Microorganisms and endotoxins

Assay methods for monitoring any degradation products may be used to justify the time limits for bulk storage. This time would include the period from when the product is formulated to the end of the filling operation. Because most lyophilized formulations do not contain a biological preservative, microbiological quality before sterilization by filtration must also be monitored. The unfiltered solution bioburden would include microorganisms and endotoxin levels. 

Dry-heat processes have two main targets microorganisms and their by-products, pyrogens or endotoxins. Depyrogenation is the process that destroys the chemical activity of these by-products. Destruction of microorganisms and endotoxins by dry heat is considered an oxidative process, which is almost a combustion. Depyrogenation requires a higher temperature than sterilization and can be summarized as follows ... 

In some cases, reverse osmosis is applied, and this removes almost all the particulates and organic materials, as well as microorganisms and endotoxins. Electrodeionization, which combines ion exchange membranes and resins, removes the last traces of dissolved ions from water under influence of a direct electric current. The last stage of the purification is ozone sterilization of water to inactivate residual microorganisms, as ozone is an efficient disinfectant (UV at 254 nm wavelength is then used to break up the spent ozone). The PW generated is then circulated to each point where it is used (point of use POU). 

Damage to the host may arise in two ways. First, multiplication of the microorganisms may cause mechanical damage to the tissue cells through interference with the normal cell metabolism, as seen in viral and some bacterial infections. Second, a toxin associated with the microorganism may adversely affect the tissues or organs of the host. Two types of toxins, called exotoxins and endotoxins, are associated with bacteria. 

E. Th. Rietschel, L. Brade, U. F. Schade, U. Seydel, U. Zahringer, S. Kusumoto, and H. Brade, in E. Schrinner, M. Richmond, G. Seibert, and U. Schwartz (Eds.), Sutface Structures of Microorganisms and Their Interaction with the Mammalian Host Bacterial Endotoxins Properties and Structure of Biologically Active Domains, p. 1. Verlag Chemie, Weinheim, 1988. 

Sodium hydroxide is one of the most extensively used chromatography CIP agents. It is readily available, inexpensive and effective. It is usually applied to a column at strengths of up to 1.0 M. At such concentrations, it quickly removes/destroys most contaminants, including microorganisms and viruses. It will also degrade endotoxin (discussed later) within minutes. 

These are produced by addition of formalin to the toxin of microorganisms and incubating them at 37°G for three to four weeks. Gertain microorganisms produce endotoxins e.g. tetanus and diphtheria. The toxins produced by these organism are detoxicated and used for the preparation of vaccine. The toxoids have lost their toxicity but antigenicity is retained. 

The main objective of validation of an analytical procedure is to demonstrate that the procedure is suitable for its intended purpose. The procedures presented in this SOP provide basic guidelines for the validation of methods for microbiological assay, estimation of the number of microorganisms, detection of indicators of objectionable microorganisms, validation of preservative efficacy testing, and validation of the sterility testing and endotoxin test (LAL test). 

Hydrophilic surfactant proteins A (SP-A) and D (SP-D), secreted by type II pneumocytes, interact specifically with a wide range of microorganisms and play important roles in the innate, natural defense system of the lung [16]. Both mRNA and protein levels of SP-A and SP-D increase dramatically in response to lung infection, injury and endotoxin challenge [17]. Type II pneumocytes also express class II major histocompatibility complex (MHC) antigens and intracellular adhesion molecule (ICAM-1), which may facilitate pulmonary immune responses [15]. 

Where the compounding is nonaseptic, careful control over the environment, materials, and equipment is still appropriate to reduce viable/nonviable levels and to reduce the potential for endotoxin. Time limits should be imposed on manufacturing operations for additional control over microorganisms and thus microbial toxins. 

The LAL and bioburden methods must be developed to isolate and quantitate bacteria and endotoxins. Rinse samples are usually tested for bacterial endotoxin (LAL) and swab and rinse samples are tested for bioburden. Isolated microorganisms should be identified to an appropriate level, whenever possible. 

Pyrogenicity. Pyrogens are bacterial endotoxins consisting of polysaccharides or proteins produced by the metabolism of microorganisms and upon... 

C. When an apparatus for column chromatography is used in the manufacturing processes, to take action necessary for preventing contamination of the apparatus by microorganisms, and to measure endotoxins, where necessary. 

When collecting and storing water, the growth of microorganisms and the increase of endotoxins as bacterial waste products must be prevented. That means it is either collected and kept at a temperamre higher than approximately 70 °C until it is processed (Water for injections in bulk), or it is sterilised, either directly or after filling into vials or ampoules (Sterilised water for injections. Aqua ad iniectabile sterilisata). See further Sect. 27.5.2. 

The harmful activity of these bacteria in the digestive tract is ascribed to enterotoxins, wbicb are classified into two groups exotoxms (toxins excreted by microorganisms into tbe surrounding medium) and endotoxins (retained by the microorganism cells but released when the cell disintegrates). 

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