Pyrogens are fever-causing agents which contaminate medicines and vaccines as a consequence of the manufacturing process.
Pyrogen-contaminated parenteral preparations can cause mild to severe clinical reactions including fever, rash, headache, myalgia, nausea, vomiting and hypotension. It is therefore vital that the level of pyrogenic contamination of parenteral preparations is carefully controlled to ensure their safety.
The most common pyrogen is endotoxin which is shed from the cell wall of Gram-negative bacteria. Medicines and vaccines produced by a process involving such bacteria are therefore at high risk of being contaminated with endotoxin.
The amount of endotoxin in a product can be quantified using one of the various methods of the bacterial endotoxins test (BET). In line with the 3Rs principle – the replacement, reduction and refinement of animal testing, the BET was a replacement for the rabbit pyrogen test (RPT). However, unlike the RPT, the BET does not detect non-endotoxin pyrogens or other pro-inflammatory contaminants of parenteral preparations which can stimulate unwanted clinical reactions.
For this reason a third test – the monocyte activation test (MAT) – was developed as a replacement for the RPT for quantifying pro-inflammatory/non-endotoxin pyrogenic contaminants of medicines and vaccines.
All three tests are described in the British and European Pharmacopoeias.
Our laboratory has considerable expertise in the detection of pyrogenic contaminants of parenteral preparations using the monocyte activation test and bacterial endotoxins test for the purposes of medicines control and research and development.
Our area was responsible for preparing the 2nd and 3rd International Standards (IS) for endotoxin as well as the new standard for the BET described in the US and European Pharmacopoeias.
The 3rd IS for endotoxin also serves as the standard for the European Pharmacopoeial MAT Methods A and B. A total of 3 x 25,000 vials of endotoxin – all with the potency of 10 000 IU of endotoxin per vial – were filled to preserve global harmonisation of unitage for this important standard.
As an Official Medicines Control Laboratory (OMCL) our area is responsible for:
The role of the pyrogen testing area in medicines control is much wider than carrying out routine testing. We advise about product testing, methodology and regulatory requirements, including setting pharmacopoeial specifications, to the pharmaceutical industry, regulatory organisations and other OMCLs to develop new policies and practices.
In particular, we work with the European Directorate for the Quality of Medicines (EDQM) through the Expert Groups for the MAT and the BET and the OMCL network.
Our work in this area includes developing novel bioassays capable of detecting unwanted intrinsic activities of parenteral preparations including vaccines and therapeutic monoclonal antibodies.
Other research areas are:
An example of this is a study we carried out in collaboration with a vaccine manufacturer to identify which component (and its batch) of a multivalent vaccine was causing problems with the vaccine’s pro-inflammatory activity.
Dr Karin Nordgren, Head of Pyrogen Science Janet SutherlandTrusha DesaiSophie MyhillDr Kata Tombacz
10/178: 3rd IS for endotoxin