05. Endotoxin

Evidence against a bacterial endotoxin masking effect in biologic drug products by limulus amebocyte lysate detection. - more The use of endotoxin as an analyte in biopharmaceutical product holdtime studies - more Normal temperature variation in New Zealand white rabbits during restraint for preliminary pyrogen test. - more Bacterial endotoxin requirements for dry powder inhalants and their excipients: are they critical quality attributes? - more Bacterial Endotoxin Testing: New Method for Demasking Endotoxin in Biopharmaceuticals - more Pyrogens, Endotoxin and the LAL test: An Introduction in Relation to Pharmaceutical Processing - more Bacterial Endotoxin in Human Disease - more LER Frequently Asked Questions - more ! NEW ! Endotoxin Limits For Parenteral Drug Products - more ! NEW ! Factors Affecting Reduction of Reference Endotoxin Standard Activity Caused by Chelating Agent/Detergent Matrices: Kinetic Analysis of Low Endotoxin Recovery - more ! NEW ! Pyrogens, Endotoxin and the LAL test: An Introduction in Relation to Pharmaceutical Processing - more ! NEW ! Development of Methods for Recovering Endotoxins from Surfaces and from Air in Production Environment of Injectable Drugs - more ! NEW ! Application of Recombinant Factor C Reagent for the Detection of Bacterial Endotoxins in Pharmaceutical Products - more 1. Evidence against a bacterial endotoxin masking effect in biologic drug products by limulus amebocyte lysate detection. Bolden JS, Claerbout ME, Miner MK, Murphy MA, Smith KR, Warburton RE PDA J Pharm Sci Technol. Abstract The inability to detect endotoxin using compendia methods is a potential safety concern for patients due to the lack of endotoxin removal capabilities at the fill-finish stage in typical aseptic biologic drug product manufacturing. We have successfully demonstrated endotoxin challenge study recovery methodology using mammalian cell-produced biologic drug products and drug substances in citrate, histidine, phosphate, and sodium acetate buffer formulations containing polysorbate, challenged with an endotoxin analyte, for up to 6 months of storage. Successful recovery was similarly demonstrated for a preserved, peptide-containing drug product formulation. To isolate a potential masking-or low-endotoxin recovery-source, additional studies were performed to evaluate factors including product manufacturing contact surfaces, drug product matrix with and without polysorbate, individual matrix components, protein concentration, reagent suppliers, an orthogonal test method, and storage conditions. In all cases, acceptable recoveries were observed. Bacterial endotoxin is known to be chemically stable at physiological conditions. Purified endotoxin in aqueous conditions is likely to self-aggregate or bind to surfaces. Neither the nature of, nor the storage conditions of, the studied formulation matrices were shown experimentally to render the challenge endotoxin biologically inactive. The results highlight the importance of appropriate study design in assessing the recovery of endotoxins. LAY ABSTRACT: Bacterial endotoxin is a Gram-negative bacterial cell wall component that is harmful to humans at threshold concentrations, and it is not expected to be in aseptically-produced pharmaceutical medicines. It has been suggested that endotoxin cannot be detected over time in certain biopharmaceutical drug product formulations containing citrate, phosphate, and polysorbate components via an unknown masking mechanism. We have generated and present data here that indicate that endotoxin can be recovered in a variety of matrices, and under various experimental conditions.

2. The use of endotoxin as an analyte in biopharmaceutical product holdtime studies J. Bolden, C. Platco, J. Dubczak, K.Z. McCullough https://www.researchgate.net/publication/282939292_ Abstract Low lipopolysaccharide recovery (LLR) has been reported in biopharmaceutical formulation matrices containing divalentcationchelating buffers and polysorbate. This poor recovery of biological activity as measured by the Bacterial Endotoxins Test (BET) led to questions regarding the utility of the Bacterial Endotoxins Test.85. as a robust predictor of pyrogenicity and patient safety. Our data demonstrate that the biological activity of laboratoryprepared endotoxin was fully recovered by Limulus amebocyte lysate (LAL) assays and was pyrogenic in rabbits when spiked into undiluted matrices containing citrate and polysorbate. In sharp contrast, the activities of spikes of bacterial lipopolysaccharide (LPS) into the same poorrecovery conditions were rapidly lost to detection by LAL reagent and by rabbit pyrogen studies. Apparently, endotoxins, which contain LPS and other cellwall components, have a conformation that allows recovery of activity to be resistant to LLR conditions and yet recoverable by LAL and pyrogen tests, demonstrating that natural endotoxins are a clinically relevant predictor of endotoxin activity, pyrogenicity, and thus, patient safety. These data plus the historical perspective on drugs formulated in chelating buffers and polysorbate support the contention that holdtime studies for parenteral drugs are not necessary because, in the unlikely case of endotoxin contamination, the BET will detect naturally occurring endotoxin through appropriate dilution and endproduct testing as required by the compendial USP.NF. The data presented herein demonstrate that endotoxins, defined as cellwall fragments of Gramnegative bacteria (GNB), are preferable to the purified LPS standards when used as analytes for endotoxin holdtime studies in undiluted biologics, should such studies be indicated.

3. Normal temperature variation in New Zealand white rabbits during restraint for preliminary pyrogen test. Caldeira C, Freitas JC, Gimenes I, Silva SA, Cabello P, Presgrave OA. http://www.ncbi.nlm.nih.gov/pubmed/25655248 Abstract The in vivo pyrogen test is the main toxicological assay used in the quality control of injectable products, especially immunobiologicals. Pharmacopoeias state that, before the main test, a preliminary test must be conducted on all animals, and must follow the same conditions as the main test. The aim of this study was to determine the normal temperature variation in New Zealand white rabbits during restraint and propose a limit value for considering an animal as suitable for testing. Results of the temperature variation in 4,689 rabbits during preliminary tests were obtained from the routine database of the Pharmacology and Toxicology Department of the National Institute of Quality Control in Health (INCQS/FIOCRUZ), Brazil. From these preliminary tests, 3,364 rabbits were considered suitable for testing according to the Brazilian Pharmacopoeia criteria (temperature variation < 0.5 °C). Results showed that about 92 per cent of the rabbits presented a normal individual temperature variation equal to or below 0.30 °C. Animals presenting a temperature variation close to the fever temperature must not be included in the main test, since they can be stressed or sick. Consequently, the temperature variation of 0.30 °C could be adopted by pharmacopoeias as a limit temperature to be considered in the preliminary test to determine which animals can be used in the main rabbit pyrogen test. Animals can be pre-tested until presenting this safe variation, in order to ensure they are healthy and minimise interference in the result.

4. Bacterial endotoxin requirements for dry powder inhalants and their excipients: are they critical quality attributes? Cundell AM PDA J Pharm Sci Technol. Abstract This review article considers whether bacterial endotoxin levels are a critical quality attribute for dry powder inhalants and their major excipient Lactose, NF. Based on international no-effect levels for workplace endotoxin exposure, the technical literature, typical indoor and outdoor airborne endotoxin levels, and manufacturing capabilities for Lactose, NF and dry powder inhalation products, a strong case is presented that bacterial endotoxin is not a critical quality attribute and endotoxin specifications are not required for inhalation products and their excipients.

5. Bacterial Endotoxin Testing: New Method for Demasking Endotoxin in Biopharmaceuticals Hyglos http://www.hyglos.de/fileadmin/media/...pdf Following the initial observations by Chen et al., leading manufacturers of biopharmaceuticals have determined that endotoxin (LPS) can be "masked" in biopharmaceutical formulations typically containing phosphate, polysorbate, and sugars (1). Being in a masked state, endotoxin is undetectable by the commonly used factor C detection methods such as the Limulus amebocyte lysate test (LAL). This phenomenon has also been denominated low endotoxin recovery (LER). As previously shown by Reich et al. (2), endotoxin masking is time dependent, meaning that a defined amount of endotoxin spiked to a sample at time point zero often cannot be recovered (detected) with commonly used methods after hours or days. This is sometimes the case even within minutes of positive product controls (PPCs) initially have indicated a positive test result. Such lack of test reliability can ultimately affect patient safety.

6. Pyrogens, Endotoxin and the LAL test: An Introduction in Relation to Pharmaceutical Processing Dr. Tim Sandle http://www.academia.edu/1802210/Pyrogens_Endotoxin_and_the_LAL_Test Pharmaceutical products which are injected into the human body are tested for pyrogenic substances. The most common, and arguably most important test, is for bacterial endotoxin. The pathological effects of endotoxin, when injected, are a rapid increase in core body temperature followed by extremely rapid and severe shock, often followed by death before the cause is even diagnosed. However, there needs to be large quantities of endotoxin within the human body for this to happen, and the endotoxin needs to be injected into the blood stream. Bacterial endotoxin is the lipopolysaccharide (LPS) component of the cell wall of Gram-negative bacteria. It is pyrogenic, and it is a risk to patients who are administered intravenous and intramuscular preparations. The purpose of the LAL test is to detect endotoxin (Guy, 2003).

7. Bacterial Endotoxin in Human Disease Michael H. Silverman, MD, FACP Marc J. Ostro, PhD http://www.biostrategics.com/ How advances in understanding the role of Gram-negative bacteria and endotoxin in infectious diseases and complications may improve the development of diagnostic and treatment options. Gram-negative bacteria and their endotoxins may be a causal or complicating factor in many serious diseases. The syndromes most commonly connected with bacterial endotoxins are sepsis and septic shock, which are systemic complications of many diseases. Systemic infections (septicemias) caused by invasive Gram-negative bacteria are a well known source of endotoxin exposure. Less well-recognized, although perhaps of greater importance, are infectious complications (such as those following trauma or surgery) that may be initiated by exposure to endogenous Gram-negative intestinal bacteria. Whatever the source, exposure to endotoxin induces a systemic inflammatory response (also called the inflammatory or sepsis cascade) that involves many interconnected cellular and plasma mediators. The inflammatory response manifests in such clinical signs as fever, increased heart and respiratory rates, and other systemic symptoms. These may be self-limiting, or the cascade can proceed to shock, organ failure and death. Currently available treatment efforts are limited to antibiotics and, in serious cases, supportive intensive medical care. Advancements in efforts to prevent or treat the systemic inflammatory response to endotoxin have been hampered by several factors. The lack of a consistent and rapid diagnostic for endotoxin exposure is one. The choice of a clinically-relevant therapeutic target is another. Halting the ongoing endotoxin stimulation of the inflammatory response at the source would seem to be more effective than inhibiting any individual component. Nevertheless, most investigational therapies have targeted individual mediators within the inflammatory cascade. A third factor is the conceptual lock that sepsis has had on pharmaceutical development. In the past eight years, at least 13 different products have failed to show the required survival benefit in sepsis trials. Surprisingly, these failures have, with rare exceptions, not stimulated radical change in pharmaceutical companies' or regulators' approach to clinical trial design or disease targets. This paper challenges the current understanding in this field. In the old model, sepsis was viewed as a unique clinical syndrome, difficult to treat, but the obvious target for therapy. The new model (see Figure 1) incorporates sepsis, but as a late-stage syndrome on a continuum of endotoxin-related diseases. The new map encompasses the entire inflammatory cascade and its clinical manifestations. Understanding such a new paradigm may open the way to improved diagnostic and therapeutic approaches that can identify at-risk patients and treat them at the appropriate stage in the inflammatory process, within the context of each patient's underlying disease.

8. LER Frequently Asked Questions Karen Zink McCullough http://www.americanpharmaceuticalreview.com/Featured-Articles/177349-LER-Frequently-Asked-Questions/ Since the initial reports of Low Endotoxin Recovery (LER) in 2013 drug manufacturers and regulators have voiced concern that there may be conditions under which the Bacterial Endotoxins Test (BET) could under-report endotoxin activity in biological drug products. Clearly, this potential risk to patient safety merits further investigation and discussion. What is LER? While conducting a hold time study on a monoclonal antibody formulated in a chelating buffer and polysorbate ("LER matrix"), Joseph Chen and Anders Vinther from Genentech/Roche reported a temperature dependent, irreversible, almost instantaneous and reproducible decline in the Limulus Amebocyte Lysate (LAL) reactivity to Control Standard Endotoxin (CSE) after its addition to the test sample (Chen and Vinther, 2013.) They coined the phrase, "Low Endotoxin Recovery" or LER to describe this phenomenon. LER has been replicated by a number of researchers including Bolden, et al (2014), Platco (2014), Burgenson (2014) and Dubczak (2014). No one disputes that low recovery of CSE activity in this type of formulation is a real phenomenon. Interestingly, these same researchers all also report that there is a lesser decline or in some cases, no decline in recovery when endotoxin is used instead of CSE as the analyte "spike" in the hold time experiments using LAL reagent for detection of activity. A summary of these data may be found in Bolden, et al (2015).

9. Endotoxin Limits For Parenteral Drug Products Dawson M White Paper vol.1 no.2 http://www.acciusa.com/pdfs/whitepapers/EndotoxinLimits_SeanJH-PR17-012.pdf Abstract: When performing endotoxin testing to determine the acceptability of a product, it is axiomatic that there must be an endotoxin limit above which the product is rejected. Prior to publication in 2012 of the FDA "Guidance of Industry: Pyrogen and Endotoxin Testing: Questions and Answers" 1, the endotoxin limits used were often those given in the monographs of the United State Pharmacopeia (USP). However, the 2012 Questions and Answers Guidance makes clear that the limits given in pharmacopeial monographs may not account for current product strengths or dosage regimes. It states that endotoxin limits should also be checked using the calculations recommended USP or AAMI standards. In addition, the February 2015 European Pharmacopoeia policy on bacterial endotoxins2 states that most new monographs for parenteral products will not include a requirement for the bacterial endotoxins test (BET) or an endotoxin specification. According to the policy, manufacturers will be responsible for determining whether the endotoxin test is necessary and, if it is, what the endotoxin limit should be. For these reasons, it is more important than ever before to understand how to calculate the endotoxin limit for a particular product.

10. Conservation status of the americanhorseshoe crab, (limulus polyphemus): A regional assessment Smith DR, Brockmann HJ, Beekey MA, King TL, Millard MJ, Zaldívar-rae J Reviews in Fish Biology and Fisheries, 2017, 27(1), 135-175. https://link.springer.com/article/10.1007/s11160-016-9461-y Abstract: Horseshoe crabs have persisted for more than 200 million years, and fossil forms date to 450 million years ago. The American horseshoe crab (Limulus polyphemus), one of four extant horseshoe crab species, is found along the Atlantic coastline of North America ranging from Alabama to Maine, USA with another distinct population on the coasts of Campeche, Yucatán and Quintana Roo in the Yucatán Peninsula, Mexico. Although the American horseshoe crab tolerates broad environmental conditions, exploitation and habitat loss threaten the species. We assessed the conservation status of the American horseshoe crab by comprehensively reviewing available scientific information on its range, life history, genetic structure, population trends and analyses,major threats, and conservation. We structured the status assessment by six genetically-informed regions and accounted for sub-regional differences in environmental conditions, threats, and management. The transnational regions are Gulf of Maine (USA), Mid-Atlantic (USA), Southeast (USA), Florida Atlantic (USA), Northeast Gulf of Mexico (USA), and Yucatán Peninsula (Mexico). Our conclusion is that the American horseshoe crab species is vulnerable to local extirpation and that the degree and extent of risk vary among and within the regions. The risk is elevated in the Gulf of Maine region due to limited and fragmented habitat. The populations of horseshoe crabs in the Mid-Atlantic region are stable in the Delaware Bay area, and regulatory controls are in place, but the risk is elevated in the New England area as evidenced by continuing declines understood to be caused by over-harvest. The populations of horseshoe crabs in the Southeast region are stable or increasing. The populations of horseshoe crabs in the Florida Atlantic region show mixed trends among areas, and continuing population reductions at the embayment level have poorly understood causes. Within the Northeast Gulf of Mexico, causes of population trends are poorly understood and currently there is no active management of horseshoe crabs. Horseshoe crabs within Mexico have conservation protection based on limited and fragmented habitat and geographic isolation from other regions, but elevated risk applies to the horseshoe crabs in the Yucatán Peninsula region until sufficient data can confirm population stability. Future species status throughout its range will depend on the effectiveness of conservation to mitigate habitat loss and manage for sustainable harvest among and within regions.

11. Factors Affecting Reduction of Reference Endotoxin Standard Activity Caused by Chelating Agent/Detergent Matrices: Kinetic Analysis of Low Endotoxin Recovery Tsuchiya M PDA J Pharm Sci and Tech 2017, 71 478-487 https://www.ncbi.nlm.nih.gov/pubmed/28928294 Abstract: Reduction of reference standard endotoxin activity was kinetically analyzed under low endotoxin recovery conditions and was considered as an apparent first-order reaction. Temperature, pH, and salt concentrations affected the rates of reduction of reference standard endotoxin activity. Temperature appeared to be the most important factor affecting low endotoxin recovery. Components of low endotoxin recovery matrices, such as citrate and polysorbate 20, showed similar low endotoxin recovery effect at concentrations commonly used. Phosphate concentrations showed negative correlation against the half-life of reference standard endotoxin activity in solutions containing phosphate buffer and polysorbate 20. Activation energy for low endotoxin recovery with naturally occurring endotoxin was higher than that with reference standard endotoxin, and this explained one of the reasons for naturally occurring endotoxin resistance to low endotoxin recovery. Lower temperature, lower pH, and a higher salt concentration are preferable to avoid low endotoxin recovery in a hold-time study. This study provides useful data for anticipation of the severity of the low endotoxin recovery effect and future hold-time studies in the biopharmaceutical field.

12. Development of Methods for Recovering Endotoxins from Surfaces and from Air in Production Environment of Injectable Drugs Fiammetta F, Carducci A, Davini G, et al. PDA J Pharm Sci and Tech 2017, 71 502-510 https://www.ncbi.nlm.nih.gov/pubmed/28819048 Abstract: The aim of this work is to develop and validate methods for quantifying endotoxins on surfaces and in the air of the manufacturing environment of injectable drugs, in order to use them to evaluate the quality of the process and the risk for the products processed therein. The method for recovering endotoxins from surfaces is a direct method that provides sampling surfaces by swabbing and extraction of endotoxins from the swabs with an appropriate diluent, while the method for airborne endotoxins provides an air-active sampling on a glass fiber filter and endotoxins extraction with an appropriate diluent.

13. Application of Recombinant Factor C Reagent for the Detection of Bacterial Endotoxins in Pharmaceutical Products Bolden J, Smith K PDA J Pharm Sci and Tech 2017, 71 405-412 https://www.ncbi.nlm.nih.gov/pubmed/28733334 Abstract: Recombinant Factor C (rFC) is non-animal-derived reagent used to detect bacterial endotoxins in pharmaceutical products. Despite the fact that the reagent was first commercially available nearly 15 years ago, the broad use of rFC in pharmaceutical industry has long been lagging, presumably due to historical single-source supplier concerns and the lack of inclusion in worldwide pharmacopeias. Commercial rFC reagents are now available from multiple manufacturers, thus single sourcing is no longer an issue. We report here the successful validation of several pharmaceutical products by an end-point florescence-based endotoxin method using the rFC reagent. The method is equivalent or superior to the compendia bacterial endotoxins test method. Based on the comparability data and extenuating circumstances, the incorporation of the end point fluorescence technique and rFC reagent in global compendia bacterial endotoxins test chapters is desired and warranted.