Presentation on theme: "Paul Ehrlich Institute, Federal Agency for Sera and Vaccines"— Presentation transcript:
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 MontagPaul Ehrlich Institute,Federal Agency for Sera and VaccinesPaul-Ehrlich-Straße 51-5963225 LangenGERMANY +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 TestLimulus-TestInjection of test sample into ear veins of 3 rabbits, monitoring of body temperature200,000 animals per year in EuropeSampling of hemolymph from animals during spawning on beach and lyophilisation, thereafter In vitro testLimulus polyphemus, North AmericaTachypleus tridentatus, Asia threatened species
4 Fever reaction in humans: Principle of alternative pyrogen testingFever reaction in humans:feverExogenousexogenousendogenousPyrogenpyrogensblood(monocyte)pyrogensendogenous pyrogens:Interleukin-1 beta (IL-1 β Interleukin-6 (IL-6) Tumor Necrosis Factor alpha (TNF-α)measuring of fever inducing mediators
8 samples blinded and sent to PEI Adverse fever reactions by an infusion solution containing gelatine negative Limulus Test as release criterion incriminated batches recalledsamples blinded and sent to PEIIL IL TNFcut off: pg/ml pg/ml pg/mlfever rabbit limulusLecture 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 !
13 European Research Project “Human(e) Pyrogen Testing” RIVMNational Institute of Public Healthand the EnvironmentThe NetherlandsNIBSCNational Institute for BiologicalStandards and ControlUnited KingdomPEIPaul-Ehrlich-InstitutFederal Agency for Sera and VaccinesGermanyNovartisSwitzerlandUniversity of BernSwitzerlandUniversity of InnsbruckAustriaUniversity of Konstanz /STZ InPuT GermanyNIPHNational Institute of Public HealthNorwayEuropeanPharmacopoeiaECVAM (EU)European Centre for theValidation of Alternative Methods
14 European Validation Study coordinated by ECVAM 10 intravenous drugs, 1000 blinded samples, spiked with different concentrations of Endotoxin, negative controlsTest principleWhole 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)RabbitSpecificity Sensitivity100.0 % %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 samplescryo-preserved bloodBlood (- 80°C, PEI)Blood (nitrogen, Konstanz)Fresh BloodRabbitspecificity sensitivity92.6 % %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
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 EUROPEEuropean 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-63. PBMC IL-6 4. MM6 IL-6 5. Human Cryopreserved Whole Blood IL-1Alternative 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 onlyon request of both Pharm. Eur. Group 6B “Blood Products” and German Pharmacopoeia Commission installation of a new Expert Group “Monocyte Activation Test” in 2006Draft chapter “Monocyte Activation Test” existingDiscussions 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 2005Pre-screen evaluation November 2005On December 16th 2005 a call for experts for a peer review evaluation was released in the Federal registerCurrent 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: