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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: [email protected]: 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
[email protected] [email protected] [email protected]
Sanofi-Aventis Deutschland GmbH Dr. Ulrich Pflugmacher Dr. Jens Solsbacher
[email protected] [email protected]
Baxter BioScience Dr. Brian Crowe Dr. Peter Turecek
[email protected] [email protected]
Institut Fresenius Dr. Jochen Dobberstein [email protected] Merck KgaA Dr. Michael Rieth [email protected] Novartis Pharma AG Dr. Peter Brügger [email protected] Schering AG Dr. Detlef Schlote
Herr Andreas Kempe [email protected]
ZLB Behring Dr. Jacques Maring [email protected] Roche Dr. Sven Deutschmann [email protected] DRK-Blutspendedienst Mannheim Dr. Xuan Duc Nguyen
Annette Schuller [email protected] [email protected]
R&D Systems GmbH Michael Stein [email protected] additionally: German Pharmacopoeia Commission Dr. Hugo Peeters [email protected] EAQ Arbeitsgruppe Mikrobiologie Prof. Dr. Dietrich Krüger [email protected] European Compliance Academy Mike Edgington [email protected]
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: