A Comparative Study of Different Methods for Endotoxin Destruction
Tim Sandle, Ph.D. Bio Products Laboratory, UK Introduction
Dry heat is the established method of depyrogenation within the pharmaceutical industry. This paper describes a series of studies undertaken to determine whether successful depyrogenation can be achieved practicably using methods other than dry heat. Depyrogenation refers to the removal or inactivation of pyrogens. In practice, depyrogenation processes are qualified by demonstrating that they are capable of reducing bacterial endotoxin to an acceptance level. Depyrogenation, like sterilization, is an absolute term that can only be theoretically demonstrated because of test insensitivity [1].
Depyrogenation of glassware is important in the production of parenteral pharmaceuticals as residual pyrogens could ultimately be injected into a patient resulting in an adverse reaction. In laboratories, it is important that glassware used for bacterial endotoxin testing is depyrogenated to minimize contamination of the test; sampling containers also need to be free of pyrogens to avoid contamination of samples and the detection of false positives [2]. The main pyrogenic substance, which poses a risk to glassware used in the production of parenteral pharmaceuticals, is endotoxin. Endotoxin is the natural heat stable lipopolysaccharide contained in the outer walls of Gram-negative bacteria [3]. It is released into the environment during bacterial cell death, lysis, growth and multiplication [4, 5]. Endotoxin is considered to be the most significant pyrogen due to its ubiquity and potency [6].
One concern with endotoxin in relation to pharmaceuticals is that they are heat stable, making them resistant to most conventional sterilization processes and thus necessitating separate tests for viable cells and endotoxin. Pharmaceutical processes and equipment are at risk from endotoxin.
Thus depyrogenation is an important factor in maintaining sterility assurance
Tim Sandle, Ph.D., is the Head of Microbiology at the Bio Products Laboratory. Dr. Sandle is
responsible for a range of microbiological tests, batch review, microbiological investigation and policy development. In addition, Dr. Sandle is an honorary consultant with the University of Manchester and is a tutor for the university’s pharmaceutical microbiology MSc course. He also runs an online pharmaceutical microbiology site (
www.pharmamicro.com). Dr. Sandle is a
chartered biologist and holds a first class honors degree in Applied Biology; a Master degree in
education; and a Ph.D. in microbiology. Dr. Sandle can be reached at:
timsandle@btinternet.com
during the preparation of pharmaceutical products. There are several different means to achieve depyrogenation (including ultrafiltration; ion exchange chromatography and the use of acid- base hydrolysis). Arguably the most common depyrogenation devices are those which operate using dry-heat (such as a depyrogenation tunnel using unidirectional hot air, which is used to prepare primary packaging articles – product vials – for aseptic filling) [7].
Requirements for depyrogenation differ amongst the regulatory bodies. The European
Pharmacopeia specifies dry heat at 250°C for 30 minutes, or 200°C for 60 minutes for depyrogenation of glass used for pyrogen tests, although there is no written requirement for the depyrogenation of glassware used for parenterals [8]. Glassware to be used for LAL testing must be depyrogenated to a level lower than the sensitivity of the test. The USP <1211> and FDA guidelines do not contain a temperature specification, but rather require a three-log reduction in endotoxin from a starting concentration of at least 1000 Endotoxin Units (EU) for depyrogenation [9]. In terms of the depyrogenation reaction, endotoxin, when dry heat inactivated, follows a linear log-reduction curve until reduction to three-logs. After this destruction continues to
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