Overview of the 3Rs Concept in Vaccine Quality Control

- Approaches and Goals -

Klaus CusslerPaul-Ehrlich-InstitutLangen, Germany

Technology and Approaches to Reduce, Refine and Replace Animal TestingApril 5-7, 2004 Ames Iowa

Klinische Endpunkte bei derTollwutimpfstoffprüfung

Testing for Safety (A.D.

1. Adspection

2. Sterilitysera ad us. hum.: sterilesera ad us. vet.: „nearly sterile“

3. Phenol content0.5 ml injected s.c. in one mouse

4. Freedom from toxins (tetanus)10 ml injected s.c. in one guinea pig

from: R. Otto (1906)Die staatliche Prüfung der HeilseraGustav Fischer Verlag Jena

Batch testing book anno 1934

Complete Documentation: 30 pages

Safety documentation: 1 page– abnormal toxicity test ( 2 guinea-pigs, 5 mice)

– sheep safety test ( 2 sheep in TAST)

– specific safety test ( 10 guinea-pigs)

– potency test ( 10 rabbits)

The safety of a product was only testedduring batch release

Licensing Documentation of a ClostridialVaccine (Germany, 1979)

Numbers of animals used for production and quality control of biologicals

Biologicals

Fundamental

Education

Diagnostic

Pharmaceuticals

Toxicity testingThe Netherlands, 2000

% of experiments with severe pain & distressPharmaceuticals : 3%

Toxicity testing : 10%

Diagnostic testing : 0%

Education & Training : 0%

Fundamental research : 5.4%

Biological products : 17%

~15-20%

The concept of the The concept of the Three Three R’sR’s

“Any procedures which do away with the use of animals altogether, lead to a reduction in the total number of animals used, or lead to less distress to the animals employed”

The Three R’s: Russell & Burch (1959)

Three R’s are:used as the red line in regulations on the

use of laboratory animals, e.g. Council Directive 86/609/EEC

adopted by regulatory bodies such as OECD, Eur.Phar., ‘WHO’, USDA, etc.

part of the policy of the European Science Foundation (ESF)

TheThe ClassicalClassical 3R 3R ConceptConceptinin

VaccineVaccine DevelopmentDevelopment and and QualityQuality ControlControl

Replacement

Reduction

Refinement

Russel & Burch (1959)

3R Concept

Animal Experiments for Licensingof Veterinary Immunobiologicals

Licensing

Safety

Efficacy

Animal Experiments for Quality Controlof Veterinary Immunobiologicals

Batch Testing

Safety(Target species)

Potency(Laboratory Animals)

Animal tests in the European Pharmacopoeia

CONVENTION ON THE ELABORATION OF A EUROPEAN PHARMACOPOEIA (Ph. Eur)

Goal:

“It is questionable whether by the turn of the century the European Pharmacopoeiawill still include tests and assays involvingwhole animals.“Closing Remark by J.-M. Spieserof his talk “Reduction of the Use of Animals for QualityControl Testing in the European Pharmacopoeia“

1st World Congress on Alternatives and Animal Use in the Life SciencesBaltimore, November 1993

How are alternativesimplemented in

European Pharmacopoeiamonographs?

Ph.Eur.: GENERAL STATEMENTSThe tests and assays described are the officialmethods upon which the standards of thePharmacopoeia are based. With the agreement of thecompetent authority, alternative methods of analysismay be used for control purposes, provided that themethods used enable an unequivocal decision to bemade as to whether compliance with the standards of the monographs would be achieved if the officialmethods were used. In the event of doubt or dispute, the methods of analysis of the Pharmacopoeia arealone authoritative.

Alternatives to Animal Testing

Development

In-house validation

Research Laboratory,

Industry

Compliance with the standards of the monograph:Comparison with the animal test

Alternatives to Animal Testing: The long way from science to practice

DevelopmentResearch Laboratory

In-house validation

Prevalidation

Validation

National Authorities, ECVAM

EDQM, ECVAM

Acceptance Regulatory Bodies

(e.g. Pharmacopoeia)

Introduction in routinequality control

Implementation

Industry and OMCLs

Validation of Alternative Methodsfor the Potency Testing of

Vaccines

The Report and Recommendations ofECVAM Workshop 31

ATLA 26, 747-761 (2000)

Replacement

The 3R Concept:

Antigen Quantification Tests

• In the US antigen quantification tests arepromoted and are under evaluation for manyvaccines.• In Europe antigen quantification tests are still rarely used, but some improvement is seen.• The major drawback is the adjuvant problem.• There is an urgent need for standardisedreference material (monoclonal antibodies)

SWINE ERYSIPELAS VACCINE (INACTIVATED)

A proposal to introduce an antigen quantification assay during the revision of the monograph was not accepted by the Expert Group 15V.

General signs of sicknessGeneral signs of sickness

HematuriaHematuria

Leptospirosisin hamsters

Healthy HamsterHealthy Hamster

CANINE LEPTOSPIROSIS VACCINE (INACTIVATED)

For vaccines without adjuvantsA suitable in vitro test may be carried out to determine antigenic components which are indicators for protection and which are specific for that serovar. The criteria for acceptance are set with reference to a batch that has been given satisfactory results in the dog challenge test.

PHARMEUROPA 14, Oct.2002

Reduction

The 3R Concept:

Possibilities for Reduction

• “Upstream“ performance of a test

• One-dilution assay instead ofmulti-dilution assays

Statistics

WORKSHOP: Statistical Aspects of Validation Studies

• P. Volkers, PEI: Assessing Agreement

• A. Daas, EDQM: Reducing the number of animals by moving frommultiple dilution assaysto single dose assays

___________________VACTRAIN Symposium, Strasbourg, 7-8 November 2002Download: www.vaccinetraining.com

Refinement

The 3R Concept:

Intracerebral Injection

Good Practice Guide for IntracerebralInjection in Mice

Recommendation of an

ECVAM/AGAATI-Workshop on the3Rs in Vaccine Quality Control of Rabies Vaccines, held in Langen, April 2002.

Annex to the Workshop Report published in ATLA 31: 429-454 (2003)

Refinement

Example:

Humane endpoints for tetanus toxinchallenge

Humane Endpoints in Vaccination Challenge Tests

Highlights

The 3R Concept:

3R Examples:Clostridial Animal Tests

9 rabbits

20 mice20 mice20 mice

20 mice

20 mice

C. perfringens CC. perfringens DC. sordelliiC. novyiC. septicum

Total AnimalsTotal Animals: 9 Rabbits 100 Mice: 9 Rabbits 100 MiceSource: Paul Source: Paul HauerHauer, CVB, CVB

Challenge test ELISAAntigen

quantification

Possibilities for reduction of animal numbers inthe erysipelas vaccine potency test

TheThe SpiritSpirit of of thethe 3Rs3Rsinin

VaccineVaccine DevelopmentDevelopment and and QualityQuality ControlControl

– Host animal• vaccination and challenge • vaccination and serology

– Laboratory animal (Model) • vaccination and challenge• vaccination and serology

– In vitro• titer or count for live vaccines • direct antigen quantitation for killed vaccines

Batch Potency Tests

Our Duty Is To:• Minimise the extent of welfare

compromise• Maximise the benefits

It is therefore important to carry out a thorough harm versus benefit analysis

Animal Ethics

New approaches

inin

VaccineVaccine QualityQuality ControlControl

The 4R Concept for Animal Tests in Vaccine Quality Control

- Reassessment

- Replacement

- Reduction

- Refinement

Reassessment:

Is the animal test still necessary?

Examples: Abnormal Toxicity TestTarget Animal Safety TestExtraneous Virus (i.c. Test)

Safety Test = Animal Test ?

A biological such as a vaccine has to be tested for safety at least once in an animal.

Deletion of an Animal TestSafety test for erysipelas vaccines in mice:

Today only in vitro

New Approach for Batch Testing

Current Approach:Each batch is unique and is tested separatly for safety and potency

New Approach:Testing for consistency of production

More effective information network

- Conferences & Meetings- Workshops and Training Courses- Internet, e.g. www.vaccinetraining.com

www.agaati.org

New approaches

Three Rs Approaches in theQuality Control of Inactivated

Rabies Vaccines

The Report and Recommendations of ECVAM Workshop 48

ATLA 31: 429-454 (2003)

VACTRAINWORKSHOP/TRAINING COURSE ONVACCINE QUALITY CONTROL WITHTHREE Rs METHODS IN 2002/2004

Training courses on

• Bacterial Vaccines for Veterinary Use • Rabies Vaccines for Human and Veterinary Use• Avian Vaccines - Extraneous Agents Testing

with PCR

For information see www.vaccinetraining.com

Independent forum to handle problems in the field of biologicals and alternativesECVAM Steering Group on BiologicalsFirst meeting November 2003

New approaches

• International harmonisation

• Mutual Recognition

New approaches

Goals

Promote the 3Rs concept in vaccine development and quality control! The replacement of animal tests has first priority, but there are also many possibilities to reduce test frequency or animal numbers and to limit pain and distress.

Goals

Revise old testing requirements, especially for traditional biologics, e.g. rabies vaccines!

Goals

Find a forum for an open discussion of animal welfare issues in the QC of biologicals.

And please, don’t forget harmonisation and mutual recognition!

Conclusions (I)

Today's stringent requirements for licensing, production and control improved the quality and safety of vaccines.

Therefore, it is the responsibility of both, regulators and industry, to reassess the need for animal safety tests regularly.

Conclusions (II)

Much progress has been achievedconcerning the development and validationof alternatives for vaccine quality control. More and more alternatives areimplemented into Ph.Eur. monographs.

However, there is still a large potencial forimprovements. New additional approachesshould facilitate further reduction of animaltesting.

THANK YOU

FOR YOUR ATTENTION