Method and detection kit for detecting pyrogen in a biological sample

Abstract

The present invention relates to a method and a detection kit for detecting a pyrogen in a biological sample, which comprises contacting immune cells, preferably the monocyte cell line THP-1 or THP-1 macrophages, with a sample that may contain a pyrogen, thereby causing the expression of immunomodulatory mediators, and after lysing the immune cells, extracting the nucleic acid of the immune cells and detecting the expression of the immunomodulatory mediators by PCR. At least one of three different cytokines / chemokines, preferably IL-1β, TNFα or CXCL8 / IL-8, is detected in parallel. The present invention can be used for detecting endotoxin and non-endotoxin pyrogen contamination in medical samples.

Description

Technical Field

[0001] The present invention generally presents an improved monocyte activation test based on nucleic acid amplification technology (NAT-MAT). The present invention will enable reliable and rapid detection of pyrogens in medical products by employing the techniques of polymerase chain reaction (PCR), quantitative real-time PCR (qPCR), and digital PCR (dPCR). The present invention includes a process of treating immune cells with the medical product to be tested, extracting nucleic acids, and detecting cytokines by PCR technology. The present invention also includes a detection kit developed for the detection of endotoxin and non-endotoxin pyrogen contamination in medical samples and which can be used for this detection. The present invention is described in the claims.

[0002] Background of the Invention An essential step in the manufacture of pharmaceuticals is the safety test. Parenteral application medical products and devices need to be tested for the presence of pyrogens, which are contaminants that cause fever. Generally, these are of biological origin, but they can also be of non-biological origin. Pyrogens can cause a systemic reaction when they enter the circulatory system of humans or other mammals. This systemic reaction is known as an inflammatory reaction or simply inflammation and is mediated by many different cells of the immune system. The immune system is an important defense mechanism that protects the body from severe infections by pathogens. The immune system fights pathogenic invaders or initiates the healing process in case of injury. The immune defense system mainly includes two mechanisms. One mechanism is the migration of immune cells, i.e., white blood cells such as B lymphocytes and T lymphocytes or monocytes / macrophages, through the blood and lymphatic systems to the site of infection. The other mechanism is the systemic increase in temperature that causes fever. Fever causes the overheating of the pathogen's environment, making this environment hostile to the pathogen. The invader itself is involved in the increase in temperature because its own compounds cause a fever reaction. As mentioned at first, these compounds are called pyrogens or pyrogenic compounds. Exogenous pyrogens and endogenous pyrogens are distinguished. Exogenous pyrogens are components of the entities of microorganisms such as bacteria, viruses, or fungi. Usually, these are found on the surface of the microorganisms, i.e., whether endotoxin lipopolysaccharides (LPS) are found in Gram-negative bacteria, lipoteichoic acids (LTA) are found on Gram-positive bacteria, or others are found in viruses or fungi. Exogenous pyrogens induce the release of endogenous pyrogens from infiltrating immune cells to the site of infection or injury. These are released by white blood cells, by malignant tumors, or by tissue trauma. Endogenous pyrogens are, for example, inflammatory mediators such as cytokines and chemokines, i.e., interleukin (IL)-1, IL-6, tumor necrosis factor (TNFα), or CXCL8 / IL-8.

[0003] Apart from its role as a pyrogen, the released inflammatory mediators attract other immune cells, migrate them to the site of infection, and release additional cytokines and chemokines. If there are too many inflammatory mediators, when the invader and, consequently, the immune response enter the circulatory system, it can potentially damage the body locally and especially systemically. Therefore, a sophisticated regulatory mechanism ensures that there are sufficient inflammatory cytokines and chemokines to fight the invader. Similarly, dysregulation of the immune response can lead to local or systemic hyperinflammation. Local and systemic hyperinflammation can lead to life-threatening septic shock. The factors contributing to the development of sepsis depend on the type and amount of exogenous or endogenous pyrogens, as well as the individual's susceptibility and predisposition to hyperinflammation. Exogenous pyrogens can be of non-biological origin, such as metal compounds in elastomers or rubber wear, which can potentially contaminate medical products.

[0004] To prevent the systemic inflammatory response from leading to hyperinflammation and systemic shock or sepsis, it is necessary to closely monitor the safety of parenterally administered pharmaceuticals, which are intravenous, intramuscular, or subcutaneous, for the presence of pyrogens. The European Pharmacopoeia describes two animal-based tests. These are the rabbit pyrogen test (RPT) and the Limulus amebocyte lysate (LAL), also known as the bacterial endotoxin test (BET). These are currently routinely used to test pharmaceutical parenteral products.

[0005] The rabbit pyrogen test is described in the European Pharmacopoeia (EP2.6.8) and the United States Pharmacopoeia (USP<151>). This is an in vivo test that examines the statistically significant elevation of body temperature in a number of rabbits. An aseptic solution of the pharmaceutical product to be tested is injected intravenously, and the mean body temperature of the animals being tested is calculated. The RPT is sensitive to many pyrogens, including endotoxins and non-endotoxin compounds, but has low sensitivity. This is in the range of nanograms of endotoxin / ml, as compared to picograms of endotoxin / ml in other pyrogen tests, such as the LAL. Furthermore, the correlation between pyrogens and the febrile response has a variable of up to 10,000-fold among rabbit species and is only roughly known. Additionally, since the EU Directive 2010 / 63 ("Protection of Laboratory Animals"), the adoption of the RTP has become stricter, and since 2018, re-approval every five years has been refused. Therefore, due to the inter-species variability in rabbits, non-sensitivity, insufficient quantitative results, ethical reasons, and especially the time-limited approval for this test, at least in Europe, the RPT will be abolished in the near future.

[0006] An alternative to RTP is LAL. This is described in the European Pharmacopoeia (EP2.6.14) and the United States Pharmacopoeia (USP<85>). It is an ex vivo test that collects amoebocyte lysate from horseshoe crabs (Limulus polyphemus). The erythrocytes in the lysate react with the bacterial endotoxins in the medical products that need to be tested by coagulation. Then, the coagulation can be measured. Limulus polyphemus lives on the east coast of the United States, and its population is strictly monitored and regulated. To remove amoebocyte lysate from the animals, the animals need to be captured and transported to the location of blood collection. After blood collection, the animals are returned to their environment and released. Depending on the estimates, 5 - 15% of these animals are estimated not to survive. This is an important issue because Limulus polyphemus has been designated as an endangered animal. Furthermore, three other Asian horseshoe crab species have already been designated as being at risk of extinction for reasons other than LAL. Since the LAL tests being carried out are increasing steadily worldwide, the availability of animals is decreasing. This could expose the safety of pharmaceuticals to risk and has led to a global debate. LAL also has other limitations in that it can only detect the endotoxin LPS from Gram-negative bacteria and cannot detect pyrogens of non-endotoxin origin (Moltz, 1993; Tilders et al., 1994; Zeisberger & Roth, 1993). As a result, the potential potency of non-endotoxin contamination in pharmaceuticals for humans is not fully reflected by LAL. For this reason, other tests, usually RFP, are also used additionally.

[0007] These two animal-based test systems have significant limitations in terms of availability, sensitivity, specificity, and ethical reasons. Other tests that are not animal-based are also needed. The requirements for these tests include high sensitivity, specificity for a wide range of pyrogens, and unlimited availability. One non-animal alternative that meets all of these requirements is the monocyte activation test. This is described in the European Pharmacopoeia (EP2.6.30). In the early 1990s, models that utilize the ability of immune cells to respond to pyrogens were described as potential alternative candidates to RPT and LAL (A et al., 1999; Eperon et al., 1997; Hartung & Wendel, 1995). Several different methods that utilize peripheral blood mononuclear cells (PBMC) from humans (Hartung & Wendel, 1995), monocyte cell lines such as MonoMac6 (MM6) (Ziegler-Heitbroc et al., 1988) or THP-1 (Auwerx, 1991; Tsuchiya et al., 1980), and the murine macrophage cell line RAW264.7 have been employed and described. THP-1 cells can be cultured and used as monocytes, which are cells in suspension and metabolically inactive but can differentiate into THP-1-derived macrophages that are more sensitive to the detection of pyrogens by an increase in the cluster of differentiation (CD)-14. The readouts are different from the cytokines IL-1β, IL-6, or TNF and the non-pyrogenic metabolites neopterin or nitrite described in the European Pharmacopoeia EP2.6.30 (Hartung et al., 2001).

[0008] The European Centre for the Validation of Alternative Methods (ECVAM) has summarized and described six different alternative pyrogen tests based on leukocyte cell lines or human whole blood.

[0009] Pyrogen tests based on leukocyte cell lines include tests using MM6 and THP-1 cells (Eperon & Jungi, 1996; Peterbauer et al., 2000; TAKTAK et al., 1991; Werner-Felmayer et al., 1995).

[0010] The MM6-based test detects IL-6 or TNF in the supernatant using a commercially available ELISA kit. The sensitivity for endotoxin standard biological reference preparation batch No. 2 was 0.125 IU / ml, while that for LAL was 0.03 IU / ml. The detection limit for LPS was 10 pg / ml. This test may be inhibited by the presence of immunoglobulin G. The MM6 test can be used in combination with LAL for the testing of some bacterial vaccines (TAKTAK et al., 1991).

[0011] Using THP-1 cells, it was shown that the detection of pyrogen from Gram-negative bacteria was correlated with the detection limit of LAL, but was superior to that of LAL for the detection of pyrogen from Gram-positive bacteria. The detection of pyrogen was determined by the measurement of TNF or neopterin (Peterbauer et al., 2000). The detection limit was 1 - 10 pg / ml endotoxin.

[0012] Pyrogen tests based on fresh human whole blood were developed to measure the production and secretion of the pro-inflammatory cytokines IL-1β, IL-6, TNF or prostaglandin E2 (PGE2) by ELISA. These tests were correlated with the amount of pyrogen present in the samples tested (Fennrich et al., 1999; Hartung & Wendel, 1996; Pool et al., 1998). Two tests have been established by endpoint detection of IL-1β or IL-6 by ELISA. The detection limits are <50 pg / ml of LPS / ml or 3 pg / ml of LPS for a 1-plate assay and 0.06 IU / ml, i.e. 6 pg / ml of LPS for a 2-plate assay, as described in Novel Pyrogen Tests Based on the Human Fever Reaction. The Report and Recommendations of ECVAM Workshop 43 (Hartung et al., 2001).

[0013] Several alternative methods equivalent to LAL are described, but these are not yet widely used routinely. Furthermore, due to the prohibition of the conduct of the rabbit pyrogen test and the threat of extinction of the horseshoe crab Limulus polyphemus, there is a need to develop reliable and widely applicable pyrogen tests to avoid endangering the safety of pharmaceuticals.

[0014] Summary of the Invention The present invention discloses an advanced in vitro pyrogen test based on a monocytic THP-1 cell line model of the monocyte / macrophage system. This model is aimed at detecting endotoxins and non-endotoxin pyrogens from medical products based on the requirements of monocyte activation assays. Instead of detecting secreted proteins by ELISA as described in the European Pharmacopoeia EP2.6.30, the expression of inflammatory cytokines is detected by measuring the amplification of messenger RNA (mRNA) using polymerase chain reaction (PCR) with either well-established quantitative real-time PCR (qPCR) or newly established digital PCR (dPCR).

[0015] For the first time, a method has been developed from the culture of monocytes / macrophages to the detection of cytokines by PCR. This system functions with either monocytes or macrophages. In the first step, the cells are cultured and either differentiated into macrophages or left as monocytes. The cells are seeded on a 96-well scale cell culture plate and combined with the medical product and tested in quadruplicate at three different dilutions. After a certain incubation time, the cells are lysed in the next step and nucleic acids are extracted. The extracts are subjected to amplification by qPCR or dPCR.

[0016] The present invention includes a primer / probe system for simultaneously detecting at least three cytokines / chemokines, IL-1β, IL-6, and IL-8. Additionally, a fourth cytokine, TNF, or an internal extraction control which is synthetic RNA can be added.

[0017] The present invention has also been developed for detecting endotoxin pyrogens and non-endotoxin pyrogens.

[0018] The present invention provides a kit comprising a cell line, a primer / probe system for the detection of the aforementioned cytokines / chemokines, and an assay system including a housekeeping gene to ensure data accuracy, two lyophilized master mixes containing or lacking reverse transcriptase for performing dPCR, and an analysis program for easy analysis of data obtained by dPCR.

[0019] Other objects and features of the present invention will be described later.

Brief Description of the Drawings

[0020]

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[0021] The present invention is based on the ability of human immune cells to respond to external stimuli such as pyrogens and express a number of pro-inflammatory immunomodulatory substances, including TNFα, IL-1β, IL-6, and IL-8. The subsequent release of these expressed pro-inflammatory cytokines and chemokines causes an immunomodulatory response of the body's immune system and results in an inflammatory reaction. The present invention is directed to measuring the gene expression of the cytokines / chemokines TNFα, IL-1β, IL-6, and IL-8 in response to an external pyrogen. For this purpose, macrophages are incubated with a medical sample together with a pyrogen standard for several hours, after which the mRNA is isolated and the gene expression is measured by polymerase chain reaction. Two methods of polymerase chain reaction (PCR) can be applied (quantitative real-time PCR or digital PCR).

[0022] The monocyte activation test presented here was developed to detect endotoxins, which are Gram-negative bacteria (e.g., Escherichia coli, Pseudomonas aeruginosa, Chlamydia trachomatis, Yersinia pestis), and non-endotoxin pyrogens such as Gram-positive bacteria (e.g., Staphylococcus aureus, Streptococcus spec., Bacillus spec., Clostridium) or fungi (e.g., Saccharomyces cerevisiae). Due to the ability of monocytes / macrophages to respond to many pyrogens, the present invention can be utilized to detect a wide variety of different biological and non-biological substances.

[0023] 1. Components of the pyrogen test The present invention can be used to detect endotoxin and non-endotoxin contamination in pharmaceuticals such as parenteral pharmaceuticals, dialysates, vaccines, intravenous solutions, and other medical or non-medical fluids that are not for oral application or that come into contact with body fluids.

[0024] A. Endogenous immunomodulatory mediators of the inflammatory response Theoretically, any immunomodulatory mediator expressed by the monocyte / macrophage system in response to pyrogens, including among others IL-1β, TNFα, IL-6 or IL-8, could be applied to detect pyrogen contamination (Maekelae et al., 2009). The cytokines IL-1β, TNFα or IL-6 are recommended as detectors in the monocyte activation assay (MAT) in the monocyte activation test of chapter 2.6.30. of the European Pharmacopoeia 07 / 2017:20630. These cytokines are known to be involved in the etiology leading to fever and septic shock (Evans et al., 2015). The role of the chemokine IL-8 in the onset of fever and sepsis has not been studied as much, but the chemokine IL-8 is massively produced by the monocyte / macrophage system (Duque & Descoteaux, 2014), and macrophages are the first cells in the immune response to express and secrete IL-8 to attract other immune cells such as neutrophils to the site of infection (Duque & Descoteaux, 2014). Neutrophils are the main mediators involved in the rapid innate host defense against microbial pathogens and are the first defense substances to produce reactive oxygen species (ROS) (Frφland, 1984). Since ROS can activate, among others, the nuclear factor κB pathway, which is an important transcription factor for cytokines and chemokines, the association between increased ROS production and the progression of inflammatory diseases has emerged as an additional factor in the inflammatory response (Naik & Dixit, 2011). Thereby, IL-8 becomes an important detector for the inflammatory response.

[0025] The present invention is based on Chapter 2.6.30 of the above-mentioned European Pharmacopoeia 07 / 2017:20630. Here, it is recommended to detect at least one of the cytokines IL-1β, TNFα or IL-6. The present invention depends on the parallel measurement of at least three cytokines / chemokines by dPCR or qPCR (IL-1β, TNFα, CXCL8 / IL-8), the housekeeping gene TATA-binding protein (TBP) to control the accuracy of the data, and an additional opportunity to add either the fourth cytokine IL-6 or the internal extraction control RNA (IK_RNA), which is a synthetic RNA that can be added before the extraction of treated monocytes / macrophages. The secreted IL-6 protein with immunoreactivity is considered to be an important cytokine in the inflammatory induction reaction, but the IL-6 mRNA in macrophages is bound by YB-1 and is easily secreted into the extracellular space, thereby reducing the IL-6 mRNA in macrophage cells, unlike TNFα mRNA (Kang et al., 2014). In different immune cell types (dendritic cells), the situation is different. Here, YB-1 functions as an IL-6 mRNA stabilizer. Therefore, the present invention provides a detector for IL-6 gene expression, which is used for the detection of pyrogen contamination in medical products and is optionally cell type-dependent.

[0026] B. Primer / probe system for the detection of endogenous immunomodulatory mediators of the inflammatory response After determining the endogenous immunomodulatory mediators of the inflammatory response, it is necessary to prepare a primer / probe system for use in the present invention. The primer / probe system has been developed to detect gene expression that detects the mRNA levels of the cytokines IL-1β, IL-6, TNFα, the chemokine CXCL-8 / IL-8, the housekeeping gene TBP, and the IK_RNA, which is a synthetic mRNA. The primer / probe system is specific for each mRNA and does not detect genomic DNA that may contaminate the sample. The primer / probe system is assumed to be amplified simultaneously by either dPCR or qPCR in one reaction.

[0027] The four primer / probe sets are essential components of the master mix containing assay reagents and reverse transcriptase provided by QIAGEN to be used with the QIAcuity-dPCR system (QIAGEN). An optional fifth primer / probe set can be added, either a primer / probe set for the detection of IL-6 or a primer / probe set for the detection of synthetic RNA (IK_RNA). The master mix can be used equally well in any qPCR system (e.g., CFX96™ Real-Time System by BioRad) that allows the simultaneous detection of at least four mRNAs.

[0028] C. Monocyte / macrophage cell line The first line in the Nucleic Acid Amplification Test Monocyte Activation Assay (NAT-MAT) is the immune cell line. The present invention relies on the monocyte / macrophage system combined with the sample to be tested. Basically, all cells that can express Toll-like receptors (TLRs) or are engineered to express TLRs can be used in the application of the present invention. TLRs are mediators of the inflammatory response to pyrogens. These are preferably of the same origin as the subject for which the use of the medicament is intended, which means human monocytes / macrophages in the case of pharmaceuticals, and cells such as cats, dogs, mice, etc. in the case of veterinary products. The cells are seeded into the wells of a 96-well cell culture plate.

[0029] One embodiment of the present invention is the monocyte cell line THP-1. The monocyte cell line THP-1 needs to be present at a cell density of at least 30,000 cells per 96-well, but can also function at cell densities of at least 40,000 cells per 96-well, 50,000 cells per 96-well, 60,000 cells per 96-well, 70,000 cells per well, 80,000 cells per well, 90,000 cells per well, or 100,000 cells per well. Other monocyte cell lines can be used equally well.

[0030] Another embodiment of the present invention is THP-1-derived macrophages. THP-1-derived macrophages have a higher ability to respond to pyrogens than monocytes. In the first step, monocytes are induced to differentiate into macrophages. The macrophages are provided by the present invention and seeded in a 96-well cell culture plate. These macrophages need to be present at a cell density of at least 30,000 cells per 96 wells, but can also function at a cell density of at least 40,000 cells per 96 wells, 50,000 cells per 96 wells, 60,000 cells per 96 wells, 70,000 cells per well, 80,000 cells per well, 90,000 cells per well, or 100,000 cells per well. THP-1-derived macrophages need to be given for at least 24 hours to adhere to the surface of the 96-well cell culture plate.

[0031] 2. Assay system The pyrogenicity assay is performed in three different containers.

[0032] A. Seeding of monocytes / macrophages and their treatment with samples and pyrogen standards Seed THP-1 monocytes or THP-1-derived macrophages in a first container, which is a 96-well cell culture plate, and incubate for different lengths of time with the sample and a series of pyrogen standard concentrations. The volume of the sample added is at least 50 μl and 150 μl or less. Ideally, the incubation time is in the range of 2 to 4 hours. Incubation of the cells with the sample is performed in the same container in which the cells were seeded.

[0033] B. Extraction to obtain mRNA for the detection of endogenous immunomodulatory mediators of the inflammatory response After completion of the incubation time, three volumes of lysis buffer are added to the 96-well cell culture plate. Subsequently, the assay vessel is subjected to an automated extraction system (e.g., KingFisher Flex from Thermo Fisher Scientific), and depending on the extraction system used (e.g., the improved AniPath-Kit from IST Innuscreen), a mixture of total DNA and total RNA or a mixture of total RNA is obtained as the eluate.

[0034] C. Quantitative polymerase chain reaction for the detection of endogenous immunomodulatory mediators of the inflammatory response Both digital PCR and real-time PCR are methods that can quantitatively measure the amount of nucleic acid. Both methods are highly sensitive and accurate. These methods are distinguished by the readout. The newly emerged digital PCR is similar to endpoint PCR, which is conventional PCR, unlike quantitative real-time PCR where the progress of the reaction is monitored. In digital PCR, a standard curve is not required for the quantitative determination of nucleic acids. For the quantification of nucleic acids amplified by real-time PCR, a standard curve or reference that enables relative quantification, i.e., a housekeeping gene, is required. Digital PCR provides an absolute quantitative determination of nucleic acids and does not require a standard curve.

[0035] The eluate is optionally subjected to dPCR or qPCR in a reaction mix containing PCR buffer, reverse transcriptase for reverse transcribing mRNA, and a primer / probe system for simultaneously detecting IL-1β, IL-8, TNFα, and the housekeeping gene TBP, by adding an additional primer / probe system for detecting either IL-6 or synthetic RNA (IK_RNA) added to the cells before extraction.

[0036] According to the requirements of the monocyte activation test in Chapter 2.6.30 of the European Pharmacopoeia 07 / 2017:20630, concentration series of pyrogen standards are added to the cell line together with the sample and amplified.

[0037] 3. Detection kit The above-described embodiments can be incorporated into a diagnostic kit for detecting pyrogen contamination in medical products or for performing a pyrogen test. This kit can detect endotoxin pyrogens from Gram-negative bacteria, and non-endotoxin pyrogens from Gram-positive bacteria, and their components (e.g., lipoteichoic acid, peptidoglycan, flagellin), and fungi or their components (e.g., zymosan).

[0038] This kit can be used to detect endotoxin and non-endotoxin contamination in pharmaceuticals such as parenteral medical products, dialysate, vaccines, intravenous solutions, and other medical or non-medical fluids that are not for oral application or that come into contact with body fluids.

[0039] 4. Interpretation of cytokine / chemokine gene expression data Interpret and analyze the gene expression data obtained by dPCR according to the requirements of the monocyte activation test in Chapter 2.6.30 of the European Pharmacopoeia 07 / 2017:20630.

[0040] A. Quantitative test using the standard curve method In the quantitative test, a pyrogen standard curve at the concentration recommended by the monocyte activation test in Chapter 2.6.30 of the European Pharmacopoeia 07 / 2017:20630 is utilized. This standard curve is generated by incubating monocytes / macrophages with the endotoxin USP reference standard. This enables the quantification of cytokine production in response to the activation of monocytes / macrophages in comparison with known endotoxin standards. Other available pyrogens can be used for the generation of the standard curve. The standard curve can be generated at any data point generated at significantly different concentrations of the standard endotoxin or other known pyrogens by using standard best-fit data analysis software. In the monocyte activation test of Chapter 2.6.30 of the European Pharmacopoeia 07 / 2017:20630, five data points in the range of R0 (untreated, negative control) and R1 to R4 are presented for generating the standard curve. The R1 data point represents 0.5 times the detection limit for the known pyrogen used in the standard curve, R2 represents 1 times the detection limit for the known pyrogen used in the standard curve, R3 represents 2 times the detection limit for the known pyrogen used in the standard curve, and R4 represents 4 times the detection limit for the known pyrogen used in the standard curve. After generating the standard curve, the equivalent endotoxin or non-endotoxin concentration of the sample to be tested can be interpolated from the standard curve. Utilizing the USP reference standard helps to normalize the obtained data to endotoxin units (EU) (per milliliter) defined by the USP / FDA, which is identical to the international unit (IU) defined by the World Health Organization and is the industry standard unit for indicating pyrogen contamination.

[0041] Definition As used herein, the term endotoxin refers to endotoxin lipopolysaccharide (LPS).

[0042] As used herein, the term "non-endotoxin pyrogen" refers to all pyrogens other than endotoxin lipopolysaccharide (LPS).

[0043] As used herein, the term "assay system" refers to any cell culture plate containing monocytes / macrophages that includes the sample to be tested, a standard, and a primer / probe system used for the detection of cytokine gene expression, gene expression, housekeeping genes, and the internal extraction control IK_RNA.

[0044] Materials and Methods Cell culture of THP-1 monocytes THP-1 monocytes (CLS Cell Lines Service GmbH) were cultured in RPMI 1640 medium (Capricorn Scientific) supplemented with sodium pyruvate (Sigma-Aldrich), HEPES (Gibco), glucose (Capricorn Scientific) to obtain 3.5 g / L glucose, heat-inactivated fetal bovine serum (Sigma-Aldrich), and β-mercaptoethanol (PAN Biotech) at a cell density of 10,000 - 50,000 cells per milliliter in an environment of 37 °C and 5% CO2.

[0045] Preparation of THP-1-derived macrophages THP-1 monocytes were cultured in RPMI 1640 medium (Sigma-Aldrich) supplemented with sodium pyruvate (Sigma-Aldrich), HEPES (Gibco), and glucose (Capricorn Scientific), heat-inactivated fetal bovine serum (Sigma-Aldrich), and β-mercaptoethanol (PAN Biotech), and containing glutamine and sodium bicarbonate. 15,000,000 THP-1 monocyte suspension cells were counted and treated in a T175 cell culture flask (area 175 cm 2 ) in an incubator at 37 °C with 5% CO2 for 4 - 8 days to differentiate THP-1 monocytes into THP-1-derived macrophages using 10 - 20 nM phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich).

[0046] Cell seeding of THP-1-derived macrophages THP-1-derived macrophages were seeded in quadruplicate 96-well plates, and each well of the quadruplicate contained RPMI 1640 medium (Sigma-Aldrich) supplemented with sodium pyruvate (Sigma-Aldrich), HEPES (Gibco), and glucose (Capricorn Scientific), heat-inactivated fetal bovine serum (Sigma-Aldrich), and β-mercaptoethanol (PAN Biotech), and containing glutamine and sodium bicarbonate. Standard cell treatment is carried out in the same matrix in which the sample to be tested is dissolved, usually in a 100 μL volume of physiological sodium chloride solution for 1 - 4 hours. Samples were diluted according to the requirements of the monocyte activation test in chapter 2.6.30 of the European Pharmacopoeia 07 / 2017:20630. The samples to be tested were applied to the cells at dilutions of undiluted, 2-fold diluted, and 4-fold diluted, and at the same dilutions spiked with intermediate-dose standards.

[0047] Extraction of mRNA from THP-1-derived macrophages after treatment with standard pyrogen and samples to be tested After the incubation time, mRNA is extracted and gene expression of cytokines and housekeeping genes is measured. For this purpose, a lysis buffer containing guanidine thiocyanate is added to the cells in the cell culture plate in an equal volume (100 μl). This causes detachment of the cells from the surface of the cell culture plate. The lysed cells are transferred to a deep well plate (KFFLX Plate Set, #845-KF-1296010, IST Innuscreen) for automated extraction in the KingFisher Flex system (Thermo Fisher Scientific). Nucleic acids (DNA and RNA) are captured by magnetic beads (#31-00641 IST Innuscreen), washed, and eluted into the elution plate with RNAse-free water (#314-00851, IST Innuscreen).

[0048] Digital polymerase chain reaction (dPCR) The extract is subjected to amplification by digital PCR (QIAcuity, QIAGEN). For this, the inventors used a one-step dPCR kit that combines reverse transcription from messenger RNA (mRNA) to complementary DNA (cDNA). A reaction mix containing a probe master mix, reverse transcriptase, a gene-specific primer / probe mix containing specific primers and probes for cytokines / chemokines IL-1β, TNFα, IL-8 and TBP, and 6 μl of the extract containing RNA up to a total volume of 12 μl is transferred to one well of an 8.5k 96-well nanoplate (#250011, QIAGEN) for amplification. The reaction mix is pipetted directly from the elution plate containing the extract to the nanoplate using the pipetting robot QIAgility (QIAGEN). Amplification is performed using digital PCR in a thermocycler QIAcuity (QIAGEN). The results are shown as copies per microliter calculated by the integrated analysis software QIAcuity Suite.

[0049] Interpretation of cytokine production data Test the medical product at different dilution degrees and quantitatively analyze the results according to the requirements of the monocyte activation test in Chapter 2.6.30 of the European Pharmacopoeia 07 / 2017:20630.

[0050] A. Standard curve method - Qualitative method First, it is necessary to confirm the verification of the standard curve consisting of R0 to R4 according to the following requirements: i) The regression of the response to log10 dose should be statistically significant (p < 0.01). ii) The regression of the response to log10 dose should not deviate significantly from linearity (p > 0.05). In the first step, for analysis, it is necessary to determine the appropriate dilution degrees of the samples without dilution and those added at 2-fold and 4-fold dilutions. In one assay, two different samples can be tested simultaneously in one 96-well cell culture plate; there are three dilution degrees for each (without dilution, 2-fold and 4-fold dilutions), and for each sample, the equivalents in each repetition should meet the following criteria: i) Calculate the average of four repetitions and calculate the recovery rate of endotoxin obtained by subtracting the average concentration without spike from the average concentration of the sample dilution with spike R3 added. This should be in the range of 50 - 200%. ii) Dilution degrees that do not meet the spike recovery criteria are not considered in subsequent analysis; if none of the dilution degrees meet these criteria, the assay cannot be analyzed and different dilution degrees need to be reexamined. iii) The samples to be investigated should comply with the requirements of the endotoxin equivalents measured in the samples without spike after prior correction for dilution degrees, and to be considered negative, the concentration should be less than the contaminant limit concentration (CLC) specified for the samples to be investigated. iv) For the assay to be valid for analysis, an additional negative control added to the PCR reaction to confirm the purity of the reagents used should require a result of 0 copies per microliter.

[0051] [Table 1]

[0052] The method according to the present invention includes the following embodiments: 1. A method for detecting endotoxin or non-endotoxin pyrogen in a sample, comprising the following steps: Seeding cryopreserved monocytes / macrophages in a 96-well cell culture plate at an appropriate cell density, Combining the sample with a monocyte / macrophage system capable of reacting with the sample with the production of a specific pro-inflammatory cytokine, After an incubation time of 2 to 4 hours, adding lysis buffer to the cells and transferring the cell culture plate containing monocytes / macrophages to an automated extraction system using magnetic beads that can bind nucleic acids and be eluted after washing and purification from cell debris, Transferring the eluate containing nucleic acid DNA / RNA to an automated pipetting system and combining it with a PCR reagent containing a buffer in which an enzyme capable of amplifying DNA can act, a primer / probe system containing primers / probes for three cytokines / chemokines IL-1β, TNFα, IL-8, a primer / probe for the housekeeping gene TBP, and the eluate, and transferring it to an 8.5k 96-well nanoplate, Transferring the nanoplate to a dPCR thermocycler, and then analyzing the gene expression of a specific pro-inflammatory cytokine, wherein an increase in the level of at least one of the three fever-related cytokines / chemokines (IL-1β, TNFα, IL-8) indicates the presence of pyrogen in the sample, A method comprising.

[0053] 2. A method for detecting endotoxin or non-endotoxin pyrogen in a sample, comprising the following steps: Seeding cryopreserved monocytes / macrophages in a 96-well cell culture plate at an appropriate cell density, Combining the sample with a monocyte / macrophage system capable of reacting with the sample with the production of a specific pro-inflammatory cytokine, After an incubation time of 2 to 3 hours, add lysis buffer to the cells and transfer the cell culture plate containing monocytes / macrophages to an automated extraction system that utilizes magnetic beads capable of binding nucleic acids and eluting after being washed and purified from cell debris. Transfer the eluate containing nucleic acid DNA / RNA to an automated pipetting system and combine it with a PCR reagent containing a buffer in which an enzyme capable of amplifying DNA can act, a primer / probe system containing primers / probes for four cytokines / chemokines, IL-1β, TNFα, IL-8, IL-6, a primer / probe for the housekeeping gene TBP, and the eluate, and transfer it to an 8.5k 96-well nanoplate. Transfer the nanoplate to a dPCR thermocycler and then analyze the gene expression of certain inflammatory cytokines / chemokines, wherein an increase in the level of at least one of the four pyrogen-related cytokines / chemokines (IL-1β, TNFα, IL-8, IL-6) indicates the presence of pyrogen in the sample. A method comprising the above.

[0054] 3. A method for detecting endotoxin or non-endotoxin pyrogen in a sample, comprising the following steps: Seeding cryopreserved monocytes / macrophages in a 96-well cell culture plate at an appropriate cell density. Combining the sample with a monocyte / macrophage system capable of reacting with the sample with the production of certain inflammatory cytokines. Adding an internal extraction control, which is a synthetic RNA molecule, to each well of the 96-well plate containing monocytes / macrophages. After an incubation time of 2 to 3 hours, add lysis buffer to the cells and transfer the cell culture plate containing monocytes / macrophages to an automated extraction system. Transfer the eluate to an automated pipetting system and combine it with a PCR reagent containing a buffer in which the enzyme can act, in addition to the enzyme capable of amplifying DNA, a primer / probe system containing primers / probes for four cytokines / chemokines IL-1β, TNFα, IL-8, IL-6, a primer / probe for the housekeeping gene TBP, and the eluate, and transfer it to an 8.5k 96-well nanoplate. Transfer the nanoplate to a dPCR thermocycler, and then analyze the gene expression of certain inflammatory cytokines / chemokines, wherein an increase in the level of at least one of three fever-related cytokines / chemokines (IL-1β, TNFα, IL-8) indicates the presence of pyrogen in the sample. A method comprising.

[0055] A method for detecting endotoxin or non-endotoxin pyrogen in a parenteral medicinal product, comprising the following steps: Seeding cryopreserved monocytes / macrophages in a 96-well cell culture plate at an appropriate cell density. Combining the medicinal product with a monocyte / macrophage system capable of reacting with the sample with the production of certain inflammatory cytokines. After an incubation time of 2 to 3 hours, add lysis buffer to the cells and transfer the cell culture plate containing monocytes / macrophages to an automated extraction system using magnetic beads that can elute after nucleic acids are bound and washed and purified from cell debris. Transfer the eluate containing nucleic acid DNA / RNA to an automated pipetting system and combine it with a PCR reagent containing a buffer in which the enzyme can act, in addition to the enzyme capable of amplifying DNA, a primer / probe system containing primers / probes for three cytokines / chemokines IL-1β, TNFα, IL-8, a primer / probe for the housekeeping gene TBP, and the eluate, and transfer it to an 8.5k 96-well nanoplate. Transferring the nanoplate to a dPCR thermocycler and then analyzing the gene expression of certain inflammatory cytokines / chemokines, wherein an increase in the level of at least one of three fever-related cytokines / chemokines (IL-1β, TNFα, IL-8) indicates the presence of pyrogen in the sample, the step A method comprising.

[0056] 5. A method for detecting endotoxin or non-endotoxin pyrogen in a parenteral pharmaceutical, comprising the following steps: Seeding cryopreserved monocytes / macrophages in a 96-well cell culture plate at an appropriate cell density, Combining the sample with a monocyte / macrophage system capable of reacting with the sample with the production of certain inflammatory cytokines, After an incubation time of 2-3 hours, adding lysis buffer to the cells and transferring the cell culture plate containing monocytes / macrophages to an automated extraction system using magnetic beads that can bind nucleic acids and be eluted after washing and purification from cell debris, Transferring the eluate containing nucleic acid DNA / RNA to an automated pipetting system and combining it with a PCR reagent containing a buffer in which an enzyme capable of amplifying DNA can act, a primer / probe system containing primers / probes for four cytokines / chemokines IL-1β, TNFα, IL-8, IL-6, a primer / probe for the housekeeping gene TBP, and the eluate, and transferring it to an 8.5k 96-well nanoplate, Transferring the nanoplate to a dPCR thermocycler and then analyzing the gene expression of certain inflammatory cytokines / chemokines, wherein an increase in the level of at least one of four fever-related cytokines / chemokines (IL-1β, TNFα, IL-8, IL-6) indicates the presence of pyrogen in the sample, the step A method comprising.

[0057] 6. A method for detecting endotoxin or non-endotoxin pyrogen in a parenteral medicinal product, comprising the following steps: Seeding cryopreserved monocytes / macrophages at an appropriate cell density in a 96-well cell culture plate, Combining the sample with a monocyte / macrophage system capable of reacting with the sample with the production of certain inflammatory cytokines, Adding an internal extraction control, which is a synthetic RNA molecule, to each well of the 96-well plate containing monocytes / macrophages, After an incubation time of 2-3 hours, adding lysis buffer to the cells and transferring the cell culture plate containing monocytes / macrophages to an automated extraction system, Transferring the eluate to an automated pipetting system and combining it with a PCR reagent containing a buffer in which an enzyme capable of amplifying DNA can act, a primer / probe system containing primers / probes for four cytokines / chemokines IL-1β, TNFα, IL-8, IL-6, primers / probes for the housekeeping gene TBP, and the eluate, and transferring it to an 8.5k 96-well nanoplate, Transferring the nanoplate to a dPCR thermocycler and then analyzing the gene expression of certain inflammatory cytokines / chemokines, wherein an increase in the level of at least one of three pyrogen-related cytokines / chemokines (IL-1β, TNFα, IL-8) indicates the presence of pyrogen in the sample, A method comprising the above steps.

[0058] The in vitro pyrogen test is performed as follows: Example 1: (a) Combining a monocyte / macrophage system with the sample to be tested in a cell culture plate, (b) Incubating the monocyte / macrophage system and the sample so that, if the sample contains pyrogen, the monocyte / macrophage system reacts by expressing inflammatory cytokines, (c) Transfer the combined monocyte / macrophage system and the sample to an automated extraction system containing magnetic beads to which the nucleic acids eluted in a later step will bind. (d) Transfer the eluate to an 8.5k 96-well nanoplate together with all the reagents necessary to amplify the gene of interest, including a primer / probe system that allows amplification of at least one of the pyrogen-related proinflammatory cytokines / chemokines IL-1β, TNFα, IL-8 and the housekeeping gene TBP. (e) Assay the amplicon for the presence of cytokines / chemokines, whereby the presence of a pyrogen is indicated if one of the cytokines / chemokines tested is elevated above baseline.

[0059] Example 2: (a) Combine the monocyte / macrophage system and the sample to be tested in a cell culture plate. (b) Incubate the monocyte / macrophage system and the sample so that, if the sample contains a pyrogen, the monocyte / macrophage system reacts by expressing proinflammatory cytokines / chemokines. (c) Transfer the combined monocyte / macrophage system and the sample to an automated extraction system containing magnetic beads to which the nucleic acids eluted in a later step will bind. (d) Transfer the eluate to an 8.5k 96-well nanoplate together with all the reagents necessary to amplify the gene of interest, including a primer / probe system that allows amplification of at least one of the pyrogen-related proinflammatory cytokines IL-1β, TNFα, IL-8, IL-6 and the housekeeping gene TBP. (e) Assay the amplicon for the presence of cytokines, whereby the presence of a pyrogen is indicated if one of the cytokines tested is elevated.

[0060] Example 3: (a) Combine the monocyte / macrophage system and the sample to be tested in a cell culture plate. (b) Incubate the monocyte / macrophage system and the sample so that, when the sample contains pyrogen, the monocyte / macrophage system reacts by expressing inflammatory cytokines. (c) Transfer the combined monocyte / macrophage system and sample to an automated extraction system containing magnetic beads to which the nucleic acid eluted in a later step binds. (d) Transfer the eluate to an 8.5 k 96-well nanoplate together with all the reagents necessary to amplify the target gene, including a primer / probe system that enables amplification of at least one of the housekeeping gene TBP and the pyrogen-related inflammatory cytokines IL-1β, TNFα, and IL-8 in the internal extraction control. (e) Assay the amplicon for the presence of cytokines, whereby the presence of pyrogen is indicated if one of the tested cytokines is elevated.

[0061] The samples of Examples 1 to 3 are pharmaceuticals, nutritional agents, or medical products.

[0062] The in vitro pyrogen test can detect two inflammatory cytokines, IL-1β, TNFα, and the chemokine CXCL8 / IL-8.

[0063] The in vitro pyrogen test may be based on three inflammatory cytokines, IL-1β, TNFα, IL-6, and the chemokine CXCL8 / IL-8.

[0064] It is also possible to detect three inflammatory cytokines, IL-1β, TNFα, and IL-8.

[0065] The in vitro pyrogen test is quality-controlled by the housekeeping gene TBP or by an internal extraction control.

[0066] In one embodiment of the in vitro pyrogen test, the monocyte / macrophage system consists of human THP-1 cells, a monocyte cell line isolated from the peripheral blood of a patient with acute monocytic leukemia.

[0067] In the in vitro pyrogen test, the monocyte / macrophage system contains a number of cells sufficient to provide at least 50,000 cells per well of the test system. Detection kits for use in the in vitro pyrogen test include, among other things, microtiter plates, dPCR nanoplates, cryopreserved monocyte / macrophage systems, PCR reagent mixes, and standard samples.

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Mitochondrial reactive oxygen species drive proinflammatory cytokine production. In Journal of Experimental Medicine (Vol. 208, Issue 3, pp. 417-420). https: / / doi.org / 10.1084 / jem.20110367 Peterbauer, A., Werner, E. R., & Werner-Felmayer, G. (2000). Weiterentwicklung eines Zellkul-turmodells zum Nachweis bakterieller Pyrogene. 455-455. https: / / doi.org / 10.1007 / 978-3-7091-6760-l_98 Pool, E. J., Johaar, G., James, S., Petersen, I., & Bouic, P. (1998). The detection of pyrogens in blood products using an ex vivo whole blood culture assay. Journal of Immunoassay, 19(2-3), 95-111. https: / / doi.org / 10.1080 / 01971529808005475 TAKTAK, Y. S., SELKIRK, S., BRISTOW, A. F., CARPENTER, A., BALL, C., RAFFERTY, B., & POOLE, S. (1991). Assay of Pyrogens by lnterleukin-6 Release from Monocytic Cell Lines. Journal of Pharmacy and Pharmacology: An International Journal of Pharmaceutical Science, 43(8), 578. https: / / doi.Org / 10.llll / j.2042-7158.1991.tb03540.x Tilders, F. J. 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Claims

1. A method for detecting pyrogens in a biological sample, wherein immune cells are brought into contact with a sample that may contain pyrogens, thereby causing the expression of immunomodulatory mediators, the method comprising extracting nucleic acids from the immune cells after lysis of the immune cells and detecting the expression of immunomodulatory mediators by PCR, characterized in that THP-1 macrophages function as immune cells.

2. The method according to claim 1, characterized in that the immunomodulatory mediator is an inflammatory mediator, preferably a cytokine or chemokine, particularly IL-1β, TNFα, or CXCL8 / IL-8.

3. The method according to claim 1, characterized in that the detection of expression by measuring the amplification of messenger RNA (mRNA) is performed using quantitative real-time PCR (qPCR) or digital PCR (dPCR).

4. The method according to claim 1, characterized by detecting at least one of three different cytokines / chemokines, preferably IL-1β, TNFα, or CXCL8 / IL-8, in parallel.

5. The method according to claim 1, characterized by adding a housekeeping gene, preferably a housekeeping gene TATA-binding protein (TBP), to control the accuracy of the data.

6. The method according to claim 1, further characterized by adding either a fourth cytokine, IL-6, or an internally extracted control RNA (IK_RNA).

7. The method according to claim 1, characterized in that the gene expression of five synthetic gene fragments is detected in parallel by five reactions.

8. The following steps: a) The step of seeding the cryopreserved THP-1 macrophages into a cell culture plate having 96 wells, b) A step of combining a sample with a THP-1 macrophage that can react with the sample, accompanied by the production of a specific pro-inflammatory cytokine. c) Add lysis buffer and transfer the cell culture plate containing THP-1 macrophages to an automated extraction system, thereby binding the released nucleic acids to the solid phase. d) A step of washing and purifying the nucleic acid bound to the solid phase, e) A step of DNA amplification of the nucleic acid and PCR to detect the gene expression of the immunomodulatory mediator. The method according to claim 1, including the method described in claim 1.

9. The method according to claim 8, further comprising adding a primer / probe system for three cytokines (IL-1β, TNFα, IL-8) or for four cytokines (IL-1β, TNFα, IL-8, IL-6) and the housekeeping gene TBP to the DNA amplification step of the nucleic acid.

10. The method according to claim 8, wherein an increase in the level of at least one of the fever-related cytokines (IL-1β, TNFα, IL-8, or IL-6) indicates the presence of pyrogen in the sample.

11. A detection kit for carrying out the method described in claim 1, a) Cultured THP-1 macrophages, b) Preferably a cell culture plate having 96 wells, c) at least one lysis buffer, and a buffer for binding the nucleic acid released by lysis, d) Solid phase, preferably magnetic beads, e) A primer / probe system containing PCR reagents for DNA amplification, primers / probes for at least three cytokines / chemokines, and primers / probes for the housekeeping gene TBP. A detection kit that includes this.

12. f) A fourth cytokine, IL-6 or internally extracted control RNA (IK_RNA) The detection kit according to claim 11, further comprising:

13. a) Pharmaceuticals, preferably parenteral medicines, dialysis solutions, vaccines or intravenous solutions, b) Non-medical fluids that are not administered orally or come into contact with bodily fluids, c) Other medical products, d) Nutritional supplements Use of the method according to any one of claims 1 to 10 or the detection kit according to claim 11 for detecting endotoxin and non-endotoxin contamination in a given location.

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