case study on pyrogenicity the long way to regulatory acceptance of alternative pyrogen testing....

Case study on pyrogenicityThe long way to regulatory acceptance of

alternative pyrogen testing.

Paul-Ehrlich-Straße 51-5963225 Langen

GERMANY

+49 (0) 6103 77-8020 +49 (0) 6103 77-1250

Email: month@pei.deHomepage: http://www.pei.de

Thomas Montag

Paul Ehrlich Institute,

Federal Agency for Sera and Vaccines

Pyrogens are fever inducing substances which must not be

contained in drugs intended for injection.

-> different sources (e.g. components of micro-organisms,

chemicals)

-> adverse reactions up to life-threatening and fatal outcomes

What are pyrogens ?

What pyrogen tests are regulated ?(European + United States + Japanese

Pharmacopoeias)Rabbit Pyrogen

TestLimulus-Test

Injection of test sample into ear veins of 3 rabbits,

monitoring of body temperature

200,000 animals per year in Europe

Sampling of hemolymph from animals during spawning on

beach and lyophilisation, thereafter In vitro test

Limulus polyphemus, North America

Tachypleus tridentatus, Asiathreatened species

ExogenousPyrogen

endogenouspyrogens

measuring of fever inducing

mediators

fever

blood(monocyte)

exogenous

pyrogens

endogenous pyrogens: Interleukin-1 beta (IL-1 βInterleukin-6 (IL-6)Tumor Necrosis Factor alpha (TNF-α)

Fever reaction in humans:

Principle of alternative pyrogen testing

Eppendorf-vial

100 µl fresh human whole blood (single/pool blood)

+ 1000 µl saline

+ 100 µl sample

Microtiter-plate

40 µl cryoblood (single/pool blood)

+ 180 µl RPMI 1640

+ 20 µl sample

Fresh blood Cryo-blood - 80°C

Incu

batio

nIn

cubati

on

ELISA ELISA

37°C8-16h

37°C 5% CO28-16h

Potency of conventional and alternative pyrogen tests

Pyrogen Rabbit Pyrogen Test

Limulus Test Alternative Pyrogen Test

Endotoxins

yes yes yes

Non-Endotoxin

Pyrogens yes no yes

Human-specific

Pyrogens no no yes

furthermore …

Examples for human-specific pyrogens

Drug Fever in humans

Limulus Test Rabbit Pyrogen Test

Whole Blood Pyrogen Test

Infusion solution

positive negative negative positive

Encephalitis

vaccine positive negative negative positive

Adverse fever reactions by an infusion solution containing gelatine

negative Limulus Test as release criterion

incriminated batches recalled

fever rabbit limulus

- - -

+ + -

+ - -

IL-1 IL-6 TNF

cut off:32.6 pg/ml 127.55 pg/ml 43.6 pg/ml

8.5 28.0 28.2

142.6 654.4 67.6

421.5 9444.0 116.7

samples blinded and sent to PEI

Lecture of Marcel Leist, lower line: “false positive result” during validation process, i.e. in comparison to established tests !!!

Potency of conventional and alternative pyrogen tests

Pyrogen Rabbit Pyrogen Test

Limulus Test Alternative Pyrogen Test

Endotoxins

yes yes yes

Non-Endotoxin

Pyrogens yes no yes

Human-specific

Pyrogens no no yes

furthermore …

The new challenge in drug safety:Advanced Medicinal Products

• Advanced Medicinal Products (Cell Based Medicinal Products, Gene Therapeutics, combinations etc.) offer fascinating opportunities in medicine.

• But: In case of Cell Based Medicinal Products, most of precautions established in pharmaceutical industry are not applicable (e.g. harvest of human cells in intensive care units).

Therefore, new strategies in safety testing necessary.

• There is no established pyrogen test at all which is applicable for Cell Based Medicinal Products.

Potency of conventional and alternative pyrogen tests

Pyrogen Rabbit Pyrogen Test

Limulus Test Alternative Pyrogen Test

Endotoxins

yes yes yes

Non-Endotoxin

Pyrogens yes no yes

Human-specific

Pyrogens no no yes

Advanced Medicinal

Products no no yes

Additionally to animal protection reasons, there is a need for alternative pyrogen tests

in medicine !

Validation of Alternative Pyrogen Tests

European Research Project “Human(e) Pyrogen Testing”

RIVMNational Institute of Public Health

and the Environment

The Netherlands

RIVMNational Institute of Public Health

and the Environment

The Netherlands

University of BernSwitzerland

University of BernSwitzerland

ECVAM (EU)European Centre for the

Validation of Alternative Methods

ECVAM (EU)European Centre for the

Validation of Alternative Methods

NIPHNational Institute of Public Health

Norway

NIPHNational Institute of Public Health

Norway

University of InnsbruckAustria

University of InnsbruckAustria

NovartisSwitzerland

NovartisSwitzerland

PEIPaul-Ehrlich-Institut

Federal Agency for Sera and Vaccines

Germany

PEIPaul-Ehrlich-Institut

Federal Agency for Sera and Vaccines

Germany

NIBSCNational Institute for Biological

Standards and Control

United Kingdom

NIBSCNational Institute for Biological

Standards and Control

United Kingdom

European Pharmacopoeia

European Pharmacopoeia

University of Konstanz /STZ InPuT Germany

European Validation Studycoordinated by ECVAM

10 intravenous drugs, 1000 blinded samples, spiked with different concentrations of Endotoxin, negative controls

Test principle

Whole Blood (IL-6, S. Poole, UK)

Whole Blood (IL-1, T. Hartung, G)

PBMC (IL-6, P. Bruegger, CH)

MonoMac-6 (IL-6, RIVM, NL)

Rabbit

Specificity Sensitivity

100.0 % 100.0 %

94.0 % 96.0 %

98.0 % 97.0 %

95.0 % 100.0 %

57.9 % 88.3 % 57.9 % 88.3 %

European Validation Study (ECVAM):

cryo-preserved Whole Blood• 120 pyrogenic samples + 30 non-pyrogenic samples

cryo-preserved blood

Blood (- 80°C, PEI)

Blood (nitrogen, Konstanz)

Fresh Blood

Rabbit

specificity sensitivity

92.6 % 96.2 %

85.2 % 99.0 %

85.7 % 99.1 %

57.9 % 88.3 %

cryo-preserved Whole Blood

ready to use, defined batches, quality controlled, tested for infection markers ….

Stability of cryo-preserved human whole blood at – 80°C

(no isolation and washing of cells after thawing)

Stimulation by different concentrations of LPS (WHO Standard)induction of IL-1 beta

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

control day 127pool 1

day 247pool 2

OD

450nm

200 pg/ml

100 pg/ml

50 pg/ml

25 pg/ml

12,5 pg/ml

6,25 pg/ml

0 pg/ml

05.08.2004

cryoblood

fresh blood

meanwhile, stability over 2 years

Good idea,man!

Collaborative study of Paul Ehrlich Institute with leading pharmaceutical companies on pyrogen test

using cryo-preserved human blood

Paul-Ehrlich-Institut Dr. Thomas Montag-Lessing Dr. Ingo Spreitzer Bettina Löschner

month@pei.de sprin@pei.de loebe@pei.de

Sanofi-Aventis Deutschland GmbH Dr. Ulrich Pflugmacher Dr. Jens Solsbacher

ulrich.pflugmacher@aventis.com jens.Solsbacher@sanofi-aventis.com

Baxter BioScience Dr. Brian Crowe Dr. Peter Turecek

brian_crowe@baxter.com peter_turecek@baxter.com

Institut Fresenius Dr. Jochen Dobberstein jochen.dobberstein@sgs.com Merck KgaA Dr. Michael Rieth michael.rieth@merck.de Novartis Pharma AG Dr. Peter Brügger peter.bruegger@pharma.novartis.com Schering AG Dr. Detlef Schlote

Herr Andreas Kempe detlef.schlote@schering.de

ZLB Behring Dr. Jacques Maring Jacques.Maring@zlbbehring.com Roche Dr. Sven Deutschmann sven.deutschmann@roche.com DRK-Blutspendedienst Mannheim Dr. Xuan Duc Nguyen

Annette Schuller x.nguyen@blutspende.de a.schuller@blutspende.de

R&D Systems GmbH Michael Stein m.stein@rndsystems.co.uk additionally: German Pharmacopoeia Commission Dr. Hugo Peeters h.peeters@bfarm.de EAQ Arbeitsgruppe Mikrobiologie Prof. Dr. Dietrich Krüger prof.krueger@t-online.de European Compliance Academy Mike Edgington mike@edgington.nl

Regulatory Acceptance of Alternative Pyrogen Tests

Tests validated by ECVAM must be endorsed by its Scientific Advisory Committee (ESAC) composed of representatives of the 25 member states from academia, industry and animal welfare organisations before they can be used within the regulatory framework.

Press release 21.3.2006 regarding Five In Vitro Pyrogen Tests:

Five cell based tests, using human cells grown in the laboratory, have been endorsed for the detection of undesired side effects of drugs, such as fever reactions arising from contaminants (pyrogens) of injectible drugs. This will save the lives of about 200,000 laboratory rabbits per year in Europe. ESAC Statement: http://ecvam.jrc.it/ft_doc/ESACstatementpyrogenicity20060321.pdf

1. Human Whole Blood IL-1 2. Human Whole Blood IL-6 3. PBMC IL-6 4. MM6 IL-6  5. Human Cryopreserved Whole Blood IL-1

Situation in EUROPEEuropean Centre for the Validation of Alternative Methods (ECVAM)

Alternative Pyrogen Tests are accepted in European Community for endotoxin detection, but still not by Pharm.

Eur.

European Pharmacopoeia Commission(European Department for the Quality of Medicines (EDQM)

• installed an Expert Group “Alternative Pyrogen Test” already in 2001, unfortunately, 1 meeting only

• on request of both Pharm. Eur. Group 6B “Blood Products” and German Pharmacopoeia Commission installation of a new Expert Group “Monocyte Activation Test” in 2006

• Draft chapter “Monocyte Activation Test” existing

• Discussions regarding pyrogen limits:

-> “false negatives”: animal test positive, alternative test negative explanation: “non-human” pyrogen (e.g. Immunoglobulins)

-> “false positives”: animal test negative, alternative test positive explanation: “human-specific” pyrogen or solution: quantification allows drug release (animal experiment gives “yes/no” answer only

Situation in United States of America:Interagency Coordinating Committee on the Validation

of Alternative Methods(ICCVAM)

ECVAM submitted Background review Documents (BRD´s) on the european study on alternative pyrogen tests to ICCVAM in June 2005

http://iccvam.niehs.nih.gov/nomsub/ecvampyro.pdf

Pre-screen evaluation November 2005

http://iccvam.niehs.nih.gov/nomsub/pyroprescrn.pdf

On December 16th 2005 a call for experts for a peer review evaluation was released in the Federal register

http://iccvam.niehs.nih.gov/docs/FR/7074833.pdf

Current state: Round table, Dr. William Stoke, ICCVAM

(role of FDA, USP ?)

Situation in Japan:

Japanese Pharmacopoeia ?NIHS (Biologicals) ?

Round table: Dr. Kojima

• Alternative Pyrogen Tests are (at least) as valid as Rabbit Pyrogen Test (RPT). Both sensitivity and specificity are significantly higher than those of RPT.

Conclusions:

• In contrast to Limulus assay, non-endotoxin pyrogens are detected securely. Furthermore, Alternative Pyrogen Tests detect certain non-endotoxin pyrogens (active in humans) which cannot be found in RPT.

• Different Alternative Pyrogen Tests are validated and can be used in parallel (Whole blood, PBMC, and monocytic cell lines are working properly).

• All components of Alternative Pyrogen Tests (at least in case of Human Whole Blood Pyrogen Test) can be standardised without problems. The respective technologies are established.

• The technology for production of cryo-preserved monocyte sources at minus 80°C (validated for whole blood) allows an economical and feasible application of Alternative Pyrogen Tests in pharmaceutical industry regarding manufacturing process, shipping, storage, and handling.

• Almost all what we have to do is, to implement the Alternative Pyrogen Tests into the regulatory

documents.

that the rabbits can say: