Pyrogen

For pyrogen testing of devices or injection assemblies, use washings or rinsings of the surfaces that come in contact with the parenterally administered textile or with the injection site or internal tissues of the patient.

From: Acute Toxicology Testing (Second Edition) , 1998

A Framework for Evaluating Nonclinical Safety of Novel Adjuvants and Adjuvanted Preventive Vaccines

P.E. Boucher , in Immunopotentiators in Modern Vaccines (2d Edition), 2017

In Vitro Tests for Pyrogens: The Limulus Amebocyte Lysate and Monocyte Activation Test Assays

Pyrogens are fever-inducing substances normally derived from microorganisms [endotoxins or lipopolysaccharide (LPS)] and when present systemically in sufficient quantity can lead to severe signs of inflammation, shock, multiorgan failure, and sometimes even expiry in humans. Testing for pyrogens is a requirement for all parenteral products including injectable vaccines, and in that location have traditionally been two tests for pyrogenicity: the rabbit pyrogen test (RPT) and the Limulus amebocyte lysate (LAL) test. In keeping with the policy of the 3Rs, the FDA has in many cases accepted the utilize of the in vitro test in lieu of the animal test for more than thirty   years. The LAL test is a highly quantitative mode to measure out contaminating endotoxin from gram-negative bacteria and is based on the clotting reaction of the hemolymph of the horseshoe crab. We note that the WHO guidance recommends that vaccine developers should provide evidence that the adjuvant or adjuvanted vaccine formulation does not interfere with LAL test. Still, the high specificity for bacterial LPS restricts the LAL exam'southward ability to monitor pyrogenicity and potential immune activation mediated by other product contaminants. xviii The test is incapable of detecting pyrogens such as lipoproteins, peptidoglycan, and lipoteichoic acids from gram-positive bacteria 9,70 and is thus an imperfect reflection of the human febrile reaction. This precludes the complete dismissal of the RPT exam in many cases.

The MAT is based on the in vitro activation of human being monocytoid cells in whole human blood or cell lines by pyrogens. The presence of pyrogens such as bacterial endotoxin stimulates the release of proinflammatory cytokines interleukin (IL)-1β, IL-six, and tumor necrosis gene (TNF)-α, which are quantified past ELISA, period cytometry, or bead-array. 46,49,75 Variants of the MAT have been standardized and validated 47,83 and were adopted past the European Pharmacopoeia in 2010. Depending on the various immunization strategies and novel vaccines and adjuvants, the use of both the MAT and the LAL test for routine lot release safe evaluation has been recommended. 69

It is noteworthy that despite the benefits offered past the utilise of validated in vitro cell culture assays, discussions with regulatory authorities are highly recommended to ensure that whatsoever MAT analysis would suffice to monitor pyrogen contamination. For case, pyrogens may induce fever contained of cytokines, by acting at TLRs. 21 Studies in mice genetically engineered to lack pyrogenic cytokines and clinical observations of fevers that frequently occur without a concomitant increment of circulating cytokines 11,43,62,72 collectively propose that the MAT assay may not e'er faithfully reflect a physiological febrile response in humans. In such scenarios, the RPT may be an indispensable or at least complementary tool to monitor febrile signals.

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Endotoxin and pyrogen testing

Tim Sandle , in Pharmaceutical Microbiology, 2016

11.10 Conclusion

Pyrogens are a concern for pharmaceutical drug products and for many of the ingredients used to formulate them. This is particularly so for products that come up into contact with human claret. Here, by far the well-nigh concerning pyrogen is bacterial endotoxin. In relation to this, the chapter has considered the risks of endotoxin to pharmaceutical processing and some of the control measures in place to reduce the risk of endotoxin contamination.

Furthermore, the affiliate has provided an introduction to endotoxin as well as to the primary method for detecting endotoxin: the LAL test. Here the chapter has examined the 3 chief types of LAL examination: gel-clot, chromogenic, and turbidimetric, as well as because alternative test methods. Such tests are an essential feature of most pharmaceutical microbiology laboratories.

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Sterilization and decontamination of surfaces by plasma discharges

F. Rossi , O. Kylián , in Sterilisation of Biomaterials and Medical Devices, 2012

6.v.1 Effect on pyrogens

Pyrogens are another mutual surface contaminant that is generally not addressed in the hospital sterilization processes. The outer coatings of spores and bacteria incorporate endotoxins (due east.g. lipopolysaccharides (LPS), peptidoglycans (PGN) sor lipoteichoic acids), which are potent modulators of the man immune organization. Their presence in the blood stream leads to physiological events such as fever, swelling or sepsis, and at higher doses to death ( Beutler et al., 2003). Endotoxins are extremely resistant to temperature and difficult to remove by conventional methods; their elimination by low-force per unit area plasma has been studied extensively in our grouping. We take shown that UV radiations in the 200–300   nm range, while having sufficient intensity to sterilize bacterial spores, does not decrease the biological action of pyrogens (Kylian et al., 2006c). Moreover, we showed that a MW low-pressure post-discharge plasma handling is able to inactivate different kinds of pyrogenic substances at low temperatures (Kylian et al., 2006b; Rossi et al, 2006; Hasiwa et al., 2008) through etching and chemical reactions with plasma radicals. These experiments were performed on different types of pyrogens, namely LPS, zymozan, Lipid A and PGN. In particular, it was plant that mixtures containing O2 and H2 are the most efficient (see Fig. vi.10), leading to two different deactivation mechanisms, every bit depicted in Fig. half-dozen.11. Analysis of the mechanisms led to the conclusion that one of these mechanisms is based on the removal of the pyrogen film by carving (oxygen containing mixtures), whereas the other proceeds by ways of chemic changes in the structure of the pyrogenic molecule (Rossi et al., 2006; Kylian et al., 2008a; Rossi et al., 2009). While depyrogenation in the case of Oii/Htwo can plain be attributed to the physical removal of contamination past etching, the loss of pyrogenic activity in the instance of Ar/ H2 is related to the volatilization of the fatty acrid chains (e.thou. C12H23O2 and CfourteenH27O2) too as the alteration of phosphoryl groups (PO, PO., POthree and PH2Oiv) (Kylian et al, 2008a; Rossi et al., 2009; von Keudell et al., 2010), which are the different components governing the bioactivity of Lipid A (Brandenburg et al., 2000; Erridge et al., 2002). It was also found that PGN could not be deactivated by post-belch treatment, for whatsoever of the different gas mixtures that we tested (von Keudell et al., 2010).

half dozen.ten. Bioactivity after plasma handling normalized to the bioactivity in discharge in pure oxygen (practical microwave power thousand   W, pressure xiii.3   Pa, total gas menstruum 100 sccm, handling time lx   s, LPS dilution i   ng/mL).

 Source: After Kylián et al., 2006b.

six.11. Relative alter of the IL-iβ response of a LPS contaminated surface treated by H2/Otwo and Ar/H2 microwave postal service discharges. By varying the composition of the discharge, different etching rates are observed, not correlated to the activity of the surface.

Other tests were performed in direct discharge. Since a chemical sputtering mechanism can be expected on other organic substances by oxidation of their carbon courage, nosotros tested different O2-based plasma discharges (Rossi et al., 2008) and found that all the compounds tested, including PGN, could be etched by an Oii-based mixture (Rossi et al., 2008, 2009).

An of import point to underline is that the sterilization of bacteria may atomic number 82 to an increase in the pyrogenic character of the surface. In the experiment presented in Fig. half dozen.12, 106 bacteria (Staphylococcus aureus) were sprayed into two-well bedroom slides (tested to be pyrogen-complimentary beforehand) and were allowed to dry. After plasma treatment, the CFU was evaluated together with the inflammatory mediator release (IL-iβ), an indicator measuring the pyrogenic activity of the surface. The results conspicuously show that the destruction of bacteria corresponds to a net increase in the pyrogenic activity of the surface; this is the result of plasma etching of the bacterial spore membrane, which releases pyrogens during the starting time stages of the handling. This consequence underlines the connectedness between sterilization and decontamination of surfaces: a express sterilization treatment may potentially lead to a contamination of the surface if the etching of the organic fabric is simply partially carried out. To our knowledge, the action of atmospheric plasma belch on pyrogens has not been reported in the literature to date.

6.12. Relationship betwixt decrease in bacteria population and increment in pyrogenicity (IL-1β release). Staphylococcus aureus numbers, 106. ICP treatment 100   Westward, thirteen.three   Pa.

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Toxicity and Safety Testing

Debra Kirchner , Susan Henwood , in The Laboratory Rabbit, Guinea Pig, Hamster, and Other Rodents, 2012

Pyrogen Test

A pyrogen is a substance causing consecration of a febrile response (elevation of trunk temperature, fever) which can be fatal in humans and animals. The causal substance is commonly a bacterial endotoxin, the lipopolysaccharide complex associated with the outer membrane of Gram-negative pathogens including, only non express to, Escherichia coli, Salmonella, and Shigella. Pyrogenicity is associated with microbiological contagion of parenteral products, including those that have been sterilized, biosynthetically produced materials, vaccines and adjuvants, and medical devices introduced into the fluid path. Regulatory agencies require evaluation of these products for pyrogenic potential, either by in vitro methods or the in vivo rabbit test, as a means to limit acceptable run a risk of febrile reaction in humans to administration, by injection, of the specified production.

The in vitro Limulus Amebocyte Lysate (LAL) test is a clotting reaction produced by a small corporeality of endotoxin in aqueous extracts of circulating amebocytes of the horseshoe crab (Limulus polyphemus). Considering the LAL examination cannot detect pyrogens except endotoxin, the USP (USP <85>, 2009) only recognizes the LAL method for assaying lipopolysaccharides produced by Gram-negative micro-organisms. Other in vitro alternatives to the rabbit pyrogenicity test accept been proposed (Hartung et al., 2001), and ECVAM and ICCVAM consider 5 in vitro methods based on cytokine release from human blood cells to be validated replacements for the evaluation of materials or products where the objective is to identify and evaluate pyrogenicity produced by Gram-negative bacterial endotoxins simply, every bit with the LAL method, not for other pyrogens.

The in vivo pyrogenicity test is conducted in good for you, adult rabbits past evaluating the rising in rectal temperature after Iv injection of a test solution (exam product reconstituted, if necessary) into the ear vein (European Pharmacopeia (EP), 2009; Gad and McCord 2008c; USP <151>, 2009). The examination is designed for products that tin can exist tolerated by rabbits at a dose not to exceed ten   ml/kg injected 4 for 4 minutes (EP) or 10 minutes (USP). No rabbit should be used for pyrogen testing more than frequently than once every 48 hours or prior to two weeks post-obit a rise in temperature of 0.half-dozen°C or more or post-obit its having been given a test specimen judged pyrogenic. Up to 1 calendar week ahead of the examination, rabbits are conditioned to all study procedures including injection per EP, and except injection per USP. The pyrogen test is conducted in three rabbits, injecting a sterile, warmed solution that may be dissolved or diluted with adequate liquids (east.g., sodium chloride). Temperatures are recorded at baseline (30 minutes prior to injection) and at 30-minute intervals upward to three hours postal service-injection, although EP and USP differ slightly in the verbal times. The UPS and EP also differ with respect to criteria for absence of pyrogenicity. The test material is not pyrogenic if no rabbit shows an individual rise in temperature of ≥0.5°C above its respective control temperature. If any rabbit's temperature increases ≥0.5°C, five other rabbits are tested. Requirements for the absence of pyrogenicity are met if not more than than iii of eight rabbits have an individual rise in temperature of ≥0.5°C and if the sum of the maximum rise in temperature of the viii rabbits does not exceed three.three°C.

Per the EP, the exam material passes if the summed response (i.e., divergence between the highest temperature 3 hours post-injection and baseline temperature for the 3 rabbits) does not exceed one.15°C, and fails if the summed response exceeds 2.65°C. If the test fails, it is repeated in three additional rabbits and their response plus the responses of the commencement 3 rabbits (i.e., summed responses of the six rabbits) determines if the production passes (summed response does not exceed 2.eighty°C) or fails (summed response exceeds iv.30°C). Up to 12 rabbits may be tested with pass/fail criteria based on whether or non the summed temperatures are less than or greater than 6.60°C.

Other findings in the rabbit resulting from smaller injection amounts can include increased capillary permeability, increased numbers of white claret cells, hemorrhages, and changes in venous pressure level. In some cases, the response to the pyrogen is a severe hemorrhagic reaction with localized necrosis, known as the Shwartzman's miracle or reaction. This response is demonstrated in rabbits by SC assistants of a bacterial pyrogen followed 24 hours later with Iv of the aforementioned pyrogen and noting if the site of the later injection turns blueish at the center and red at the periphery. Pyrogen tolerance develops when the animals are given repeated injections of a pyrogen. A reduced sensitivity to the aforementioned and other pyrogens develops and the febrile response diminishes.

In some cases, a blended testing approach is recommended. For example, the US Eye for Veterinary Medicine requires testing the first 3 batches manufactured using both the LAL and rabbit pyrogen tests to determine whether other types of pyrogenic substances are present (PPM 1240.4122, 2000). This arroyo takes into account the possibility that pyrogenic materials present in the product are not lipopolysaccharides. Testing the commencement iii commercial batches demonstrates if pyrogen contagion other than lipopolysaccharides is present in the final drug product. If rabbit pyrogen testing in the start three commercial lots is negative, the LAL test can exist utilized for release testing.

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Streptococcal superantigenic toxins

Thomas Proft , John D. Fraser , in The Comprehensive Sourcebook of Bacterial Poly peptide Toxins (Third Edition), 2006

Pyrogenicity and augmentation of endotoxin activity

Pyrogenicity, or the induction of fever, is believed to exist a common feature of all streptococcal superantigens, although this has not been experimentally confirmed for all toxins. The IV injection of rabbits with either SPE A or SPE C resulted in elevated temperatures upward to four–5 h subsequently a thirty–sixty min latency every bit reported past Schuh et al. (1970). The minimal pyrogenic dose of SPE A and SPE C adamant 3–4 h after injection ranged between 0.1 and 0.7 μg/kg. The ability to cause fever in rabbits has as well been reported for SPE J (McCormick et al., 2001a) and for SMEZ (Müller-Alouf et al., 2001). It has been suggested that pyrogenicity is due to SAg-induced release of IL-i and TNF-α past macrophages and the result of these cytokines on the hypothalamus (Fast et al., 1989).

Streptococcal SAgs can heighten the host's susceptibility to lethal endotoxic shock. Rabbits that received SPE C alone showed fevers only, but those given both SPE C and endotoxin showed initial fever followed by hypothermia, labored breathing, diarrhea, evidence of vascular collapse, and finally death (Schlievert, 1982). Susceptibility of rabbits to lethal shock by endotoxin was enhanced by every bit much as fifty,000-fold. It has been suggested that endotoxin and superantigen might act synergistically as a double-striking, resulting in toxic shock in the absenteeism of a typical focus of bacterial infection (Llewelyn and Cohen, 2002).

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RADIOCHEMICAL METHODS | Pharmaceutical Applications

R.E. Stringer , in Encyclopedia of Analytical Science (2nd Edition), 2005

Pyrogenicity Testing

Bacterial endotoxins (pyrogens) are polysaccharides from bacterial membranes. They are water soluble, heat stable, and filterable. If they are present in a preparation and administered to a patient they can cause fever and also leukopenia in immunosuppressed patients. To minimize the chances that pyrogens are nowadays information technology is of import that preparations are manufactured and dispensed nether aseptic conditions and that all consumables and equipment used accept been heat treated and known to be pyrogen free. Most licensed products are guaranteed pyrogen free. Yet, the Pharmacopoeiae state that certain radiopharmaceutical preparations are required to comply with the test for pyrogens and it may be necessary to test for pyrogens in unlicensed products or intermediates used in manufacture. This test can either be performed in rabbits or a test for bacterial endotoxin employing limulus amoebocyte lysate may be used. These tests can exist carried out in the radiopharmacy or by a quality control testing laboratory.

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Strategies for large scale production and optimized stability of pharmaceutical liposomes developed for parenteral use

Eastward.C.A. VAN WINDEN , ... D.J.A. CROMMELIN , in Medical Applications of Liposomes, 1998

3.6 Apyrogenic and sterile production of liposomes

Pyrogens can cause fever and shock. Mutual sources for pyrogens are microorganisms, in particular those producing endotoxins (lipopolysacharides). It is extremely difficult to remove all pyrogens from formulated liposome dispersions. 121 Depyrogenation of fluids (including organic solvents holding the lipids) is possible by ultrafiltration through filters with cut-offs of x kDa. 121 Therefore, the manufacturer should check the quality of the raw materials, and pattern the liposome conception process in such a style that the generation of pyrogens past micro-organism growth or contact with contaminated equipment during the production procedure is avoided. The reader is referred to the book of Pearson 122 and pharmacopeia 123 , 124 for further detailed information about pyrogens and pyrogenicity tests.

A production is considered sterile if the gamble to find a unit that is contaminated with living microorganisms is less than 1 in 106 sterilized units of that product. 123 , 124 Recently, our group has published an overview of the different sterilisation techniques which may be considered to sterilise liposome dispersions. 125 It was stressed in this article that sterility tin can not be guaranteed by testing the final product, merely should be assured by validated, well-defined grooming procedures.

Sterility will be achieved if a low caste of contamination (100 or less colony forming units (cfu)/ml) is combined with an effective sterilisation step immediately after finishing the preparation procedure. An effective sterilisation procedure gives at least a x 12 fold reduction of the test organism known to be highly resistant to that particular sterilisation method (worst-case supposition). The post-obit approaches to achieve sterile liposomes take been considered: (1) autoclaving 125–128 (2) loftier pressure sterilisation, 129 (3) utilize of ethylene oxide 130 , (4) γ-irradiation 131–133 and (5) filtration. 121 , 134

Autoclaving (121°C, 15 minutes) is a preferred sterilisation method for several reasons. First of all, it is relatively simple and has been extensively validated. In addition, autoclaving can be applied to the end product. Under neutral, buffered pH conditions, liposomes without encapsulated agents or with oestrus-stable, bilayer interacting (lipophilic) agents can exist sterilized. 127 , 135 Oxidation of egg phospholipids is not a trouble when using EPC with a depression peroxide value. 127 Withal, in other cases the chemical and physical stability of the liposomes and the drug during the oestrus treatment tin exist insufficient. Autoclaving may not be adequate for liposomes (1) in a bones or acrid medium, or (2) loaded with a water soluble, non-bilayer interacting drug which tin leak out of the liposomes. 127 , 135 Nonetheless, autoclaving of liposomes may still be an option if the free drug does not interfere with the desired therapeutic outcome, or can be loaded later the autoclaving process (eastward.g., by active loading techniques).

High pressure sterilisation 129 (e.g., five hours at 60°C and 2.5 × 10eight Pa or 21 hours at forty°C and 2 × 108 Pa) could be an attractive option for sterilising temperature sensitive and high pressure resistant drugs such as proteins. Nevertheless, application of this technique is hindered by its limited efficacy against the spores of Bacillus stearothermophilus.

For the sterilisation of estrus labile drugs, several other options exist. Treatment with ethylene oxide has been applied to freeze-dried cakes. 130 However, its sterilising capacity for freeze-dried liposome dispersions has not been proven yet. Moreover, the possibility that toxic residues remain in the cakes has to exist excluded.

The apply of γ-irradiation 131–133 as a sterilisation technique is still nether debate. 124 Later on γ-irradiation of aqueous liposome dispersions with the sterilisation dose of 25 kGy, which is mostly used for this purpose co-ordinate to Pharmacopeias such as the U.S.P. and B.P., besides much degradation of the selected liposomal phospholipids has been institute. 136 More than studies are necessary to evaluate this method in combination with a powerful antioxidant and/or freeze-drying or freezing in the presence of a safety and effective cryoprotectant.

Filtration through filters with a pore size of ≤0.22 μm is non a sterilisation technique to be considered as a showtime option, because it is non the last footstep in the production procedure. This calls for carefully validated production protocols and well-trained personnel. According to the USP the probability of non production-related contagion may be about 10−3 during an hygienic performance, 123 much higher than the contamination level accustomed later rut sterilization (10−6). In spite of these disadvantages, filtration is still widely used to produce parenteral products that can not exist sterilized with other techniques. 121 , 134 The major advantage of filtration is that information technology is non destructive (except maybe for inducing leakage) for small liposomes. To minimise initial contamination it is recommended to routinely filtrate media used for the preparation of liposomes and the liposomes "in statu nascendi" at different stages of the production process through filters with a pore size of ≤0.22 μm, even when autoclaving is used as a final sterilisation technique. In a written report by Sorgi and Huang 87 it was found that the loss of lipid during filtration may depend on the choice of filter material and the selected lipids. A eighteen% loss of DOPE (i,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine) was observed after filtration of small (<0.two μm) liposomes consisting of DOPE and DC-Chol (3β[Due north-(N′N'-dimethylaminoethane)-carbamoyl]cholesterol) through 0.2 μm filters equanimous of cellulose acetate. However, the employ of nylon or cellulose nitrate filters reduced the loss of DOPE to circa iii%.

It would be interesting to study a combination of unlike approaches to reach acceptable reduction factors that are not achievable with one approach lone. The susceptibility of microorganisms to a sure sterilisation technique depends on the subpopulation. This susceptibility pattern is different for the different sterilization techniques. Therefore, treatment of liposome dispersions with more than i technique under relatively balmy weather, might outcome in an acceptable contagion level and piffling damage to the liposomes. For example, a low dose of γ-irradiation (±5–ten kGy) in combination with filtration might be a suitable sterilisation method for those dispersions which tin not be sterilised past autoclaving.

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Bacterial superantigens and superantigen-like toxins

Ries J. Langley , ... Thomas Proft , in The Comprehensive Sourcebook of Bacterial Protein Toxins (Fourth Edition), 2015

Pyrogenicity and augmentation of endotoxin activity

Pyrogenicity, or the induction of fever, is believed to be a mutual feature of all streptococcal SAgs, although this has not been experimentally confirmed for all toxins. The IV injection of rabbits with either SPE-A or SPE-C resulted in elevated temperature upwardly to 4–5   h after a 30–60   min latency, as reported by Schuh et al. [220]. The minimal pyrogenic dose of SPE-A and SPE-C determined 3–4   h after injection ranged between 0.1 and 0.vii   μg/kg. The ability to crusade fever in rabbits has besides been reported for SPE-J [51] and SMEZ [221]. It has been suggested that pyrogenicity is due to SAg-induced release of IL-1 and TNF-α by macrophages and the result of these cytokines on the hypothalamus [222]. SAgs tin can enhance the host'southward susceptibility to lethal endotoxic daze. Rabbits that received SEC alone experienced only fever, but those given both SEC and endotoxin showed initial fever, followed by hypothermia, labored animate, diarrhea, evidence of vascular collapse, and finally death [223].

Susceptibility of rabbits to lethal shock by endotoxin was enhanced by as much as fifty,000-fold. It has been suggested that endotoxin and SAg might human action synergistically as a double hit, resulting in toxic stupor in the absence of a typical focus of bacterial infection [224]. It has been suggested that this synergy results from enhanced pattern recognition of endotoxin, and this is based on the observation that SAg signaling increases expression of toll-similar receptor 4 (TLR4), the pattern recognition receptor for LPS [225]. Furthermore, several SAgs, including the streptococcal SAgs SPE-A and SMEZ, were able to upregulate TLR2 on the surface of chief man monocytes. This was dependent on SAg-binding to MHC grade II, simply did not involve signaling by ligation to TLR2. TLR2 upregulation was associated with an increment in the proinflammatory response to TLR2 ligands, but only at loftier ligand concentrations [226]. More recently, it was demonstrated that SAgs also raise the proinflammatory cytokine response of the TLR2 agonist bacterial lipoprotein (BLP) in a mouse model, which might also contribute to SAg-mediated toxic daze [227].

A report past Schlievert and colleagues has shown that IFN-γ derived from TSST-ane-activated T cells plays an of import role in hypersensitivity to endotoxin [228]. Farther testify for the involvement of T cells in the cytokine storm came from a study with a TcR binding mutant of TSST-1 (H135A), as this mutant failed to increase sensitivity to endotoxin [229].

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Pyrogenicity and bacterial endotoxin

Tim Sandle , in Sterility, Sterilisation and Sterility Assurance for Pharmaceuticals, 2013

2.two Pyrogenicity

The term pyrogen is derived from the Greek word pyrexia. Pyrogens can be either internal (endogenous) or external (exogenous) to the body. All endogenous pyrogens are cytokines, molecules that are a part of the innate immune arrangement (e.g. interleukin 1 (α and β) and interleukin 6 (IL-6)). Exogenous pyrogens tin can enter the claret stream via injection of pharmaceutical preparations (parenteral products). The most common type is bacterial endotoxin. Although a 'pyrogen' is introduced into the body as an exogenous agent, its presence causes the release of endogenous factors, that is, the immunological response within the trunk is the same [ 5].

In the early days of the pharmacopoeia, drug substances were classed as apyrogenic or pyrogenic based, from 1942 and until the 1980s, solely on the 'pyrogen examination', whereby a quantity of the drug was injected into 3 rabbits and the temperature response of the rabbits was noted. The rabbit pyrogen test was first described by Florence Seibert in 1925 [6].

The rabbit examination is no longer widely used and has been largely been replaced, for the testing of parenteral drug products, by the LAL test, particularly inside Europe under the requirements of the European Pharmacopoeia Commission [vii] and the European Medicines Agency [8]. The LAL test is a method of the Bacterial Endotoxin Test (BET) for detecting the presence, and to go some way to determining the level, of Gram-negative bacterial endotoxins in a given sample or substance. Current editions of the Pharmacopoeia behave statements to the effect that where the term apyrogenic or pyrogen-costless is used, it should be interpreted as pregnant that samples of the product will comply with a limit for bacterial endotoxin.

It was not until the early twentieth century that an understanding began to emerge in which bacteria could be classified into pyrogenic and not-pyrogenic types, correlatable to their Gram stain. Gram staining is a method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative). It is based on the chemical and concrete properties of their jail cell walls. Primarily, information technology detects peptidoglycan, which is present in a thick layer in Gram-positive bacteria. A Gram-positive results in a purple/bluish colour, while a Gram-negative results in a pink/reddish color.

Gram-negative leaner were plant to be pyrogenic, Gram-positive bacteria were generally not and killed cultures of Gram-negative bacteria were comparable to live cultures in their ability to induce fevers. Information technology was found that the injection of living or killed Gram-negative cells into experimental animals caused a wide spectrum of non-specific pathophysiological reactions, such as fever, changes in white blood prison cell counts, disseminated intravascular coagulation, hypotension, shock and expiry.

Thus, by the 1920s it was apparent that sterility in parenteral pharmaceuticals could be no guarantee of non-pyrogenicity, and that if pyrogenicity was to be avoided, information technology was imperative to avoid bacterial contamination at every stage of manufacture of parenteral pharmaceuticals.

In recognition that the causative agent of pyrogenicity was filterable and heat stable, efforts were applied to place its chemical limerick. Trichloracetic acid and phenol-water extractions of leaner were institute to be constructive in isolating the pyrogenic element from bacteria. These extracts were chemically identifiable as LPS, or what is commonly described as bacterial endotoxin.

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Parenteral preparations

Mangal Shailesh Nagarsenkar , Vivek Vijay Dhawan , in Remington (Twenty-3rd Edition), 2021

29.7 Pyrogens (endotoxins) and depyrogenation

The most potent pyrogenic substances (endotoxins) are constituents of the cell wall of Gram-negative bacteria (e.k., Pseudomonas sp., Salmonella sp., Escherichia coli). Gram-positive bacteria produce peptidoglycans, whereas fungi product β-glucans, both of which can cause nonendotoxin pyrogenic responses. Endotoxins are LPS that be in high molecular weight aggregate forms. However, the monomer unit of LPS is less than x,000   Da, enabling endotoxin to hands pass through sterilizing 0.2 μm filters. The lipid portion of the molecule is responsible for the biological activeness. LPS targets circulating mononuclear cells (monocytes and macrophages) that produce proinflammatory cytokines, such as interleukin 2, interleukin six, and tissue necrosis gene. Exotoxin A, peptidoglycan, and muramuyl peptides also mimic the activity of LPS and induce cytokine release. Pyrogens, when nowadays in parenteral drug products and injected into patients, can crusade fever, chills, hurting in the dorsum and legs, and malaise. They tin can crusade serious discomfort and, in the seriously ill patient, shock-similar symptoms that can exist fatal. Intrathecal administration is most chancy if contaminated with pyrogens followed by intravenous, intramuscular, and subcutaneous.

Water is probably the greatest potential source of pyrogenic contamination, since water is essential for the growth of microorganisms and frequently contaminated with Gram-negative organisms. Therefore WFI should be used for compounding the product or rinsing product contact surfaces, such every bit tubing, mixing vessels, and condom closures. Rinsed equipment and supplies are left wet and improperly exposed to the surround, there is a high take a chance they will become pyrogenic. WFI storage weather condition must be such that microorganisms are not introduced and subsequent growth is prevented. Other potential sources of contamination are containers and equipment. Pyrogenic materials attach strongly to glass and other surfaces, especially condom closures. Residues of solutions in used equipment often become bacterial cultures, with subsequent pyrogenic contagion. Since drying does not destroy pyrogens, they may remain in equipment for long periods. Adequate washing reduces contamination, and subsequent dry-estrus treatment can return contaminated equipment suitable for use. Aseptic processing guidelines require validation of the depyrogenation procedure by demonstrating at to the lowest degree three-log reduction in an applied endotoxin challenge. Solutes may also be a source of pyrogens. Manufacturing of bulk chemicals may involve the use of pyrogenic h2o for process steps, such as crystallization, precipitation, or washing. Bulk drug substances derived from cell culture fermentation will almost certainly be heavily pyrogenic.

Pyrogens can be destroyed by heating at loftier temperatures. A typical process for depyrogenation of glassware and equipment is maintaining a dry-rut temperature of 250°C for 45   minutes. Exposure of 650°C for 1   minute or 180°C for 4   hours, likewise, will destroy pyrogens. The usual autoclaving cycle will non do so. Heating with stiff alkali or oxidizing solutions destroys pyrogens. It has been claimed that thorough washing with detergent will render glassware pyrogen-gratuitous, if afterwards rinsed thoroughly with pyrogen-gratis water. Safety stoppers cannot withstand pyrogen-destructive temperatures, and so reliance must be on an constructive sequence of washing, thorough rinsing with WFI, prompt sterilization, and protective storage to ensure adequate pyrogen command. Similarly, plastic containers and devices must exist protected from pyrogenic contamination during industry and storage, since known ways of destroying pyrogens affect the plastic adversely. It has been reported that anion-exchange resins and positively charged membrane filters remove pyrogens from water. Also, although contrary osmosis (RO) membranes will eliminate them, the most reliable method for their elimination from water is distillation. Other in-process methods for their destruction or elimination include selective extraction procedures and conscientious heating with dilute alkali, dilute acid, or mild oxidizing agents. Although ultrafiltration now makes pyrogen separation on a molecular-weight basis possible and the process of tangential menstruation is making large-scale processing more than applied, use of this technology is limited, except in biotechnological processing.

Through understanding the means past which pyrogens may contaminate parenteral products, their command becomes more doable. Compounding and manufacturing processes should be carried out as expeditiously as possible, preferably planning completion of the procedure, including sterilization, within the maximum allowed fourth dimension, according to process validation studies. Aseptic processing guidelines require institution of time limitations throughout processing for the chief purpose of preventing the increase of endotoxin (and microbial) contamination that, subsequently, cannot be destroyed or removed. Preferably, no more products should be prepared than tin be processed completely within one working day, including sterilization.

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