Improved Monocyte Activation Test

Abstract

The present invention is particularly in the field of in vitro assays for detecting pyrogens. The present invention provides an improved animal-free approach by providing an improved monocyte activation test. The improved test allows for reduced reagent consumption and improved throughput.

Description

[Technical Field]

[0001] [Field of the Invention] The present invention is in the field of in vitro assays, particularly in the field of pyrogen and endotoxin detection. The present invention provides an improved animal-free approach by providing an improved monocyte activation test. The improved test allows for reduced reagent consumption and improved throughput.

[0002] [Background of the invention] Detecting the presence of pyrogens and endotoxins is highly relevant for quality and safety testing of pharmaceutical compositions and medical devices due to the ability of pyrogens and endotoxins to cause severe adverse reactions in patients. Traditionally used detection assays include the rabbit pyrogen test (RPT) and the limulus amebocyte lysate assay (LAL), also known as the bacterial endotoxin test (BET). These tests are costly, time-consuming, and require the use of test animals. Furthermore, the limulus amebocyte lysate assay is generally limited to the detection of Gram-negative bacteria only and is prone to false positives.

[0003] An alternative to the above assays is the Monocyte Activation Test (MAT), which does not require the use of animals and is more representative of the human immune response. The MAT is a test governed by specific regulatory guidelines for sample preparation, testing, and result analysis established by the European Pharmacopoeia (Ph. Eur.; European Pharmacopoeia 10th Edition, 2019, Monograph 2.6.30, the Council of Europe). Constrained by these guidelines, currently used protocols have low throughput, are costly, and require the use of large amounts of reagents and sample volumes. Therefore, there remains a need for improved animal-free detection assays for pyrogens and endotoxins. Furthermore, there remains a need for improved monocyte activation tests. There remains a need to improve the throughput of monocyte activation tests. There remains a need for further miniaturization of monocyte activation tests. There remains a need to reduce the cost of monocyte activation tests. There remains a need to reduce the material consumption of monocyte activation tests.

[0004] [Summary of the Invention] In a first aspect, there is provided a method for detecting a pyrogen in a sample, comprising the steps of: i) providing one or more samples; ii) contacting the sample with peripheral blood mononuclear cells (PBMCs) in an incubation medium; iii) determining the response of the PBMCs; Including, A method is provided wherein the incubation medium has a volume of at most 175 μL / sample.

[0005] In some embodiments, the method is a monocyte activation assay. In some embodiments, the incubation medium has a volume of 20-150 μL, preferably 30-140 μL, and more preferably 50-110 μL. In some embodiments, the contacting in step ii) is performed in a standardized 384-well plate. In some embodiments, the incubation medium comprises 1-4 vol.%, e.g., about 2%, human culture medium supplement. In some embodiments, the response of the PBMCs determined is the release of proinflammatory cytokines, such as IL-6, IL-1 beta, IL-8, TNF-alpha, MCP-1, IL-10, IFN-alpha, IFN-beta, IFN-gamma, IFN-lambda, prostaglandin release, or high mobility group protein release. In some embodiments, the response of the PBMCs is determined by an ELISA assay. In some embodiments, the PBMCs have a density of at most 500×1000 cells / cm. 2 , preferably at most 250 x 1000 cells cm 2 In some embodiments, the PBMCs are present at a density of about 10 x 1000 cells / cm. 2 ~Approx. 250×1000 cells / cm 2 , preferably about 50 x 1000 cells / cm 2 ~Approx. 150×1000 cells / cm 2 , more preferably about 90 x 1000 cells / cm 2 ~Approx. 130×1000 cells / cm 2 In some embodiments, the response of PBMCs determined in multiple duplicate samples has a coefficient of variation of at most 20%.

[0006] In some embodiments, in step i), a further control sample, preferably a lipopolysaccharide sample, is provided, the control sample preferably comprising from about 0.005 to about 15 endotoxin units / mL.

[0007] In some embodiments, at least 50, preferably at least 97 samples are provided. In some embodiments, the incubation medium has a volume of 80-120 μL, and the PBMCs are present at a concentration of about 90-130×1000 cells / cm. 2 It exists at a density of

[0008] In another aspect, there is provided a method of releasing a pharmaceutical composition or medical device for use, said method comprising subjecting a sample derived from the pharmaceutical composition or medical device to the method of the first aspect.

[0009] In another aspect, a kit-of-parts is provided that includes a pyrogen or endotoxin standard, PBMCs, and one or more 384-well plates.

[0010] [Description of the Invention] In one aspect, there is provided a method for detecting a pyrogen in a sample, comprising: i) providing one or more samples; ii) contacting the sample with peripheral blood mononuclear cells (PBMCs) in an incubation medium; iii) determining the response of the PBMCs; Including, A method is provided in which the incubation medium has a volume of at most 175 μL / sample. This method is attractive because it does not require the use of test animals. This method has high predictive power.

[0011] [Pyrogen] A pyrogen is a substance capable of triggering an immune response in a subject by activating a cascade of immunological processes, typically characterized by an increase in internal body temperature (fever) outside of normal levels. The biological activity of a pyrogen is its ability to cause fever in a subject, alternatively referred to herein as its pyrogenicity. A pyrogen can be an exogenous pyrogen. "Exogenous" or "foreign" pyrogen refers to a pyrogen that originates outside the subject's body. A pyrogen can be an endotoxin. Endotoxins, such as lipopolysaccharide (LPS), are cellular components of bacteria, such as gram-negative bacteria, and constitute the major component of their outer cell wall. The presence of endotoxins in a subject's bloodstream is associated with multiple adverse symptoms, including fever, hypotension, nausea, shivering, and shock, and can lead to complications such as disseminated intravascular coagulation (DIC), endotoxic shock, and adult respiratory distress syndrome (ARDS).

[0012] The pyrogen can be a non-endotoxin pyrogen (NEP), which includes microbe-associated molecular patterns (MAMPs) and pathogen-associated molecular patterns (PAMPs), including, for example, flagellin, peptidoglycan, lipoproteins, lipoteichoic acid, fibroblast-stimulating lipopeptide-1, macrophage-activating lipopeptide-2, viral pyrogens, yeast pyrogens, and fungal pyrogens (e.g., yeast polysaccharides or fungal polysaccharides).

[0013] The pyrogen can be a product-related or process-related impurity present in the pharmaceutical composition or on the surface (e.g., of a medical device). Examples of pyrogenic impurities are chemical agents, such as polyadenylic acid, polyuridylic acid, polybionosinic acid, dinitrophenol, trinitrophenol, 4,6-dinitro-o-cresol, N-phenyl-p-naphthylamine, aldo-α-napthylamine, metals, and nanoparticles (typically <1 nm), as well as any other impurity that exhibits pyrogenic properties.

[0014] The pyrogen may be a damage-associated molecular pattern (DAMP), which refers to a biomolecule typically released by dying or damaged cells. Examples of DAMP pyrogens include biglycan, decorin, versican, hyaluronan, fibronectin, tenascin, uric acid, S100 proteins, ATP, GTP, F-actin, cyclophilin A, histones, HMGB1, HMGN1, IL-1a, IL-33, SAP130, DNA, RNA, mtDNA, TFAM, formyl peptides, mROS, calreticulin, defensins, heat shock proteins, and any other biomolecule released by cells exhibiting pyrogenicity.

[0015] The pyrogen may be a vehicle component of the pharmaceutical composition. Examples of such components include excipients, solvents, dispersion media, coating agents, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonicity agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gelling agents, binders, excipients, disintegrating agents, lubricants, sweeteners, flavoring agents, dyes, etc.

[0016] The pyrogen can be an endogenous pyrogen. "Endogenous" or "endogenous" pyrogen means a pyrogen produced by a subject's body after contact with an exogenous pathogen. An endogenous pyrogen can be associated with an inflammatory response. An endogenous pyrogen can be a DAMP. Examples of endogenous pyrogens include cytokines and chemokines.

[0017] In some embodiments, the pyrogen is an exogenous pyrogen. In some embodiments, the pyrogen is an endotoxin. In some embodiments, the endotoxin is a cellular component, preferably a lipopolysaccharide of Gram-negative bacteria. "Gram-negative" bacteria refers to bacteria that do not retain crystal violet staining as used in standard Gram staining methods. In some embodiments, the Gram-negative bacteria is a pathogenic bacteria. Examples of pathogenic Gram-negative bacteria include bacteria of the genera Escherichia, Salmonella, Shigella, Pseudomonas, Neisseria, Haemophilus, Bordetella, and Vibrio. In some embodiments, the pyrogen is a non-endotoxin pyrogen (NEP). In some embodiments, the pyrogen is a product- or process-related impurity present in or on a pharmaceutical composition. In some embodiments, the pyrogen is a damage-associated molecular pattern (DAMP). In some embodiments, the pyrogen is a vehicle component of the pharmaceutical composition.

[0018] In some embodiments, the pyrogen is an endogenous pyrogen. In some embodiments, the endogenous pyrogen is associated with an inflammatory response. In some embodiments, the endogenous pyrogen is a damage-associated molecular pattern (DAMP). In some embodiments, the endogenous pyrogen is a cytokine. In some embodiments, the endogenous pyrogen is a chemokine.

[0019] [Step i) Providing samples] In step i) of the method, one or more samples are provided. The samples can be taken (derived) from an original source, for example, from a product such as a pharmaceutical composition or a medical device. The sample taken can also be a subsample from the original sample (or from another subsample). The sample can also be or be taken from a subsample resulting from dilution or concentration of the original sample.

[0020] The sample may also be a replicate of an original sample or subsample. Replicate samples are preferably identical. When a sample is a replicate of an original sample or subsample, at least two, at least three, or at least four, preferably at least four, replicates of the original sample or subsample are provided. Replicates may, for example, be prepared individually or may result from taking equal subsamples from the original sample or subsample. The sample is preferably a liquid sample, more preferably an aqueous sample.

[0021] The sample may be taken from a pharmaceutical composition to be tested for the presence of a pyrogen or endotoxin, e.g., a therapeutic composition, a diagnostic composition, or a composition for preventing a disease or condition or reducing its symptoms (e.g., a vaccine). The pharmaceutical composition may be in any form, e.g., it may be a pharmaceutical composition suitable for topical, transdermal, intravenous, intramuscular, intraperitoneal, intraparenchymal, subcutaneous, intra-articular, intraadipose, oral, intrahepatic, intravisceral, intraauricular, intrathoracic, intracardiac, intraocular, or intratracheal administration, or administration via inhalation. Intraocular administration is preferred. In some embodiments, the pharmaceutical composition is a vaccine.

[0022] Samples can be taken from surfaces to be tested for the presence of pyrogens or endotoxins, for example, from the surface of a medical device or equipment. Examples of medical devices and equipment include bedpans, cannulae, cardioverters, defibrillators, catheters, dialysis machines, electrocardiographs, enema machines, endoscopes, gas cylinders, gauze sponges, surgical scissors, hypodermic needles, syringes, infection control equipment such as masks, gowns, face shields, and goggles, instrument sterilizers, kidney dishes, nasogastric tubes, surgical scalpels, nebulizers, ophthalmoscopes, otoscopes, pipettes, proctoscopes, radiographers, sphygmomanometers, thermometers, tongue depressors, infusion kits, tuning forks, ventilators, watches, etc. Such samples can be taken, for example, by rinsing the surface to be tested with a solution (e.g., water or a buffer solution), collecting the rinsing liquid, and using it to prepare the sample.

[0023] In some embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least At least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, at least 99, at least 100, at least 132, at least 164, at least 196, at least 228, at least 260, at least 292, at least 324, at least 356, or at least 384 samples are provided. In some embodiments, at least 50, preferably at least 97 samples are provided. In some embodiments, at least 96 samples are provided. In some embodiments, at least 384 samples are provided.Advantageously, the method according to the invention can be practiced using 384-well plates.

[0024] In some embodiments, in step i), a further control sample is provided. The control sample may contain a pyrogen (positive control) or may be pyrogen-free (negative control). Including a pyrogen positive control sample with a known pyrogen concentration may allow for improved accuracy of quantification. For example, a standard curve may be generated using multiple samples containing different pyrogen concentrations. The control sample may be a sample from a (reference) standard. Such standards are described later in this specification.

[0025] In some embodiments, the control sample is an endotoxin sample, preferably a lipopolysaccharide (LPS) sample. Endotoxin is typically measured in endotoxin units / mL (EU / mL). 1 EU / mL is approximately equivalent to 0.1 to 0.2 ng endotoxin / mL of solution, preferably 0.15 ng / mL. In embodiments where the control sample is an endotoxin sample, preferably a lipopolysaccharide sample, the control sample preferably contains about 0.005 to about 15 endotoxin units / mL. In some embodiments, the control sample contains about 0.005 to about 1 endotoxin unit / mL. In some embodiments, the control sample contains about 0.008 to about 0.5 endotoxin units / mL, or about 0.01 to about 0.4, preferably about 0.05 to about 0.3, and more preferably about 0.1 to about 0.2 EU / mL.

[0026] [Step ii) Contacting with PBMCs] Step ii) involves contacting one or more samples with peripheral blood mononuclear cells (PBMCs) in an incubation medium. In some embodiments, contacting is with whole peripheral blood or a fraction thereof containing PBMCs. In some embodiments, contacting is with isolated PBMCs. PBMC-containing blood fractions and isolated PBMCs can be obtained by standard methods, for example, using density gradient centrifugation.

[0027] In some embodiments, the contacting is with a PBMC cell line. In some embodiments, the contacting is with PBMCs obtained from a single donor. In some embodiments, the contacting is with PBMCs obtained from pooled whole peripheral blood from multiple donors. When PBMCs are obtained from a single or multiple donors, the donors are preferably certified according to standardized guidelines, more preferably as described in sections 5-3, 5-4, 5-5, 6-3, and / or monograph 2.6.30 of the European Pharmacopoeia (Ph.Eur.; European Pharmacopoeia 10th Edition, 2019, the Council of Europe).

[0028] In some embodiments, contacting is performed with fresh PBMCs. In a preferred embodiment, contacting is performed with cryopreserved PBMCs. Cryopreservation of PBMCs can be performed according to standard procedures, for example, as described in standard handbooks such as Hubel, A., 2018: Preservation of Cells: A Practical Manual, 1st Edition, Wiley-Blackwell, NJ, USA.

[0029] In some embodiments, the PBMCs are from a mammal, preferably from a human. In some embodiments, the PBMCs are leukocytes. In some embodiments, the PBMCs are macrophages. In preferred embodiments, the PBMCs comprise or are monocytes, preferably mammalian monocytes, more preferably human monocytes. In some embodiments, the macrophages or monocytes are derived from pluripotent stem cells. PBMCs are well known and well characterized, and generally comprise 10-20% monocytes.

[0030] The method is preferably a monocyte activation test. More preferably, the monocyte activation test follows the guidelines laid down in monograph 2.6.30 of the European Pharmacopoeia.

[0031] The contacting with PBMCs in step ii) can be the addition of the sample to a medium containing PBMCs. It can also be the addition of a medium containing PBMCs to the sample. It can be performed in any suitable receptacle, such as a microplate (with one or more wells), a tube (e.g., an Eppendorf tube), a flask (e.g., an Erlenmeyer flask), a bottle (e.g., a Schott bottle), a fermenter, etc. In some embodiments, the contacting is performed in a standardized 96-well plate. In a preferred embodiment, the contacting is performed in a standardized 384-well plate. Standardized well plates are widely available from suppliers. Suitable standards are ANSI / SLAS standards, preferably all five: 1-2004 (R2012), 2-2004 (R2012), 3-2004 (R2012), 4-2004 (R2012), and 6-2012 (R2012).

[0032] When using 96-well or 384-well plates, a single sample can be placed in each well. The present invention uniquely allows for the use of 384-well plates. The use of 384-well plates has the advantage of allowing for higher detection throughput (since more samples can be tested simultaneously) while reducing reagent requirements and overall costs. In standardized use of well plates for MAT, a given amount of wells is required for control or reference samples. This limits the amount of wells available for actual test samples. 384-well plates have a better test-to-control ratio because, after allocating wells to the required control samples, more wells remain available for test samples. In this regard, the following may be a conventional allocation of wells in a 96-well plate using four replicates per data point:

[0033] [Table 1]

[0034] Alternatively, the following is applicable if the NEP control is omitted at 2 dilutions per test sample.

[0035] [Table 2]

[0036] It should be noted that the sample of product being analyzed may result in multiple samples being provided to the method for detecting pyrogens. As an example, in the table above, a single test sample 1 results in multiple samples when each well is considered to contain a provided sample. This difference, when not explicitly indicated, will be clear from the context.

[0037] PBMCs are preferably cultured at 100 cells / cm for the contacting step. 2 It can exist at a specific density measured in units (cm 2 (wherein refers to the growth area, which is preferably the cross-sectional area of ​​the well, with preferred wells being flat-bottom wells). Density of PBMCs in the context of the present disclosure refers to the density of PBMCs used per contact sample. Those skilled in the art will be able to determine the growth area (cm), for example by using a cell counter or by using microscopic techniques. 2 ), cell concentration (cells / mL), and volume (mL) of cells / cm in a receptacle, preferably a well of a standardized 384-well plate. 2 The density of the unit can be readily calculated. This density is a commonly used parameter, and one skilled in the art will appreciate that dying cells may be present in the population. In some embodiments, PBMCs, which preferably contain monocytes, have a density of at most 500 x 1000 cells / cm. 2 , preferably at most 250 x 1000 cm 2 It exists at a density of

[0038] In some embodiments, PBMCs, preferably containing monocytes, have a density of about 10 x 1000 cells / cm 2 ~Approx. 250×1000 cells / cm 2 , preferably about 50 x 1000 cells / cm2 ~Approx. 150×1000 cells / cm 2 , preferably about 60 x 1000 cells / cm 2 ~Approx. 140×1000 cells / cm 2 of preferably about 70 x 1000 cells / cm 2 ~Approx. 130×1000 cells / cm 2 , preferably about 80 x 1000 cells / cm 2 ~Approx. 120×1000 cells / cm 2 , more preferably about 90 x 1000 cells / cm 2 ~Approx. 130×1000 cells / cm 2 , in some embodiments, about 10×1000 cells / cm 2 ~Approx. 115×1000 cells / cm 2 In some embodiments, the PBMCs, which preferably contain monocytes, are present at a density of 110 x 1000 cells / cm. 2 or approximately 110 x 1000 cells / cm 2 It exists at a density of

[0039] In some embodiments, PBMCs, preferably comprising monocytes, are present at a density of about 560 cells / well to about 14,000 cells / well, preferably about 2,800 cells / well to about 8,400 cells / well, preferably about 3,360 cells / well to about 7,840 cells / well, preferably about 3,920 cells / well to about 7,280 cells / well, preferably about 4,480 cells / well to about 6,720 cells / well, and more preferably about 5,040 cells / well to about 7,280 cells / well. In some embodiments, PBMCs, preferably comprising monocytes, are present at a density of 6,160 cells / well or about 6,160 cells / well. Conventional 384-well plates have a density of about 0.056 cm. 2 6160 cells / well has a growth area of ​​approximately 110 x 1000 cells / cm 2 It can be considered that

[0040] The incubation medium may have a specific volume, preferably measured in mL or μL, at the contacting step. The volume of the incubation medium in the context of the present disclosure refers to the volume per sample. In the provided methods, the incubation medium has a volume of at most 175 μL / sample. In some embodiments, it has a volume of at most 150 μL / sample.

[0041] In some embodiments, the incubation medium has a volume of 20 to 150 μL, preferably 30 to 140 μL, and more preferably 50 to 110 μL. In some embodiments, the incubation medium has a volume of 80 to 120 μL. In some embodiments, the incubation medium has a volume of 40 to 130 μL, preferably 60 to 120 μL, more preferably 70 to 115 μL, more preferably 75 to 105 μL, and more preferably 85 to 105 μL.

[0042] In some embodiments, the incubation medium has a volume of 20 to 100 μL, preferably 30 to 100 μL, and more preferably 50 to 100 μL. In some embodiments, the incubation medium has a volume of 80 to 100 μL. In some embodiments, the incubation medium has a volume of 100 μL or about 100 μL.

[0043] In some embodiments, the incubation medium has a volume of 20-150 μL, and the PBMCs, preferably monocytes, are cultured at a density of about 10×1000 cells / cm. 2 ~Approx. 250×1000 cells / cm 2 In some embodiments, the incubation medium has a volume of 20-150 μL, and the PBMCs, which are preferably monocytes, are present at a density of about 50×1000 cells / cm. 2 ~Approx. 150×1000 cells / cm 2 In some embodiments, the incubation medium has a volume of 20-150 μL, and the PBMCs, which are preferably monocytes, are present at a density of about 90×1000 cells / cm. 2~Approx. 130×1000 cells / cm 2 It exists at a density of

[0044] In some embodiments, the incubation medium has a volume of 30-140 μL, and the PBMCs, preferably monocytes, are cultured at a density of about 10×1000 cells / cm. 2 ~Approx. 250×1000 cells / cm 2 In some embodiments, the incubation medium has a volume of 30-140 μL, and the PBMCs, which are preferably monocytes, are present at a density of about 50×1000 cells / cm. 2 ~Approx. 150×1000 cells / cm 2 In some embodiments, the incubation medium has a volume of 30-140 μL, and the PBMCs, which are preferably monocytes, are present at a density of about 90×1000 cells / cm. 2 ~Approx. 130×1000 cells / cm 2 It exists at a density of

[0045] In some embodiments, the incubation medium has a volume of 80-120 μL, and the PBMCs, preferably monocytes, are cultured at a density of about 10×1000 cells / cm. 2 ~Approx. 250×1000 cells / cm 2 In some embodiments, the incubation medium has a volume of 80-120 μL, and the PBMCs, preferably monocytes, are present at a density of about 50×1000 cells / cm. 2 ~Approx. 150×1000 cells / cm 2 In some embodiments, the incubation medium has a volume of 80-120 μL, and the PBMCs, which are preferably monocytes, are present at a density of about 90×1000 cells / cm. 2 ~Approx. 130×1000 cells / cm 2 It exists at a density of

[0046] In some embodiments, the incubation medium has a volume of 50-110 μL, and the PBMCs, preferably monocytes, are cultured at a density of about 10×1000 cells / cm. 2 ~Approx. 250×1000 cells / cm 2In some embodiments, the incubation medium has a volume of 50-110 μL, and the PBMCs, which are preferably monocytes, are present at a density of about 50×1000 cells / cm. 2 ~Approx. 150×1000 cells / cm 2 In some embodiments, the incubation medium has a volume of 50-110 μL, and the PBMCs, which are preferably monocytes, are present at a density of about 90×1000 cells / cm. 2 ~Approx. 130×1000 cells / cm 2 It exists at a density of

[0047] In some embodiments, the incubation medium has a volume of 20-100 μL, and the PBMCs, preferably monocytes, are cultured at a density of about 10×1000 cells / cm. 2 ~Approx. 250×1000 cells / cm 2 In some embodiments, the incubation medium has a volume of 20-100 μL, and the PBMCs, which are preferably monocytes, are present at a density of about 50×1000 cells / cm. 2 ~Approx. 150×1000 cells / cm 2 In some embodiments, the incubation medium has a volume of 20-100 μL, and the PBMCs, which are preferably monocytes, are present at a density of about 90×1000 cells / cm. 2 ~Approx. 130×1000 cells / cm 2 It exists at a density of

[0048] In some embodiments, the incubation medium has a volume of 30-100 μL, and the PBMCs, preferably monocytes, are cultured at a density of about 10×1000 cells / cm. 2 ~Approx. 250×1000 cells / cm 2 In some embodiments, the incubation medium has a volume of 30-100 μL, and the PBMCs, which are preferably monocytes, are present at a density of about 50×1000 cells / cm. 2 ~Approx. 150×1000 cells / cm 2In some embodiments, the incubation medium has a volume of 30-100 μL, and the PBMCs, which are preferably monocytes, are present at a density of about 90×1000 cells / cm. 2 ~Approx. 130×1000 cells / cm 2 It exists at a density of

[0049] In some embodiments, the incubation medium has a volume of 50-100 μL, and the PBMCs, which are preferably monocytes, are cultured at a density of about 10×1000 cells / cm. 2 ~Approx. 250×1000 cells / cm 2 In some embodiments, the incubation medium has a volume of 50-100 μL, and the PBMCs, which are preferably monocytes, are present at a density of about 50×1000 cells / cm. 2 ~Approx. 150×1000 cells / cm 2 In some embodiments, the incubation medium has a volume of 50-100 μL, and the PBMCs, which are preferably monocytes, are present at a density of about 90×1000 cells / cm. 2 ~Approx. 130×1000 cells / cm 2 It exists at a density of

[0050] In some embodiments, the incubation medium has a volume of 80-100 μL, and the PBMCs, preferably monocytes, are cultured at a density of about 10×1000 cells / cm. 2 ~Approx. 250×1000 cells / cm 2 In some embodiments, the incubation medium has a volume of 80-100 μL, and the PBMCs, which are preferably monocytes, are present at a density of about 50×1000 cells / cm. 2 ~Approx. 150×1000 cells / cm 2 In some embodiments, the incubation medium has a volume of 80-100 μL, and the PBMCs, which are preferably monocytes, are present at a density of about 90×1000 cells / cm. 2 ~Approx. 130×1000 cells / cm 2 It exists at a density of

[0051] In some embodiments, the incubation medium has a volume of 100 μL or about 100 μL, and the PBMCs, which are preferably monocytes, are cultured at a density of 110×1000 cells / cm. 2 or approximately 110 x 1000 cells / cm 2 It exists at a density of

[0052] The incubation medium can be any medium suitable for culturing PBMCs, preferably mammalian PBMCs, more preferably human PBMCs, and most preferably human monocytes. Preferably, the incubation medium follows the guidelines established in monograph 2.6.30 of the European Pharmacopoeia (ibid.). A preferred incubation medium is RPMI (Roswell Park Memorial Institute) medium, more preferably RPMI 1640 medium, which is commercially available, for example, from ThermoFisher Scientific (Waltham, MA, USA). However, other suitable media, such as DMEM (Dulbecco's Modified Eagle's Medium), EMEM (Eagle's Minimum Essential Medium), Ham's F-10 or F-12 medium, and Iscove's Modified Dulbecco's Medium (IMDM), may be contemplated and are all commercially available. Further examples of suitable media are provided later in the Examples section of this specification.

[0053] The incubation medium can be supplemented to optimize the incubation and subsequent PBMC response determined in step iii). Thus, in some embodiments, the incubation medium comprises 0.1-15 vol%, preferably 0.1-10 vol%, and more preferably 1-4 vol% media supplement. A preferred media supplement is a human media supplement. In some embodiments, the incubation medium comprises 1 or about 1 vol% media supplement. In some embodiments, the incubation medium comprises 2 or about 2 vol% media supplement. In some embodiments, the incubation medium comprises 3 or about 3 vol% media supplement. In some embodiments, the incubation medium comprises 4 or about 4 vol% media supplement. As used herein, "media supplement" refers to an additional media component capable of promoting PBMC activity, e.g., cytokine production, preferably human PBMC. In some embodiments, the media supplement is fetal bovine serum (FBS). In some embodiments, the media supplement is human AB serum (AB).

[0054] The one or more samples to be contacted with peripheral blood mononuclear cells (PBMCs) are incubated for a duration sufficient to elicit a response in the PBMCs, preferably monocytes. In some embodiments, the duration of incubation is at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, or at least 24 hours. Preferably, the duration of incubation is at least 16 hours. In some embodiments, the contacting is carried out for at most 72 hours, preferably at most 36 hours, preferably at most 30 hours, more preferably at most 24 hours, even more preferably at most 18 hours, and most preferably at most 16 hours.

[0055] Preferably, the incubation is carried out under cell culture conditions, preferably human cell culture. Preferred conditions are a temperature in the range of 30-42°C, e.g., about 37°C, and a CO2 level of 0-8%, e.g., about 5%. In a preferred embodiment, the incubation is carried out at 37°C and a 5% CO2 level for at least 16 hours. Following incubation, the incubated sample may be used directly in step iii) or may be frozen and step iii) may be carried out at a later time.

[0056] Step iii) Determining the response of PBMCs In step iii), the response of PBMCs, preferably mammalian PBMCs, more preferably human PBMCs, most preferably human monocytes, is determined. The response of PBMCs can be activation of PBMCs. The response of PBMCs can be expression of surface activation markers. Examples of surface activation markers include CD80, CD86, CD11c, CD38, CD282, and CD64. The response of PBMCs can be production and / or secretion of inflammatory cytokines, preferably secretion. Examples of inflammatory cytokines include IL-6, IL-1 beta, I-6, IL-8, TNF-alpha, MCP-1, IL-10, IFN-alpha, IFN-beta, IFN-gamma, and IFN-lambda, preferably IL-6, IL-1 beta, I-6, IL-8, TNF-alpha, MCP-1, IL-10, more preferably IL-6. The response of PBMCs can be production and / or secretion of prostaglandins, preferably secretion, an example of which is PGE2. The response of the PBMCs can be the production and / or excretion of high mobility group proteins, preferably excretion, an example of which is HMGB1. The response can be the production and / or excretion of neopterin. In some embodiments, the response of the PBMCs that is determined is the expression of surface activation markers.

[0057] In some embodiments, the determined response of PBMCs is the production and / or excretion, preferably the excretion, of inflammatory cytokines such as IL-6, IL-1 beta, 1-6, IL-8, TNF-alpha, MCP-1, IL-10, IFN-alpha, IFN-beta, IFN-gamma, IFN-lambda, prostaglandins, or high mobility group proteins. Those skilled in the art will appreciate that determining the PBMC response can also involve the combined determination of the production and / or excretion, preferably the excretion, of multiple inflammatory cytokines, prostaglandins, and / or high mobility group proteins. In a preferred embodiment, the determined response of PBMCs is the production and / or excretion, preferably the excretion, of IL-6.

[0058] Determination of the PBMC response can be performed immediately after the contacting step in the same or a different receptacle, or the incubation mixture can be stored, optionally frozen, and used for response determination at a different time point. Generally, the PBMC response correlates with the detection of the pyrogen. Sometimes no response is detected, in which case the pyrogen is not detected at all.

[0059] The determination of the PBMC response can be performed, for example, by quantitative PCR, flow cytometric techniques (e.g., FACS analysis), or by immunoassay, preferably an ELISA assay. Those skilled in the art are familiar with how to perform such immunoassays, and descriptions thereof can be found in standard handbooks, such as "The Immunoassay Handbook: Theory and Applications of Ligand Binding, ELISA and Related Techniques," 2013, 4th Edition, Ed. Wild, D., Elsevier Science, NL (incorporated herein by reference in its entirety). Commercially available ELISA kits are also available, such as the MabTech ELISA basic IL-6 kit (HRP, MabTech AB, Nack Strand, SE). ELISA assays are particularly advantageous for use in the methods of the present invention, as they allow for high-throughput testing of multiple samples.

[0060] Thus, in some embodiments, the response of PBMCs is determined by ELISA assay. In some embodiments, the ELISA assay is performed using an antibody against IL-6, IL-1 beta, 1-6, IL-8, TNF-alpha, MCP-1, IL-10, IFN-alpha, IFN-beta, IFN-gamma, IFN-lambda, prostaglandins, or high mobility group proteins. In a preferred embodiment, the ELISA assay is performed using an antibody against IL-6 (anti-IL-6). Such an antibody is commercially available, for example, as clone 13A5 from MabTech AB, Nack Strand, SE. In some embodiments, the ELISA assay is performed in a standardized 384-well plate. Examples of the application of ELISA in the context of the present disclosure are provided later in the Examples section of this specification.

[0061] The detection methods of the present disclosure demonstrate low inter-measurement variability, particularly when testing multiple duplicate samples. Thus, in some embodiments, the response of PBMCs determined from multiple duplicate samples has a coefficient of variation of at most 30%. In some embodiments, the response of PBMCs determined from multiple duplicate samples has a coefficient of variation of at most 25%, or 24%, 23%, 22%, or 21%. Preferably, the response of PBMCs determined from multiple duplicate samples has a coefficient of variation of at most 20%. More preferably, the response of PBMCs determined from multiple duplicate samples has a coefficient of variation of at most 15%. Even more preferably, the response of PBMCs determined from multiple duplicate samples has a coefficient of variation of at most 10%, and most preferably at most 9.5%.

[0062] The detection methods of the present disclosure allow for the detection of pyrogens or endotoxins in samples taken from various products intended for therapeutic use or treatment, preferably for therapeutic use or treatment in humans. Examples of such products are pharmaceutical compositions, medical devices, and medical equipment, as previously described herein. If no pyrogens or endotoxins are detected in / on the product, it can be safely released (cleared) for use. In this context, release can be considered as making the product available to the general public, certifying that the product complies with certain (applicable) standards.

[0063] Thus, in one aspect, there is provided a method for releasing a product, preferably a pharmaceutical product, for use, said method comprising subjecting a sample derived from the product to a method for detecting pyrogens or endotoxins as previously described herein. The product is preferably released only when no or low pyrogen levels are detected. This may depend on which specifications are complied with.

[0064] In some embodiments, a method is provided for releasing a pharmaceutical composition or medical device for use, said method comprising subjecting a sample derived from the pharmaceutical composition or medical device to a method for detecting a pyrogen or endotoxin previously described herein.

[0065] The present disclosure further provides a kit of parts. Preferably, the kit is suitable for carrying out the method of the present invention. The kit may include a pyrogen or endotoxin standard of known concentration. A preferred standard is a lipopolysaccharide (LPS) standard. The pyrogen or endotoxin standard may be prepared according to European Pharmacopoeia guidelines (Monograph 2.6.30, supra) or may be obtained pre-prepared from a commercial source, for example, from the European Directorate for the Quality of Medicines & Healthcare (EDQM; e.g., Ph.Eur. Reference Standards: Orders and Catalogue provided by EDQM). Examples of pyrogen standards are provided later in the Examples section of this specification.

[0066] The pyrogen or endotoxin standard may correspond to one or more samples. Preferably, the multiple samples each contain a different concentration of the pyrogen or endotoxin, allowing for the construction of a standard curve. Exemplary standard concentrations for endotoxin, preferably LPS, are 0.5 EU / ml, 0.25 EU / ml, 0.125 EU / ml, 0.06 EU / ml, 0.03 EU / ml, 0.016 EU / ml, and 0.008 EU / ml. Multiple replicate (identical) samples may be provided, preferably at least two, more preferably at least three, and most preferably at least four samples.

[0067] The kit may comprise PBMCs, preferably monocytes, more preferably mammalian monocytes, most preferably human monocytes. Suitable PBMCs are described herein above.

[0068] The kit may include one or more 384-well plates, which allow for increased test throughput while minimizing reagent and overall costs compared to standard pyrogen detection methods, for example, compared to monocyte activation tests that utilize 96-well plates.

[0069] Thus, in one aspect, a kit-of-parts is provided that includes a pyrogen standard, PBMCs, and one or more 384-well plates. In some embodiments, the kit is a Monocyte Activation Assay (MAT) kit. In some embodiments, the kit further includes an incubation medium, optionally in combination with a (human) medium supplement as described above herein. In some embodiments, the (human) medium supplement is at a concentration of 1-4 vol%, e.g., about 2%.

[0070] In some embodiments, the kit is suitable for simultaneously testing at least three identifiable products, e.g., identifiable pharmaceutical compositions, medical devices, or medical equipment, for the presence of pyrogens. In some embodiments, the kit is suitable for simultaneously testing at least four identifiable products. In some embodiments, the kit is suitable for simultaneously testing at least five identifiable products. In some embodiments, the kit is suitable for simultaneously testing at least six identifiable products. In some embodiments, the kit is suitable for simultaneously testing at least seven identifiable products. In some embodiments, the kit is suitable for simultaneously testing at least eight identifiable products. In some embodiments, the kit is suitable for simultaneously testing at least nine identifiable products. In some embodiments, the kit is suitable for simultaneously testing at least ten identifiable products. Testing is preferably performed in accordance with the European Pharmacopoeia guidelines for the detection of pyrogens and endotoxins (Monograph 2.6.30, supra).

[0071] "Parallel" testing refers to the testing of samples corresponding to identifiable products on a single plate, allowing for increased testing throughput and minimization of reagent and overall costs.

[0072] In some embodiments, the incubation medium for each sample being tested per well has a volume of at most 175 μL. In some embodiments, it has a volume of at most 170, 165, 160, 155, or 150 μL. In some embodiments, the incubation medium for each sample being tested per well has a volume of 20-150 μL, preferably 30-140 μL, more preferably 40-130 μL, more preferably 50-120 μL, more preferably 60-115 μL, more preferably 70-110 μL, more preferably 80-105 μL, or more preferably 90-100 μL. In some embodiments, the incubation medium for each sample being tested per well has a volume of 20-100 μL, preferably 30-100 μL, or more preferably 50-100 μL. In some embodiments, the incubation medium for each sample being tested per well has a volume of 80-120 μL. In some embodiments, the incubation medium for each sample being tested per well has a volume of 80-100 μL, hi some embodiments, the incubation medium for each sample being tested per well has a volume of at or about 100 μL.

[0073] In some embodiments, the PBMCs are at most 500 x 1000 cells / cm 2 , preferably at a density of at most 250 x 1000 cells cm. In some embodiments, PBMCs are present at a density of about 10 x 1000 cells / cm. 2 ~Approx. 250×1000 cells / cm 2 , preferably about 50 x 1000 cells / cm 2 ~Approx. 150×1000 cells / cm 2 , more preferably about 90 x 1000 cells / cm 2 ~Approx. 130×1000 cells / cm 2 In some embodiments, the PBMCs are present at a density of 110 x 1000 cells / cm. 2 or approximately 110 x 1000 cells / cm 2 It exists at a density of

[0074] In some embodiments, the incubation medium for each sample tested per well has a volume of 80 to 120 μL, and the PBMCs are present at a concentration of about 90 to about 130×1000 cells / cm. 2 In some embodiments, the incubation medium for each sample tested per well has a volume of from about 100 μL, and the PBMCs are present at a density of 110×1000 cells / cm. 2 or approximately 110 x 1000 cells / cm 2 It exists at a density of

[0075] Generally, the above volumes can be considered the total volume present in the well. In some embodiments, incubation medium is added in an amount such that the total volume described above is achieved.

[0076] [General definition] In this document and the claims, the verb "to comprise" and its conjugations are used in their open-ended sense, meaning to include the items that follow it but not to exclude items not specifically listed. Additionally, the verb "to consist" may be substituted with "to consist essentially of." That is, each method or component defined herein may include additional steps or components other than those specifically set forth, provided that these additional steps or components do not alter their unique characteristics. Additionally, a reference to "an element" by the indefinite article "a" or "an" does not exclude the possibility that more than one element may be present, unless the context clearly requires that only one element be present. Thus, the indefinite article "a" or "an" typically means "at least one."

[0077] As used herein, "at least" a particular value means greater than or equal to the particular value. For example, "at least 2" is understood to be the same as "2 or greater," i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, ..., etc.

[0078] The word "about" or "approximately" when used in connection with a numerical value (e.g., about 10) preferably means that the value may be the given value or may be 5%, preferably 1%, greater or less than the given value. As used herein, the term "and / or" indicates that one or more of the specified cases may occur alone or in combination with at least one of the specified cases, up to all of the specified cases. Various embodiments are described herein. Each of the embodiments specified herein may be combined together unless otherwise indicated.

[0079] All patent applications, patents, and publications cited herein are incorporated herein by reference in their entirety. The present invention is in no way limited to the methods and materials expressly described. The following examples further illustrate the present invention. These are provided for illustrative purposes only and should not be construed as limiting the scope of the present invention. [Brief explanation of the drawings]

[0080] [Figure 1A] Effect of cell density on IL-6 production in a monocyte activation assay. PBMCs were seeded into 384-well plates at the indicated cell densities (×1000 cells / cm²) in a total volume of 33 µL. The x-axis represents cell density (×1000 cells / cm²). The y-axis represents IL-6 per 1000 cells. Four replicates (symbols) and the mean (horizontal line) are plotted. The figure title indicates the total MAT volume. [Figure 1B] As 1A except 50 μL total MAT volume was used. [Figure 1C]As 1A except 66 μL total MAT volume was used. [Figure 1D] As 1A except 100 μL total MAT volume was used. [Figure 2] Plot of optical density (OD) absorbance values ​​(y-axis) at each concentration of LPS (x-axis) in EU / ml for each density (×1000 cells / cm, increasing from left to right). The dotted line represents 0.1 OD. The average of three experiments with four replicates each is depicted. [Figure 3] Signal-to-noise ratio (bars) plotted against density (1000 cells / cm2). Signal-to-noise ratio was calculated by dividing the OD at 0.016 EU / ml by the OD of the blank. [Figure 4] Coefficient of variation (CV) at various cell densities. The average CV% of four replicates for each LPS concentration (EU / ml) was calculated and then averaged per density (1000 cells / cm). Plots show the mean (bars) and standard deviation (error bars) of three different experiments. [Figure 5] Relative gain (y-axis) plotted as a percentage of the optical density (OD) at 0.032 EU / ml LPS normalized to the optical density in a 100 microliter assay volume. The x-axis represents cell density in 1000 cells / cm. Error bars represent the standard deviation of triplicate experiments. [Figure 6] Average CV% across different assay volumes. The average CV% of four replicates for each LPS concentration (EU / ml) was calculated and then averaged per assay volume (microliter) and density (1000 cells / cm2). The plot shows the mean (bars) and standard deviation (error bars) of three different experiments (55 / 110 / 220 from left to right). The pattern represents cell density in 1000 cells / cm2. [Figure 7]Optical density (OD) plotted against LPS concentration (EU / ml). The x-axis is a logarithmic scale. The gray line represents the standard curve at a density of 110 (×1000 cells / cm2) and a 100 microliter assay volume. The black line represents the standard curve at a density of 220 (×1000 cells / cm2) and a 66 microliter assay volume. Error bars represent the standard deviation of four replicates. [Figure 8] Curve slope. Bars represent the curve slope of the 4-parameter logistic curve. Left: LPS standard curve at 110 density (×1000 cells / cm2) and 100 microliter assay volume. Right: LPS standard curve at 220 density (×1000 cells / cm2) and 66 microliter assay volume. [Figure 9] Average CV%. The average CV% of four replicates for each LPS concentration (EU / ml) was calculated and then averaged per volume / density combination. Left: Average CV% for the LPS standard curve with 110 density (×1000 cells / cm2) and 100 microliter assay volume. Right: Average CV% for the LPS standard curve with 220 density (×1000 cells / cm2) and 66 microliter assay volume.

[0081] [Example] Materials and Methods [Control preparation] Lipopolysaccharide (LPS) was obtained from EDQM (batch 5.1) and handled as directed by EDQM. LPS was rehydrated in 5 mL LAL reagent water (LRW, Lonza Bioscience, Basel, CH) by vortexing for 30 min and diluted to a stock concentration of 10 endotoxin units per milliliter (EU / ml) by vortexing for 3 min in LRW. An LPS reference endotoxin curve (RSE) was then generated via serial dilutions and subsequent resuspension in RPMI 1640 (Thermo-Fisher Scientific, Waltham, MA, USA).

[0082] [Cell thawing] A vial of PBMCs (10 million PBMCs / ml) was quickly thawed in a water bath set at 37°C and resuspended by the gradual addition of pre-warmed (37°C) RPMI medium containing 4% human culture medium supplement (Mediatech, Manassas, VA, USA).

[0083] [Cell density] Samples of 0.2 EU / ml LPS were plated onto 384-well microplates (Thermo-Fisher Scientific, Waltham, MA, USA) at 50% of the final volume to achieve various final volumes as indicated (per experiment). Subsequently, human medium supplement (HMS) was added at a 1:1 ratio to the cell suspension at various cell concentrations as indicated (per experiment), resulting in a final concentration of 2% (vol. / vol.). The final LPS concentrations corresponded to two-fold serial dilutions starting from 0.1 EU / ml obtained by resuspension in RPMI on the plate. Cells were cultured with LPS for 20 hours ± 1 hour in an incubator (Binder (CB60), Tuttlingen, Germany) set at 37°C and 5% CO2, after which IL-6 concentrations were measured by ELISA as described below. IL-6 per 1000 cells was calculated by interpolating the measured optical density (OD) with a linear regression model of the IL-6 standard curve and dividing the total IL-6 produced by the total number of cells in the well.

[0084] Thirty-three microliters of the LPS standard curve (0.064 EU / ml, 0.032 EU / ml, 0.016 EU / ml, 0.008 EU / ml, and 0.004 EU / ml) were plated onto a 384-well microplate. Cryopreserved peripheral blood mononuclear cells (PBMCs) were thawed and resuspended in RPMI medium containing 4% human culture medium supplement. The cell suspensions were serially diluted (dilution factor 2) to concentrations of 1514 cells / microliter, 757 cells / microliter, 378 cells / microliter, 189 cells / microliter, 94.7 cells / microliter, and 47.4 cells / microliter. Thirty-three microliters of each cell suspension was added to the plate, resulting in a density of approximately 440,000 cells / cm. 2 , 220,000 cells / cm 2 , 110,000 cells / cm 2 , 55,000 cells / cm 2 , 27,500 cells / cm 2 , and 13,700 cells / cm 2 The final cell densities were 0.032 EU / ml, 0.016 EU / ml, 0.008 EU / ml, 0.004 EU / ml, and 0.002 EU / ml at each density. The final concentration of HMS was 2% in each well.

[0085] [MAT incubation volume] Samples for the LPS standard curve (concentrations: 0.064 EU / ml, 0.032 EU / ml, 0.016 EU / ml, 0.008 EU / ml, and 0.004 EU / ml) were plated onto a 384-well microplate in three different volumes (16.7 microliters, 33 microliters, and 50 microliters). Cryopreserved PBMCs were thawed and reconstituted in RPMI medium containing 4% human medium supplement. The cell suspension was diluted to different cell concentrations and added to the plate in a 1:1 ratio to achieve 55,000 cells / cm in each assay volume (33 microliters, 66 microliters, and 100 microliters) with a final HMS concentration of 2%. 2 , 110,000 cells / cm 2 , and 220,000 cells / cm 2A final cell density of 0.1% was obtained.

[0086] [MAT] Different volumes of samples for the LPS standard curve were added to the culture plate together with the resuspended PBMCs (at a 1:1 ratio) and incubated in a 37°C, 5% CO incubator for 16 hours. The final concentrations of LPS were 0.5 EU / ml, 0.25 EU / ml, 0.125 EU / ml, 0.06 EU / ml, 0.03 EU / ml, 0.016 EU / ml, and 0.008 EU / ml. The final concentration of HMS was 2%.

[0087] [ELISA] ELISA plates were coated with IL-6 capture antibody (clone 13A5, MabTech AB, Nack Strand, SE) at a concentration of 1:2000 diluted in phosphate-buffered saline and incubated overnight at 4°C. ELISA was performed using the MabTech ELISA basic IL-6 kit (HRP) according to the manufacturer's protocol (MabTech). Absorbance was measured at a wavelength of 450 nanometers using a Thermo-Scientific absorbance plate reader. Background at 630 nanometers was subtracted. Supernatant (16.7 microliters) was diluted 1 + 1 with incubation buffer and added to the ELISA plate.

[0088] [statistics] Statistical analyses, including four-parameter / five-parameter logistic regression, were performed using Graphpad Prism 8 (GraphPad Software, San Diego, CA, USA).

[0089] [result] [LPS response at different densities and volumes at 0.1EU / ml] Increasing cell density showed improved IL-6 response at different MAT volumes per 1000 cells at a concentration of 0.1 EU / ml, with densities (×1000 cells / cm) at 50 μL, 66 μL, and 100 μL. 2A rapid increase was observed at a density of 227 (×1000 cells / cm) using a 33 μL volume in MAT. 2 ) at a density of 152 (×1000 cells / cm 2 ), whereas a 3-fold increase in signal was observed compared to 303 density (×1000 cells / cm 2 ) showed a slight decrease in IL-6 per cell (Fig. 1A-D).

[0090] [Cell density and LPS response] At higher densities, stronger LPS signals (OD) were estimated at each LPS concentration, but at a density of 440 (×1000 cells / cm 2 At 0.032 EU / ml, the background signal (0.00 EU / ml) also increased to 0.12 OD (Figure 2). Furthermore, the signal-to-noise ratio, calculated by dividing the signal at 0.032 EU / ml by the background signal, increased with increasing density, reaching a density of 110 (×1000 cells / cm). 2 ) the optimum (signal to noise 6.4) was reached.

[0091] Furthermore, although the LPS signal and signal-to-noise ratio increased with density (Fig. 3), replicate variability also increased, as shown in Fig. 4, at 440 densities (×1000 cells / cm). 2 ) the average CV% increases to 28.5%. 2 ), the signal-to-noise ratio was the highest among the densities tested, but the average CV% was the lowest.

[0092] [Effect of volume on LPS signaling] A MAT volume of 33 microliters showed a significant increase in the LPS response, as depicted in Figure 5, which shows a relative gain of 0.032 EU / ml normalized to 100 μL. The relative gain also increased when plating at higher cell densities, reaching a density of 55 (×1000 cells / cm) at a MAT volume of 33 μL. 2 ) at 219%, 110 density (×1000 cells / cm 2 ) at 304% and 220 densities (×1000 cells / cm 2) resulted in an average relative gain of 387%. However, smaller volumes showed increased variability between replicates (Figure 6). At each density, the 100 μL MAT volume showed the lowest CV% (55 density (×1000 cells / cm)). 2 ) at 11.8%, 110 density (×1000 cells / cm 2 ) at 13.3%, and 220 density (×1000 cells / cm 2 The pattern of increasing CV% with increasing density was observed for each assay volume, but at 110 densities (×1000 cells / cm), the CV% was 18.6%. 2 ) was the exception, with a density of 220 (×1000 cells / cm) in all test assay volumes. 2 ) showed a lower CV%.

[0093] [LPS standard curve] Two configurations: cell density 220 (×1000 cells / cm 2 MAT volume was 66 μL and cell density was 110 (× 1000 cells / cm) 2 An LPS standard curve was generated using a MAT volume of 100 μL (Figure 7). Four-parameter logistic regression yielded a correlation coefficient of 0.99 (66 μL / 220 density (×1000 cells / cm)). 2 )) and 0.98 (100 μL / 100 density (×1000 cells / cm 2 Figure 8 shows the curve slopes for both plots, resulting in an approximately two-fold difference in slope between the two configurations. Additionally, the average CV% was 66 μL / 220 density (×1000 cells / cm). 2 ) MAT is 100 μL / 110 density (×1000 cells / cm 2 ) were significantly higher than those in the control group (23.4% and 9.4%, respectively) (Figure 9).

Claims

1. A method for detecting a heat source in a sample, i) A step of providing one or more samples, ii) The step of bringing the sample into contact with peripheral blood mononuclear cells (PBMCs) in incubation medium, iii) The step of determining the response of the PBMC, Includes, The incubation medium has a volume of at most 175 μL / sample.

2. The method according to claim 1, wherein the method is a monocyte activation test.

3. The method according to claim 1, wherein the incubation medium has a volume of 20 to 150 μL.

4. The method according to claim 1, wherein the incubation medium has a volume of 50 to 110 μL.

5. The method according to claim 1, wherein step ii) is performed with a standardized 384-well plate.

6. The method according to claim 1, wherein the incubation medium comprises 1 to 4 vol. -% medium supplement.

7. The method according to any one of claims 1 to 6, wherein the determined response of the PBMC is the efflux of inflammatory cytokines, the efflux of prostaglandins, or the efflux of high-mobility group proteins.

8. The method according to any one of claims 1 to 6, wherein the response of the PBMC determined is the efflux of IL-6, IL-1 beta, IL-8, TNF-alpha, MCP-1, IL-10, IFN-alpha, IFN-beta, IFN-gamma, IFN-lambda, prostaglandin efflux, or high mobility group protein efflux.

9. The method according to any one of claims 1 to 6, wherein the response of the PBMC is determined by an ELISA assay.

10. The method according to any one of claims 1 to 6, wherein the PBMCs are present at a density of at most 500 × 1000 cells / cm².

11. The aforementioned PBMC has approximately 10 × 1000 cells / cm² ２ The method according to any one of claims 1 to 6, wherein the cells are present at a density of approximately 250 × 1000 cells / cm².

12. The method according to any one of claims 1 to 6, wherein the PBMC is present at a density of about 50 × 1000 cells / cm² to about 150 × 1000 cells / cm².

13. The method according to any one of claims 1 to 6, wherein the response of the PBMC determined by multiple identical samples has a coefficient of variation of at most 20%.

14. The method according to any one of claims 1 to 6, wherein a further control sample is provided in step i).

15. The method according to claim 14, wherein the further control sample is a lipopolysaccharide sample containing about 0.005 to about 15 endotoxin units / mL.

16. The method according to any one of claims 1 to 6, wherein at least 50 samples are provided.

17. The incubation medium has a volume of approximately 80 to 120 μL, and the PBMCs are approximately 90 to 130 × 1000 cells / cm². ２ The method according to any one of claims 1 to 6, which exists at a density of .

18. A method for releasing a pharmaceutical composition or medical device to be used, the method comprising subjecting a sample derived from the pharmaceutical composition or medical device to the method according to any one of claims 1 to 6.

19. A kit of parts including a pyrogen standard or endotoxin standard, PBMC, and one or more 384-well plates.

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