Pyrogens endotoxin lipopolysaccharide

Excipients derived from bacterial transformation or enzymatic processes, such as sugars (e.g., mannitol or dextrose), pose special concerns due to potential contamination by endotoxins. Endotoxins, or pyrogens, are lipopolysaccharides from gram-negative bacteria that can induce severe fever upon parenteral administration. Parenteral products have different limits for allowable endotoxin levels.57... 

Ultrafiltration is used to remove pyrogens and other microorganisms from high purity water. Pyrogens are lipopolysaccharides (also known as endotoxins) with molecular sizes ranging from 20,000 dalton (-0.005 pm) up to about 200,000 dalton ( 0.1 pm) produced from bacterial cell walls. Pyrogens induce fever when injected into animals or humans and cannot be removed by autoclaving or microfiltration.l H ... 

Fever is the most common manifestation. The thermoregulatory centre in the hypothalamus regulates body temperature and this can be affected by endotoxins (heat-stable lipopolysaccharides) of Gram-negative bacteria and also by a monokine secreted by monocytes and macrophages called interleukin-1 (IL-1) which is also termed endogenous pyrogen. Antibody production and T-cell proliferation have been shown to be enhanced at elevated body temperatures and thus are beneficial effects of fever. 

Endotoxins are bacterial cell envelope constituents that, when present in pharmaceutical products, cause pyrogenic reactions sometimes resulting in lethality. The toxicity of endotoxins is directly related to their chemical composition. However, the viability of the organism is irrelevant since endotoxin derived from dead or live microbes is equally active. The classical endotoxin is lipopolysaccharide (LPS). However, peptidoglycan (PG) also displays endotoxin-like activities. LPS is found only in gram-negative bacterial outer membranes, while PG is present in the cell... 

Pharmaceutical products can be sterilized by steam sterilization, dry-heat sterilization, filtration sterilization, gas sterilization, and ionizing-radiation sterilization. The USP provides monographs and standards for biological indicators required to test the validity of the sterilization process. These products must also be tested for pyrogens—fever-producing substances that arise from microbial contamination most likely thought to be endotoxins or lipopolysaccharide in the bacterial outer cell membrane. 

Pyrogenic contamination is detected using two tests. In the older method, rabbits are injected with product samples, and rectal temperature is measured. Compendial limits are established with respect to how much temperature increase is permitted before the product is judged to be free or contaminated with pyrogens. The newer method involves a relatively simple in vitro technique called the Limulus Amebocyte Lysate (LAL) test. It is based on the high senstivity of amebocytes of the horseshoe crab (Limulus) to the lipopolysaccharide component of endotoxins originating from Gramnegative bacteria. The LAL test is now the USP method of choice with endotoxin limits established for most SVIs. ... 

Purified endotoxin consists only of lipopolysaccharide. This itself has three distinct chemical regions an inner core called lipid A. an intermediate polysaccharide layer, and an outer polysaccharide side chain. Lipid A, a highly substituted disaccharide of gluct amiiie, is responsible for pyrogenicity and other immunological and biological properties associated with endotoxin. 

Removal of endotoxin from manufacturing equipment or materials can be done by appropriate washing and rinsing procedures. Pyrogen-free water can be obtained by several means. The oldest and most effective method is distillation —the heavyweight lipopolysaccharide molecules (MW of around 10 ) are left behind when water is rapidly boiled in a still. Reverse osmosis can remove 99.5 to 99.9% of water s endotoxin load in a single pass. For these reasons, distillation and reverse osmosis are the only two methods of preparing Water for injection allowed in the pharmacopoeias. The pharmacopoeial requirement for Wtuer for injection is that there should be no more than 0.25 EU (endotoxin units)/mL. 

Endotoxin is a term often used synonymously with lipopolysaccharide (LPS), a major component of the Gram-negative bacterial cell wall. Endotoxin has been estimated to make up between 3 and 4% of the dry weight of Escherichia coli K12. The LPS molecule consists of a Lipid A component anchored in the outer membrane. This is attached to a series of saccharide units specific for different strains of Gram-negative bacteria. LPS is extremely heat stable and needs to be heated to 180 °C for three hours to ensure inactivation. When injected into the bloodstream, it activates the alternative complement pathway and produces a pyrogenic reaction. Studies on laboratory animals have shown that it can produce severe reactions when inhaled in microgram quantities. ... 

The gram-negative cell wall contains only a shallow peptidoglycan layer. On the outer side of this layer is the outer membrane, a complex stmcture consisting of four major components phospholipids, lipopolysaccharide, proteins (e.g., porins), and lipoprotein. Lipolysaccharide (endotoxin) is responsible for the pyrogenicity of the gram-negative bacteria. 

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