IABS
13.05 13.10
Rob van Zeeland, CEO Intravacc
13.10 13.30
Hans van Dongen (Ministry Economic Affairs, NL) with video

welcome from State Secretary Mrs.Sharon Dijksma, Ministry of
Economic Affairs, NL
13.30 15.30
Session 1: 3Rs alternatives and safety testing
13
13.30 13.55
3Rs into action (Coenraad Hendriksen, Intravacc, NL)
13.55 14.20
Time to apply 3 Rs to virus testing? (Rebecca Sheets, Grimalkin

Partners, USA)
14.20 14.45
Waiving the target animal batch safety test at MSD Animal Health
(Harrie Glansbeek, MSD Animal Health, NL)
14.45 15.10
Abnormal Toxicity Testing Roots and evolution of first animal safety

tests for biological (Klaus Cussler, Paul Ehrlich Institut, D)
15.10 15.30
Viral safety of vaccines: how to streamline the Adventitious Testing

Package? (Laurent Mallet, Sanofi Pasteur, F)
15.30 16.00
Coffee break
16.00 17.25
Session 2: 3Rs opportunities and barriers

Chairs : Ian Ragan (NC3Rs)/ Nora Dellepiane (SII)
16.00 16.25
Feedback from the satellite meeting on harmonisation (Catrina Stirling,

Zoetis, UK)
16.25 16.50
BSP130: Validation of cell line assays for toxicity and antigenicity

testing of Clostridium septicum vaccine antigens (Keith Redhead, Chair
EPAA clostridial toxoids working group, UK)
16.50 17.15
Human Rabies vaccine potency testing; the test for G-protein : report
of the pre-collaborative study and future strategies (Jean-Michel
Chapsal, Chair EPAA Rabies working group, F)
14
17.15 17.40
Regulatory acceptance and use of 3R models in rabies vaccines

quality control (Jean-Marie Schiffelers, USBO- Utrecht University, NL)
17.40 18.00
A competition ELISA for potency testing of equine anti rabies sera as

an alternative assay for the Mouse Neutralisation test (Sylvie
Morgeaux, Agence Nationale de Scurit du Mdicament et des
Produits de Sant, F)
18.30 19.30
Get-Together in The Pub
19.30 -
Dinner (buffet) in restaurant
Thursday, September 17, 2015
08.30 10.35
Session 3: 3Rs alternatives, new ideas, pitfalls and solutions

Chairs: Arnoud Akkermans (RIVM)/ Paul Stickings (NIBSC)
08.30 08.50
Proteomic analysis of bovine tuberculin purified derivates (Elisabeth

Balks, Paul Ehrlich Institut, D)
08.50 09.10
Alternative method for Rabies Immunoglobulin Mouse Potency testing

(Emmanuelle Coppens, Sanofi Pasteur, F)
09.10 09.35
The promise and perils of complex data analysis (Stanley Deming,

Statistical Designs, USA)
09.35 10.00
Comparison and bridging of bioassays: doing more with less using

Bayesian statistics (Perceval Sondag, Arlenda, B)
10.10 10.25
Use of Risk Assessment to effect replacements of animal-based

toxicity tests for vaccines by in vitro tests (Blaise Descampe, GSK, B)
15
10.25 11.00
Coffee break
11.00 12.05
Session 4: Consistency Approaches

Chairs : Carmen Jungbaeck (PEI)/Joris Vandeputte (IABS)
16
11.00 11.20
Evolution of veterinary vaccines an industry perspective on

consistency (Vaughn Kubiak, Zoetis, BE)
11.20 11.40
What do we talk about when we talk about Replacement (Juan

Arciniega, FDA, USA)
11.40 12.00
The Ph.Eur.s stance on consistency testing and 3Rs (Karl-Heinz

Buchheit, EDQM, F)
12.00 12.20
Development of international standards for Biological products by

WHO-perspective on animal testing and consistenct testing (Dialiang
Lei, WHO, CH)
12.20 -13.30
Lunch (walking lunch outside the Lamoraalzaal)
13.30 15.10
Session 5 : Consistency approaches, continued

Chairs: Carmen Jungbaeck (PEI)/Joris Vandeputte (IABS)
13.30 13.55
The use of production platforms in vaccine manufacturing (Jody

French, Harris Vaccines, USA)
13.55 14.20
Antigenic fingerprinting of diphtheria toxoid adsorbed to alum based

adjuvants (Gideon Kersten, Intravacc, NL)
14.20 14.45
A model of the human artificial lymph node (HuALN) for testing

biopharmaceuticals and vaccines (Christoph Giese, ProBiogen, D)
14.45 15.10
Use of physico-chemical methods for consistency testing (John

Hoogerheide, Zoetis, US)
15.10 15.30
Coffee break
17
15.30 18.00
Session 6: Workshops (parallel)

Workshop 1: 3Rs alternatives* : Room 559 (Chair: Lukas Bruckner,
IVI, CH)
Workshop 2: Consistency testing** : Room 558 (organised by
the National Institute for Public Health and the
Environment (RIVM, NL))
19.30 -
Conference Dinner : Restaurant ZilteZoen
* Members Panel : Lukas Bruckner (chairman), Marlies Halder (EURL-ECVAM, I),

Perceval Sondag, (Arlenda), Laurent Mallet (Sanofi Pasteur, F), Marie-Jeanne
Schiffelers (UU, NL), Keith Redhead , UK and Juan Arciniega (FDA, USA)
** Moderators : Carmen Jungbaeck (PEI, D), Laura Viviani (GSK, CH), Eddy
Rommel, (Rommel Consulting Partners,B), Ton van der Stappen (MEB, NL), Ian
Ragan (NC3Rs, UK)
18
Friday, September 18, 2015
09.00 10.45
Session 7: Consistency testing in practice

Chairs: Keith Redhead/ Sylvie Uhlrich (Sanofi-Pasteur)
09.00 09.25
3Rs and consistency testing in emerging economies : progress and

global expectations (Nora Dellepiane, Serum Institute of India, India)
09.25 09.45
Functional in vitro testing of vaccines within the consistency approach:

a proof-of-principle using whole cell Bordetella pertussis vaccine
(Marieke Hoonakker, Intravacc, NL)
09.45 10.05
10.05 10.25 Moving towards consistency approach for Diphtheria

Tetanus Pertussis potency assays (Sylvie Uhlrich, Sanofi Pasteur, F)
10.05 10.25
Diphtheria and consistency testing: the results of a comparison study

(Bernard Metz, Intravacc, NL)
10.25 11.10
Coffee break
11.10 13.00
Session 8: The way forward

Chairs: Catrina Stirling (Zoetis)/ Ian Ragan (NC3Rs)
11.10 11.50
Feed back from workshops by rapporteurs
11.50 12.20
The way forward (Catrina Stirling, Zoetis, UK & Ian Ragan, NC3Rs,
UK)
19
12.20 12.35
Take home message (Karl-Heinz Buchheit, EDQM, F)
12.35
Closure of conference (Coenraad Hendriksen, Intravacc, NL)
13.00
Lunch
ABSTRACTS
20
3RS INTO ACTION
Coenraad Hendriksen
Institute for Translational Vaccinology (Intravacc) & Utrecht University, Faculty of Veterinary
Medicine
This year we celebrate the 130st anniversary of the rabies vaccine. In 1885 Louis Pasteur
injected a small boy, named Joseph Meister, bitten by a rabid dog with a product which
development had been entirely based on his work in animals. The approach meant a
blueprint for the role of animal models in vaccine development and quality control in the 130
years that followed. This presentation will start with a quick scan of historical landmarks,
highlighting the traditional link between vaccines and laboratory animals.
Although the concept of 3Rs was introduced by Russell and Burch in their book The
Principles of Humane Experimental Technique as early as in 1959, it took almost three
decades before the 3Rs found there way to vaccine development and testing. Actually one of
the first times the 3Rs were mentioned was at the IABS symposium of 1985 entitled
Reduction of Animal Usage in the Development and Control of Biological Products
(Dev.Biol.Stand., 64, 1986). It was particular thanks to three drivers: Peter Knight of
Welcome Research UK, Jorn Lyng of the State Serum Institute (DK) and Hans Kreeftenberg
of RIVM (NL) that the 3Rs came to the political and regulatory agenda. Institutions such as
the European Pharmacopoeia, WHO and ECVAM became involved and at various levels
3Rs activities started. At an ECVAM organised expert workshop in 1994 several general and
specific recommendations were made to speed up 3Rs activities. Now, 20 years after the
workshop, it is encouraging to note that most of the recommendations have been turned into
daily practice.
21
Early work on 3Rs started from a one-to-one approach: developing a 3Rs alternative to
replace an existing animal test, all within the existing paradigm of vaccine lot release testing.
Although this approach has been successful, animal use for vaccine development and quality
successful, control remained extensive, estimated to be now about 15%of total animal use
for biomedical research and testing. Work is not done yet.
For several reasons we are now moving to another level of 3Rs development. The objective
is no longer replacing an individual animal test by a 3R alternative, but modifying the concept
of lot release testing. The base line of this approach is replacement of safety and potency
testing by a set of non-animal models demonstrating consistency in production and product
quality. This approach starts from the assumption that subsequent lots of vaccine produced
can be compared to a clinical/historical lot, which is thoroughly tested and has a well defined
profile. The consistency approach has come into reach by improvements in production and
control: optimised production processes, a tight protocol for in-process testing using
innovative analytical and cell-based techniques and a state-of-the-art quality monitoring
system (GMP, QA). In my presentation I will elaborate on the principle of consistency testing,
the various techniques that can be used and the way forward.
22
TIME TO APPLY 3 RS TO VIRUS TESTING?
Rebecca Sheets
Grimalkin Partners, USA
For decades, the biologicals industry has relied on mid-century clinical diagnostics methods
to screen for adventitious viruses. The standard tissue culture tests, using cytopathic effects
and hemadsorption and/or hemagglutination as read-outs and the in vivo tests, using
death/survival, evidence of viral infection, and hemagglutination as read-outs have been
used without validation according to the ICH guidance. These compendial methods need
only be verified, but they were never really optimized, standardized, and validated, including
inter-laboratory reproducibility, as would be expected today. Data from a research study to
evaluate the performance of the tissue culture and in vivo tests for breadth and sensitivity will
be presented. These data suggest that the in vivo methods are not as broad nor sensitive as
imagined. New genomic methods have been developed, are being introduced, and soon will
become validated for this purpose (testing for adventitious viruses).
But, how can one
demonstrate that the new methods are comparable or better, when the old methods have
unknown performance parameters? The presenter suggests a pathway forward and that it is
time to apply the 3 Rs to virus testing.
23
WAIVING THE TARGET ANIMAL BATCH SAFETY TEST

AT MSD ANIMAL HEALTH
Harrie Glansbeek, Imke Kross, Kim van Asseldonk, Wim Hesselink

MSD Animal Health, Wim de Krverstraat 35, 5831 AN Boxmeer, the Netherlands.
Harrie.Glansbeek@merck.com
In 2012 the Ph. Eur. announced a milestone step with regards to animal welfare. An update
to the Ph. Eur. was published in October 2012 requiring the target animal batch safety test
(TABST) to be discontinued for all veterinary vaccines, with the exception of a very small
number of vaccines (the test is now titled Residual toxicity test).
In order to cope with the complexities of waiving the TABST of 130 vaccines produced on 5
international production sites and marketed within and outside of the EU (> 100 non-EU
countries), a cross-functional project team was developed at MSD AH consisting of members
from Supply Chain, Regulatory Affairs, Planning, Quality and Global Tech Support. Since the
majority of Merck Animal Health vaccines are not dedicated to the EU market, but rather sold
globally, it was not possible to simply remove the TABST from the specifications without
contacting other impacted markets. For this reason, as well as for ethical reasons, it was
decided early on to try to implement the waiving of the TABST in as many non-EU countries
as possible. As a result of the ensuing inventarisation in these markets, products were
assigned to 4 scenario groups, designed to best balance reduction of animal use with
feasibility and logistic efforts. Criteria for scenarios included percentage of the demand
coming from countries accepting the discontinuation, number of batches produced per year,
and whether the TABST test was coupled to another animal test or not.
Results and conclusions:
Project was far more complex than initially expected
24
Reduction of total animal costs (animal facilities + resources) was not significant and are
outweighed by
Extra costs to business due to stock building of batches with and without TABST plus
implementation costs
BUT
Appr. 80% of non-EU countries accepted discontinuation of TABST
75% of batches of products in scope can be released without safety test
Reduction of > 600 animal tests/year, resulting in a decrease of animal use of > 3000
animals/year
ABNORMAL TOXICITY TESTING ROOTS AND EVOLUTION OF FIRST ANIMAL SAFETY

TESTS FOR BIOLOGICALS
Klaus Cussler
Paul-Ehrlich-Institut, Langen, Germany
The Abnormal Toxicity Test (ATT) is an animal-based general safety test which is required in
the European Pharmacopoeia and with minor modifications in most other regulatory
requirements for medicines of biological origin. Mice and guinea pigs are injected with a fixed
dose of the product to test for untoward toxic reactions in general. When the discussions
about animal welfare and the proposal for the 3R concept reached the area of biologicals
the necessity of the ATT was one of the first topics on the agenda. However, no consensus
could be achieved to delete the test. Furthermore, no valid alternatives could be developed
as the purpose of the test was not specified. Indeed, no official information on the purpose
and the history of the ATT was published since the test was mentioned for the first time in
The International Pharmacopoeia in 1951 when the WHO started to develop

internationally accepted guidance for the testing of sera and vaccines.
25
During studies on the history of governmental quality control of sera and vaccines in
Germany documents could be retrieved which described a mouse test already established in
1894 to measure the content of phenol used as a preservative in diphtheria serum. A safety
test in guinea pigs was introduced some years later after lethal accidents occurred due to
contamination of antiserum with tetanus toxin. Both tests are the first animal safety tests as
documented in published the German guidance on batch testing of immune sera in 1906.
The development and modifications of these tests in the following years within the German
regulatory requirements clearly document how both animal tests were modified until they
evolved as ATT.
It is hoped that the information about the history and the purpose of the mouse and the
guinea pig safety test is able to promote the discussion about the ATT and allow the waiving
of the test animal, hopefully at the global level.
26
VIRAL SAFETY OF VACCINES: HOW TO STREAMLINE THE ADVENTITIOUS AGENT

TESTING PACKAGE?
Laurent Mallet
Sanofi Pasteur - 1541 avenue Marcel Merieux, 69280 Marcy lEtoile France
Laurent.Mallet@sanofipasteur.com
Testing for adventitious agents is one major pillar to ensure the viral safety of Vaccines. The
recent contamination case of a rotavirus vaccine by a porcine circovirus has highlighted the
limitations
of
the
current
testing
package.
Moreover
current
developments
and
implementation of new broad molecular methods such as High Throughput Sequencing, as

well as a recent study (Gombold et al. 2014) illustrating the poor sensitivity of in vivo
adventitious agent tests are new elements supporting the need for a change in adventitious
agents testing strategy implemented for cell banks and seed lots used for vaccine
manufacturing. The presentation will discuss the use and development of HTS as a
promising platform to address these elements as well as potential approaches for
streamlining the adventitious agent testing package including the removal of in vivo tests for
adventitious agents.
27
Catrina Stirling : report of the workshop held September 15/16
28
BSP130: VALIDATION OF CELL LINE ASSAYS FOR TOXICITY AND ANTIGENICITY

TESTING OF CLOSTRIDIUM SEPTICUM VACCINE-ANTIGENS
K. Redhead, A. Daas, M-E. Behr-Gross, L. Bruckner

Vaccine & Assay Consultancy Ltd, United Kingdom
keithredhead@virginmedia.com
Veterinary clostridial vaccines in general make up less than 4% of the veterinary vaccine
market but their QC testing accounts for approximately 40% of veterinary immunological
animal usage (1). The majority of clostridial vaccines are based on the toxins that these
bacteria produce. The toxins are chemically inactived to yield toxoids which are used as the
main antigens in the vaccines. Animals, mainly mice, guinea pigs and rabbits, are used
throughout the manufacturing process to check toxicity, antigenicity, safety and potency. In
addition to the large numbers of animals used, many of these tests are the most severe
allowed in several national pharmacopoeia and often death is the end-point.
In the past, most work on trying to reduce and/or replace animal usage in this field has
centered on alternatives for potency testing. However, more recent efforts, supported by
NC3Rs, concentrated on the development of cell line alternatives for the in-process toxicity
and antigenicity tests for the toxins and toxoids. These in vitro assays have proved to be
more rapid, sensitive and precise than the original mouse tests (2). Progress was so
impressive that in March 2013 EPAA approved the start of an international collaborative
study, coordinated by EDQM, to evaluate the transferability and the performance of these
alternative methods compared with the current in vivo mouse tests, using Cl. Septicum as a
model vaccine. Eleven laboratories, including manufacturers and public laboratories, in eight
countries participated in the study. The outcomes of this study and the resulting
recommendations are presented.
References
29
(1) Animal Usage in Quality Control Tests for Batch Release of Immunological Veterinary
Medicinal Products (IVMPs) via the UK form 2007-2012. Veterinary Medicines
Directorate. (https://www.gov.uk/government/organisations/veterinary-medicinesdirectorate)
(2) Redhead K, Wood K and Jackson K. Testing of veterinary clostridial vaccines: From
mouse to microtitre plate. Developments in Biologicals (Langen). Basel, Karger 2011;
134: 45-50
HUMAN RABIES VACCINE POTENCY TESTING; THE TEST FOR G-PROTEIN : REPORT
OF THE PRE- COLLABORATIVE STUDY AND FUTURE STRATEGIES.
Jean Michel
CHAPSAL EPAA, jmchapsal@gmail.com
Numerous studies have shown that immunization with the native immunogenic form of the
rabies glycoprotein G results in the production of neutralizing antibodies and protection
against lethal challenge. In human rabies vaccines, antigen quantification is used at final bulk
stage, allowing definition of the vaccine final antigen content. Following an NICEATM,
ICCVAM meeting (1), an EPAA project meeting in 2012 focused on gaps in technical
knowledge and validation of in vitro G antigen quantification methods and proposed solutions
for the replacement of the NIH test. Regulators and manufacturers stressed that the NIH test
should be replaced and emphasized that the current in vivo assay should not be used for
correlation, since it is highly variable and therefore a concordance strategy should be
followed. The ELISA methods under development (2) should be able to discriminate between
potent and sub-potent batches: agreement study. An International Working Group was
formed to coordinate a more harmonized approach of the alternative assay developments
30
through the acquisition and distribution of a common set of rabies vaccines. A protocol was
established to allow preliminary comparison of different ELISA methods. Results from these
studies presented mid 2015 at a group workshop has indicated the good agreement of the
ELISA methods with the NIH test. An ELISA method was selected and should be evaluated
for his ability to detect strains used in rabies vaccine manufacturing around the world.
Results of this study should form the basis for an EDQM collaborative study leading to the
replacement of the NIH test.
References
(1) Report on the international workshop on alternative methods for human and veterinary
rabies vaccine testing: State of the science and planning the way forward William Stokes,
Richard McFarland, Jodie Kulpa-Eddy, Donna Gatewood, Robin Levis, Marlies Halder, Gayle
Pulle, Hajime Kojima, Warren Casey, Alexander Gaydamaka, Timothy Miller,Karen Brown,
Charles Lewis, Jean- Michel Chapsal, Lukas Bruckner,Sunil Gairola, Elisabeth Kamphuis,
Charles E. Rupprecht, Peter Wunderli, Lorraine McElhinney, Fabrizio De Mattia, Koichiro
Gamoh, Richard Hill, David
Reed, Vivian Doelling, Nelson Johnson, David Allen, Lori Rinckel and Brett Jones Biological,
2012, 40, 5, p369-381
(2) A relevant in vitro ELISA test in alternative to the in vivo NIH test for human rabies
vaccine batch release. Richard Gibert; Corinne Jallet; Bertrand Poirier; Nol Tordo; Monique
Alberti; Sylvie Morgeaux Vaccine 2013;31(50):6022-9.
31
REGULATORY ACCEPTANCE AND USE OF 3R MODELS IN -RABIES- VACCINE

QUALITY CONTROL
Marie-Jeanne Schiffelers
Utrecht University School of Governance
m.j.w.a.schiffelers@uu.nl
The use of animals in batch release testing of vaccines very often is a regulatory obligation
and represents around 80% of the total number of animals used in the vaccine industry.1
This heavy reliance on animal experimentation meets serious ethical, scientific and economic
objections. Additionally the use of 3R models is stimulated through European legislation.
Nonetheless, the acceptance and use of available 3R methods is highly challenging, raising
the question which factors influence the acceptance and use of 3R models for regulatory
purposes and how to optimise this process? To examine the influencing variables and to
define optimizing options, this presentation focusses on a survey (1.) and a case study (2.)
regarding rabies vaccine potency testing to elucidate the factors influencing the acceptance
and use of 3R models for rabies vaccine potency testing purposes in general and the Serum
Neutralisation Test developed by the Paul Ehrlich Institut in Germany, in particular. The
findings are put into the broader perspective of technology acceptance. Through this
additional step, the broader mechanism behind the existing inertia is described and input is
given for the discussion between regulatory authorities and industry on how to enhance the
regulatory acceptance and use of 3R models.
References
http://www.evm-vaccines.org/pdfs/vaccines_and_animal_welfare_fin.pdf
32
1. Schiffelers, M.J., Blaauboer, B., Bakker, W. and Hendriksen, C. (2014). Replacing the
NIH test for rabies vaccine potency testing: a synopsis of drivers and barriers,
Biologicals 42, 4, 205-217. Available at:
http://dx.doi.org/10.1016/j.biologicals.2014.04.001
2. Schiffelers, M.J.W.A., Blaauboer, B.J, Bakker, W.E., Hendriksen C.F.M. (2015).
Regulatory acceptance & use of serology for inactivated veterinary rabies vaccines: a
process reconstruction and lessons learned. ALTEX (accepted manuscript)
A COMPETITIVE ELISA FOR POTENCY TESTING OF EQUINE ANTI RABIES SERA AS

AN ALTERNATIVE ASSAY FOR MOUSE NEUTRALISATION TEST
Sylvie Morgeaux1, Jehanara Korimbocus, Nicolas Dehay, Franois Cano

1 Agence Nationale de Scurit du Mdicament et des Produits de Sant Department,
Control Directorate, Batch Release and Marketing Surveillance of Biological Products
Department, 321 avenue Jean Jaurs, F-69007 Lyon, France
Introduction:
In case of a bite by a rabies infected animal, WHO recommends a prophylactic treatment
including the administration of anti-rabies immunoglobulin of human or equine origin.
According to international regulation, quality control of highly purified F(ab)2 fragments
produced from Equine Rabies Immunoglobulin (F(ab)2 ERIGs) requires potency testing by
the in vivo MNT prior to marketing. However, the 3Rs strategy for animal testing required by
33
the European Directive encourages the replacement of the in vivo potency test by an in vitro
assay. In this context, a competitive ELISA method (c-ELISA) has been developed by ANSM
where the F(ab)2 ERIGs are in competition with the D1 clone monoclonal antibody
recognizing the trimeric native form of the site III of the rabies glycoprotein.
Results and discussion:
The c-ELISA has been optimised and validated in terms of precision and linearity. Seven
dilutions were selected for potency testing corresponding to a concentrations range between
0.067 IU/ml and 0.758 IU/ml for both references and samples of F(ab)2-ERIGs.
The precision of the method was estimated as 0.056

log10IU/mlfortheWHOISand0.070log10IU/mlfortheF(ab)2-ERIGsbatch.Linearity
wasdemonstrated for the range between 430 and 861 IU/ml for the F(ab)2-ERIGs products.
A control chart based on the titres of WHO IS versus the European reference HBRP1 was
set up on the basis of 85 assays in order to analyse the consistency of the c-ELISA method
and to validate the assay of the day. The lack of trends and the fact that no points are out of
the control limits, allow us to consider that the c-ELISA method is suitable to monitor the
consistency of the F(ab)2 ERIGs production. Around 50 batches of F(ab)2 ERIGs
including commercial potent batches, a high potent batch and sub-potent batches with
respect to MNT were tested and included in a correlation study between the data of the cELISA and the manufacturers MNT. There was a statistically significant linear relationship
between the MNT and the c-ELISA results at the 95% confidence level.. This supports that
the D1 clone, which recognises the major immunodominant site III epitope of the
glycoprotein, is able to quantify the potential of the F(ab)2-ERIGs to neutralise the rabies
virus and consequently to assess their potency in humans.
Conclusion:
Our c-ELISA method for the potency testing of anti-rabies F(ab)2 ERIGs has been validated
for the parameters linearity and precision that are required for a quality control for this
product. As the c-ELISA method is able to discriminate between potent and sub-potent lots
with regard to the official MNT, this is a potential alternative test to the MNT. Moreover, since
this c-ELISA method design is not dependent on the origin of the immunoglobulins, it could
34
also be used theoretically for the quantification of rabies immunoglobulin from other animal
species notably for rabies immunogenicity assay in mice or for human immunoglobulins in
the replacement of the RRFTI
35
PROTEOMIC ANALYSIS OF BOVINE TUBERCULIN PURIFIED PROTEIN DERIVATIVES
E. Balks1, F. Mohs1, J. Trsemeier2 and A. Reuter3

1Veterinary
Medicine, Paul-Ehrlich-Institut, 63225 Langen, Germany; 2 Biostatistics, Paul-
Ehrlich-Institut, 63225 Langen, 3Allergology, Paul-Ehrlich-Institut, 63225 Langen, Germany

Elisabeth.Balks@pei.de
Rationale: PPD tuberculins are heat treated products derived from culture material of
mycobacteria. Tuberculins reveal a delayed hypersensitivity in individuals sensitized to
mycobacteria and are thus diagnostic tools. According to European Pharmacopoeia
requirements batch potency needs to be tested in guinea pigs. Because of ethical concerns
and poor reproducibility of these tests, there is an urgent need for in vitro alternatives.
Therefore, we are evaluating mass spectrometry to characterize tuberculins and to assess
batch to batch consistency regarding the presence and abundance of relevant proteins.
Results: So far, we examined 6 batches of bovine PPD tuberculins originating from three
manufacturers and the WHO International Standard Purified Protein Derivative (PPD) of
Mycobacterium bovis. All PPDs had passed in vivo potency testing. In total we identified 35
proteins. A subset of 7 proteins was present in all batches and all technical replicates
examined. Four of these have been explicitly identified to be markers of tuberculin potency
(Whelan et al., 2010; Stavri et al., 2012) and/or identity (Lyashchenko et al., 1998; Whelan et
al., 2010; Stavri et al., 2012; Souza et al., 2012) by others. Additional findings include 2 heat
shock proteins and acyl carrier protein.
Conclusions: Mass spectrometry holds potential to assess batch quality and batch to batch
consistency of bovine tuberculins and to further characterize the rather heterogeneous
product group of tuberculins.
36
Funded by the German Federal Ministry of Education and Research

3R-methods to replace and refine legally required animal tests for immunologicals
(0316009A C).
References
Lyashchenko, K. P. Pollock, J. M., Colangeli, R., Gennaro, M. L. (1998). Infect Immun 66,
5344-5349
Souza, I. I. F., Melo, E. S., Ramos, C. A. et al. (2012). Springerplus 1, 77.
Stavri, H., Bucurenci, N., Ulea, I. et al. (2012). Indian J Med Res 136, 799-807.
Whelan, A. O., Clifford, D., Upadhyay, B. et al. (2010). J Clin Microbiol 48, 3176-3181.
37
ALTERNATIVE METHOD FOR RABIES IMMUNOGLOBULIN MOUSE POTENCY

TESTING
Emmanuelle Coppens
Sanofi Pasteur - 1541 avenue Marcel Mrieux, 69280 Marcy lEtoile France
Emmanuelle.coppens@sanofipasteur.com
Potency of antirabies immunoglobulin is assessed by performing the MNT: Mouse

Neutralization Test. This test is a lethal test which consists in a first step of seroneutralisation
in which Immunoglobulin is incubated in contact with a live virus suspension followed by a
second step of injection of this suspension into mice. The read out of the test is the number
of surviving animals enabling to determine a titer. An alternative in vitro method has been
developed by Sanofi Pasteur. This new method is based on the same 2 steps as the in vivo
method, the titer being determined by ELISA titration of residual virus. This method is
particularly interesting as it mimics the in vivo method and ELISA is based on the use of 2
different monoclonal antibodies: one for the capture of the viral antigen and the second for its
quantification. Validation process of this alternative method will be described.
38
THE PROMISE AND PERILS OF COMPLEX DATA ANALYSIS"
Stanley N. Deming, Ph.D.

Statistical Designs, Houston, TX 77075
From a primitive systems theory point of view, pharmaceutical manufacturing processes

have inputs (e.g., raw materials, process variables, environmental conditions), transforms
(e.g., chemistry or biochemistry), and outputs (e.g., product, waste, cost). With today's
modern sensors, computerized data acquisition, and inexpensive digital storage, it's not
unusual to record (as a function of time) hundreds of attributes from the system's many
inputs, transforms, and outputs.
Statistical process control charts (or trend charts) are often used to display and monitor the
measured attributes. There is sometimes an assumption that if the measured attributes show
consistency -- that is, if they behave well within limits -- then the unmeasured attributes must
be consistent, as well. This assumption can be justified if there is historical evidence that at
least one of the measured attributes is mechanistically linked to the (now) unmeasured
attribute of interest (e.g., relative potency) and can, in fact, be used as a surrogate for the
unmeasured attribute. In the bsence of such historical evidence, the assumption is weak. A
non-pharmaceutical example will be given for which the assumption was unfortunately not
justified, and it will be suggested that a multivariate (principal components) approach to data
analysis might strengthen the way the consistency approach us used.
39
COMPARISON AND BRIDGING OF BIOASSAYS: DOING MORE WITH LESS USING

BAYESIAN STATISTICS
Perceval Sondag and Bruno Boulanger

Arlenda SA, Chausse verte 93, 4470 Saint-Georges, Belgium
Contact : perceval.sondag@arlenda.com
European Partnership for Alternative Approaches to Animal Testing (EPAA) was created in
2005 and promotes the implementation of test methods to replace, reduce and refine (Three
Rs alternatives) the use of laboratory animals to understand chemicals properties. Several
European legislations already require or strongly encourage the replacement of animal
testing.
Challenges for the companies are double: new in vitro assays need to be developed, and
should be proven equivalent to the in vivo assays. Bridging studies are conducted to
demonstrate that a new method will provide equivalent method to a reference one, and are
based on statistical tools, such as Orthogonal Regression.
When developing alternative methods to animal testing, Bayesian statistics allow to
incorporate prior knowledge in statistical models. That is, the knowledge on the well-known in
vivo assay can be used in order to demonstrate equivalence more efficiently and with less
samples.
This talks gives an overview on statistical tools for method comparison and present how
Bayesian statistics can be used to reduce uncertainties.
40
USE OF RISK ASSESSMENT TO EFFECT REPLACEMENTS OF ANIMAL-BASED

TOXICITY TESTS FOR VACCINES BY IN VITRO TESTS
Blaise Descampe
GSK
For years now, Biosafety officers in pharmaceutical companies have applied risk assessment
approaches in order to define appropriate containment of micro-organisms in production
areas to both protect workers and environment. To achieve this task, they pioneered in
developing models to take the right decision. ICH, in 2005, developed a guideline addressing
technical requirement for registration of pharmaceuticals for human use: Quality Risk
Management known as ICH Q9.
What lessons can we learn from application of these tools to question like the risk
underpinning the replacement of animal based assay by in vitro method or even the waiver of
animal testing?
This presentation is attempting to sensitize pharmaceutical companies and regulatory
agencies to use a systematic approach in considering file variations or submissions that
propose the replacement of animal testing. It provides with an overview of tools principles
and proposes some worked example on actual release animal testing in the vaccine
manufacturing field.
Risk is the combination of the probability of occurrence of harm and the consequences
(severity) of that harm. Beginning by proper risk identification is key yet often neglected.
Animal toxicity test are used to detect absence of reversion to its toxin form of chemically
detoxified native toxins. This feature, part of the target antigen profile of the vaccine product,
should be maintained throughout the life cycle of the product such that this quality attribute
41
remain consistent with the one used in clinical studies. Proposal of risk identification
statement could be as follow: There is a risk of intoxication of the patient that receive vaccine
due to the failure of detection of residual or reversion to toxicity following replacement of an
in vivo method by an in vitro method or due to the waiving of in vivo assay from the control
strategy.
Unclear mechanistic understanding of in vivo assay is sometimes viewed as a major
regulatory hurdle to propose and / or approve replacement model. Yet, taking a step back
and shedding a broader light on the overall risk of implementing replacement method may
help the decision and the acceptance of the risk.
Risk analysis must encompass the potential immediate and potentially delayed harm to the
patient Risk evaluation should take into consideration, the manufacturing process and its
validation including stability data, the extent of available safety record data base, the quality
system in place, and the control strategy. This latest element is key to understand from one
process to another the barriers that are in place to both reduce probability and eventually
consequences of the potential harm.
Keywords: risk management, replacement, vaccine, toxicity, patient.
42
EVOLUTION OF VETERINARY VACCINES AN INDUSTRY PERSPECTIVE ON

CONSISTENCY
Vaughn Kubiak
Zoetis Belgium SA
This evolution then requires a balance between the key Development and Manufacturing
drivers. Development Drivers include the pursuit of products that address unmet medical
needs, development of products using a speed to market approach, and delivery of a range
of solutions to meet individual customer needs. These drivers create unique products with
unique
manufacturing
expectations
and
demand
tight
process
development/transfer/qualification timelines. Manufacturing Drivers include robust processes

using compatible technologies/assets, predictable costs, options for process scale-up and
yield/shelf life improvements, and the ability to introduce new technology where cost-justified.
These drivers create a strong expectation for process knowledge, benchmark indicators to
support process improvements and new technologies, and a regulatory environment that
facilitates timely process adjustments and efficient, compliant change control.
Historically, the movement towards the Consistency Approach has been justified by a
strong industry commitment to reduce animal use for product testing/release, as well as the
need for reproducible batch release. To be truly transformational, however, we need to think
about these principles more-broadly and further extend the benefit from a collective animal
health perspective. Examples will be shared during the presentation and include starting
material replacement, adjustment/optimisation of antigen production parameters, and
confirmation of proper finished product formulation.
43
WHAT DO WE TALK ABOUT WHEN WE TALK ABOUT REPLACEMENT?
Juan Arcinega
FDA, USA
More than fifty years have passed since the seminal publication of Russell and Burch (The
Principles of Humane Experimental Technique, Methuen, London. 1959), and some

regulatory authorities may still be accused of excessive caution when deciding whether to
approve an alternative to an animal-based assay included in a license dossier. The main
concern associated with this decision is that adoption of the alternative (a refinement, a
reduction, but especially, a replacement) may lead to degradation of the quality of the
information provided by the animal-based test to be modified or replaced. This concern may
be allayed, at least in part, by critical consideration of the underlying reason for the original
test, and how well the proposed alternative meets that essential regulatory need. I will
attempt to convey at least some ideas of what Replacement is (the act of replacing one
thing with another, especially something that is newer or better) and is not, in the context of
the 3 Rs, and what a regulatory authority may be expecting in support of non-animal
replacement tests, using examples of recent (and no so recent) efforts to eliminate the use of
animal-based testing for vaccines.
44
THE PH. EUR.S STANCE ON CONSISTENCY TESTING AND 3RS
Karl-Heinz Buchheit
European Directorate for the Quality of Medicines & HealthCare (EDQM), Council of Europe,
Strasbourg, France
The European Pharmacopoeia (Ph. Eur.) sets legally binding quality standards for all
medicinal products in the European Union (EU) and all states who have signed the Ph. Eur.
Convention. The Ph. Eur. seeks to reduce animal usage wherever possible. To assess
compliance with the Ph. Eur. not all tests in monographs need to be performed. With the aim
to minimise animal usage as much as possible, assurance that a product is of Ph. Eur.
quality can also be obtained on the basis of its design, control strategy and data derived, e.g.
from validation studies of the manufacturing process, with agreement of the competent
authorities. Furthermore with the agreement of the competent authorities, alternative
methods (e.g. 3Rs methods) may be used. By means of the Biological Standardisation
Programme (BSP), alternative methods applying the 3Rs principles are elaborated for the
Quality Control (QC) of biologicals, in collaboration with industry and public Official Medicines
Control Laboratories (OMCLs). In conclusion, the 3Rs approach and the application of the
consistency approach are supported by the Ph. Eur.; the BSP actively promotes the
validation of 3Rs methods for the QC of biologicals.
45
DEVELOPMENT OF INTERNATIONAL STANDARDS FOR BIOLOGICAL PRODUCTS BY

WHO -PERSPECTIVE ON ANIMAL TESTING AND CONSISTENCY TESTING
Dianliang Lei
Technologies, Standards and Norms, Department of Essential Medicines and Health
Products, World Health Organization
WHO has played a key role for over 60 years in developing WHO guidelines and
recommendations to assure the quality, safety and efficacy of biological products as well as
establishing the international standards/references preparations necessary to standardize
biological materials or assay methods. The Expert Committee on Biological Standardization
(ECBS) established in 1947 has overall responsibility to endorse and adopt both written and
measurement standards. The written standards are developed through a consultation
process with participation of regulators, industry and academia. The standards or
recommendations developed through the Committee published by WHO provide guidance
for national regulatory authorities (NRA), manufacturers and product developer. If an NRA so
desires, these WHO recommendations may be adopted as definitive national requirements.
Modifications to these standards may be justified and made by the NRA however, it is
recommended that modifications to these recommendations be made only on condition that
the modifications ensure that the vaccine is at least as safe and efficacious as that prepared
in accordance with the recommendations set out in the Recommendations.
During the development of WHO Recommendations and standards 3R principles are
followed to reduce, replace and refine the use of animal in the quality control testing such as
innocuity test, toxicity test, potency test etc. Developing of in vitro alternatives to the animal
testing is highly encouraged by WHO in its Recommendations. Moreover, in the spirit of
minimizing animal testing worldwide, agreements between the national country laboratories
of importing countries and the producing/exporting countries in a mutual recognition or
46
collaborative agreement, in order to utilize the results of animal test already performed by
other laboratory.
47
THE USE OF PRODUCTION PLATFORMS IN VACCINE MANUFACTURING
Jodi French
Head of Manufacturing and Regulatory Affairs, Harrisvaccines, Inc.
1102 Southern Hills Dr., Ste 101, Ames, Iowa, USA
jfrench@harrisvaccines.com
Production platforms provide consistency in manufacturing by allowing uniform testing

methods and standard manufacturing equipment, which directly correlates to product safety.
Production platforms are capable of responding rapidly to new and emerging diseases as
well as diseases known to frequently mutate due to genetic shift or drift. The manufacturing
process is identical for all platform products and only varies by the gene sequence inserted
into the platform. Live viruses or bacteria are not required or a part of the manufacturing
process. Once the sequence of a specific pathogen has been identified from a diagnostic
lab, the gene is inserted into the platform to produce a safe and effective vaccine. Previously
used in humans, this technology has an exceptional safety profile, leading the way to
regulatory changes for animal biologics.
Harrisvaccines set precedence for production
platform vaccines when the USDA CVB published a guidance document, Veterinary Services
Memorandum 800.213, describing licensure guidance for Production Platforms based on
non-replicating
and
nonviable
biological
products.
http://www.aphis.usda.gov/animal_health/vet_biologics/publications/memo_800_213.pdf.
The Production Platform, based off of AlphaVirus technology, was approved as a vaccine
option when the USDA CVB granted licensure of Swine Influenza Vaccine, RNA (SIV) in
2012. Subsequently, each product formulated under the same technology uses the SIV
vaccine approval for supportive data leading to a quicker turn around for licensure and
limited animal field safety studies for new products. A full field safety study was conducted
for SIV licensure.
Based on those results a limited field safety protocol was approved
through USDA CVB for Porcine Epidemic Diarrhea Vaccine, RNA.
48
The potential exists for field safety exemption for production platform vaccines, contributing
to a reduction in animal usage in the field.
In 2013, Harrisvaccines, Inc. was the first
company to license a vaccine for Porcine Epidemic Diarrhea Virus in the Unite States. A
product application was submitted in September of 2013 and a Conditional License was
granted in June of 2014.
With the proven safety, performance and the consistency in manufacturing, we are currently
working with USDA CVB on an exemption to target animal safety, a requirement for serial
release.
Veterinary Memorandum 800.116 describes guidance for animal safety testing
exemption.
USDA CVB will consider an exemption to the regulations for products that
document consistency in the manufacturing process and product safety. This exemption is
part of satisfying International Cooperation on Harmonization of Technical Requirements for
Registration of Veterinary Medicinal Products (VICH) guidance.
49
ANTIGENIC FINGERPRINTING OF DIPHTHERIA TOXOID ADSORBED TO ALUM BASED

ADJUVANTS
G. Kersten, J. Westdijk, B. Metz, N. Spruit, W. Tilstra, J. van der Gun, C. Hendriksen
Panels of monoclonal antibodies against different epitopes are being used to probe bulk
antigens. More recently methods have been described to perform ELISA or FACS based
assays with antigens adsorbed to aluminium-type adjuvants
The antigenic quality of diphtheria toxoid (DTd) adsorbed to aluminium phosphate and
aluminium hydroxide was determined and compared with non-adsorbed toxoid (starting
material as well as toxoid desorbed from aluminium salts). A panel of monoclonal antibodies
was used, covering six epitopes, distributed over the antigen. One epitope disappeared
almost completely but was re-established after desorption of the antigen. The results
indicates that DTd is adsorbed to aluminium salt in a preferred orientation and not randomly.
The antigenic profile of DTd could also be determined in combination vaccines, containing
DTd, tetanus toxoid and inactivated poliovirus. The antigenic fingerprinting of DTd
demonstrates consistency of production on antigen level in combination vaccines containing
an aluminium-based adjuvant.
50
THE MODEL OF THE HUMAN ARTIFICIAL LYMPH NODE (HUALN) FOR TESTING
BIOPHARMACEUTICALS AND VACCINES
Christoph Giese and Annika Lubitz

ProBioGen AG, Goethestr. 54, 13086 Berlin (Germany)
As modern biopharmaceuticals show a very high degree of species-specificity, animal

models are inadequate to assess drug safety and drug efficacy. Drawbacks in the
pharmaceutical arena during the last years have raised significant concerns about the
predictability of animal models for immune reactivity, assessing e.g. immunoregulation and
immunogenicity.
In particular for the development of peptide-based vaccines additional challenges of
specificity, epitope selection, patient variability and restrictions have to be solved. Relevant
in-vitro methods and predictive tools are mandatory for rational and effective vaccine design,
the selection, combination, and dosing of peptides, the carrier and adjuvant formulation.
The Human Artificial Lymph Node Model (HuALN) is a micro physiological system (MPS)
mimicking immunity in a continuously perfused 3D culture system and suitable for long-term
treatment (e.g. 28 d) and repeated dosing. The MPS serves as a human micro organoid
lymph node model for induction or modulation of cellular and humoral immune responses.
The implementation of stromal cells improves organoid formation. The HuALN model is
designed for testing immunomodulation (e.g. MoA of checkpoint modulators), to assess
unwanted immunogenicity reactions (e.g. ADA formation, sensitization) or efficacy of
vaccines, adjuvants and formulation. T cell responses and shifts in the TH1/TH2 pathway are
continuously monitored by cytokine secretion profiles. The induction of primary humoral
responses is demonstrated by B cell activation, plasma cell formation and antibody secretion
51
profiles for IgM and IgG. Cells can be harvested from 3D matrix at the end of the MPS
culture time and used for flowcytometric analysis and functional tests, e.g. ELISPOT assays.
We will introduce into the HuALN model and present recent data in the relevant applications
of vaccine testing, immunomodulation and immunogenicity assessment.
Together with DC/T cell assays, MHC class I and class II peptide-binding assays using
PBMCs and dendritic cells a set of in-vitro assays based on human cells are available to
improve vaccine development and manufacturing in the concept of the 3Rs.
Recent publications:
Giese C, Lubitz A (2015; in press):
Human Artificial Lymph Node model (HuALN).
In Vohr (Editor) Encyclopedic Reference of Immuno-Toxicology, Springer
Giese C and Marx U (2014):

Human immunity in vitro - Solving immunogenicity and more.
ADDR 69-70:103-122
Seifert M, Lubitz A, Trommer J, Koonig D, Korus G, Marx U, Volk HD, Duda G, Kasper G,
Lehmann K, Stolk M, Giese C (2012):
Crosstalk between immune cells and mesenchymal stromal cells in a 3D bioreactor system.
Int J Artif Organs 35(11):986995
Giese C, Lubitz A, Demmler CD, Reuschel J, Bergner K, Marx U (2010):

Immunological substance testing on human lymphatic micro-organoids in vitro.
J Biotechnol 148:3845
52
USE OF PHYSICO-CHEMICAL METHODS FOR CONSISTENCY TESTING
John G. Hoogerheide
PhD, Research Fellow, Zoetis
We present examples of the application of sensitive and selective physico-chemical methods

for determining levels of antigens and adjuvants in formulated vaccines. These results are
used to establish the consistency of the manufactured product. Gas chromatography with
flame ionization detection is used for the determination of mineral oil. High performance
liquid chromatography (HPLC) with charged aerosol detection is used for a variety of
adjuvant components, including an oil, a poloxamer, a sterol, a glyocolipid, and a quaternary
amine. HPLC with tandem mass spectrometry (LC-MS/MS) of antigens is applied to both
identification and quantitation.
53
3RS AND CONSISTENCY TESTING IN EMERGING ECONOMIES: PROGRESS AND

GLOBAL EXPECTATIONS
Dr. Nora Dellepiane, Consultant

Quality and Regulation of Biological Products
Mobile +41 79 475 5460, e.mail dellepianen@outlook.com
Increasing numbers of vaccines are being produced in developing countries and especially
by manufacturers from emerging economies. Serum Institute of India Ltd is a long term
collaborator on 3R initiatives and consistency testing in vaccines. My presentation will give
an overview of 3Rs initiatives by way of successful case studies at Serum Institute of India
Ltd. Implementation of consistency based approaches in vaccine testing needs global
harmonization to develop newer tools, technologies and interfaces to bring closer
communication between different stakeholders such as compendia, regulators, academia
and industry. DCVMN can play an important role for such initiatives. My presentation will give
an overview of global expectations for implementation of such approaches.
54
FUNCTIONAL IN VITRO TESTING OF VACCINES WITHIN THE CONSISTENCY

APPROACH: A PROOF-OF-PRINCIPLE USING A WHOLE CELL BORDETELLA
PERTUSSIS VACCINE
M.E. Hoonakker1,3, L. Verhagen1,3, W. Han3, A Sloots1, C.F.M. Hendriksen1,2

1
Institute for Translational Vaccinology (Intravacc), P.O. Box 450, 3720 AL Bilthoven,
The Netherlands
2
Utrecht University, Faculty of Veterinary Medicine, Department Animals in Science
and Society, P.O. Box 80.166, 3508 TD Utrecht, The Netherlands

3
Centre for Immunology of Infectious Diseases and Vaccines, National Institute for
Public Health and the Environment, Bilthoven, The Netherlands
Batch release testing of several classical vaccines is still based on animal models that are
costly, time-consuming, sometimes of questionable relevance and pose concern regarding
animal welfare, while accounting for approximately 10% of the animal use. The consistency
approach is based on the integrated strategy of thorough process- and productcharacterization and in vitro testing of vaccine quality rather than potency testing using
animal models. Though a panel of physico-chemical assays has been developed for
diphtheria vaccines, these assays do not assess the capacity of the vaccines to induce
immune responses and they are not applicable for complex vaccines such as whole cell
pertussis vaccines. To assess the quality of whole cell Bordetella pertussis vaccines (wP) by
this approach we used physico-chemical and immuno-chemical assays in combination with
several functional in vitro assays to evaluate various product-parameters of wP vaccines. We
analysed a panel of experimental wP vaccines of varying quality that were produced by
deliberate modulation of the production process, resulting in altered expression of virulence
proteins that are regarded as important for the induction of protective immunity. Testing of
55
these vaccines of high, intermediate and low quality in the conventional in vivo challenge
model, the Kendrick test, revealed that the manipulation of the production process had
resulted in vaccines with potencies corresponding with their expected qualities. Using ELISA
and mass spectrometry, we demonstrated that the vaccine of a low quality contained
significantly less of the virulence proteins than the vaccine of high quality. In agreement
with the in vivo potencies of the vaccines, the low quality vaccine induced activation of
innate immune cells (MM6 and dendritic cells) of reduced magnitude compared to the high
quality vaccine. Interestingly, the high quality vaccine evoked a significantly higher
response in HEK293-hTLR4 cells compared to the vaccine of low quality, while vaccine
quality had no effect on the response of HEK293-hTLR2 cells. The study provides a proof-ofprinciple that a combination of physico-chemical, immuno-chemical and functional
immunological assays can be used to asses whole cell Bp batch consistency in vitro.
56
MOVING TOWARDS CONSISTENCY APPROACH FOR DIPHTHERIA TETANUS

PERTUSSIS POTENCY ASSAYS
Sylvie Uhlrich
Sanofi Pasteur - 1541 avenue Marcel Merieux, 69280 Marcy lEtoile France
Sylvie.Uhlrich@sanofipasteur.com
For Tetanus and Diphtheria potency tests, the replacement of the challenge tests by a single
immunogenicity assay involving antibodies titration using non-animal methods e.g. ELISA
has been described in European Pharmacopeia for several years as an alternative for the
release of combination vaccines. Pertussis activity is already assessed with an
immunogenicity test where the lot is compared to a reference vaccine.
The implementation of such immunogenicity assays for the routine control of vaccines is
difficult and not always successful mainly due to the variability of these in vivo tests and their
current design.
The limitations of current in vivo immunogenicity assays involving a reference vaccine and
the alternative approaches based on unidose assays (Geometric mean titer - GMT- of
antibody responses) as consistency tools will be presented. For Pertussis vaccines, the
unidose assay with GMT read-out is already accepted in North America and described in
WHO recommendations. Establishment of the specifications for the antibody response to
each antigen claimed to contribute to efficacy will be discussed, including the issues
encountered when using subpotent batches.
Reduction and refinement of the in vivo methods currently used for vaccines quality control
are important improvements and their complete replacement with in vitro consistency assays
will be the ultimate target for the future.
57
DIPHTHERIA AND CONSISTENCY TESTING: THE RESULTS OF A COMPARISON

STUDY
Bernard Metz
Intravacc, NL
Currently, animal tests are used to assess the quality of newly produced diphtheria vaccines.
The replacement of the in vivo potency test is a major hurdle, because the development of
functional alternatives is very complicated. The consistency approach offers an alternative
strategy to prove the quality of vaccines. The detoxification of diphtheria toxin by
formaldehyde and the adsorption of diphtheria toxoid to alum adjuvants determine largely the
ultimate efficacy and safety of the vaccine. For that purpose, physicochemical and
immunochemical methods are used to monitor these crucial steps in the production of
diphtheria vaccines.
Serum Institute of India, Bilthoven Biologicals and Intravacc decided to characterize twenty
diphtheria toxoids (purified bulks) and final vaccines. A panel of consistency tests (circular
dichroism, ELISA, flocculation test, fluorescence, and TNBS assay) was used to determine
58
the variation between toxoids in antigenicity, purity and protein structure. Based on the
dataset, control charts can be drawn providing mean and range of a quality characteristic for
diphtheria vaccines. The animal experiments to determine the quality of diphtheria vaccines
can be reduced or even omitted if once consistency of intermediate and final products has
been demonstrated.
59
ABSTRACTPOSTERS
60
POSTER
DEVELOPMENT OF A CELL-BASED ASSAY FOR TETANUS TOXIN.
Shalini Rajagopal, Paul Stickings, Thea Sesardic

Division of Bacteriology, National Institute for Biological Standards and Controls, South
Mimms, Potters Bar, Hertfordshire, EN6 3QG, UK
Shalini.Rajagopal@nibsc.org
Background:
Tetanus toxin (Ttx) is a highly potent neurotoxin, and the inactivated toxin (toxoid) is used in
production of tetanus vaccines. Currently, safety testing of tetanus vaccines and functional
assessment of tetanus antitoxins requires the use of in vivo assays. An in vitro assay that
can detect tetanus toxin, especially one which incorporates all stages of in vivo toxin action
(i.e. receptor binding, translocation and enzyme action) would be highly desirable and could
fully replace some existing in vivo assays. The aim of this study was to develop a highly
sensitive in vitro assay for the detection of tetanus toxin based on the cleavage of the
intracellular target for Ttxn (vesicle associated membrane protein, VAMP-2) in differentiated
mouse embryonic stem cells.
Methods:
Mouse embryonic stem cells (E14TG2a) were cultured in the presence of leukemia inhibitory
factor, differentiated into motor neurons after the formation of embryoid bodies (EBs) and
treated sequentially with retinoic acid, Sonic hedgehog/purmorphamine, brain-derived and
glial-derived neurotrophic factors.
For final differentiation, EBs were dissociated to form
single cells, and cultured in the presence of nerve growth factor-. Uptake of Ttx by neuronal
cells was determined by use of Ttx heavy chain (Hc) conjugated to mCherry. Neuronal cells
were treated with a range of Ttx concentrations (for 24 h) and cell lysates were examined for
VAMP-2 expression by western blotting. The specificity of VAMP-2 cleavage by Ttx was
confirmed by neutralisation with tetanus antitoxin.
61
Results:
Differentiated neuronal cells were competent for both binding and uptake of Hc conjugated to
mCherry, with positive staining in both the cell bodies and neuronal processes and staining
was prevented by co-incubation with unlabelled Hc.
TTx treatment resulted in a dose
dependent reduction in expression of VAMP-2, with sensitivity comparable to that seen in a

mouse bioassay. VAMP-2 cleavage was prevented by co-incubation of Ttx with either equine
or human polyclonal tetanus antitoxin.
Conclusions:
These preliminary results suggest that a cell based assay using differentiated embryonic
stem cells might be suitable for use in a number of applications that are based on the
detection of tetanus toxin function.
62
POSTER
PERTUSSIS SEROLOGICAL POTENCY TEST: LINKS TO THE PROTECTION AND

SIGNIFICANCE FOR VACCINE LOT RELEASE
Mario Landys Chovel, Niurka Gutirrez, Tatiana Mahy, Lucy Herrera, Aleida Mandiarote
Finlay Institute, Avenida 27, No. 19805, P.O. Box 16017, La Lisa, La Habana, Cuba.
E-mail: mlandys@finlay.edu.cu and mlchovel@gmail.com
Although Mouse Protection Test (MPT) this test has been deeply criticized, it still remains
being the golden standard for Pertussis Potency in vaccines. Pertussis Serology Potency
Test (PSPT) seems to be the most suitable alternative to MPT and a correlation between
both methods has been described. Nonetheless, some issues related to the relevance of the
antibodies for protection remain rather unclear. The present Paper aims to evaluate the
relevance of antibodies for protection during PSPT, by combining immunological and
biological tests. Several whole cell Pertussis vaccine batches were tested in parallel by MPT
and PSPT. Sera were tested for total and specific antibodies (PT, FHA, PRN and FIMB 2 and
3) by whole-cell and specific antigen ELISAs, respectively. The complement activating,
neutralizing and bactericidal capacities were evaluated, as well as the subclasses. The
functionality of antibodies produced during PSPT was evaluated by using an in vitro
opsonophagocytosis assay. All batches were also characterized by a 2D-electrophoresis
procedure and by antigen ELISA using monoclonal antibodies. MPT and PSPT correlated
and were able to discriminate potent and the sub-potent vaccines batches in an equivalent
way. Specific antibody responses, neutralizing, complement activating and bactericidal titres
showed poor correlations regarding MPT and PSPT titres, but a strong correlation against
the opsonophagocytosis assay was obtained. 2-D electrophoresis and antigen ELISAs
provided relevant information on the antigen profile of our vaccines with interesting links to
the PSPT results. Relevant information obtained from the combination of immunological and
biological assays was provided about the relationship between the total antibodies raised by
wP component in vaccines and protection, thus supporting the possibility of replacing MPT
by PSPT in a near future.
63
POSTER
POTENCY DETERMINATION OF INACTIVATED RABIES VACCINES USING A

SEROLOGICAL POTENCY ASSAY BASED ON THE FAVNT SERONEUTRALISATION
TECHNIQUE
Alexandre Servat*, Sbastien Kempff, Valre Brogat, Estelle Litaize, Jean-Luc Schereffer
and Florence Cliquet
*French Agency for Food, Environmental and Occupational Health Safety (ANSES), Nancy
Laboratory for Rabies and Wildlife, Technople Agricole et Vtrinaire, Domaine de
Pixrcourt, CS 40009, 54220 Malzville, France. E-mail: alexandre.servat@anses.fr
The mouse challenge test is still the reference method for the potency determination of
human and animal inactivated rabies vaccines and is still widely used throughout the world.
This test suffers from many disadvantages: expensive, time consuming, use of a large
number of mice, significant animal distress and high variability. Recently the European
Pharmacopoeia has recognised the use of a serological potency assay (SPA) as an
alternative method to the challenge test. This new test is based on the determination of the
rabies neutralising antibody titres in vaccinated mice using a modification of the Rapid
Fluorescent Focus Inhibition Test (mRFFIT). In the present study, we evaluated the
performance of this SPA on a large collection of rabies vaccines currently assessed in our
laboratory. The Fluorescent Antibody Virus Neutralisation test (FAVNt) was used in parallel
to the mRFFIT and results were compared to the mouse challenge test. Our results
64
demonstrated that the SPA is capable of estimating the potency of vaccines that are
formulated with a comfortable margin above the minimum of 1 IU/dose. For low potency
vaccines, this new method demonstrated some limitations due to recurrent invalidation of the
assay. We have also demonstrated a better sensitivity of the FAVNt and the importance to
minimize the risk of detecting non-responders in vaccinated mice. The SPA, implemented in
our laboratory in 2014 for batch release of rabies inactivated vaccines for veterinary used,
has significantly decreased the number of animals used in experimental procedures.
65
POSTER
FURTHER DEVELOPMENT OF A CHO CELL ASSAY FOR DETECTION OF PERTUSSIS

TOXIN IN ADJUVANTED ACELLULAR PERTUSSIS PRODUCTS AT SANOFI PASTEUR
Nelson, Sue; Fowler, Meaghan; Vydelingum, Seeven; Jeyanthan, Kajaparan; Siu, Karen
Sanofi Pasteur. 1755 Steeles Ave. West Toronto ON, Canada. M2R 3T4
sue.nelson@sanofipasteur.com
Two in vitro methods (Direct and Indirect) for detection of pertussis toxin (PTx) in adjuvanted
acellular Pertussis (aP) Vaccines using CHO Cells have been previously assessed for their
transferability in 13 international laboratories (EDQM study BSP114 phase 2). The Direct
method (developed by the US Federal Drug Administrations Center for Biologics Evaluation
and Research) utilized vaccine samples diluted prior to their addition to CHO cells to reduce
the non-specific cytotoxic effect. The Indirect Method (developed at Health Canadas
Biologics and Genetic Therapies Directorate) employed 0.4m polycarbonate tissue culture
inserts to prevent direct contact between adjuvant in the reconstituted vaccine sample pellet
and the CHO cells thereby preventing cytotoxicity. Further development of a CHO cell assay
for detection of PTx in adjuvanted aP products has been carried out at Sanofi Pasteur (SP).
The developed method combines aspects of both the Indirect and Direct Methods such that
0.4m polycarbonate tissue culture inserts are used to test 50L of whole vaccine in a 24
well plate seeded with 2.0104 CHO-K1 cells per well. Wells with 10 clusters are defined as
positive and 10 clusters are detected in SP adjuvanted aP product matrices spiked with PTx
(BRP-1) at 1IU/mL. Additional characterization of a complex SP adjuvanted aP product
matrix using this method demonstrated detection of 10 clusters in samples spiked with PTx
at 1IU/mL, 0.5IU/mL and 0.25IU/mL. The sensitivity of the method is well within the targeted
range of 1 IU per single human dose and can assess PTx in a whole vaccine sample, with a
semi-quantitative read-out.
66
POSTER
PROGRESS ON ESTABLISHING VICH GUIDELINES ON WAIVING CRITERIA FOR

GENERAL BATCH SAFETY TESTS OF VETERINARY VACCINES
Marlies Halder
European Commission Joint Research Centre, IHCP, ST Unit/EURL ECVAM; 21027 Ispra (VA),
Italy; email: marlies.halder@ec.europa.eu
General batch safety tests for veterinary vaccines as the Laboratory Animal Batch Safety
Test (LABST) or the Target Animal Batch Safety Test (TABST) are supposed to demonstrate
that a vaccine does not cause abnormal local or systemic reactions. They have been
introduced decades ago during the development of the first veterinary vaccines. However,
over the last 25 years, the relevance of the TABST and LABST was questioned due to the
introduction of more specific safety tests, strict control of starting material and the
introduction of Good Manufacturing Practice. Retrospective analysis of LABST and TABST
data revealed that the two tests are no longer relevant and not able to detect problematic
batches. The LABST (or abnormal toxicity test) had been removed from European
Pharmacopoeia monographs for veterinary vaccines in 1997, and the TABST in a stepwise
approach until its complete deletion in 2013.
In 2008, Europe proposed to The International Cooperation on Harmonisation of Technical
Requirements for Registration of Veterinary Medicinal Products (VICH) to aim at
harmonisation of general batch safety tests across the VICH regions (USA, Japan, Europe)
in order to minimise the need to perform separate studies for regulatory authorities of
different countries. However, due to the great divergence in requirements between the
regions it was concluded to adopt a phased approach with the first step to harmonize the
criteria on data requirements for waiving of the TABST for inactivated vaccines in regions
where it is required, and the respective VICH GL50 came into force in 2014. A comparable
guideline for live vaccines is under development and discussions on steps towards waiving
possibilities for LABST are ongoing.
67
POSTER
USE OF ANTIBODY-DEPLETED HUMAN SERUM AS COMPLEMENT SOURCE IN THE

SERUM BACTERICIDAL ASSAY TO ASSESS VACCINES AGAINST SALMONELLA
ENTERICA SEROVAR TYPHI
Helene B Juel*, Matthew K Siggins, Leanne Marsay, Peter Hart, Calman A MacLennan, and
Andrew J Pollard
*Oxford Vaccine Group, Department of Paediatrics, University of Oxford and the NIHR Oxford
Biomedical Research Centre, Churchill Hospital, Old Road, Oxford OX3 7LJ, United Kingdom.
Email: Helene.juel@paediatrics.ox.ac.uk
Background
Salmonella enterica serovar Typhi (S. Typhi) is estimated to cause 26.9 million cases of typhoid
fever per year. The lack of an effective vaccine remains a serious problem in developing
countries, and development of new vaccines is hindered by the lack of a suitable animal model
and correlates of protection. The Oxford Vaccine Group has set up a human challenge model to
test the efficacy of new vaccines against S. Typhi.
The serum bactericidal assay (SBA) measures antibody-mediated complement-dependent
bacterial killing by sera. Several studies using animal sera as an exogenous complement source
for the S. Typhi SBA have been published, but animal sera have several limitations, including
68
inherent toxicity to S. Typhi, and the ethical implications of using large numbers of baby rabbits
to produce serum batches. However, human serum is highly bactericidal to S. Typhi, possibly
because of the presence of natural antibodies against Gram-negative bacilli. The method of
Siggins and MacLennan depletes human serum of these antibodies by incubating with whole
Salmonella.
Methods
Antibodies in sera from 11 healthy volunteers were depleted using S. Typhi (Quailes strain).
Sera and bacteria (log phase, stationary, or heat-killed) were mixed and incubated at 4C for 1560 min with regular inversion, and 1-5 depletion cycles were tested.
The complement activity of the depleted sera was assessed using the sheep erythrocyte lysis
assay, and the S. Typhi-specific IgM and IgG antibody content was quantified by ELISA.
The antibody-depleted serum was tested for suitability in the S. Typhi SBA. Bacteria (six
different growth conditions) were mixed with antibody-depleted serum (complement source) and
heat-inactivated test serum (antibody source). After 30-90 min incubation, bacteria were plated
onto tryptone soya agar plates and incubated overnight.
Results
Ten of 11 donors showed suitability as complement sources for the S. Typhi SBA after antibody
depletion. The optimised protocol for antibody depletion of serum included three cycles of 30
min depletions, each using 1011 CFU stationary phase S. Typhi per mL of serum. This depletion
had no adverse effect on the complement activity, and reduced the content of S. Typhi-specific
antibodies by 25-75%.
The method was scaled up to deplete the serum from a 450mL blood donation, yielding enough
complement to assess approximately 3600 serum samples for bactericidal activity. A donor with
consistently high SBA titres was chosen as positive control.
The optimal conditions for the S. Typhi SBA were log phase bacteria incubated with complement
and/or test serum for 60 min at 37C. Bacterial survival in 25% complement increased from 1%
to around 80% survival upon antibody depletion. Addition of heat-inactivated test serum
significantly decreased survival.
Conclusions
69
Protocols for 1) antibody-depletion of human serum for use as complement source and 2) the
SBA suitable for the detection of S. Typhi bactericidal antibodies were successfully optimised,
effectively replacing animal sera with human serum as complement source.
These protocols allow us to assess sera from S. Typhi vaccination and challenge studies to
establish whether serum bactericidal activity may be used as a correlate of protection for S.
Typhi vaccines.
POSTER
AN IN VITRO ASSAY SYSTEM AS AN ALTERNATIVE TO CURRENT MURINE HISTAMINE

SENSITIZATION TEST FOR ACELLULAR PERTUSSIS VACCINES.
Kevin Markey, CT Yuen, Catpagavalli Asokanathan, Alex Douglas-Bardsley and Dorothy Xing
70
National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters
Bar, EN6 3QG, UK. Kevin.Markey@nibsc.org
The histamine sensitization test (HIST) is a safety test for the batch release of acellular
pertussis vaccines (ACV) and it monitors residual toxic pertussis toxin (PTx) or reversion to
toxicity of the toxoid. HIST is a lethal test requiring large numbers of animals and it is difficult
to standardise. Therefore, there is an urgent need to develop an in vitro alternative to this
test. An in vitro test system has been developed as a potential alternative to HIST. This test
system examines both functional domains of PTx (enzymatic and carbohydrate binding
domains) using a combination of enzyme coupled-HPLC and carbohydrate-binding ELISA. A
previous international collaborative study demonstrated that the test system is transferable
between laboratories and is suitable for differentiating three types of commercially available
ACV products which supported its use as a potential alternative to the HIST. Further method
optimisation has been made on ACV products spiked with PTx. A good dose response in the
spiked vaccine samples was observed and sensitivities equal to that of HIST were detected.
Confocal microscopy has demonstrated that the ability of PTx and PTd to translocate into
mammalian cells is proportional to the binding activity indicating the clinical relevance of this
test. Furthermore, the activities of the in vitro systems also correlated with the mouse foot
pad swelling assay. Here we report on the assay system with more data on various ACV
products and suggest possible validation steps.
71
POSTER
THE IMPLEMENTATION PROBLEM: BARRIERS TO AND OPPORTUNITIES FOR THE

USE OF NON-ANIMAL VETERINARY VACCINE POTENCY TESTS.
Jeffrey Brown, Dr. Amy Clippinger, Dr. Gilly Stoddart

PETA International Science Consortium Ltd.
Society Building, 8 All Saints Street, London N1 9RL
jeffreyb@piscltd.org.uk
As new strategies emerge for the consistent production and testing of biologics batches
using non-animal methods, currently available non-animal approaches have still not been
widely implemented. In some cases, available non-animal methods have not been
implemented at all, despite significant public investments toward validation. Resistance to
consistency measures also contributes to the difficulty in implementing non-animal batch
tests for manufacturers and regulators alike. Referencing the non-animal methods validated
for use by the U.S. Department of Agriculture Center for Veterinary Biologics (CVB), we
describe progress and obstacles influencing the implementation of these methods since
2008. Our work in this area has shown that non-governmental organizations operating
outside of the direct regulation or manufacturing of biologics are uniquely poised to identify
and resolve implementation issues not directly addressed by manufacturers, regulators, or
cooperative efforts between these groups. Here, we present our work towards eliminating
barriers to the implementation of non-animal methods. With particular focus on leptospira
vaccine batch potency testing, we discuss the challenges and opportunities faced by
manufacturers and regulators in the pursuit of integrating consistency measures and nonanimal batch tests into biologics production and testing guidelines.
72
POSTER
IN VITRO ANTIGEN ELISA FOR QUALITY CONTROL OF DIPHTHERIA AND TETANUS

VACCINES
L. Coombes, R. Tierney, P. Rigsby, D. Sesardic and P. Stickings

National Institute for Biological Standards and Control, Division of Bacteriology, Blanche Lane,
South Mimms, Potters Bar, Hertfordshire, EN6 3QG, UK
Email: Laura.Coombes@nibsc.org
For conventional vaccines containing diphtheria and tetanus components, in vivo potency
assays are gold standard methods to confirm biological activity. Antigen and adjuvant are
the major components contributing to vaccine potency although their precise contribution to
the measured potency of a vaccine is difficult to predict and will be influenced by other
factors. Consistency of production is also an important criteria for vaccine manufacture and
the amount of antigen and degree of adsorption are critical factors that should be monitored
in final vaccine products. Traditional immunochemical methods can be used to identify the
presence of antigen in a vaccine, but do not necessarily provide quantitative information in
support of consistency of production. We have developed a simple and sensitive Enzyme
Linked Immunosorbent Assay (ELISA) to quantify diphtheria and tetanus antigens in
combined vaccine products and measure the degree of adsorption to adjuvant. The assay
has been applied to various combined vaccine final products and is robust, specific and
highly sensitive, with a limit of quantification of approximately 0.001 Lf/ml for both diphtheria
and tetanus antigens.
Compared to in vivo potency assays, in vitro assays are often
inherently less variable and are therefore better suited to providing information on product
consistency and batch-to-batch variation. In routine use as a consistency test for a
pentavalent vaccine, the antigen ELISA has a GCV for total diphtheria antigen content of
12.1% compared to 38.8% for diphtheria potency (challenge assay, n=22) and a GCV of
7.3% for tetanus antigen content compared to 40.4% for potency (challenge assay, n=22).
The in vitro antigen ELISA may also be applied to predict the stability of diphtheria and
tetanus vaccines and we have observed a correlation between the antigen content and
immunogenicity for some artificially degraded and real-time aged vaccine samples. For
73
adsorbed products a change in antigen content as determined by ELISA may be due to a

change in the strength of association between antigen and adjuvant affecting the efficiency of
desorption in the assay, rather than a change in antigen integrity. As such, the use of the
assay as a stability indicator would need to be validated for specific products. In conclusion,
the antigen assay is an excellent tool for characterisation of final vaccine lots, providing
important information on batch to batch variation and consistency of production. This assay
can be used as part of a panel of in vitro tests to provide a more informative product profile to
help assure the quality of diphtheria and tetanus vaccines.
74
POSTER
REPORTER CELL LINES FOR ASSESSMENT OF PERTUSSIS TOXIN-INDUCED C-AMP

IN ACELLULAR PERTUSSIS VACCINES AS AN ANIMAL-FREE ALTERNATIVE TO THE
HIST
M.E. Hoonakker1,3, L. Verhagen1,3, A Sloots1, C.F.M. Hendriksen1,2

1
Institute for Translational Vaccinology (Intravacc), P.O. Box 450, 3720 AL Bilthoven, The
Netherlands
2
Utrecht University, Faculty of Veterinary Medicine, Department Animals in Science and
Society, P.O. Box 80.166, 3508 TD Utrecht, The Netherlands

3
Centre for Immunology of Infectious Diseases and Vaccines, National Institute for Public
Health and the Environment, Bilthoven, The Netherlands
Safety is a characteristic that is essential for vaccine quality and has to be monitored to
guarantee consistent vaccine production. Safety testing for pertussis toxin (PTx) in acellular
vaccine batches relies on the histamine sensitisation test (HIST). Within this test, mice
receive an injection with an acellular pertussis vaccine followed by a challenge with a fixed
dose of histamine. This in vivo test is based on the empirical finding that PTx reduces the
lethal histamine dose. Studies into the mechanism of this test showed that decreased
contractile properties of arteries and increase blood pressure both contribute to PTx induced
histamine sensitization. Intracellularly, PTx ADP-ribosylates Gi proteins, leaving an
ineffective protein that is unable to inhibit its target enzyme adenylate cyclase. This results in
accumulation of cAMP, which disturbs intracellular signalling pathways. It is likely that PTx
mediated ADP ribosylation of Gi proteins is involved in the regulation of the vasoconstriction
of vascular smooth muscle cells.
Based on this information, the cell-based in vitro cAMP assay was developed, as an
alternative to the HIST. In this assay, the cAMP response of a rat vascular smooth muscle
cell line to PTx is studied. Since binding, internalization and ADP ribosylation are necessary
for enhancing cAMP levels, the assay assesses aspects crucial for toxicity. The assay
proved to be specific for PTx and was able to detect up to 25 ng of PTx (Hoonakker et al.
75
2010). However, standardization using a commercially available cAMP ELISAs proved

challenging and we therefore aimed to develop reporter cell lines allowing direct
quantification of toxin activity. Using a commercially available plasmid, four clonal reporter
cell lines were generated, based on A10 and CHO cells. These reporter cells produce a
variant of luciferase under the control of a cAMP responsive element and detected PTx in
amounts as small as 0.2 IU/mL (1.3 ng PTx/mL). Preliminary results demonstrate that PTx
can also be detected within a complete acellular pertussis vaccine. Taken together, we
demonstrated that cAMP responsive reporter cell lines are functional and sensitive tools for
the detection of PTx and form a potential in vitro alternative to animal-based safety testing of
acellular pertussis vaccines.
76
POSTER
PHOTOGRAPHIC EVALUATION OF SKIN LESIONS IN BATCH POTENCY TESTING OF

TUBERCULINS A REDUCTION APPROACH
E. Balks1, C. Dumke2, F. Mohs1, K. Cussler2 and A. Hoffmann1

1
Veterinary Medicine, Paul-Ehrlich-Institut, Langen, Germany
2 Safety
of Medicinal Products and Medical Devices, Paul-Ehrlich-Institut, Langen, Germany
Elisabeth.Balks@pei.de
Rationale: Tuberculins are biological products derived from culture material of mycobacteria.
When injected intradermally, tuberculins are capable to evoke delayed hypersensitivity in
individuals infected by mycobacteria of the same or closely related species. Tuberculins are
thus used for diagnostic purposes in human and veterinary medicine. To assess batch
potency, compendial methods stipulate evaluation of skin lesions in previously sensitized
guinea pigs (e.g., Monograph 01/2008 0536. Tuberculin Purified Protein Derivative, Bovine.
European Pharmacopoeia, 8th edition). Apart from ethical concerns, the major drawback of
this procedure is the poor reproducibility of test results. Here, we evaluated software assisted
digital photography and infrared thermography as potential reduction alternatives to on-site
manual measurements of lesions.
Results: Software assisted digital photography is suited to assess local reactivity in tuberculin
batch potency testing. It allows for repeated measurements and thus helps to meet European
Pharmacopoeia requirements for test validity in terms of precision of the potency estimate. It
also facilitates documentation of test results in a QM environment. In contrast, infrared
thermography did not reflect skin reactivity.
Conclusions: Software assisted digital photography is suited to reduce the number of test
repeats and thus the number of animals used for tuberculin batch potency testing. Since
widely covered by the current wording of the relevant monographs, immediate
implementation is possible.
77
Funded by the German Federal Ministry of Education and Research: 3R-methods to replace
and refine legally required animal tests for immunologicals
(0316009A C).
78
POSTER
CONSISTENT PRODUCTION OF WHOLE CELL PERTUSSIS VACCINES: PRODUCT

QUALITY OF VACCINES EXAMINED BY GENE AND PROTEIN EXPRESSION ANALYSES
Bernard Metz, Joost Uittenbogaard, Jeroen Pennings and Arno van der Ark
Intravacc, NL
Consistent production is an important element to guarantee efficacy and safety whole cell
pertussis (wP) of vaccines. In this study, product quality of pertussis vaccines examined by
gene and protein expression analyses. Three continuous cultivations of Bordetella pertussis
were performed on a chemically defined medium to obtain uniform protein expression. Then,
magnesium sulphate was added to downregulate the expression of virulence proteins of B.
pertussis. Changes in gene expression and antigen composition were measured by gene
arrays and mass spectrometry. The analyses revealed high similarity between the three
biological triplicates, but significant changes between samples of consecutive time points.
Magnesium sulphate disturbed the steady state conditions of B. pertussis cultivations. Gene
expression of virulence factors was instantly downregulated after the addition. However, the
presence of virulence proteins in B. pertussis diminished slowly. In conclusion, gene arrays
and mass spectrometry are valuable tools that can demonstrate consistent production of
whole cell vaccines.
79
Bio-sketches
80
Arciniega, Juan
Dr. Juan L. Arciniega is a Microbiologist at the Center for Biologics Evaluation and Research
of the US Food and Drug Administration.
He received an undergraduate degree in
chemistry, bacteriology and parasitology in 1982, a Master of Science degree in clinical

biology in 1985 and a Doctor of Science degree in the same specialty in 1987, all from the
National School of Biological Sciences in Mexico City.
Before joining the Laboratory of
Pertussis at CBER in 1989 as a Visiting Fellow, he worked for nine years in different
capacities at the Mexican National Public Health Laboratory, most recently as Underdirector
for Biological Control, in charge of two departments with responsibility for sanitary testing of
food and biologics. He was also a founding member of the Permanent Commission of the
Mexican Pharmacopoeia (Biologics and Bioassay and Statistics Committees).
His research and regulatory activities have focused on pertussis, diphtheria and anthrax
vaccines and the development of alternatives to animal tests. Currently Dr. Arciniega is a
member of the Laboratory of Respiratory and Special Pathogens, Division of Bacterial,
Parasitic, and Allergenic Products, where he works as a CMC reviewer with special
emphasis on methods development, validation and quality control.
Additionally, he
participates in other regulatory activities such as batch release, and acts as a liaison with the
US Pharmacopeia (General Chapters Biological Analysis Expert Committee).
He has
published more than 20 papers in his field and cooperated with the World Health
Organization and the Pan American Health Organization as a Temporary Advisor, and with
the European Center for the Validation of Alternative Methods (ECVAM) and the European
Directorate for the Quality of Medicines and Healthcare (EDQM). Dr. Arciniega has mentored
several young Latin American vaccine regulators and other scientists.
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Balks, Elisabeth
(Paul-Ehrlich-Institut, Federal Institute for Vaccines and Biomedicines, Germany)
Professional career:
1987
graduated in veterinary medicine from the University of Giessen/Germany
1995
doctorate in veterinary medicine

(Occurrence, diagnosis and pathogenic relevance of avian haematozoa)
1988 1989 Diagnostic microbiology at the Institute for Hygiene and Infectious Diseases in
Animals, University of Giessen
1989 - 1998 Clinical microbiology and pathology at the Institute for Poultry Diseases,
University of Giessen
1996
Degree of specialist veterinarian in the field of avian medicine
1998
Degree of specialist veterinarian in the field of microbiology
Since 1998 Paul-Ehrlich-Institut, Veterinary department,

Main fields of activity:
Pre-marketing assessment and quality control of bacterial, fungal and parasitological

veterinary vaccines and immune sera (national and European level, active member of
the Official Medicines Control Laboratory Network)
Quality management (internal auditor)
Research projects on the development and validation of alternatives to animal testing

in vaccine quality control
Current focus: Leptospira vaccines and tuberculins
82
Bruckner, Lukas
Dr. Lukas Bruckner got his degree in veterinary medicine from the University of Berne
in Switzerland in 1982.Before his recent retirement, he worked as the head of the
Vaccine Control Department at the Institute of Virology and Immunology (IVI), the
competent authority for licensing and batch release for immunological veterinary
medicinal products in Switzerland. One of his main interests was in in vitro alternatives
to classical in vivo tests for the quality control of vaccines.Lukas still participates in
several activities of the European Directorate for the Quality of Medicines & Health
Care, the European Pharmacopoeia Commission, as well as the expert groups on
vaccines and sera for veterinary and human use. These activities allow Lukas to
actively promote the 3R philosophy in the regulatory framework."
Buchheit, Karl-Heinz
Dr. Karl-Heinz Buchheit obtained his degree in pharmacy from the Goethe University in
Frankfurt/M. (Germany) and his Ph.D. in pharmacology in 1984 from the same university.
From 1984 until 1999 he worked as research scientist and group leader in preclinical
pharmacology for Novartis/Sandoz in Basel (Switzerland). From December 1999 to August
2013, he was Deputy Head of the Department Biological Standardisation, OMCL Network &
HealthCare at the EDQM (Council of Europe, Strasbourg, France) and secretary of the
Steering Committee of the Biological Standardisation Programme. Since August 2013 he is
Head of the same department which is responsible for the activities of the EDQM in the fields
of biological standardisation, the OMCL network, blood transfusion, organ-, tissue and cell
transplantation, pharmaceutical care, anti-counterfeit measures and consumer health
protection.
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Chapsal, Jean-Michel
Dr. Jean Michel Chapsal has been working for 30 years at Sanofi Pasteur. Sanofi Pasteur is
the vaccines division of Sanofi Aventis group and the largest company in the world devoted
entirely to human vaccines. Dr Chapsal has led a number of units, including Research and
Development, Downstream Processing and Analytical Services-Regulatory Affairs Interface.
He has also served recently as Director of Global Analytical Services. His areas of expertise
include technical method development for vaccine control, application of emerging
technologies in vaccinology, 3Rs alternative methods development, and analytical services in
biochemistry, microbiology.
Dr Chapsal received a PhD in Biochemistry from Compigne University, France. He also
completed the general course of Virology at the Institut Pasteur in Paris and received an
advanced postgraduate diploma in Virology at Universit Paris Diderot. In 1986, he
completed a postdoctoral fellowship in the laboratory of Dr Lenore Peirera at the University of
California, San Francisco.
Dr Chapsal has been a member of the Working Group on Sera and Human Vaccines of the
French Agency for the safety of Health Products (ANSM) and of the French Scientific Interest
Group on Alternative methods (GIS) for the past 6 years. He is co-chair of the International
NIH Test Replacement Working Group in the frame of the consistency Vaccine Project of the
European Partnership for Alternative Approaches to Animal Testing (EPAA). He is member
of Francopa, the French subgroup of ECOPA (European Consensus Platform for
Alternative), and was co-chair of the international Histamine Test Replacement Working
Group.
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Coppens, Emmanuelle
DVM
Analytical Excellence in vivo- Quality Control department
Sanofi Pasteur France
1997 Doctor in Veterinary Medicine Toulouse Veterinary School (France)
1997-1998 Pierre Fabre- Animal Ressources Department
1998- now: Sanofi Pasteur Quality Control Laboratories
Head of immunology and safety laboratory up to 2006
Head of histology laboratory and in vivo Analytical Excellence up to now
Expertise-scope of activity:
Laboratory animals, AAALAC accreditation
Vaccine in vivo testing, combination vaccines, live viral vaccines especially OPV and
neurovirulence
3Rs
Analytical improvement and validation
Regulatory requirements and compliance
Cussler, Klaus
From 1976 to 1981 I studied Veterinary Medicine at the University of Giessen, Germany.
After several years as a veterinary surgeon in general veterinary practice I decided to go into
research. In 1986 I received a PhD in Immunology at the University of Mainz and became a
Doctor of Veterinary Medicine (Dr. vet. med.). In March 1987 I received a position in the
veterinary Division of the Paul-Ehrlich-Institut, (Germany). The numerous animal tests
required for licensing and batch testing of bacterial vaccines soon focused my research on
3R activities. Based on several national and European research projects and the cooperation
with other institutions some progress could be achieved in the veterinary field.
Today I am responsible for pharmacovigilance of veterinary immunological medicinal
products.
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Dellepiane, Nora
Education:
Diploma in Biology (1977) at the Faculty of Exact and Natural Sciences, University of Buenos
Aires- Argentina
Doctorate with specialization in Microbiology in the same University (1984).
Diploma in Health Systems Management at the London School of Tropical Medicine and
Hygiene, United Kingdom (2006).
After initial training in diagnostics of enteric diseases, she specialized in the field of vaccine
production, quality control and quality assurance in which she worked for more than 30
years. Adviser to WHO since 1993, joined the organization in 1996 as the Scientist
responsible for the Vaccines Prequalification Programme, which she led for 17 years.
Appointed at WHO as Coordinator of the Regulatory Systems Strengthening team since
September 2013. She is currently and independent consultant focused on vaccine quality
and regulation of Biological Products. Contracted by Serum Institute of India LTD as Senior
Regulatory Advisor since June 2015.
Deming, Stanley
Stanley N. Deming, Ph.D. is president of Statistical Designs, Houston, TX, through which he
consults and offers short courses in the areas of experimental design, optimization, and the
statistical analysis of laboratory data, with emphasis on bioassays. Dr. Deming received his
B.A. degree in chemistry from Carleton College, Northfield, MN, and his M.S. and Ph.D.
degrees in analytical chemistry from Purdue University, West Lafayette, IN. He has been on
the chemistry department faculties of Emory University (1970-1974) and the University of
Houston (1974-2001) where he is Professor Emeritus. Over his academic career he was the
major research advisor for 16 Ph.D. students and 19 M.S. students. Since 1975 he has
taught (with Stephen L. Morgan) over 650 short courses.
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Dr. Deming has been the author or co-author of approximately 100 papers and three books,
including Experimental Design: A Chemometric Approach (Elsevier) and Sequential Simplex
Optimization: A Technique for Improving Quality and Productivity in Research, Development,

and Manufacturing (CRC Press). He has served on the editorial boards of the Journal of
Chemometrics (1987-2003), Critical Reviews in Analytical Chemistry (1996-2002), and
Chemometrics and Intelligent Laboratory Systems (1987-1993). His web site is
http://www.statisticaldesigns.com.
Descampe, Blaise
Blaise Descampe, Doctor in Veterinary Medicine, hold a Master in Management.
He has hold different position in GSK quality control for 12 years.
He was responsible for GSK Vaccine quality control test on animal and headed the
AAALAC accredited animal facility in GSK Belgium.
He was Vaccine QC representative at 3Rs regional group reporting 3Rs efforts

successes and projects.
He represented Belgium in independent international working group (FELASA) active

in EU directive discussions on laboratory animal pain level.
He co-Chair workshop session of International Workshop on Alternatives to the

Murine Histamine Sensitization Test (HIST) for Acellular Pertussis Vaccines:
Progress and Challenges in the Replacement of HIST" in Prague Aug. 2014.
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French, Jodi
Head of Manufacturing and Regulatory Affairs
Before joining Harrisvaccines in September 2010, Jodi served as business manager and
alternate USDA liaison for a small biologics firm in Ames. As head of manufacturing and
regulatory affairs for Harrisvaccines, she manages our production staff and is responsible for
the implementation of processes required for vaccine production using our SirraVax
technology. Jodi also initiates and manages USDA correspondence regarding both product
and facility licensure and leads all regulatory interactions.
Giese, Christoph
QC-related skills: Binding and cell-based bioactivity assays, assay development, qualification
and validation, biosimilarity assessment, comparability excercise
Current technological and development activities:
Immune cell based assays, tissue engineering, HuALN model, organoid models,
mesenchymal stem cells, in vitro methods according to the 3R concept
Professional Experience
Since 2008
Director of the Departments Cell and Tissue Services and Quality Control,
ProBioGen AG
2006-2008
Director of the Department Cell and Tissue Services at ProBioGen AG
2000-2006
Group Leader Tissue Engineering and Cell Selection, Screening and

Automatization at ProBioGen AG
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1999-2000
Postdoc at the Institute for Biochemistry

Group of Prof. Dr. Manfred Sernetz,
Faculty for Veterinary Medicine,
Justus-Liebig-University of Giessen
1994-1999
Scientific assistant, group of Prof. Dr. Manfred Sernetz
Education
1994-1999
PhD Thesis (Dr. rer. nat., Final grade summa cum laude) Biotechnology,
Tissue Engineering, in-vitro testing, engineering of the bovine Corpus Luteum
(Artificial Organ) at the Institute for Biochemistry Faculty for Veterinary
Medicine, Justus Liebig-University of Giessen
1988-1994
Justus-Liebig-University of Giessen
and Wolfgang-Goethe-University of Frankfurt/Main
Study of Biology (Diploma degree)
Awards
Tierschutz Forschungspreis 2007
award for alternatives to animal testing by German federal government
(Bundesministerium fr Ernhrung, Landwirtschaft und Verbraucherschutz,

BMELV) for the development of a human artificial human lymph node model
for in vitro testing of drugs)
PETA Progress Award 2008
Tierfreundlichster Wissenschaftler 2008, by PETA Deutschland e.V.
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Glansbeek, Harrie
Dr. Harrie Glansbeek is a senior scientist in Department of Analytical Technical Support of
MSD Animal Health (Boxmeer, the Netherlands). He is working in this department since 2011
where he is mainly involved in the improvement of existing QC tests and the development of
new QC tests. During the last 2 years he was responsible for the development, validation
and implementation of several new in vitro potency tests to replace current animal potency
tests.
Harrie is active in vaccine R&D for more than 18 years. In the period 1997-2001 he was a
project leader in the Department of Infectious Diseases and Immunology of the Veterinary
Faculty of the University Utrecht (Utrecht, the Netherlands). He joined Intervet International
(currently MSD Animal Health; Boxmeer, the Netherlands) in 2001 where he worked as a
project leader in the R&D department. Between 2005 and 2010 he was a project leader in
the R&D department of Nobilon International (Boxmeer, the Netherlands) where he worked
on the development of human vaccines.
Halder, Marlies
European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)
Dr. Halder studied veterinary medicine at the University of Munich (Germany) and obtained a
PhD from the University of Munich in 1987 for research on crustaceans and fish diseases.
She worked for several years as a research scientist at the University of Munich and the
Akademie fr Tierschutz, Neubiberg, Germany. Dr. Halder joined the European Commission
in October 1995 and currently holds a position as a senior scientist at the European
Commissions Joint Research Centre, Institute for Health and Consumer Protection, Systems
Toxicology Unit/EURL ECVAM in Ispra, Italy. She is responsible for EURL ECVAM's
activities on replacement, reduction and refinement related to the quality control of
biologicals and environmental toxicity testing of chemicals.
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Hendriksen, Coenraad
Coenraad Hendriksen, DVM, PhD, studied veterinary medicine at Utrecht University and
received a post-doc training in Laboratory Animal Science. He obtained his PhD in 1989 on a
thesis entitled Alternatives to Animal Testing in Diphtheria and Tetanus Research. Currently
he is animal welfare officer at the Institute for Translational Vaccinology (Intravacc) and
programme manager on 3R projects. Running projects include the development of in vitro
and analytical models in batch release testing of vaccines, the international validation of a
serological potency model, as well as PhD projects on animal welfare related aspects.
Hendriksen also holds a chair on Alternatives to Animal Use at the Faculty of Veterinary
Medicine, Utrecht University, since 2000. He is member of several national and international
committees.
Hoogerheide, John
Dr. Hoogerheide is a research fellow at Zoetis in Kalamazoo, Michigan, USA. He leads a
multidisciplinary analytical team in characterizing proteins, monoclonal antibodies, and other
macromolecules. The group applies modern analytical techniques to solve difficult analytical
challenges, including antigen and adjuvant measurements in complex samples such as
vaccines.
Dr. Hoogerheide received a PhD in analytical chemistry from Michigan State University. His
career has carried him from Schering-Plough (now Merck), to The Upjohn Company,
Pharmacia, Pfizer, and finally to Zoetis, the animal health company spun off from Pfizer in
2013. His experience spans analysis of both pharmaceuticals and biopharmaceuticals in
both human and animal health.
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Hoonakker, Marieke
Marieke Hoonakker MScreceived her master degree in Biomedical Science from the
University of Utrecht in 2009. In 2011 she finalized a survey on the current status of
alternatives in the vaccine field and the results were published in the report entitled
Vaccines, Animal experiments and Alternatives. In 2011 she started with her PhD study
under the supervision prof. Hendriksen. The project aims at setting up and improving in vitro
methods for safety and quality testing of vaccines in general, and Bordetella pertussis
vaccines in particular. Part of the work is establishing immunological in vitro assays for the
consistency approach and optimization of the PTx-cAMP assay for detection of pertussis
toxin in acellular pertussis vaccines. Proof of principle for both assays has been described in
articles published in peer reviewed journals.
Jungbck, Carmen
Dr. Carmen Jungbck is Head of Section International Collaboration and Batch Control,
Poultry Viruses, in the Veterinary Department at the Paul-Ehrlich-Institut (PEI), the Federal
Institute for Vaccines and Biomedicines, Langen, Germany. She holds a PhD (1979) in
veterinary medicine from the Tierrztliche Hochschule Hannover, Germany. From 1978 to
1981, she worked as veterinary surgeon, mainly for small animals. In 1981, she joined the
Veterinary Department at the Paul-Ehrlich-Institut. 1986 she became Head of the Veterinary
Virology Section. The major topics of her work are testing, licensing and official batch release
of immunological veterinary medicinal products (IVMPs) for animals, especially viral vaccines
for poultry. This activities include testing of vaccines in the target species e.g. by challenge
before licensing according to the Ph Eur monographs as part of the assessment of the
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application, practical testing of final product batches for freedom of extraneous agents,
sterility and potency, research related to testing and alternatives to animal testing, especially
for extraneous agents testing, antigen quantification, potency testing. She is member of the
Immunologicals Working Party (IWP) of the CVMP and of the Joint CVMP/CHMP ad hoc
expert group on 3Rs (JEG 3Rs) at the European Medicines Agency (EMA). At the European
Directorate for the Quality of Medicines & Health Care (EDQM), she was chair of the General
European OMCL Network (GEON) Advisory Group (2011-2015),she is member of the
Veterinary Batch release Network (VBRN) Advisory Group and member of Expert Group 15V
at Ph Eur. She has contributed to a large number of guidelines and monographs and has
published a number of scientific papers.
Kersten, Gideon
Gideon Kersten is a vaccinologist with a special interest in vaccine delivery and vaccine
characterization. He studied biochemistry at Leiden University and obtained his PhD at
Utrecht University. He started his career at the Institute for Public Health and the
Environment in Bilthoven, The Netherlands which later became the Netherlands Vaccine
Institute. Currently he is at the new Institute for Translational Vaccinology (Intravacc) in
Bilthoven. At Intravacc he is responsible for the vaccine formulation activities and assay
development. He is also responsible for QC activities in projects with GMP status. He is
appointed as professor in vaccine delivery at Drug Delivery Technology at Leiden University.
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Kubiak, Vaughn
Vaughn Kubiak has over 30 years of experience in global animal health, with a primary focus
on development, licensure, and maintenance of global veterinary biologicals. He has helped
develop conventional and innovative immunological veterinary medicinal products for all
major species during his career. Vaughn has worked for a number of global animal health
companies, with positions in R&D, QA/QC, regulatory affairs, product management, and
commercial organisations. Vaughn has spent the last 13 years with Zoetis Inc., where he
has held management positions in US Regulatory Affairs, Biologicals Process Development,
and Biological Analytical Development. Since 2009, he has been responsible for the
European, Middle East, and African Biological Regulatory Affairs team in Sandwich, England
and then Zaventem, Belgium. He holds a Bachelor of Science and a Master of Science in
Microbiology from the Ohio State University and Emory University, respectively.
Lei, Dianliang
Dr Dianliang Lei is currently working in the Department of Essential Medicines and Health
Products of World Health Organization, Geneva, Switzerland responsible for the
development of international standards for biological products to ensure the quality, safety
and efficacy of the products since 2003. He has been in charge of development of WHO
Guidelines for Lot Release of Vaccines by Regulatory Authorities, WHO Recommendations
for acellular pertussis vaccines, Recommendations for DTP based combined vaccines,
Manual for establishment of national standards, Manual for laboratory testing of DTP
vaccines, Guidelines for post-approval changes of vaccines, GMP for biologicals and
establishment of measurement standards.
He had been working in the field regulation and quality control of vaccine in the National
Institute for the Control of Pharmaceutical Products, Beijing China for more than 20 years till
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2003. His work was focused on the regulation system for vaccines in China, especially on the
national requirements (pharmacopeia), standards and lot release system. He was the
executive member of Chinese Pharmacopoeia and the deputy director of the Institute.
He holds a Ph D in medical science from Osaka University, Japan in 1996.
Mallet, Laurent
Dr. Laurent MALLET obtained his Master Degree of Science in Biochemistry from Claude
Bernard Lyon University in France. He completed his PhD work in Virology and Molecular
Biology under the co-direction of Pr Michle Aymard (National Reference Center for
Enterovirus, Lyon, France) and Dr. Franois Pelloquin (Sanofi Pasteur, formerly Pasteur
Mrieux Connaught). He obtained his PhD in 1996. After several positions within Sanofi
Pasteur in France and in Canada, he is currently Head of Analytical R&D Europe.
He is also a member of several expert committees, Group 15 Sera and Vaccines at
European Pharmacopoeia and part of the French Pharmacopoeia Committee Biological
Products and Innovative Therapies. Recently, he has been involved in several WHO
working groups (e.g. IPV, Dengue and Yellow Fever vaccines) including the WHO Study
Group on Cell Substrates and the associated Adventitious Agent sub-group.
Metz, Bernard
Bernard Metz was born on November 18th 1972 in Dokkum, The Netherlands. In 1993 he
started to study Chemistry of the Faculty of Mathematics and Natural Sciences, University of
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Groningen. He graduated in 1998. From 1999 until 2000, he was involved as research
associate in the project entitled Random mutagenesis of gonadotrophins in Dictyostelium
discoideum (a collaboration between the Department of Biochemistry, University of

Groningen and Organon, Oss). In the period between 2000 and 2004, he was working as a
graduate student on his thesis Structural Characterisation of diphtheria toxoid. This project
was a collaboration between the Department of Pharmaceutics of the Faculty of
Pharmaceutical Sciences of the Utrecht University and the Unit Research and Development
of the Netherlands Vaccine Institute in Bilthoven (NVI). Since 2004, he is working at R&D of
the NVI focussing on test development and characterisation of vaccines. From 2013, Bernard
is working as a research scientist at Intravacc (formerly part of NVI).
Morgeaux, Sylvie
Dr. S. Morgeaux worked and prepared her PhD in the Rabies Unit at Pasteur Institute in
Paris and obtained her PhD on Animal Viruses in Paris VII University. In 1995, she joined
ANSM (formerly Agence du Mdicament) as a scientist in charge of the control and release
of various live and inactivated viral vaccines. During eleven years she was the Head of the
Viral Vaccine Control Unit and was also a Temporary Adviser at the Expert Committee on
Biological Standardization meeting for vaccines of WHO, at others working groups and for
NRAs assessment and training for vaccines control and lot release. Presently, she is in
charge of the release of Rabies, Hepatitis A vaccines, equine anti-rabies immunoglobulins
and of the 3Rs strategy at the Batch Release and Market Surveillance for Biological Products
Department of the Laboratory Control Division of ANSM. She is also involved as an expert in
the regulation assessment of Rabies, Hepatitis A and B, Varicella, Tick-Born, Japanese
Encephalitis, Rotavirus, Papillomavirus and Smallpox vaccines. Dr S. Morgeaux is a member
of the European Pharmacopoeia Group 15, of the EDQM Drafting Group for human vaccines
and of the Technical Committee of EPAA.
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Ragan, Ian
Dr Ian Ragan is a neuropharmacologist and an independent consultant in the biomedical
sector. He spent nearly 20 years in the pharmaceutical industry, most recently with Eli Lilly
as Executive Director, Neuroscience Research, Europe, and Executive Director, European
Scientific Affairs. He was the Lilly representative on the Research Directors Group of the
European Federation of Pharmaceutical Industries and Associations (EFPIA). Until recently
he was the co-ordinator of the European Partnership for Alternative Approaches to Animal
Testing (EPAA) project on the application of the 3Rs and the consistency approach for
improved vaccine quality control. Currently he is a Board Member of the National Centre for
the 3Rs and Director of the UK National Autism Project.
Dr C. Ian Ragan 51 Slaidburn St London SW10 0JW UK Office: +442073514985 Mobile:
+44(0)7793314727
Redhead, Keith
Keith Redhead was, from 1980, a Senior Scientist at the National Institute for Biological
Standards and Control, UK, responsible for research on Diphtheria, Tetanus and Pertussis
vaccines. It was the large numbers of mice necessary for the control testing of Pertussis
vaccines that first stimulated his interest in alternative testing methods. Since 1995, he has
been a researcher at a number of companies working on bacterial veterinary vaccines, with
particular emphasis on clostridial products. Once again the high number of animals used in
this area has driven his interest in applying the principles of the 3Rs to vaccine research and
testing. He is on various 3Rs advisory bodies, a member of the British Pharmacopoeia Panel
of Experts, a Project Leader of the EPAA Working Group for Clostridial Vaccines and has
authored over 70 scientific publications.
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Rommel, Eddy
Eddy Rommel is Doctor in Veterinary Medicine and Master in Animal Production. He is also
graduated in lab animal sciences and in Business Administration.
Eddy has been involved in animal research, In-vivo Quality Control of vaccines and
Regulatory Affairs for the last 30 years, starting his career as research assistant at the
University of Lige, then moving to GlaxoSmithKline Biologicals where he was Associate
Director In-vivo Quality Control and responsible for animal research before being in charge of
a regulatory affairs department.
Since 2002 Eddy is Founder and Managing Director of Rommel Consulting Partners,
providing consultancy services in preclinical development of Biologicals and Advanced
Therapies, in Quality Control of vaccines for human and veterinary use and in lab animal
sciences.
Eddy is member of the Vaccines Technical Committee of EPAA Vaccines Project and of
several Belgian and European committees and professional associations.
Schiffelers, Marie-Jeanne
Marie-Jeanne Schiffelers has a masters degree in Environmental Policy Sciences
and is a senior consultant and lecturer at the Utrecht University School of
Governance.
She works as a consultant and researcher in the field of policy
science, organizational change and technology transitions.

She has been involved in several projects in the field of acceptance and use of 3R
methods for regulatory purposes e.g.:
Project manager research Regulatory Animal Testing (2004 -2005)
Member research team Evaluatie Besluit Biotechnologie bij Dieren (20052006)
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Member research team Impact Assessment for the revision of EU Directive

86/609 on the protection of animals used for experimental and other scientific
purposes (2006-2007)
PhD research on the acceptance and use of 3R models for regulatory

purposes (since 2009)
In 2008 she was rewarded with the ALTEX prize 2008 for the article: Factors that
Stimulate or Obstruct the Implementation of 3Rs in the Regulatory Process.
Currently she is finishing a PhD degree on the Acceptance and Use of 3R Methods
for Regulatory Purposes under the supervision of Prof. Dr. Coenraad Hendriksen
(Netherlands Vaccine Institute), Prof. Dr. Bas Blaauboer (Institute for Risk
Assessment Sciences, Utrecht University) and Dr. Wieger Bakker (Depa rtement
Bestuurs en Organisatiewetenschap Universiteit Utrecht).
Sheets, Rebecca
Consultant for Grimalkin Partners
Adjunct Professor at Catholic University of America
Board member of the International Alliance for Biological Standardization; Vice

President for Human Biologicals; immediate past co-chair of the Human Vaccines
Committee
In 2013, retired from the U.S. Public Health Service (Captain)
Vaccine Scientific and Regulatory Specialist at the National Institute of Allergy and
Infectious Diseases at the National Institutes of Health (2002-2013)
o
Div. of AIDS & Vaccine Research Center
Subject matter expert on vaccine cell substrates and vaccine pre-clinical

safety assessment, including toxicology
Scientific Reviewer in the Viral Vaccines Branch of the Division of Vaccines and
Related Products Applications, Office of Vaccines Research and Review, CBER/FDA
(1993-2002)
B.S. degree in Biology from the California Institute of Technology
M.S. degree in Cellular, Viral, and Molecular Biology from the University of Utah
School of Medicine
Ph.D. in Pathology from the University of Southern California School of Medicine
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Sondag, Perceval
Perceval holds a Bachelors Degree in Physical Therapy, followed by a Masters Degree in
biostatistics, both obtained in the Universit Catholique de Louvain in Belgium. After working
in Quality Control and Operational Research in Hospital setting, he joined Arlenda in 2013
and specialized in Bayesian Modeling and Nonclinical Statistics, mostly applied in Quality by
Design, Bioassays and method validation.
Stirling, Catrina
Dr Catrina Stirling is associate director of regulatory affairs at Zoetis. Dr Stirling has a
research science background with a degree in Virology from Edinburgh and a PhD in
Veterinary Immunology conducted at the Pirbright Institute followed by 4 years post-doctoral
work on vaccine development at Pirbright. Dr Stirling then joined the Veterinary Medicines
Directorate (VMD) in the UK before moving to Industry in 2007 to join Pfizer (now Zoetis). Dr
Stirling is an expert in biologicals regulatory affairs with extensive development and
registration experience and has been involved with 3Rs projects both internally and
externally through EPAA and other regulatory bodies through her regulatory career.
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Uhlrich, Sylvie
Dr Sylvie UHLRICH received her PhD in Biochemistry in 1986 from University of Paris and
was appointed by Institut Merieux as Research scientist.
She developed her carrier in Analytical science and assay development at Sanofi Pasteur.
As head of analytical R&D biochemistry platform during several years, she was deeply
involved in the development of 3Rs alternative approaches for Diphtheria, Tetanus,
Pertussis, Hepatitis B, Hepatitis A and Rabies vaccine.
She is currently Analytical expert in charge of strategic alignment at Sanofi Pasteur (based in
Lyon), part of the French Pharmacopeia Committee for Biological Products and Innovative
Therapies and industry coordinator in European imi2 project for consistency approach to
quality control in vaccine manufacture.
Van Dongen, Hans

Education
Agricultural University Wageningen (1982), agricultural engineer in rural development and
land use
Professional career
1983-1990: policy maker in the field of agro-environmental measusres, rural development
and land use (Ministry of Agriculture and Fisheries)
1990-1993: assistant secretary of the Council of Ministers (Ministry of General Affairs)
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1993-2004: advising and executive officer in Bureau Secretary General, Department for
International Affairs and Department for Food Quality and Animal Health (Ministry of
Agriculture, Nature and Fisheries)
2004-2012: Head of Unit Animal Health (Ministry of Agriculture, Nature and Food Quality)
2012-2015: Head of Unit Animal Welfare, including animal testing and alternatives (Ministry
of Economic Affairs)
Van der Stappen, Ton

Ton is a Senior Pharmaceutical Assessor at the Medicines Evaluation Board (MEB) in the
Netherlands. His main activities comprise assessment of product quality aspects of
marketing authorisation applications for biopharmaceuticals/biological medicinal products
and providing scientific advise to pharmaceutical companies at national (MEB) and European
level (Committee for Human Medicinal Products (CHMP), European Medicines Agency)
Ton has been an appointed member of the CHMP Biologics Working Party since 1998 and
has been rapporteur of several CHMP/BWP Quality guidance documents. As of 2009, he is
also a member of the Biological Standardisation Programme Steering Committee of the
European Directorate for the Quality of Medicines & HealthCare (EDQM).
Dr.A.J. van der Stappen, Section Pharmacology, Toxicology and Biotechnology (FTBB),
Medicines Evaluation Board
CBG, Graadt van Roggenweg 500, 3531 AH Utrecht, The Netherlands
P.O. Box 8275, 3505 RG Utrecht, The Netherlands, @ aj.vd.stappen@cbg-meb.nl
+31 88 224 8263, +31 6 52756491 (cell phone))
102
Van Zeeland, Rob
??????????????????????????????????????????
Viviani, Laura
Laura Viviani studied Philosophy and Communication of Science in Milano and Trieste (Italy)
before joining Novartis in 2009. Starting from 2012 in Novartis Vaccines, she has been
involved into the development and implementation of the 3R Program within the organization
working with the management and the scientists to embed the 3R culture into the everyday
activities. She has been representing the organization to international conferences and
forums. On March 2015, Novartis Vaccines became a GSK company and she continues to
work in the 3R culture promotion into the new organization.
103
PARTICIPANTS
104

Iabs Conference 3RS and Consistency Testing in Vaccine Lot Release Testing

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IABS CONFERENCE

3RS AND CONSISTENCY TESTING IN VACCINE LOT

VACCINE LOT RELEASE TESTING

Egmond aan Zee, The Netherlands

Welcome from the Organisers

Intravacc, The Netherlands

WHERE INNOVATION IN VACCINE QUALITY CONTROL MEETS ANIMAL WELFARE.

Local Organising Committee

Juan Arciniega (FDA)

Arnoud Akkermans (RIVM)

Lukas Bruckner (IVI)

Johan van der Gun (BBio)

Karl-Heinz Buchheit (EDQM)

Marieke Hoonakker (Intravacc)

David Dusek (USDA)

Gideon Kersten (Intravacc)

Marlies Halder (EURL-ECVAM)

Fabrizio de Mattia (MSD-AH)

Richard Isbrucker (Health Canada)

Bernard Metz (Intravacc)

Suresh Jadav (SII)

Laurent Mallet (Sanofi Pasteur)

Marieke Schimmel (Intravacc)

Eddy Rommel (Rommel Consulting Partners)

Abbigail Charlet (IABS)

Thea Sesardic (NIBSC)

Rebecca Sheets (IABS)

About the Region

Egmond aan Zee (www.vvvhartvannoordholland.nl)

About Alkmaar (www.vvvalkmaar.info)

About Amsterdam (www.amsterdam.info)

Egmond aan Zee

How to get to Hotel Zuiderduin from Amsterdam (Schiphol) airport

BY PLANE + TRAIN + BUS/TAXI

For those traveling by train: * dont forget to validate your ticket

Taxibedrijf P.L. Zwart B.V.

Map of conference venue/hotel

Wednesday, 16 September and Friday, 18 September: Buffet lunches will be served

Thursday, 17 September : walking lunch will be served in the conference lobby

Hotel Zuiderduin & Facilities

The full participation includes:

Admission to the conference activities

The Get-Together Party on Wednesday evening

The Congress Dinner on Thursday evening

Two nights at Hotel Zuiderduin (16 18 September)

Breakfast (17/18 September), Lunches (16/17/18 September), Dinner (16 September,

Wednesday, September 16, 2015

Registration will be open from 10:30. Coffee will be available

Lunch (buffet) in restaurant

Opening of conference (Lamoraalzaal)

John Petricciani, chairman IABS

Rob van Zeeland, CEO Intravacc

Hans van Dongen (Ministry Economic Affairs, NL) with video

Session 1: 3Rs alternatives and safety testing

3Rs into action (Coenraad Hendriksen, Intravacc, NL)

Time to apply 3 Rs to virus testing? (Rebecca Sheets, Grimalkin

Abnormal Toxicity Testing Roots and evolution of first animal safety

Viral safety of vaccines: how to streamline the Adventitious Testing

Session 2: 3Rs opportunities and barriers

Feedback from the satellite meeting on harmonisation (Catrina Stirling,

BSP130: Validation of cell line assays for toxicity and antigenicity

Regulatory acceptance and use of 3R models in rabies vaccines

A competition ELISA for potency testing of equine anti rabies sera as