Limulus Within Arthropoda

Arthropoda has been the most successful of multicellular life forms, comprising three-fourths of all known living and extinct organisms, an estimated 1 million species. By some estimates, 10 times that amount remain undiscovered (4). Distinguishing features of Arthropoda include their modular body parts, including segmented bodies and jointed appendages (antennae, legs, feeding appendages, etc.), and chitinous exoskeleton. They also demonstrate a dizzying array of life cycle adaptations that...

Case

October 1996 35 newborns died in a small hospital in Roraima, Brazil (44). The deaths were > 3X the normal death rate for the hospital nursery. The Brazilian Ministry of Health requested the CDC's assistance in an investigation which revealed that locally manufactured intravenous solutions were contaminated with endotoxin. Sampling of parenteral fluids was tested by the LAL method with the following results. All cultures of these solutions were negative for bacterial growth. However, 6 of 13...

Overview Of Common Testing Protocols

The types of testing protocols used in developing a new method include (i) solubility protocols (see Attachments A and B to this chapter for example protocols), (ii) pNIC protocols, and the (iii) full validation protocol. The tests performed in this sequence are cumulative. In simple terms, the NIC test varies the sample concentration while keeping the endotoxin concentration fixed (none and 2A) while the I E test varies the endotoxin concentration (to mimic the standard curve) while...

Objective And Purpose Of Risk Assessment

According to ICH Q9 Quality Risk Management Effective quality risk management can facilitate better and more informed decisions . Moreover appropriate risk management permits the utilization of information to leverage resource in areas which can realize the greatest improvement in product quality assurance. All risk assessment techniques and tools may be regarded as decision tools the sole objective of any risk assessment should be to facilitate and assist in decision-making. Prior to embarking...

Endotoxin Alert Level For Active Pharmaceutical Ingredients

It is impossible to render materials absolutely pyrogen free because endotoxin is stable, highly potent, and ubiquitous in nature. Therefore, an EL represents the maximum safe amount of endotoxin that is allowed in a dose of a specific parenteral product. When a product contains endotoxin less than its EL, it may be labeled nonpyrogenic. The compendial EL for a product is calculated from the K M formula where K, the tolerance limit, varies with the type of product and route of administration,...

Early Characterization Of

The ability of hemocytes to coagulate in the presence of gram-negative bacteria or their endotoxins is not restricted solely to Limulus and has been demonstrated in the lobster, crab, and oyster (17) as well as insects (18) (although getting enough blood volume for a lysate from an oyster might prove difficult). In a subsequent study, Levin et al. (19) developed a sensitive assay for endotoxin in human plasma using the material lysed from Limulus amebocytes. As little as 0.0005 mg (0.5 ng) of...

The Bacterial Endotoxin Testing Assay Development Process

The need for a new bacterial endotoxin test (BET) typically begins with a call from a development scientist with a new compound. Perhaps it is a compound prepared for an animal toxicology study or perhaps it is a lot prepared in the development laboratory (a so-called lab-lot). The early lots of drug substance or drug product will not be used in people, but there is a need to establish their safety to insure that the studies being performed are not skewed in some manner by the presence of...

Toll And Tolllike Receptors

The fact that virtually all organisms are born knowing that endotoxin from gramnegative bacteria is the enemy speaks to both the power of endotoxin as a host defense activator and to the universality (and thus the necessity of conservation) of the host's solution to endotoxin detection and elimination. It seems strange, at least to me, that an outside prokaryotic contaminant should have gene-encoded triggers to its presence in metazoans, but this is exactly what has happened and remains, in all...

Endotoxin Limits For Intrathecal Drugs In Pharmacy Compounding

Injectables prepared in compounding pharmacies are usually formulated from bulk nonsterile powders and are usually produced individually rather than in batches. It is difficult to assign an EL to compounded injectables because the pharmacy may not know the prescribed dose. There are safeguards that a pharmacy may TABLE 1 Endotoxin Tolerance or Allowable Limit by Type of Parenteral Material TABLE 1 Endotoxin Tolerance or Allowable Limit by Type of Parenteral Material Human or veterinary drugs...

Viewing Variability in Process

It is interesting to view the entire LAL test system as a process whether looking to automate or not. The variability associated with the kinetic BET can be a cause of frustration. Generally, the source of variability resides 1. in assays for substances that retain a degree of interference that has not been eliminated by the method developed, 2. inherently in the LAL test method including reagents, pipettes, labware, etc., 3. and in the linear regression (LR) standard curve plot and resultant...

Prokaryotic Biodiversity And Prokaryotic Phylogenetics

The number and types of prokaryotes that have been studied are very few in comparison to the total number present in the biosphere. Some have been exhaustively studied (EC), whereas others are only inferred from environmental gene sequence recoveries (2) and some, if not most (3), cannot be cultured at all. Given that most bacteria occurring in nature are unculturable and thus remain as virtual, almost hypothetical, genetically inferred ghosts (4), and are therefore unknowable in a traditional...

Kinetic LAL Techniques

Until we update the guideline the following guidance and the lysate manufacturers approved procedures can be used. The kinetic LAL techniques should be done according to the lysate manufacturers recommended procedures, that is, sample lysate ratio, incubation temperature and times, measurement wavelength, and so on. Instrumentation other than the one recommended by lysate manufacturer can be used. The performance characteristics (slope, y-intercept and correlation coefficient), for the lysate...

Validation Philosophy And Ancillary Considerations

Parenteral vials have been found to be a source of endotoxin contamination in the past but with the current use of shrink-wrapping instead of paper packaging this problem has largely disappeared. A team at Squibb (24) monitored 3200 incoming vials (prior to depyrogenation treatment) over an eight-month period and found only two that had responses at the detection limit of the lysate (nevertheless an unacceptable contamination rate). Furthermore, the demonstration that it rarely happens is not...

Medicinal Hygiene And Wound Infection

It was not until the mid to late 1800s that even the most basic practices of hygiene were applied to medical procedures. In a Paris hospital between 1861 and 1864,1226 of 9886 pregnant women died a few days after childbirth. The deaths were attributed to puerperal fever. The situation was similar the world over. In Boston in 1843, Oliver Wendall Holmes taught that germs on physicians' and midwives' hands caused puerperal fever (4). Other doctors largely dismissed his ideas. Ignaz Semmelweiss...

Introduction To The New Biology

This chapter contains a review of recent knowledge that has come about from the research into the superphylum Arthropoda, which includes Limulus and Droso-philaa. Limulus served early on as a significant tool for medical research in large part due to the simplicity of its blood system and to its obvious primitive status (i.e., living fossil) among nature's creatures. More recently, Drosophila has dominated phylogenetic studies for similar reasons, but also due to its small size, ease of...

Limulus Amebocyte Lysate Assay Development Validation and Regulation

Eli Lilly & Company, Indianapolis, Indiana, U.S.A. Historically, large volume parenteral manufacturers have been foremost in developing tests for Bacterial Endotoxin assays due to the criticality ofeven minute endotoxin concentrations in solutions administered in large doses. However, many of today's problems revolve around the recovery of control standard endotoxin spike, the difficulty of which is exacerbated by the chemical nature of the small volume drug materials being validated rather...

Department Of Health And Human Services Public Health Service

Subject Statement Concerning Glucans and LAL-Reactive Material in Pharmaceuticals and Medical Devices It has been reported to the LAL Task Force that information has been circulated concerning FDA's position on glucans defined LAL-reactive material (LAL-RM) in pharmaceuticals and medical devices. Glucans are defined as polysaccharides composed only of recurring units of glucose, such as, glyco-gen, starch, and cellulose. FDA is not aware of data indicating that glucans or LAL-RM are common in...

Info

ANoted in 1991 that cytoplasmic domain of IL-1 was homologous to Toll (TLR1). bAssociated with Leptospira interrogans and Porphyromonas gingivitis. c Trypanosoma is a protozoan parasite. Abbreviations IL, Interleukin LPS, lipopolysaccharides LTA, lipoteichoic acid PAMP, Pathogen Associated Microbial Patterns TLR, Toll-like receptors. aNoted in 1991 that cytoplasmic domain of IL-1 was homologous to Toll (TLR1). bAssociated with Leptospira interrogans and Porphyromonas gingivitis. c Trypanosoma...

The Horseshoe Crab

In 1885, Howell (6) observed that the blood of L. polyphemus, the horseshoe crab, formed a solid clot when withdrawn from the animal. A year later, Loeb (7) reported that, when the blood of Limuli were collected and exposed to a foreign substance, they underwent liquefaction followed by coagulation. This was the first in a series of papers (8,9) that Loeb published detailing various aspects of coagulation with particular reference to amebocytes, the only circulating cell found in the blood of...

Significant Nonendotoxin Microbial Pyrogens

Significant nonendotoxin microbial pyrogens include cellular wall membrane components from both gram-negative and gram-positive bacteria, fungi, virus, mycobacteria, mycoplasma, and spirochetes, as well as exotoxins from Staphylococcus and Streptococcus, notably enterotoxins from Staphylococcus aureus and exotoxins from Streptococcus pyogenes. Significant microbial constituents capable of producing a host response that may include fever are listed in Table 1. The dose (or level of infection) is...

Quantifying Risk From Endotoxin In Pharmaceutical Manufacture

A number of well established risk assessment tools and techniques are available and have been effectively used to discern risks of bioburden ingress, retention, and proliferation within pharmaceutical manufacturing processes 7 . These also lend themselves to assessing the risks of endotoxins ingress and retention and include 1. Failure Mode and Effect Analysis FMEA 19-21 2. Failure Mode Effects and Critical Analysis FMECA 22 3. Hazard Analysis and Critical Control Points HACCP 23,24 Ubiquity...

Endotoxin Assay Development Cases Studies

A few assay development projects are noteworthy due to either the complexity of the problem presented and or the protracted nature of the solution. Piluso and Martinez 17 developed a quantitative LAL method of testing liposome material. Piluso and Martinez 17 considered that with their particular product there can be no assurance that endotoxin has not incorporated within the forming liposome unless specific analysis is executed, because the function of the liposome as a drug delivery vehicle...

Written Requirements For Depyrogenation Processes

Log Endotoxin Reduction

The requirements for depyrogenation validation processes are somewhat vague and subject to interpretation. A short reference occurs in the USP, Chapter 1211 Sterilization and Sterility Assurance of Compendial Articles, Dry-Heat Sterilization section as follows Since dry heat is frequently employed to render glassware or other containers free from pyrogens as well as viable microbes, a pyrogen challenge, where necessary, should be an integral part of the validation program, for example, by...

References

Utilization of the Limulus amebocyte lysate test for pyrogen testing large volume parenterals, administration sets, and medical devices. Bull Parenter Drug Assoc 1977 31 3 127-133. 2. Federal Register. 1977 42 57749-57750. 3. Mascoli CC, Weary ME. Limulus amebocyte lysate LAL test for detecting pyrogens in parenteral injectable products and medical devices advantages to manufacturers and regulatory officials. J Parenter Drug Assoc 1979 33 2 81-95. 4. Health Industry...

Are Aggregates Or Monomers More Biologically Active

In water-based environments endotoxin aggregates and the type of aggregates that form are based upon the chemical structure of the underlying LPS molecules. Many interrelated physical factors affect the pyrogenicity endotoxicity of endotoxins in solution by affecting the ability to form aggregates as well as the underlying conformation geometrical arrangement of those aggregates. The resulting changes affect the availability of the endotoxic lipid A portion of the LPS molecule to interact with...

Lal Test Method Validation For Medical Devices General Requirements

No pre-clearance is needed prior to use of the LAL test as an end product test routine testing if it is used according to the FDA Guideline on the Validation of the LAL Test. LAL test method validation requirements are listed in the FDA Guideline on the Validation of the LAL Test 14 1. Use of FDA licensed LAL reagent in all validation Including in-process and end product LAL testing. 2. Initial qualification of the LAL laboratory analyst Each analyst using a single lot of LAL and a single lot...

Preparation Of Medical Devices Test Samples Sampling

The sampling criteria for selection of medical device end product units for bacterial endotoxin testing using the LAL tests are based on the premise that the medical device manufacturing process is in control and in compliance with the medical devices Quality System Regulation QSR requirements. The selection of medical device end product or finished units for testing shall be based on criteria defined in a predetermined sampling plan. The sampling plan should specify the device sample size, the...

Overview Of Hemolymph Coagulation In Limulus And Tachypleus

Hemolymph Clotting

Invertebrates lack adaptive immune systems and rely on innate immunity to antigens common to pathogenic organisms. Nakamura et al. have extensively studied the hemolymph blood system of the Japanese horseshoe crab T. tridentatus and found amebocytes contain two types of granules large L and small S , which contain the clotting factors, proteins, and antimicrobials that are released into the crab's plasma via a process called degranulation 28,29 . Regardless of the relative simplicity of the...

Historical Development Of Depyrogenation Methods

Endotoxin is notoriously resistant to destruction by heat, desiccation, pH extremes, and various chemical treatments. The validation of endotoxin destruction or removal in the manufacture and packaging of parenteral drugs given its enduring nature and likelihood of occurrence is a critical concern to drug and device manufacturers. Lipopolysaccharide LPS requires dry heat of treatment around 250 C for half an hour to an hour to achieve destruction and standard autoclaving will not suffice. Many...

Resolving Test Interferences

Given that the LAL assay in its many forms is a water-based assay derived from a sensitive physiological environment blood of the horseshoe crab , it is not too surprising that as one ventures farther from such an aqueous environment the results often correspondingly deteriorate. The Catch-22 of such testing resembles the contradiction presented by endotoxin itself in that an increase in water content of a eThe Lilly laboratory uses a conservative approach in that when beginning, middle, and...

Depyrogenation Concepts Borrowed From Sterilization Validation

Methods and mechanisms of proving the depyrogenation of various items have been largely borrowed from sterilization processes and modified to compensate for the thermal and chemical stability of LPS as compared to whole organisms. Depyrogenation processes like sterilization methods may involve the construction of D death or destruction in the case of endotoxin since it is not alive values but for regulatory purposes, the use of a three-log reduction validation serves as the official empirical...

Depyrogenation Indicator And Recovery Variability

It is common knowledge in the endotoxin-testing lab that depyrogenation validation recoveries of applied endotoxin is a highly variable process dependent upon many difficult to control variables. An analogous variability in sterilization validation BI recovery has been reported by Shintani and Akers 28 . They list several BI variables including carrier material, primary packaging material, culture medium, different sport suspensions but not BI resistance , retrieval technique, and population...

Setting Endotoxin Specifications

The beginning of this chapter has dealt with the scientific aspects of developing a good endotoxin test this half of the chapter will focus on the scientific-regulatory interface LAL manufacturer's and foreign and domestic regulators and regulatory requirements. Both the Guideline on Validation of the LAL Test 26 and the FDA Interim Guidance for Human and Veterinary Drug Products and Biologicals 27 provide detailed descriptions on the means of satisfying the agency's requirements. While it is...

Overview

Williams 1 and Cooper 2 have published several methods for assigning endotoxin specifications to nonfinished drug ingredients. These methods will be detailed herein. The first method has been tagged a control strategy in that it seeks to estimate relevant limits by which to confine or control potential contaminants. There are cases, especially unique ingredients, that require one to develop relevant levels of endotoxin for targeting specifications. A second method, referred to here as the...

Risk In Aseptic Parenteral Manufacture

A generalized scheme for the aseptic manufacture of parenteral product presentations within a stoppered vial is illustrated in Figure 2. The left-hand box defined by a broken line encompasses nonsterile operations and materials within the formulation and preparation process steps and operational stages. This box therefore includes all those processes, which actively control or remove endotoxins. Processes and operations in this area of the schematic are vulnerable to challenge from both...

Ionizing Radiation Gamma Radiation

Several studies have been reported in which ionizing radiation with 60Co was utilized to reduce the toxicity of bacterial endotoxin. Bertok and Szeberenyi 47 describe the use of a 60Co-irradiated endotoxin preparation, TOLERIN that significantly decreased the endotoxin's lethal and hypotensive effects in a dose-related manner. Endotoxin's ability to activate the complement system was also affected, and immunoadjuvant properties and ability to stimulate nonspecific resistance were retained....

Endotoxin Relevance and Control Overview

Eli Lilly amp Company, Indianapolis, Indiana, U.S.A. The importance of endotoxin contamination control in parenteral manufacturing becomes apparent when confronted with four aspects of its existence. The first is its ubiquity in nature, the second is the potent toxicity it displays relative to other pyrogens, the third is its stability and the fourth is the relative likelihood of its occurrence in parenteral solutions. NOMENCLATURE CLASSIFICATION OF PYROGENS AND ENDOTOXIN The first order of...

Historical Development And Regulation Of Parenteral Dosage Forms

The manner of origin of most dosage forms is largely unknown. Early man may have fashioned primitive injections modeled after venomous snakes or insect bites and stings natural puncture injections . East Asians were inoculated for the prevention of small pox by pricking with needles dipped in pus centuries before the technique was used in Western cultures. Jenner used the same technique in 1796 using a cowpox sore 48 . Sir Christopher Wren was the first to inject a drug in 1657, a process later...

Overview Of Prominent Lal Tests

According to a Parenteral Drug Association PDA survey Current Practices in Endotoxin and Pyrogen Testing in Biotechnology 55 , as of 1990 the gel-clot assay was the most used assay 77 with the remainder, the kinetic chromogenic and turbidimetric LAL assays, representing less than one-third of users responding. Most of respondents 71 indicated for the continued use of the rabbit pyrogen test for at least some final product testing however, was very seldom indicated for any other sample tests....

Regulatory Provisions that Permit Initiation of End Product Testing with LAL

The regulatory provisions that a firm must meet before using the LAL test as an end product test are not the same for all categories of products because of the different applicable statutory provisions and regulations. These provisions are as follows Human Drugs subject to New Drug Applications NDAs or Abbreviated New Drug Applications ANDAs . Antibiotic Drug Applications, and animal drugs subject to New Animal Drug Applications NADAs , and Abbreviated New Animal Drug Application. For these...

Pyrogen Test Development

In the mid-1920s, Seibert 1 completed a series of classic studies, which proved conclusively that injection fevers associated with intravenous therapy resulted from heat-stable, filterable bacterial products that are commonly referred to as pyrogens. To ascertain the presence or absence of febrile responses caused by her test solutions, Seibert selected the rabbit as her animal test model, a choice that was later proven to be fortuitous 2 . Since that time, many other species have been shown to...

Threshold Pyrogenic Dose

Limulus Amebocytes

Closely tied to the concept of a standard endotoxin is the idea of a TPD for such a standard endotoxin. The establishment of a defined, specific endotoxic level has allowed the concept to be established that a certain amount of endotoxin is allowable and a certain amount of endotoxin should not be delivered into the bloodstream or cerebrospinal fluid. The advent of LAL allowed the quantitation of endotoxin as a contaminant. In turn, quantitation has allowed for the creation of specific and...

Test Animal

The USP lists several requirements for test animals. Pyrogen test rabbits must be both healthy and mature. The rabbits most widely used in pyrogen testing today are albino strains, especially New Zealand Whites, which are most commonly used in the United States. They are easily handled and trained for the pyrogen test procedure. Their ears are slightly shorter than those of Belgian Whites which are more commonly used in Europe and Canada . Upon request, animal vendors will select long-eared...

Medical Device Standards

Prior to the current 1987 FDA Guideline on Validation 45 , the endotoxin standards used for drugs and devices were different. The designation of different endo-toxins as standards resulted from administrative determinations made by the two different FDA bureaus involved. The choice of the E. coli 055 B5 preparation by the Bureau of Medical Devices was the result of a cooperative effort between the bureau and the members of the medical device industry for the express purpose of developing...

Fever Therapy And Injection Fever

Given the new-found tools of inducing and relieving fever in a controlled manner, physicians began to experiment with provoking the fever response as a form of therapy for given indications in which alternative remedies were ineffective. Rosenblum in Odessa 1876 used artificial fever for psychiatric disorders 4 . Julius Wagner von Jauregg, a Vienna neuropathologist, received the 1927 Nobel prize in medicine for his systematic studies of fever as a therapy for paralytic patients 4 . Fever...

Contamination Control Philosophy In Parenteral Manufacturing

Endotoxin is a concern for people only when it comes into contact with the circulatory system. The two relevant mechanisms for such contact involve infection and medically invasive techniques including injection or infusion of parenteral solutions. A notable exception to limiting the concern for endotoxin to blood contact is the effect that minute, almost undetectable, quantities of endotoxin may have upon cell cultures in pharmaceutical manufacturing. Modern methods of manufacturing...

Li Et Al. Journal Of Biotechnology 1998 P. 5478-5483 Vol. 180

Structures of gram-negative cell walls and their derived membrane vesicles. J Bacteriol 1999 181 16 4725-4733. 2. Raetz C et al. Gram negative endotoxin an extraordinary lipid with profound effects on eukaryotic signal transduction. FASEB 1991 5 12 2652-2660. 3. Raetz C. Biochemistry of endotoxins. Annu Rev Biochem 1990 59 129-170. 4. Galanos C, Luderitz O. Lipopolysaccharide properties of an amphipathic molecule. In Rietschel ET, ed. Handbook of Endotoxin, Chemistry of...

Limulus Scorpion And Spiderlikeness

There is much historical and recent evidence of the relatedness of Limulus to spiders and scorpions arachnids as fellow Chelicerates. Lavrov et al. 32 describe the early efforts to categorize Limulus Limulus polyphemus is one of the extant species of Xiphosura horseshoe crabs , one of the two extant major lineages of chelicerates the other lineage, Arachnida . Originally thought to be crustaceans hence the common name , xiphosuransf were recognized as aquatic chelicerates late in the 19th...