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Send Orders for Reprints to [email protected] Applied Clinical Research, Clinical Trials & Regulatory Affairs, 2017, 4, 4-15

REVIEW ARTICLE ISSN: 2213-476X eISSN: 2213-4778

Microbial and Non-microbial Pyrogens in Healthcare Products: Risks, Quality Control and Regulatory Aspects

Nasib Singh1,*, Tanuja Mishra2, Karan Singh3 and Joginder Singh4 1

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Department of Microbiology, Biotechnology2, and Chemistry3, Eternal University, Baru Sahib, H.P., India; Department of Microbiology, Lovely Professional University, Phagwara, Punjab, India

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Results: Analysis of the published scientific literature indicates that endotoxins from Gram-negative bacteria are the most common pyrogenic contaminants in various biopharmaceutical drugs and healthcare products which are difficult to remove due to heat stable nature. Endotoxins and other pyrogens trigger adverse reactions in human body ranging from mild influenza-like symptoms and fever to organ failure, septic shock and even death. Considering their significant health risks, highly sensitive and accurate detection of pyrogens, particular endotoxin, and their removal to the satisfaction of regulatory requirements is a key aspect in the development of biopharmaceutical and healthcare products. Pyrogen testing is based on either a 100 year-old Rabbit Pyrogen Test (RPT), the most widely used Limulus Amoebocyte Lysate (LAL) test or the broad spectrum and biologically relevant Monocyte Activation Test (MAT).

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DOI: 10.2174/2213476X03666160530151 854

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Received: April 28, 2016 Revised: May 24, 2016 Accepted: May 26, 2016

Methods: We searched the scientific databases for peer-reviewed research articles using keywords pyrogen, endotoxin, lipopolysachharide, lipoteichoic acid, depyrogenation, endotoxin detection etc.

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ARTICLE HISTORY

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Abstract: Background: Pyrogens are fever causing microbial and non-microbial constituents of variable chemical composition. Their presence in dialysis fluids, parenteral drugs, biopharmaceuticals, cosmetics and on solid medical devices is considered a major health concern worldwide. The main aim of this review is to summarize the scientific literature accumulated during the last few decades concerning health risks of pyrogens and regulatory requirements for quality control in parenterals and healthcare devices.

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Conclusion: Analyses of the available literature indicates significant progress towards understanding the nature, mechanism of action and health risks of pyrogens. However, there is still a need to develop universally acceptable, sensitive and integrated detection assays, to emphasize the minimization of animals use and to strengthen the regulatory framework in biopharmaceutical and healthcare production facilities.

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Keywords: Fever, endotoxin, lipoteichoic acid, rabbit pyrogen test, limulus amoebocyte lysate test, monocyte activation test, depyrogenation. 1. INTRODUCTION

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The term pyrogen (Greek pyros = fire) was coined by Burdon-Sanderson in 1875 to designate the substances which cause fever when injected into the body [1]. Florance Seibert in 1923 repeatedly linked the source of these hyperthermizing substances to dead bacteria and bacterial metabolites [2]. His group also made a significant contribution by isolating a Gram-negative bacterium from distillated water named Pyrogenic bacterium. The report published by Eli Menkin in *Address correspondence to this author at the Department of Microbiology, Akal College of Basic Sciences, Eternal University, Baru Sahib- 173101, Sirmour, India; Tel: +91-7307957476; E-mail: [email protected]

2213-4778/17 $58.00+.00

1943 that certain pus derived substances, called ‘pyrexin’ can induce fever is considered among earliest reports about endogenous or granulocytic pyrogens [3]. Microbial components or chemical compounds that induce an inflammatory response leading to rise in the body temperature in humans or test animals when applied in very small quantities via parenteral route are called pyrogens [4, 5]. Occurrence of sporadic fevers of unknown etiology in patients after injection of sterile solutions kept the microbiologist and pharmacist baffled for several decades. These health complications, termed as non- infections fever, injection fever, saline fever or distilled water fever, are now known to occur due to the entry of pyrogens into the body through parenteral route via

© 2017 Bentham Science Publishers

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Pyrogens in Biopharmaceutical and Healthcare Products

Fig. (1). The sources and types of pyrogens commonly encountered in pharmaceutical and healthcare products.

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Persistent bacteraemia due to Gram-negative bacteria releases endotoxin in the body and may be fatal [1, 3, 6, 16]. Human body not only encounters fever-causing substances from exogenous sources but there are conclusive evidences of existence of endogenous pyrogens which are produced within the body [3, 6, 17]. The role of IL-1, TNF- and IL-6 in induction of fever is now well established [1, 3, 18, 19]. Healthcare and biopharmaceutical products that require strict pyrogenic testing as mandated in US, EU and other Pharmacopoeia are parenterals (water for injections, dialysis fluids, injectables), blood preparation, medical devices, recombinant drugs (insulin, interferons, interleukins), pharmaceutics and foods/ beverages. Further, mandatory endotoxin testing is also applicable to defence, textile industry, transportation/warehousing utilities, agriculture and environmental industries [20, 21]. Estimated valuation of pyrogen test market was $462.38 million in 2014 which is expected to attain $800 million mark by year 2019 with a compound annual growth rate of about 12% [22].

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Pyrogens are either the products of microbial origin such as endotoxin (lipopolysaccharide, LPS), exotoxins, peptidoglycan and lipoteichoic acid or non-microbial substances such as endogenous pyrogens and non-living substances (Fig. 1). Their main sources are Gram-negative and Grampositive bacteria, fungi, viruses and several non-microbial substances [2, 7, 8]. The most potent, widely known and well characterized pyrogens are endotoxins which are derived from dead or living Gram-negative bacteria [9]. Another major source of pyrogens is Gram-positive bacteria which contain lipoteichoic acid (LTA). Other pyrogenic substances are peptidoglycan, exotoxins, fungal products (mannans, glucans, zymosan), viruses, parasites (phosphoinpsitol) and small particles of packaging materials [2, 10-12]. Endotoxins are the products of Gram-negative bacteria majority of which belongs to family Enterobacteriaceae. These bacteria include E. coli O113:H10 (WHO standard), E. coli O55, E. coli O127, Salmonella, Pseudomonas aeruginosa, Klebsiella pneumoniae, Shigella flexneri, Hafnia alvei etc. [2]. The major sources of non-endotoxin microbial pyrogens (e.g. LTA, peptidoglycan) are Staphylococcus, Streptococcus, Micrococcus, Listeria monocytogenes, Campylobacter etc. [13]. Apart from these, role of endogenous pyrogens and non-microbial exogenous pyrogens is well characterised in inflammatory reactions and fever development [1-3]. Pyrogens are not destroyed by usual sterilization methods and are encountered in sterile injectables, dialysis fluids, parenterals, recombinant biopharmaceuticals and invasive medical devices (catheter or infusion device) and production equipments/containers [1]. Pyrogens are usually heat stable and can withstand pH changes. Conventional sterilization and

decontamination methods such as membrane filtration, autoclaving or adsorption techniques fail to remove pyrogens from water, parenteral solutions and other healthcare products [13-15].

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intravenous fluids, dialysis fluids and water for injection [3, 6].

2. EXOGENOUS PYROGENS OF MICROBIAL ORIGIN 2.1. Endotoxin or Lipopolysaccharides (LPS) Endotoxin (Greek endo=within) was first defined by Richard Pfeiffer in 1992 as a heat-stable toxic substance that was released upon disruption of microbial envelopes (reviewed in 9, 14, 23]. LPS is an integral component of the outer membrane of most Gram-negative bacteria [9, 24].

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NH3 O O P O O O P O O

O

H3C(H2C)10

H3C(H2C)10

C H2

C O CH C C H2 O

NH

O

H C C C OH H2 O

H C C C H OH 2 O

KDO

Hep

KDO

Glc

Hep

Gal

Gal

Glc

NGa

NGc

NGc

NGa

Gal

NGa

NGa

Gal

Repeating unit

NGc

n = 4 - 40

O HO

N H

Hep

Gal

O KDO

O

O P O OH

O O P O O

CH2 H3C(H2C)10

O

Gal

O

NH3

Inner Core

Outer Core

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Lipid A

O

O O P O OH

Lipid

O-Antigen

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O P O CH C C O O H2 O

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H3C(H2C)10

H3C(H2C)10

OH

C O

H3C(H2C)12

Polysaccharide

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of amino sugars such as N-acetyl-d-glucosamine, dglucosamine, N-acetyl-d-galactosamine and d-galactosamine exists [28, 29]. It has a repeating oligosaccharide unit of 2 to 6 monosaccharides mainly glucose, galactose, mannose, rhamnose and fucose which contribute to its structural heterogeneity [29, 31]. Control Standard Endotoxin recommended by WHO and reference standard endotoxin approved in USP and EP is derived from E. coli O113:H10. Endotoxin Unit (EU) reflects the biological activity of an endotoxin. For example, 100 pg of the standard endotoxin EC-5 and 120 pg of endotoxin from E. coli O111:B4 have activity of 1 EU [32].

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These are exceptionally stable to heat and pH variations. LPS is non-essential for the survival of bacteria but contribute significantly towards their virulence properties as it is exposed on the cell surface and interact with receptors leading to a signaling cascade [23, 25]. There are about 2 million lipopolysaccharide molecules on a single E. coli cell. LPS is released during cell division or cell death [9, 26]. Chemically, endotoxins are complex lipopolysaccharides with molecular weight ranges between 2000 and 20,000 Da. However, as LPS tends to form large aggregates, molecules of size as large as 1-4 million Da are commonly detected [15, 27-30].

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Fig. (2). General structure of the endotoxin (lipopolysaccharides) of Gram-negative bacteria. Lipid A domain is highly conserved region whereas O-antigen is responsible for species-specific variability. Lipopolysaccharide complex is anchored in the outer membrane of cell wall by its lipid A moiety. Abbreviations, KDO: 3-deoxy-d-manno-oct-2-ulosonic acid, Hep: Heptulose, Glc: Glucose, Gal: Galactose, NGa: Galactosamine and NGc: Glucosamine.

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LPS comprises of three components: lipid A (a hydrophobic moiety), a hydrophilic core oligosaccharide and a repeating hydrophilic polysaccharide called O-antigen (Fig. 2). The biological activity of LPS is due to its lipid A component [9, 28, 29]. The latter is a hydrophobic phosphoglycolipid membrane anchor comprising of (16)-linked dimers of D-gluco-configured pyranosidic hexosamine residues [29]. This disaccharide unit contains phosphoryl groups and (R)-3-hydroxy fatty acids attached through ester and amide linkages. The variation in hexosamine, acyl chain length and number of the acyl groups impart specificity to lipid A [28, 29]. Core polysaccharide’s inner core contains 3 deoxy--D-manno-octulosonic acid (KDO) to which attached are heptulose (ketoheptose) monosaccharides [9, 26, 28, 29]. It connects lipid A with the carbohydrate part by acid-labile -ketosidic linkage. The inner core glycan residues are typically phosphorylated. The outer core contains hexoses such as glucose, galactose, and N-acetylglucosamine and shows higher structural diversity [9, 29]. The O-antigen is most heterogenic part of LPS and contains pyranose form

2.2. Lipoteichoic Acid (LTA) Constituents of the Gram-positive cell wall, which lacks endotoxin, are known to act as inducers of cytokine release in human monocyte/macrophages. LTA is a complex surface-associated pyrogen found in the cell wall of Grampositive bacteria [33-36]. It is a glycolipid consisting of a hydrophilic head on outer surface and a hydrophobic diacylglycerol part present in the membrane [35, 37, 38]. Chemically, LTA is a polyglycerol-phosphate polymer comprising of alternating units of glycerol or ribitol and phosphoric acid [34-36]. LTA of S. aureus shares structural similarity with Bacillus anthracis, group A and B streptococci, Enterococcus and Listeria monocytogenes [39, 40]. Its release from the cell is induced by -lactam antibiotics, lysozyme and leucocytic peptides. Just like endotoxin, LTA is a potent pyrogen and present serious health risks as evident by manifestations of inflammatory reactions, septic shock and organ failures in human body [37]. LTA is attached to a lipid in cell wall and

Pyrogens in Biopharmaceutical and Healthcare Products

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act as pathogen-associated molecular-patterns (PAMP) which is recognized by TLR2 leading to the activation of NF-kB pathway [35]. It stimulates release of inflammatory mediators such as IL-1, TNF-, IL-6 and IL-8 [36, 41].

also reported as pyrogenic substances [2, 54]. Heat killed suspension of cell wall-, LPS- and LTA-deficient Acholeplasma laidlawii act as a potent activator of macrophages [53].

2.3. Peptidoglycan (PG)

3. EXOGENOUS PYROGENS OF NON-MICROBIAL ORIGIN

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Non-microbial pyrogens, also called synthetic pyrogens, are often encountered in the healthcare and pharmaceutical industries, although these possess much lesser health threats as compared to highly potent endotoxins or LTA [2, 3]. As extensively reviewed by Mazzotti et al. [4] and Hasiwa et al. [2], synthetic bacterial lipoprotein (Pam3CSK4), synthetic TLR-agonists, synthetic immunoadjuvants, diesel exhaust, particulates (dust, asbestos, silica, turpentine, nanoparticles), media components, plastic materials, medical devices, serum albumins, urate crystals, plant lectins, drugs (bile salts, steroids, dapsone, bleomycin, colchicine) etc. may also induce inflammatory reactions (an in vivo indicator of pyrogenicity) in the human body [1, 2, 4]. Airborne pyrogens present in bioaerosols, cigarette smoke and metalworking fluids possess severe health risks such as asthma and chronic obstructive pulmonary disease [1, 55]. However, the minimum dose for pyrogenicity and mechanism of action are either not fully understood or the information available is not comprehensive enough to establish conclusive correlations.

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2.4. Short Bacterial DNA Fragments

2.6. Exotoxins

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Various structural components of yeasts and molds have been recognized as fever inducing substances [2, 4, 47]. Surface structres of fungal cell wall i.e. glycans, zymosan, curdlan, laminarin, lichenan etc. are known to activate immune system [48]. Zymosan can stimulate IL-1 release at 5 g/ml concentration [10]. Fungal spores from more than 44 different species showed pyrogenic activity when evaluated by human whole blood MAT [10]. Candida albicans, S. cervisiae, Alternaria alternata, Cladosporium cladosporoides, Penicillium crustosum, Aspergillus versicolor, Chlorophyllum molybdites, Pluteus cervinus, and Coprinus micaceus are known to induce the release of IL-1, an endogenous pyrogen [2, 10, 12].

Physiologists and molecular researchers were of the view that it is highly unlikely for exogenous pyrogens directly to act on thermoregulatory center of the brain leading to fever. Further research to identify the connecting links resulted in discovery of endogenous pyrogens, also called granulocytic or leukocytic pyrogens [3]. Endogenous pyrogens are usually not found in healthcare products as these are released only within the body by polymorphonuclear leukocytes in response to endotoxins and other exogenous pyrogens. Endogenous pyrogens are cytokines represented by interleukin1 (IL-1), TNF- (cachectin), IL-6 and IFN- (6, 16, 5659). IL-1 was the first cytokine to be conclusively associated with fever in 1970s by Phyllis Bodel and Elisha Akins [2, 60]. Recombinant cytokines were shown to induce fever at subnanomolar doses in experimental animals. IL-1 is the most powerful endogenous pyrogen followed by TNF- and IL-6 [61]. These cytokines stimulate the release of prostaglandin E2 (PGE2) in thermoregulatory centers of the hypothalamic region of the brain through highly regulated signaling cascades leading to rise in the body temperature and consequently the fever [59, 62, 63].

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2.5. Fungal Components

4. ENDOGENOUS PYROGENS

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Bacterial DNA (bDNA) consists of many unmethylated deoxycytosine-deoxyguanosine phosphate (CpG) motifs which are reportedly known to activate immune responses in host [2, 45] and thus act as pyrogen. Unmethylated CpG motifs more commonly exist in bDNA than in vertebrate DNA [46]. These are detected in water and fluids which may induce inflammation in the body [7, 45].

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PG, also called murein layer, is ubiquitously present in cell wall of most bacteria (except mycoplasma) with several folds higher amount in Gram-positive bacteria compared to Gram-negative bacteria [42, 43]. It protects the cell from bursting by forming a rigid mesh-like structure around the cell membrane. PG is a complex heteropolymer of Nacetylglucosamine and N-acetylmuramic acid residues cross linked via 1-4-glycosidic bonds [43, 44]. These long glycan chains are further cross-linked by short peptides to make long and continuous bagshaped macromolecular structure. Studies have found that the contamination of healthcare products especially dialysis fluids by PG results in pyrogenic effects as confirmed by the release of IL-1, TNF- and IL-6 in MAT assays [reviewed in 5]. However, PG is a weak pyrogenic compared to endotoxin.

Exotoxins secreted by Staphylococcus and Streptococcus are also pyrogenic in nature [49-51]. Streptococcal pyrogenic exotoxin B and mitogenic exotoxin Z are known to stimulate cytokines in human mononuclear leukocytes including IL-1 [52]. However considerably higher levels of these products are required to elicit an endotoxin-comparable pyrogenic response [2]. 2.7. Other Microbial Pyrogens Viral components (envelope proteins, DNA and RNA), phosphoinositol (parasites), lipoarabinomannans (mycobacteria), endotoxin associated proteins and bacterial porins are

5. PYROGENS: MECHANISM OF ACTION AND HEALTH EFFECTS Pyrogen-contaminated parenteral preparations, biopharmaceuticals and healthcare products can cause several adverse reactions such as rash, headache, myalgia, nausea, vomiting and hypotension, shock, disseminated coagulation, organ failure and even death [3, 23, 59, 64-66]. The low doses of pyrogen lead to mild influenza-like symptoms and lowering of blood pressure whereas high dose usually results in life-threatening systemic reactions. Our current under-

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pyrogens, requirement of large numbers of test animals and unsuitability for certain biochemical e.g. chemotherapeutics, immunosuppressive drugs, human blood components and human stem cells [1, 2, 76-78]. Use of RPT is limited to testing of parenteral drugs during the earlier development phase in combination with LAL test. Moreover, as the 4Rs- replacement, reduction, refinement and rehabilitation strategies are being adopted for scientific research and industrial research & development process; RPT is poised to be replaced with non-animal based alternate pyrogen testing assays [79]. 6.2. Limulus Amebocyte Lysate (LAL) Test

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Due to several conspicuous shortcomings of RPT, search for better pyrogen detection assays was a top pursuit of pharmaceutical and healthcare industries. This lookout was culminated in the form of an in vitro test that does not directly use living organisms but rather use their specific cell type without organism being harmed considerably. In 1885, Howell [80] and Loeb [81] observed that hemolymph of horseshoe crab coagulates in the presence of foreign substances, though identity of these substances remained elusive. About 50 years later, F. Bang was successful to recognize and correlate the coagulation of Limulus blood to the existence of bacterial endotoxin [82]. It was J. Cooper’s group who explored its practical utility towards endotoxin detection in pharmaceuticals [83]. This test is now known as Limulus Amebocyte Lysate (LAL) test. This in vitro pyrogen test employs lysates of amebocyte blood cells from Limulus polyphemus, the Atlantic horseshoe crab. These are used for collecting hemolymph by puncture, and thereafter returned back into the seawater but suffer 10-20% mortality rate [84, 85].

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6.1. Rabbit Pyrogen Test (RPT)

RPT was developed by E.C. Hort and W.J. Penfold in 1912 [73]. Following its refinement by F.B. Seibert in 1925, it has been accepted into European, US Pharmacopeia and other major Pharmacopoeias in subsequent years (Table 1). It was also the first US FDA approved method for detection of endotoxin [30]. RPT involves injecting rabbits intravenously with test samples and then determining their body temperature pattern by rectal measurements since it resembles that of humans [15, 30, 74, 75]. It is applicable to test the pyrogenicity of both endotoxin and non-endotoxin pyrogens in biological products though sensitivity towards nonendotoxin pyrogens in either low or false negative results are obtained [30, 76]. RPT has many drawbacks which include non-quantitative nature, lack of positive control, time consuming, expensive, false negative results with non-endotoxin

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LAL test principle involves degranulation and destruction of the amebocytes cells of horseshoe crab circulating in the hemolymph in the presence of endotoxin [30, 76]. It is also known as Bacterial Endotoxins Test (BET) and indirect animal test [76]. LAL tests are performed by gel-clot, turbidimetric and chromogenic techniques [30, 86]. LAL test is several times more sensitive than the RPT method and can detect endotoxin as low as 0.03 EU/ml compared to RPT test which has detection limit of 0.5 EU/ml [87, 88]. The final guidelines on LAL testing were issued by FDA in 1987 which were subsequently withdrawn in 2011 and as a result US pharmacopeia general chapter 85 bacterial endotoxins test is now considered as approved document for LAL testing. These documents are also included in other major Pharmacopoeias. The 2012 guidelines of FDA have also mentioned about recombinant horseshoe crab factor C assay. Gel clot method of BET based on F18-FDG decay is a rapid 60 min test approved by FDA [89]. Several commercial LPS detection LAL kits are now available viz. (i) Endpoint chromogenic LAL assay (Thermo Scientific, Sigma, GenScript, Charles River Laboratories), (ii) Kinetic turbidimetric LAL assay (Lonza BioScience), and (iii) Kinetic chromogenic LAL assay (Lonza BioScience) [30]. Although LAL test is limited to the detection of endotoxins, still it enjoyed wide acceptance globally and was recommended as a valid replacement for RPT partly due to better sensitivity over RPT and partly due to the fact that bacterial endotoxins are the main pyrogens of interest to pharmaceutical, healthcare

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Pyrogen testing is an indispensable safety measure for pharmaceuticals, biotherapeutics, recombinant therapeutic products, cosmetics, medical devices, blood preparations, stem cells therapy products, other healthcare products and for food and water security. It is a mandatory regulatory requirement prior to their release for clinical and health applications since pyrogen’s presence can result in life threatening adverse reactions viz. shock, coagulation of proteins, multi-organ failures and even death. The main methods recommended in different pharmacopeias and regulatory agencies for pyrogen detection are described below.

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6. METHODS FOR DETECTION OF PYROGENS

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standing of the mechanisms of fever mostly revolves around bacterial endotoxin. Specialized pattern recognition receptors (PRRs) are harboured by monocytes and macrophages which assist in recognition of pathogen-associated microbial patterns (PAMPs) including LPS, LTA and other microbial pyrogens [2, 64, 65, 67]. When LPS binds to LPS binding protein (LBP), CD14 and TLR4-MD2 complex, a cascade of intracellular reactions is initiated which culminate in the release of pro-inflammatory cytokines IL-1, TNF- and IL-6 [24, 25, 39, 59, 68-71]. The latter binds to the receptors on organum vasculosum of the laminae terminalis of brain and initiate the expression of the cyclooxygenase-2 (COX-2) which mediates the synthesis of a lipid prostaglandin (PGE2), a potent hyperthermic mediator of febrile response [6, 47]. The latter are the central mediators of the coordinated responses leading to fever through a change in the set-point of body temperature in hypothalamus [19]. The central role of COX-2 was established in the studies where COX-2 deficient mice failed to develop fever when administrated with pyrogenic cytokines [72]. Minute levels of LPS (0.1 EU/ml) are sufficient enough to upregulate the expression of inflammatory genes in human cells [24]. The first phase of LPS-induced fever starts within 30 minutes after exposure to the pyrogen. During the second and subsequent phases between 90 min and 12 h after LPS administration brain cells increase production (called upregulation) of enzymes involved in PGE2 synthesis [59]. Thus, fever starts about an hour before the PGE2-synthesizing enzymes COX-2 and microsomal PGE synthase-1 are upregulated in the brain suggesting that fever must be triggered by PGE2 produced peripherally outside the brain [59].

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Pyrogens in Biopharmaceutical and Healthcare Products

Table 1.

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Comparison of three major assays for endotoxin detection in healthcare and biopharmaceutical products.

Parameter

Year of development

Rabbit Pyrogen Test (RPT)

Limulus Amoebocyte Lysate (LAL) test or Bacterial Endotoxin Test (BET)

Monocyte Activation Test (MAT)

1912: Developed by Hort and Penfold,

1964: Developed by Levin and Bang,

1912: Incorporated in BP,

1995: Developed Wendel,

1925: Refinement by F.B. Seibert,

1971: Cooper used it for pharmaceuticals,

1942: Incorporated in USP

1987: FDA guidelines released,

by

Hartung,

and

2010: Incorporated in EP

Included in USP chapter 85 Rabbit

No

No

Animal products

Yes

Yes, Limulus polyphemus blood extract

No

Nature of test

Fail/Pass test

Quantitative

Quantitative

Type of cell used

No

Only lysate of amebocyte blood cells used

Whole blood, PBMC, Mono Mac 6

Test controls

No

Yes

Yes

Results variability

High

Low

Low

Test duration

Long (several days)

End points

Rise in temperature

Pyrogens which can be detected

Endotoxin, Lipoteichoic acid, Yeasts, Molds

Detection limits

0.5 EU/ml

0.03 EU/ml

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Endotoxin only

Three

Release of IL-1 and IL-6

Broad spectrum of pyrogens can be tested. e.g. Endotoxin, Lipoteichoic acid. Yeasts, Fungal spores, Viruses, parasites, solid medical devices, cell therapy preparations

10 pg/ml

Five i. Human whole blood/IL-1

ii. Turbidimetric

ii. Cryo human blood/IL-1

iii. Chromogenic

iii. Human whole blood/IL-6

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i. Gel-clot

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products and medical devices industries [76]. More than 90% of pyrogen detection is performed by LAL test [1]. 6.3. Monocyte Activation Test (MAT)

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Very short

Chromogenic LAL: 0.005 EU/mL

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Clot formation

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Variants of test available

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Animals used

Whereas RPT has inherent drawback of poor sensitivity, LAL test’s non-applicability to non-endotoxin pyrogens limits its wider applications in pyrogen testing of healthcare products especially the dialysis fluids. In the search for an in vitro non-animal based pyrogen detection assay, Hartung and Wendel [90, 91] developed a whole human blood based assay for pyrogen detection. It was introduced into the European Pharmacopoeia (EP chapter 2.6.30) in 2010 as the most suitable test for pyrogen testing [1, 92]. MAT is applicable to pyrogens originated from Gram-positive and Gram-negative bacteria, yeasts, viruses and parasites [93]. A study by Gimenes et al. [36] revealed higher sensitivity of MAT over RPT in detection of LTA in Brazil. Further, inclusion of both positive and negative controls makes MAT a highly sensitive and reliable assay for pyrogen detection [1, 36].

iv. Human PBMCs/IL-6 v. Mono Mac/IL-6

MAT is an in vitro quantitative test in which pyrogens activate the monocytes in human whole blood resulting in the release of IL-1, IL-6 and TNF- [2, 3, 47, 76, 94, 95]. Fresh human whole blood (heparinized) is diluted in pyrogen-free saline and incubated with the test samples. Monocytes produce pro-inflammatory cytokines in dose-dependent manner which are measured by ELISA [47]. It is highly sensitive as indicated by detection of as low as 10 pg/ml of endotoxin in a test sample [2]. Biological mechanism of MAT is quite similar to human fever reaction [77]. As living animals or animal products are not used, MAT attracted extensive applications in biopharmaceuticals and healthcare industries to detect the spectrum of pyrogenic substances of microbial or non-microbial origin [93]. The various advantages of MAT over other two pyrogen tests are extensively reviewed by Gimenes et al. [36] and Hartung [1]. Five different variants of MAT are validated and approved by regulatory bodies [European Centre for the Validation of Alternative Methods (ECVAM) and ECVAM Sci-

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(i) Human whole blood/IL-1 in vitro pyrogen test (ii) Cryopreserved human whole blood/IL-1 in vitro pyrogen test (iii) Human whole blood/IL-6 in vitro pyrogen test (iv) Human PBMCs/IL-6 in vitro pyrogen test (v) Human Mono Mac 6/IL-6 in vitro pyrogen test

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Most of the non-microbial exogenous pyrogens are not amenable to LAL testing but compatible with the whole blood MAT [1]. Cellular & gene therapy products and advanced-therapy medicinal products cannot be tested by either RPT or LAL. However, MAT is found to be suitable for the detection of pyrogens in cell therapy products. [reviewed in 1, 2]. Commercial MAT kits which were marketed earlier or currently available include Pyrocheck (DPC Biermann, Germany), Endosafe IPT (Charles-River, USA), PyroDetect (Biotest, Germany) and PyroDetect (Merck-Millipore, Germany). These kits fulfil the requirements of various Pharmacopoeias i.e. USP , EP 2.6.30 and relevant regulatory bodies. Disappointingly, MAT failed to gain wide acceptability in commercial pyrogen test market (