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Paul Ehrlich Institute, Federal Agency for Sera and Vaccines

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1 Paul Ehrlich Institute, Federal Agency for Sera and Vaccines
Case study on pyrogenicity The long way to regulatory acceptance of alternative pyrogen testing. Thomas Montag Paul Ehrlich Institute, Federal Agency for Sera and Vaccines Paul-Ehrlich-Straße 51-59 63225 Langen GERMANY  +49 (0)  +49 (0) Homepage:

2 What are pyrogens ? 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

3 What pyrogen tests are regulated
What pyrogen tests are regulated ? (European + United States + Japanese Pharmacopoeias) Rabbit Pyrogen Test Limulus-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, Asia threatened species

4 Fever reaction in humans:
Principle of alternative pyrogen testing Fever reaction in humans: fever Exogenous exogenous endogenous Pyrogen pyrogens blood (monocyte) pyrogens endogenous pyrogens: Interleukin-1 beta (IL-1 β Interleukin-6 (IL-6) Tumor Necrosis Factor alpha (TNF-α) measuring of fever inducing mediators

5 Fresh blood Cryo-blood - 80°C Incubation ELISA Eppendorf-vial
100 µl fresh human whole blood (single/pool blood) 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 Incubation ELISA 37°C 8-16h 37°C 5% CO2

6 Potency of conventional and alternative pyrogen tests
furthermore …

7 Examples for human-specific pyrogens

8 samples blinded and sent to PEI
Adverse fever reactions by an infusion solution containing gelatine negative Limulus Test as release criterion incriminated batches recalled samples blinded and sent to PEI IL IL TNF cut off: pg/ml pg/ml pg/ml fever rabbit limulus Lecture of Marcel Leist, lower line: “false positive result” during validation process, i.e. in comparison to established tests !!!

9 Potency of conventional and alternative pyrogen tests
furthermore …

10 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.

11 Potency of conventional and alternative pyrogen tests
Additionally to animal protection reasons, there is a need for alternative pyrogen tests in medicine !

12 Validation of Alternative Pyrogen Tests

13 European Research Project “Human(e) Pyrogen Testing”
RIVM National Institute of Public Health and the Environment The Netherlands NIBSC National Institute for Biological Standards and Control United Kingdom PEI Paul-Ehrlich-Institut Federal Agency for Sera and Vaccines Germany Novartis Switzerland University of Bern Switzerland University of Innsbruck Austria University of Konstanz / STZ InPuT Germany NIPH National Institute of Public Health Norway European Pharmacopoeia ECVAM (EU) European Centre for the Validation of Alternative Methods

14 European Validation Study coordinated 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 % % 94.0 % % 98.0 % % 95.0 % % 57.9 % % 57.9 % %

15 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 % % 85.2 % % 85.7 % % 57.9 % % ready to use, defined batches, quality controlled, tested for infection markers ….

16 meanwhile, stability over 2 years
Stability of cryo-preserved human whole blood at – 80°C (no isolation and washing of cells after thawing) meanwhile, stability over 2 years

17 Good idea, man!

18 Collaborative study of Paul Ehrlich Institute with leading pharmaceutical companies on pyrogen test using cryo-preserved human blood

19 Regulatory Acceptance of Alternative Pyrogen Tests

20 European Centre for the Validation of Alternative Methods (ECVAM)
Situation in EUROPE European Centre for the Validation of Alternative Methods (ECVAM) 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 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: 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 Alternative Pyrogen Tests are accepted in European Community for endotoxin detection, but still not by Pharm. Eur.

21 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

22 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 Pre-screen evaluation November 2005 On December 16th 2005 a call for experts for a peer review evaluation was released in the Federal register Current state: Round table, Dr. William Stoke, ICCVAM (role of FDA, USP ?)

23 Situation in Japan: Japanese Pharmacopoeia. NIHS (Biologicals)
Situation in Japan: Japanese Pharmacopoeia ? NIHS (Biologicals) ? Round table: Dr. Kojima

24 that the rabbits can say:
Conclusions: Alternative Pyrogen Tests are (at least) as valid as Rabbit Pyrogen Test (RPT). Both sensitivity and specificity are significantly higher than those of RPT. 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:

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