iabs conference 3rs and consistency testing in vaccine lot release testing

7/21/2019 IABS CONFERENCE 3RS AND CONSISTENCY TESTING IN VACCINE LOT RELEASE TESTING

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IABS CONFERENCE

3RS AND C ONSISTENCY T ESTING IN V ACCINE LOT

RELEASE T ESTING

Egmond aan Zee, The Netherlands

September 16 – 18, 2015



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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 theprocess 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



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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.



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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)



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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

http://www.amsterdam.info/oude-kerk/





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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



7

Egmond aan Zee

The Netherlands

T. + 31727502000

[email protected]

www.zuiderduin.nl

mailto:[email protected]


http://www.zuiderduin.nl/






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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/white-

checked 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: * don’t 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



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Taxibedrijf P.L. Zwart B.V.

De Hoefsmid 37

1851 PZ Heiloo

T: 072 – 533 13 13/ I: http://www.taxizwart.nl/ E: [email protected]

(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

http://www.taxizwart.nl/



http://www.taxizwart.nl/



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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



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Bowling Squash Fitness room Bike rental



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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.



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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



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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)



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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 Sécurité du Médicament et des

Produits de Santé 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)



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11.00 – 12.05 Session 4: Consistency Approaches

Chairs : Carmen Jungbaeck (PEI)/Joris Vandeputte (IABS)



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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)




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15.30 – 18.00Session 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)



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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)


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)



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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



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Session 1: 3Rs alternatives and safety testing

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.



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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 producedcan 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.



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TIME TO APPLY 3 R’S 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 R’s to virus testing.



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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 Körverstraat 35, 5831 AN Boxmeer, the Netherlands.

[email protected]

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






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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 Internat ional Pharmacopoeia in 1951 when the WHO started to develop

internationally accepted guidance for the testing of sera and vaccines.



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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.



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VIRAL SAFETY OF VACCINES: HOW TO STREAMLINE THE ADVENTITIOUS AGENT

TESTING PACKAGE?

Laurent Mallet

Sanofi Pasteur - 1541 avenue Marcel Merieux, 69280 Marcy l’Etoile – France

[email protected]

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, aswell 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.






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Session 2: 3Rs opportunities and barriers

Catrina Stirling : report of the workshop held September 15/16



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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

[email protected]

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






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(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-medicines-

directorate)

(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, [email protected]

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







31

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; Noël Tordo; Monique

Alberti; Sylvie Morgeaux Vaccine 2013;31(50):6022-9.



32


REGULATORY ACCEPTANCE AND USE OF 3R MODELS IN -RABIES- VACCINE

QUALITY CONTROL

Marie-Jeanne Schiffelers

Utrecht University School of Governance

[email protected]

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 thisadditional 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

1 http://www.evm-vaccines.org/pdfs/vaccines_and_animal_welfare_fin.pdf






33

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 Morgeaux

1

, Jehanara Korimbocus, Nicolas Dehay, François Cano

1 Agence Nationale de Sécurité du Médicament et des Produits de Santé Department,

Control Directorate, Batch Release and Marketing Surveillance of Biological Products

Department, 321 avenue Jean Jaurès, 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





34

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/mlfortheWHOISand±0.070log10IU/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 c-

ELISA and the manufacturer’s 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



35

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



36

Session 3: 3Rs alternatives, new ideas, pitfalls and solutions

PROTEOMIC ANALYSIS OF BOVINE TUBERCULIN PURIFIED PROTEIN DERIVATIVES

E. Balks

1, F. Mohs 1 , J. Trösemeier 2 and A. Reuter 3

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

Ehrlich-Institut, 63225 Langen, 3 Allergology, Paul-Ehrlich-Institut, 63225 Langen, Germany

[email protected]

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.






37

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.



38


ALTERNATIVE METHOD FOR RABIES IMMUNOGLOBULIN MOUSE POTENCY

TESTING

Emmanuelle Coppens

Sanofi Pasteur - 1541 avenue Marcel Mérieux, 69280 Marcy l’Etoile – France

[email protected]

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.






39


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.



40


COMPARISON AND BRIDGING OF BIOASSAYS: DOING MORE WITH LESS USING

BAYESIAN STATISTICS

Perceval Sondag and Bruno Boulanger

Arlenda SA, Chaussée verte 93, 4470 Saint-Georges, Belgium

Contact : [email protected]

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.







41


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



42

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 itsvalidation 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.



43

Session 4: Consistency Approaches

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.



44


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 R’s, 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.



45


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 aimto 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.



46


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



47

collaborative agreement, in order to utilize the results of animal test already performed by

other laboratory.



48

Session 5 : Consistency approaches, continued

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

[email protected]

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.



http://www.aphis.usda.gov/animal_health/vet_biologics/publications/memo_800_213.pdf

http://www.aphis.usda.gov/animal_health/vet_biologics/publications/memo_800_213.pdf




49

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.



50


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.



51


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



52

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):986 – 995

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 148:38 – 45



53


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 quaternaryamine. HPLC with tandem mass spectrometry (LC-MS/MS) of antigens is applied to both

identification and quantitation.



54

Session 7: Consistency testing in practice

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 [email protected]

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 IndiaLtd. 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.








55


FUNCTIONAL IN VITRO TESTING OF VACCINES WITHIN THE CONSISTENCY

APPROACH: A PROOF-OF-PRINCIPLE USING A WHOLE CELL BORDETELLA

PERTUSSIS VACCINE

M.E. Hoonakker

1,3 , L. Verhagen 1,3 , W. Han 3 , A Sloots 1 , C.F.M. Hendriksen 1,2

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 product-

characterization and in vitro testing of vaccine quality rather than potency testing usinganimal 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



56

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 r educed 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-of-

principle that a combination of physico-chemical, immuno-chemical and functional

immunological assays can be used to asses whole cell Bp batch consistency in vitro .



57


MOVING TOWARDS CONSISTENCY APPROACH FOR DIPHTHERIA TETANUS

PERTUSSIS POTENCY ASSAYS

Sylvie Uhlrich

Sanofi Pasteur - 1541 avenue Marcel Merieux, 69280 Marcy l’Etoile – France

[email protected]

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 therelease 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 assayswill be the ultimate target for the future.






58


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



59

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.



60

ABSTRACTPOSTERS



61

POSTER

DEVELOPMENT OF A CELL-BASED ASSAY FOR TETANUS TOXIN.

Shalini Ra jagopal,

Paul Stickings, Thea Sesardic

Division of Bacteriology, National Institute for Biological Standards and Controls, South

Mimms, Potters Bar, Hertfordshire, EN6 3QG, UK

[email protected]

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 inhibitoryfactor, 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-β. 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.






62

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.



63

POSTER

PERTUSSIS

SEROLOGICAL

POTENCY

TEST:

LINKS

TO

THE

PROTECTION

AND

SIGNIFICANCE FOR VACCINE LOT RELEASE

Mario Landys Chovel, Niurka Gutiérrez, Tatiana Mahy, Lucy Herrera, Aleida Mandiarote

Finlay Institute, Avenida 27, No. 19805, P.O. Box 16017, La Lisa, La Habana, Cuba.

E-mail: [email protected] and [email protected]

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 MPTby PSPT in a near future.











64

POSTER

POTENCY DETERMINATION OF INACTIVATED RABIES VACCINES USING A

SEROLOGICAL POTENCY ASSAY BASED ON THE FAVNT SERONEUTRALISATION

TECHNIQUE

Alexandre Servat , Sébastien Kempff, Valère 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, Technopôle Agricole et Vétérinaire, Domaine de

Pixérécourt, CS 40009, 54220 Malzéville, France. E-mail: [email protected]

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 parallelto the mRFFIT and results were compared to the mouse challenge test. Our results



65

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.



66

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

[email protected]

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 Administration’s 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 Canada’s

Biologics and Genetic Therapies Directorate) employed 0.4µm 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

0.4µm polycarbonate tissue culture inserts are used to test 50µL of whole vaccine in a 24

well plate seeded with 2.0×104 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.






67

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: [email protected]

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 atharmonisation 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.







68

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: [email protected]

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 sourcefor the S . Typhi SBA have been published, but animal sera have several limitations, including







69

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 15-

60 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



70

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



71

National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters

Bar, EN6 3QG, UK. [email protected]

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.







72

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

[email protected]

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 toconsistency 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 non-

animal batch tests into biologics production and testing guidelines.






73

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: [email protected]

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 andimmunogenicity for some artificially degraded and real-time aged vaccine samples. For







74

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.



75

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. Hoonakker

1,3

, L. Verhagen 1,3 , A Sloots 1 , C.F.M. Hendriksen 1,2

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 toguarantee 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 G i 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 assayproved to be specific for PTx and was able to detect up to 25 ng of PTx (Hoonakker et al.



76

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.



77

POSTER

PHOTOGRAPHIC EVALUATION OF SKIN LESIONS IN BATCH POTENCY TESTING OF

TUBERCULINS – A REDUCTION APPROACH

E. Balks

1

, C. Dumke 2 , F. Mohs 1 , K. Cussler 2 and A. Hoffmann1

1 Veterina ry Medicine, Paul-Ehrlich-Institut, Langen, Germany

2

Safety of Medicinal Products and Medical Devices, Paul-Ehrlich-Institut, Langen, Germany

[email protected]

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 batchpotency, 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.






78

Funded by the German Federal Ministry of Education and Research: 3R-methods to replace

and refine legally required animal tests for immunologicals

(0316009A – C).



79

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.



80

Bio-sketches



81

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 ofPertussis 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



83

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 August2013, 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.



84

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 Compiègne 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 Université 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.



85

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 vaccineproduction, 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.



87

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.



88

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



89

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/MainStudy of Biology (Diploma degree)

Awards

“Tierschutz Forschungspreis 2007”

award for alternatives to animal testing by German federal government

(Bundesministerium für Ernährung, 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.



90

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 für Tierschutz, Neubiberg, Germany. Dr. Halder joined the European Commission

in October 1995 and currently holds a position as a senior scientist at the European

Commission’s 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.



91

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.



92

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.

Jungbäck, Carmen

Dr. Carmen Jungbäck 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 Tierärztliche 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



93

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.



94

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, safetyand 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



95

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 Michèle Aymard (National Reference Center for

Enterovirus, Lyon, France) and Dr. François Pelloquin (Sanofi Pasteur, formerly Pasteur

Mérieux 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” atEuropean 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



96

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 Médicament) 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 ProductsDepartment 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.



97

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 hasbeen 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.



98

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 Liège, 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 master’s degree in Environmental Policy Sciencesand 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’ (2005 -

2006)



99

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 (Departement

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

o 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



100

Sondag, Perceval

Perceval holds a Bachelor’s Degree in Physical Therapy, followed by a Master’s Degree in

biostatistics, both obtained in the Université 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.



101

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)



102

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, @ [email protected]

+31 88 224 8263, +31 6 52756491 (cell phone))



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Van Zeeland, Rob

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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.



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