Improved monocyte activation test using human platelet serum.

Abstract

The present invention is in the field of in vitro assays, particularly in the field of pyrogen and endotoxin detection. The present invention provides improved compositions suitable for use in animal-free tests, for example, in monocyte activation tests. The improved compositions allow for increased sensitivity, improved dynamic range, and improved fidelity of test responses.

Description

Detailed Description of the Invention

[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 improved compositions suitable for use in animal-free tests, such as monocyte activation tests. The improved compositions allow for increased sensitivity, reduced variability, improved dynamic range, and improved fidelity of test responses.

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

[0003] An alternative to the above assay 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 has been selected by the European commission to replace the Rabbit Pyrogen Test (RPT) by 2025. It is listed in the European Pharmacopoeia (Ph.Eur.; European Pharmacopoeia 10th Edition). thThe test is governed by detailed regulatory guidelines for sample preparation, testing, and analysis of results established in the Council of Europe's Monograph 2.6.30, Edition 2019. Constrained by these guidelines, currently used protocols have low throughput, are costly, and require the use of large amounts of reagents and sample volumes.

[0004] To date, classical MAT is typically performed using fetal bovine serum (FBS) or human AB (hAB) serum as a medium supplement (Ph.Eur.; European Pharmacopoeia, supra). However, while FBS provides good reactivity for endotoxin detection, it is poor at detecting low concentrations of non-endotoxin pyrogens. Conversely, hAB serum has been shown to provide good sensitivity for non-endotoxin pyrogens, but its ability to detect endotoxins is reduced compared to, for example, FBS. Therefore, MAT assays using fetal bovine serum and MAT assays using hAB serum are required to detect the full spectrum of pyrogens with sufficiently high sensitivity (Molenaar-de Backer 2021, ALTEX - Alternatives to animal experimentation, 38(2); 307-315). Therefore, there remains a need for improved pyrogen and endotoxin detection assays that are animal-free. Furthermore, there remains a need for improved monocyte activation tests. There is also a need to improve the sensitivity of monocyte activation tests. There is a need to reduce the cost of monocyte activation tests. There is a need to improve the reliability of monocyte activation tests. There is a need to simplify monocyte activation tests. There is a need to reduce the need to perform multiple parallel tests.

[0005] [Summary of the Invention] The inventors have discovered that human platelet lysate (hPL) is a human-based media supplement that unexpectedly exhibits high sensitivity to both endotoxins and multiple non-endotoxin pyrogens, thus providing assays with advantages over other media supplements. For example, as shown in the Examples, it was found that assays using either one of the commonly used supplements, hAB serum or FBS, cannot effectively detect both endotoxins and non-endotoxin pyrogens at low concentrations. However, assays using hPL consistently demonstrated strong sensitivity to both endotoxins and non-endotoxin pyrogens, particularly compared to FBS and hAB.

[0006] Thus, in a first aspect, there is provided a method for detecting pyrogens in a sample, comprising: i) providing one or more samples; ii) contacting the sample with peripheral blood mononuclear cells (PBMCs) in an incubation medium containing human platelet lysate (hPL); iii) determining the response of the PBMCs; A method is provided that includes:

[0007] In some embodiments, the method is a monocyte activation test. In some embodiments, the incubation medium has a volume of up to 300 μL or up to 250 μL per sample, preferably 20-250 μL, more preferably 30-175 μL, and most preferably 50-110 μL. In some embodiments, the contacting in step ii) is performed in a standardized 96-well or 384-well plate, preferably a 96-well plate. In some embodiments, the incubation medium comprises 0.05-20 vol.%, preferably 0.5-4 vol.%, more preferably 0.8-3 vol.%, even more preferably 1-2.5 vol.%, and most preferably 1.2-2.2 vol.%, e.g., about 2 vol.%, of human platelet lysate. In some embodiments, the response of PBMCs that is determined is the excretion of inflammatory cytokines, such as IL-6, IL-1β, IL-8, TNF-α, MCP-1, IFN-α, IFN-β, IFN-γ, IFN-λ, prostaglandins, or high mobility group proteins. In some embodiments, the response of PBMCs is higher than the response of PBMCs in comparative methods that differ only in the replacement of human platelet serum with human AB serum or with fetal bovine serum, or the absence of human platelet serum. In some embodiments, the response of PBMCs is determined by ELISA assay. In some embodiments, PBMCs are cultured at up to 500×1000 cells / cm. 2 , preferably up to 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. 350×1000 cells / cm 2 , optionally about 300 x 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 2In some embodiments, the limit of quantification of lipopolysaccharides is less than 0.01 EEU / mL, and / or the limit of quantification of triacylated lipopeptides is less than 0.1 ng / mL, and / or the limit of quantification of bacterial proteins is less than 1 ng / mL, preferably less than 0.5 ng / mL. In some embodiments, in step iii), the determined responses of PBMCs for multiple identical samples have a coefficient of variation of up to 30%. In some embodiments, the incubation medium has a volume of about 80 to about 120 μL, and the PBMCs are present at a density of about 90 to about 130×1000 cells / cm. 2 In a further aspect, there is provided a method for releasing a pharmaceutical composition or medical device for use, the method comprising subjecting a sample from the pharmaceutical composition or medical device to the method of the first aspect. In a further aspect, there is provided a kit of parts comprising a pyrogen or endotoxin standard and a vial containing human platelet lysate and optionally containing PBMCs.

[0008] [Description of the Invention] In one aspect, there is provided a method for detecting pyrogens in a sample, comprising: i) providing one or more samples; ii) contacting the sample with peripheral blood mononuclear cells (PBMCs) in an incubation medium containing human platelet lysate (hPL); iii) determining the response of the PBMCs; A method is provided that includes:

[0009] This method is interesting because it has advantages over methods currently available in the art: it has no requirement for the use of test animals, it has improved sensitivity to endotoxins and / or non-endotoxin pyrogens, it has an improved range of response to endotoxins and / or non-endotoxin pyrogens, it has improved reliability of PBMC response determination, it has low variability between assays, and therefore results in lower costs. The steps of the method are preferably performed in numerical order.

[0010] [Pyrogen] Pyrogens are well known in the art. Pyrogens are substances that can induce an immune response in a subject by activating a cascade of immunological processes, typically characterized by an increase in the body's internal 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. The pyrogen may be an exogenous pyrogen. "Exogenous" or "external" pyrogen refers to a pyrogen originating from outside the subject's body. The pyrogen may also be an endotoxin, preferably lipopolysaccharide (LPS). Endotoxins, such as lipopolysaccharides, are cellular components of bacteria, such as gram-negative bacteria, and constitute the main 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 acute respiratory distress syndrome (ARDS). The pyrogen may be a non-endotoxin pyrogen (NEP). Non-endotoxin pyrogens include microbe-associated molecular patterns (MAMPs) and pathogen-associated molecular patterns (PAMPs), including, for example, bacterial cell components such as bacterial proteins (e.g., flagellin), peptidoglycan, lipoproteins, lipoteichoic acid, fibroblast-stimulating lipopeptide 1, macrophage-activating lipopeptide 2, viral pyrogens, yeast pyrogens, and fungal pyrogens (e.g., yeast or fungal polysaccharides). In some cases, the flagellin may be derived from a gram-positive bacterium, such as Bacillus subtilis.

[0011] Pyrogens can be product- or process-related impurities present in pharmaceutical compositions or on surfaces (e.g., of medical devices). Examples of pyrogenic impurities are chemicals 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), and any other impurities that exhibit pyrogenic properties.

[0012] Pyrogens may be damage-associated molecular patterns (DAMPs), which typically refer to biomolecules released by dead 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 biomolecules released by pyrogenic cells. Pyrogens may also be Toll-like receptor 1 / 2 (TLR1 / 2) agonists. Pyrogens may also be media components of pharmaceutical compositions. 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, disintegrants, lubricants, sweeteners, flavoring agents, dyes, etc. The pyrogen may be a triacylated lipopeptide. The pyrogen may be an endogenous pyrogen. "Endogenous" or "internal" pyrogens refer to pyrogens produced by a subject's body after contact with an exogenous pathogen. Endogenous pyrogens may be associated with an inflammatory response. Endogenous pyrogens may be DAMPs. Endogenous pyrogens may be Toll-like receptor 1 / 2 (TLR1 / 2) agonists. Examples of endogenous pyrogens include cytokines and chemokines.

[0013] 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 of Gram-negative bacteria, preferably lipopolysaccharide. "Gram-negative" bacteria refers to bacteria that generally do not retain the crystal violet stain used in standard Gram staining, unlike "Gram-positive" bacteria, which generally retain the stain. In some embodiments, the Gram-negative bacteria are pathogenic or potentially pathogenic bacteria. Examples of pathogenic or potentially 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 cellular component of a Gram-positive bacterium, preferably a bacterial protein (e.g., flagellin). In some embodiments, the Gram-positive bacterium is pathogenic or potentially pathogenic. Examples of pathogenic or potentially pathogenic Gram-positive bacteria include bacteria of the genera Streptococcus, Staphylococcus, Corynebacterium, Listeria, Bacillus (e.g., Bacillus subtilis), and Clostridium. 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 media component of a pharmaceutical composition. In some embodiments, the pyrogen is a Toll-like receptor 1 / 2 (TLR1 / 2) agonist. In some embodiments, the pyrogen is a triacylated lipopeptide. 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. In some embodiments, the endogenous pyrogen is a Toll-like receptor 1 / 2 (TLR1 / 2) agonist.

[0014] [Step i) Sample provision] In step i) of the method, one or more samples are provided. The sample may be taken (derived) from an original source, for example, a product such as a pharmaceutical composition or a medical device. The sample may also be a subsample taken from the original sample (or another subsample). The sample may also be, or be taken from, a subsample resulting from dilution or concentration of the original sample.

[0015] The sample may also be a duplicate of the original sample or subsample. The duplicate samples are preferably intended to be identical, more preferably they are identical. When the sample is a duplicate of the original sample or subsample, at least two, at least three, or at least four, preferably at least four, duplicates of the original sample or subsample are provided. The duplicates 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.

[0016] The sample may be taken from a pharmaceutical composition to be tested for the presence of a pyrogen, e.g., a therapeutic composition, a diagnostic composition, or a composition for preventing or reducing the symptoms of a disease or condition (such as a vaccine). The pharmaceutical composition may be in any form. In some embodiments, the pharmaceutical composition is a vaccine.

[0017] Samples can be taken from surfaces to be tested for the presence of pyrogens, such as the surface of a medical device or equipment. Examples of medical devices and equipment include bedpans, cannulas, 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, sterilization equipment, cesspits, nasal tubes, scalpels, nebulizers, ophthalmoscopes, otoscopes, pipettes, rectoscopes, radiographers, blood pressure monitors, thermometers, tongue depressors, blood transfusion 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 for sample preparation.

[0018] 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 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 6, 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 carried out using a 384-well plate.

[0019] In some embodiments, in step i), an additional control sample is prepared. The control sample may contain a pyrogen standard (positive control) or may not contain pyrogen (negative control). Inclusion of a pyrogen positive control sample with a known pyrogen concentration may increase the accuracy of quantification. For example, a standard curve may be prepared 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.

[0020] In some embodiments, the control sample is an endotoxin sample, preferably a lipopolysaccharide (LPS) sample. Endotoxin is typically measured in endotoxin units per mL (EEU / mL or EU / mL, where EEU is "equivalent endotoxin unit"). 1 EEU / mL (EU / mL) is equivalent to about 0.1 to 0.2 ng of endotoxin / mL of solution, preferably 0.15 ng / mL. In embodiments where the control sample is an endotoxin, 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, more preferably about 0.1 to about 0.2 EU / mL.

[0021] In some embodiments, the control sample is a non-endotoxin pyrogen (NEP) sample, e.g., a cellular component such as a bacterial protein (e.g., flagellin). In some embodiments, the control sample is a triacylated lipopeptide. In some embodiments, the control sample is a Toll-like receptor 1 / 2 (TLR1 / 2) agonist. In some embodiments, the Toll-like receptor 1 / 2 (TLR1 / 2) agonist in the non-endotoxin control sample is a synthetic molecule. Such molecules are commercially available, e.g., PAM3CSK4 (CAS No.: 112208-00-1). In some embodiments, where the control sample is a non-endotoxin pyrogen sample, the control sample preferably contains about 0.01 to about 125 ng / mL of non-endotoxin pyrogen. In some embodiments, the control sample contains about 0.1 to about 20 ng / mL, preferably about 0.1 to about 15 ng / mL, and more preferably about 1 to about 10 ng / mL of non-endotoxin pyrogen.

[0022] [Step ii) Contact with PBMCs] In step ii), one or more samples are contacted with peripheral blood mononuclear cells (PBMCs) in an incubation medium containing human platelet lysate (hPL). In some embodiments, the contacting is with whole peripheral blood containing PBMCs, or a fraction thereof. In some embodiments, the contacting is with isolated PBMCs. PBMC-containing blood fractions and isolated PBMCs can be obtained by standard methods, for example, using leukocyte depletion and / or density gradient centrifugation.

[0023] In some embodiments, the contacting is with a PBMC cell line. In some embodiments, the contacting is with immortal or non-immortal cells, preferably non-immortal cells. 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, Council of Europe).

[0024] In some embodiments, the contacting is with fresh PBMCs. In preferred embodiments, the contacting is with cryopreserved PBMCs. Cryopreservation of PBMCs may be performed according to standard procedures, e.g., as described in standard handbooks such as Hubel, A., 2018: Preservation of Cells: A Practical Manual, 1st Edition, Wiley-Blackwell, NJ, USA. In some embodiments, the PBMCs are mammalian, preferably 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. Preferred PBMCs are non-immortal PBMCs, preferably non-immortal monocytes.

[0025] The method is preferably a monocyte activation test or part of a monocyte activation test. More preferably, the monocyte activation test follows the guidelines set out in the European Pharmacopoeia monograph 2.6.30 (supra).

[0026] The contacting with PBMCs in step ii) can be the addition of one or more samples to a medium containing PBMCs. It can also be the addition of a medium containing PBMCs to one or more samples. It can be performed in any suitable container, such as a microplate (having one or more wells), a tube (such as an Eppendorf tube), a flask (such as an Erlenmeyer flask), a bottle (such as a Scott bottle), a fermenter, etc. In a preferred embodiment, the contacting is performed in a standardized 96-well plate. In some embodiments, the contacting is performed in a standardized 384-well plate. Standardized well plates are widely available from commercial suppliers. Suitable standards are the ANSI / SLAS standards, preferably all five: 1-2004 (R2012), 2-2004 (R2012), 3-2004 (R2012), 4-2004 (R2012), and 6-2012 (R2012).

[0027] When 96-well or 384-well plates are used, a single sample can be placed in each well. The present invention advantageously allows for the use of 384-well plates. The use of 384-well plates has the additional advantage of allowing for higher detection throughput (when more samples can be tested simultaneously) and reducing reagent requirements and overall costs. In the 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 / 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:

[0028] [Table 1]

[0029] Alternatively, the following can be applied, with two dilutions per test sample and omitting the NEP control:

[0030] [Table 2]

[0031] It should be noted that a sample of the product to be analyzed may result in multiple samples being provided in the method for detecting pyrogens. For example, in the table above, a single test sample 1 results in multiple samples, where each well is recognized as containing a provided sample. This distinction would be apparent if not made explicit from the context.

[0032] The PBMCs, which preferably contain monocytes, are preferably cultured at a concentration of 10 ... 2 Measured in units (cm 2 refers to the growth area, which is preferably an area that is a cross section of the well; preferred wells are flat-bottom wells). Density of PBMCs in the context of this disclosure refers to the density of PBMCs used per contacted sample. Those skilled in the art will appreciate that the number of cells / cm within a container, preferably within the well of a standardized 96-well or 384-well plate, can be present at a specific density. 2 The density in units of growth area (cm) can be determined, for example, by using a cell counter or by using microscopic techniques. 2 ), cell concentration (cells / mL) and volume (mL). This density is a commonly used parameter, and one skilled in the art will appreciate that some dead cells may be present in the population. In some embodiments, PBMCs, preferably including monocytes, are present at a density of up to 500 x 1000 cells / cm. 2 , preferably up to 250 x 1000 cm 2 It exists at a density of

[0033] In some embodiments, PBMCs, preferably containing monocytes, have a density of about 10 x 1000 cells / cm 2~ about 350 x 1000 cells, preferably about 50 x 1000 cells / cm 2 ~Approx. 150×1000 cells / cm 2 , preferably about 60 x 1000 cells / cm 2 ~Approx. 140×1000 cells / cm 2 , 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, 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

[0034] The incubation medium may have a specific volume during the contacting step, preferably measured in mL or μL. The volume of the incubation medium in the context of the present disclosure refers to the volume per sample. In some embodiments, the incubation medium has a volume of up to 300 μL per sample or up to 250 μL per sample, preferably up to 250 μL per sample. In preferred embodiments, it has a volume of up to 200 μL per sample, up to 175 μL per sample, or up to 150 μL per sample, preferably up to 250 μL or about 250 μL per sample. In some embodiments, the incubation medium has a volume of 20-250 μL, preferably 30-175 μL, more preferably 50-110 μL.

[0035] 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.

[0036] 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. In some embodiments, the incubation medium has a volume of 1 to 99 μL, preferably 30 to 70 μL, for example, 33 μL or 66 μL.

[0037] 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, 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, 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

[0038] In some embodiments, the incubation medium has a volume of 30-140 μL and the PBMCs, preferably monocytes, are cultured at a density of approximately 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, 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, 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

[0039] In some embodiments, the incubation medium has a volume of 80-120 μL and the PBMCs, preferably monocytes, are cultured at a density of approximately 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, 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

[0040] In some embodiments, the incubation medium has a volume of 50-110 μL and the PBMCs, preferably monocytes, are cultured at a density of approximately 10×1000 cells / cm. 2 ~Approx. 250×1000 cells / cm 2 In some embodiments, the incubation medium has a volume of 50-110 μL and the PBMCs, 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 50-110 μL and the PBMCs, 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

[0041] 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, 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, 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

[0042] In some embodiments, the incubation medium has a volume of 30-100 μL and the PBMCs, preferably monocytes, are cultured at a density of approximately 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, 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 30-100 μL and the PBMCs, 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

[0043] In some embodiments, the incubation medium has a volume of 50-100 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 50-100 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 50-100 μL and the PBMCs, 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 80-100 μL and the PBMCs, preferably monocytes, are cultured at a density of approximately 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, 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, 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 100 μL or about 100 μL, and the PBMCs, 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 0.32 cm 2 110 × 1000 cells / cm in a 96-well plate with wells having a surface area of 2equals approximately 35,200 cells / well.

[0046] The incubation medium containing human platelet lysate (hPL) may 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 set forth in the European Pharmacopoeia monograph 2.6.30 (see above). A preferred incubation medium is RPMI (Roswell Park Memorial Institute) medium, more preferably RPMI 1640 medium, which is widely available commercially. However, other suitable media may be considered, such as DMEM (Dulbecco's Modified Eagle's Medium), EMEM (Eagle's Minimum Essential Medium), Ham's F-10 or F-12 medium, Iscove's Modified Dulbecco's Medium (IMDM), and α-MEM (Minimum Essential Medium α), all of which are commercially available. Further examples of suitable media are provided in the Examples section herein below. In some embodiments, the incubation medium is not α-MEM.

[0047] As used herein, human platelet lysate (hPL) has its common meaning in the art. It refers to a media supplement obtained by lysis of human platelets. In some embodiments, hPL is autologous with respect to PBMCs. In some embodiments, hPL is allogenic with respect to PBMCs. Human platelet lysate can be prepared by standard methods available in the art, for example, as described in Burnouf et al., 2016, Biomaterials 76:371-387, Mohamed et al., 2020, Blood Res 55:35-43, and Guiotto et al., 2020, J Transl Med 18:351. Human platelet lysate, e.g., platelet concentrate, can be prepared from fresh or stored whole blood. Platelet concentrate can be prepared using standard methods; for example, it can be prepared from anticoagulated whole blood according to the buffy coat method or PRP (platelet-rich plasma) method, or it can be prepared using platelet apheresis. Typically, the buffy coat method involves centrifugation of whole blood followed by pooling of four buffy coat units plus one plasma unit from four donors. After a second centrifugation step, the platelet concentrate is filtered through a leukocyte-depleting filter and stored. Typically, the PRP method involves pooling of four or five blood units followed by centrifugation to separate the blood cells from an upper layer consisting of platelets mixed with plasma. Platelet apheresis typically involves processing the blood through an apheresis machine that uses centrifugation to remove platelets.

[0048] hPL can be prepared from platelet concentrates by repeated freeze / thaw cycles, e.g., one to five freeze cycles at temperatures between about -30°C and about 80°C, followed by thawing at 37°C. As another example, it can be prepared by sonicating the platelet concentrate for up to 30 minutes, e.g., at a frequency of about 20 kHz. Sonication can optionally be combined with repeated freeze / thaw cycles. As another example, it can be prepared by direct platelet activation in the platelet concentrate, including the addition of calcium salts (e.g., CaCl), which activate the platelet thrombin cascade and cause platelet lysis. Alternatively, platelet lysis can be induced by solvent / detergent treatment. In some cases, preparation of human platelet lysate involves the addition of an anticoagulant, e.g., heparin, to whole blood or platelet concentrates. In some cases, once the human platelets have been lysed, a further centrifugation step is performed to remove platelet fragments. The human platelet lysate can be used fresh (i.e., immediately after preparation), or alternatively, a stored lysate, e.g., a human platelet lysate frozen after thawing, can be used.

[0049] Human platelet lysate can also be obtained from commercial vendors, such as those provided by Mediatech (Manassas, VA, USA), StemCell Technologies GmbH (Koeln, Germany; catalog number 06960), Sigma-Aldrich (St Louis, MO, USA; SCM141 and SCM142), and others. In some embodiments, the hPL comprises human platelet lysate from about 7 to about 90 mg / mL of total protein.

[0050] Preferably, the human platelet lysate contains one or more growth factors. In some embodiments, the growth factors are selected from PDGF, PDGF-AA, PDGF-AB, PDGF-BB, TGFβ, VEGF, bFGF, EGF, BDNF, IGF-1, and HGF, preferably PDGF-AA, PDGF-AB, PDGF-BB, TGFβ, VEGF, and IGF-1. In some embodiments, the PDGF concentration in the hPL is about 0 to 70 ng / mL, preferably about 10 to about 70 ng / mL. In some embodiments, the PDGF-AA concentration in the hPL is about 0 to about 240 ng / mL, preferably about 0.01 to about 240 ng / mL. In some embodiments, the PDGF-AB concentration in the hPL is about 0 to about 580 ng / mL, preferably about 0.01 to about 580 ng / mL. In some embodiments, the PDGF-BB concentration in hPL is about 0 to about 23 ng / mL, preferably about 0.01 to about 23 ng / mL. In some embodiments, the TGFβ concentration in hPL is about 0 to about 250 ng / mL, preferably about 0.04 to about 250 ng / mL. In some embodiments, the VEGF concentration in hPL is about 0 to about 0.8 ng / mL, preferably about 0.15 to about 0.8 ng / mL. In some embodiments, the bFGF concentration in hPL is about 0 to about 5.5 ng / mL, preferably about 0.04 to about 5.5 ng / mL. In some embodiments, the EGF concentration in hPL is about 0 to about 20 ng / mL, preferably about 0.01 to about 20 ng / mL. In some embodiments, the BDNF concentration in hPL is about 0 to about 100 ng / mL, preferably about 0.03 to about 100 ng / mL. In some embodiments, the IGF-1 concentration in hPL is about 0 to about 500 ng / mL, preferably about 0.1 to about 500 ng / mL. In some embodiments, the HGF concentration in hPL is about 0 to about 2.6 ng / mL, preferably 0.1 to about 2.6 ng / mL.

[0051] A highly preferred hPL is PLTMax Human Platelet Lysate, available from Millipore (Product No. SCM141; Catalog No. 6D0352). Preferably, the hPL has a pH of 6.8-7.8, a total protein content of 4.0-6.5 g / dL, endotoxin USP <10 EU / mL, no Mycoplasma detected, and a sterility test negative for growth. Preferably, the hPL has been sterile filtered, e.g., with a 0.2 μm filter. These highly preferred hPLs preferably have a PDGF concentration of at least about 5 ng / mL, preferably about 10 to about 70 ng / mL.

[0052] In some embodiments, the incubation medium contains 0.05 to 20 vol.%, preferably 0.2 to 10 vol.%, more preferably 0.5 to 5 vol.%, even more preferably 1 to 4 vol.%, most preferably 0.5 to 2.5 vol.%, for example, about 2 vol.% human platelet lysate. In some embodiments, it contains 0.1 to 15 vol.%, preferably 0.1 to 10 vol.%, more preferably 1 to 4 vol.%, most preferably 0.5 to 2.5 vol.%, for example, about 2 vol.% human platelet lysate. In a preferred embodiment, the incubation medium contains 0.05 to 20 vol.%, preferably 0.5 to 4 vol.%, more preferably 0.8 to 3 vol.%, even more preferably 1 to 2.5 vol.%, most preferably 1.2 to 2.2 vol.%, for example, about 2 vol.% human platelet lysate. In some embodiments, the incubation medium comprises 1 or about 1 vol.% human platelet lysate. In preferred embodiments, the incubation medium comprises 2 or about 2 vol.% human platelet lysate. In some embodiments, the incubation medium comprises 3 or about 3 vol.% human platelet lysate. In some embodiments, the incubation medium comprises 4 or about 4 vol.% human platelet lysate. In preferred embodiments, the incubation medium does not comprise 1.5-2.5 vol.% or 4.5-5.5 vol.% hPL, and in particular, the incubation medium does not comprise or consist of α-MEM containing 1.5-2.5 vol.% or 4.5-5.5 vol.% hPL.

[0053] The one or more samples to be contacted with peripheral blood mononuclear cells (PBMCs) are incubated for a time sufficient to induce a PBMC, preferably monocyte, response. 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, contacting is carried out for up to 72 hours, preferably up to 36 hours, preferably up to 30 hours, more preferably up to 24 hours, even more preferably up to 18 hours, and most preferably up to 16 hours.

[0054] The incubation is preferably carried out under cell culture conditions, preferably human cell culture conditions. 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 CO2 level of 5% for at least 16 hours. After 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.

[0055] [Step iii to determine the response of PBMCs)] In step iii), the response of PBMCs, preferably mammalian PBMCs, more preferably human PBMCs, and 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 excretion, preferably excretion, of cytokines, e.g., inflammatory or anti-inflammatory cytokines, most preferably inflammatory cytokines. Examples of inflammatory cytokines include IL-6, IL-1β, IL-2, IL-8, IL-12, IL-17, IL-18, TNF-α, MCP-1, IFN-α, IFN-β, IFN-γ, and IFN-λ, preferably IL-6, IL-1β, IL-8, TNF-α, MCP-1, and more preferably IL-6. Examples of anti-inflammatory cytokines include IL-4, IL-10, IL-11, IL-13, and TGF-β. IL-6 may be thought to exert an anti-inflammatory role under some circumstances, and therefore, in some embodiments, IL-6 is an example of an anti-inflammatory cytokine. The response of PBMCs may be the production and / or excretion of prostaglandins, preferably excretion, an example of which is PGE2. The response of PBMCs may be the production and / or excretion of high mobility group proteins, preferably excretion, an example of which is HMGB1. The response may be the production and / or excretion of neopterin. In some embodiments, the response of PBMCs that is determined is the expression of surface activation markers.

[0056] In some embodiments, the PBMC response that is determined is the production and / or excretion, preferably excretion, of inflammatory cytokines such as IL-6, IL-1β, IL-8, TNF-α, MCP-1, IFN-α, IFN-β, IFN-γ, IFN-λ, prostaglandins, or high mobility group proteins. Those skilled in the art will appreciate that determining the PBMC response can include combined determination of the production and / or excretion, preferably excretion, of multiple inflammatory cytokines, prostaglandins, and / or high mobility group proteins. In a preferred embodiment, the PBMC response that is determined is the production and / or excretion, preferably excretion, of IL-6.

[0057] The determination of the response of PBMCs can be carried out directly after the contacting step in the same or a different container, or the incubation mixture can be stored, optionally frozen, and used for determining the response at a different time point. Generally, the response of PBMCs correlates with the detection of pyrogens. If no response is detected, it is possible that no pyrogens are detected. The determination of the response of PBMCs can be carried out, for example, by quantitative PCR, flow cytometry techniques (such as FACS analysis), or immunoassays, preferably ELISA assays. Those skilled in the art know how to perform such immunoassays, and a description thereof can be found in The Immunoassay Handbook: Theory and Applications of Ligand Binding, ELISA and Related Techniques, 2013, 4. th The methods for determining IL-6 activity can be found in standard handbooks, such as "The Journal of Clinical Chemistry, Vol. 1, No. 1, pp. 111-115, 1999," 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 when used in the methods of the present invention, as they allow for high-throughput testing of multiple samples.

[0058] 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-1β, IL-8, TNF-α, MCP-1, IFN-α, IFN-β, IFN-γ, IFN-λ, 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 antibodies are commercially available, for example, clone 13A5 from MabTech AB, Nack Strand, SE. In some embodiments, the ELISA assay is performed in a standardized 96-well or 384-well plate, preferably a 96-well plate. Examples of the application of ELISA in connection with the present disclosure are provided in the Examples section hereinafter.

[0059] In some embodiments, the response of PBMCs is higher than the response of PBMCs in a comparison method (assay) that differs only in that human platelet serum is replaced with a different medium supplement (or in that no medium supplement is used). "Medium supplement," as used herein, refers to an additional medium component that can promote a response of PBMCs, preferably human PBMCs, and more preferably human monocytes, such as activation, expression of surface activation markers, cytokine production, prostaglandin production, or high mobility group protein production. Preferably, the comparison method differs only in the replacement of human platelet serum with human AB serum (hAB) or fetal bovine serum (FBS), or the absence of human platelet serum. Human AB serum and fetal bovine serum are used herein in their common sense and can be obtained from commercial vendors, such as the FBS catalog provided by VWR Avantor (Radnor, PA, USA) and hAB provided by Merck (West Point, PA, USA).

[0060] In some embodiments, where the pyrogen is an endotoxin, preferably a lipopolysaccharide, the response of PBMCs is higher than the response of PBMCs in a comparative method that differs only in the replacement of human platelet serum with human AB serum (hAB) or fetal bovine serum (FBS), preferably human AB serum. In some embodiments, where the pyrogen is a non-endotoxin pyrogen, preferably a triacylated lipopeptide or a bacterial cell component, more preferably a Toll-like receptor 1 / 2 (TLR1 / 2) agonist (e.g., PAM3CSK4) or a bacterial protein (e.g., flagellin), the response of PBMCs is higher than the response of PBMCs in a comparative method that differs only in the replacement of human platelet serum with human AB serum (hAB) or fetal bovine serum (FBS), preferably fetal bovine serum. A "higher" PBMC response may be at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 250%, or at least 300% higher than a comparison method that differs only in the replacement of human platelet serum with a different media supplement, preferably with hAB or FBS (or in that no media supplement is used). PBMC responses can be determined as previously described herein. For example, in embodiments in which the determined PBMC response is IL-6 production and / or excretion, preferably excretion, a higher PBMC response means that more IL-6 is produced and / or excreted by PBMCs.

[0061] In some embodiments, the sensitivity of the method for pyrogen detection is higher than a comparative method that differs only in that human platelet serum is replaced with a different media supplement, preferably hAB or FBS (or in that no media supplement is used). Sensitivity can preferably be expressed using the limit of quantitation (LoQ), which defines the lowest concentration of pyrogen in a sample that is detectable by a method. A lower LoQ indicates increased sensitivity by a method. Those skilled in the art will understand that the exact calculation used in determining the LoQ may vary based on the assay used to assess PBMC response. For example, when a spectrophotometric immunoassay such as ELISA is used, the LoQ is preferably calculated as the mean OD of the blank + 10, determined at approximately 450 nm. * It can be determined by identifying the first average signal (OD) of the sample that exceeds a cutoff value, which can be defined as the standard deviation of the blank. Optionally, the background OD determined at 630 nm can be subtracted from the signal before analysis. Further examples of LoQ determination are provided in the Examples.

[0062] In some embodiments, the limit of quantitation for endotoxin, preferably lipopolysaccharide, is less than 0.1 EEU / mL, preferably less than 0.05 EEU / mL, more preferably less than 0.02 EEU / mL, and most preferably less than 0.01 EEU / mL. In some embodiments, the limit of quantitation for triacylated lipopeptides, preferably Toll-like receptor 1 / 2 (TLR1 / 2) agonists (e.g., PAM3CSK4), is less than 1 ng / mL, preferably less than 0.5 ng / mL, more preferably less than 0.2 ng / mL, and most preferably less than 0.1 ng / mL. In some embodiments, the limit of quantitation for bacterial cell components, preferably bacterial proteins (e.g., flagellin), is less than 2 ng / mL, preferably less than 1.5 ng / mL, more preferably less than 1 ng / mL, and most preferably less than 0.5 ng / mL. Preferably, the limit of quantitation is determined by spectrophotometric immunoassay, more preferably ELISA, and even more preferably ELISA, at about 450 nm.

[0063] In some embodiments, the LoQ of an endotoxin, preferably lipopolysaccharide, is lower than the LoQ exhibited by a comparative method that differs only in the substitution of human platelet serum with human AB serum (hAB) or fetal bovine serum (FBS), preferably human AB serum. In some embodiments, the LoQ of a non-endotoxin pyrogen, preferably a triacylated lipopeptide or bacterial cell component, more preferably a Toll-like receptor 1 / 2 (TLR1 / 2) agonist (e.g., PAM3CSK4) or a bacterial protein (e.g., flagellin), is lower than the LoQ exhibited by a comparative method that differs only in the substitution of human platelet serum with human AB serum (hAB) or fetal bovine serum (FBS), preferably fetal bovine serum. A "lower" LoQ is at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 250%, or at least 300% lower than the LoQ exhibited by a comparative method that differs only in the replacement of human platelet serum with a different media supplement, preferably hAB or FBS (or in that no media supplement is used).

[0064] The detection methods of the present disclosure exhibit low variability between measurements, especially when multiple identical samples are tested. Thus, in some embodiments, the determined responses of PBMCs for multiple identical samples have a coefficient of variation (ratio of standard deviation to the average measurement) of up to 30%. In some embodiments, the determined responses of PBMCs for multiple identical samples have a coefficient of variation of up to 25%, or 24%, 23%, 22%, or 21%. Preferably, the determined responses of PBMCs for multiple identical samples have a coefficient of variation of up to 20%. More preferably, the determined responses of PBMCs for multiple identical samples have a coefficient of variation of up to 15%. Even more preferably, the determined responses of PBMCs for multiple identical samples have a coefficient of variation of up to 10%, and most preferably, up to 9.5%.

[0065] The detection method of the present disclosure further exhibits low variability between measurements of multiple samples of different concentrations. This variability can be determined, for example, by constructing a PBMC response / pyrogen concentration curve (e.g., using pyrogen standards as described later in this specification) and applying nonlinear regression analysis. Thus, in some cases, the concentration curve is a standard curve. Typically, a four- or five-parameter logistic (4PL or 5PL) regression model is applied in bioassay analysis, preferably ELISA. Such analyses are well known to those skilled in the art and can be performed using commercially available software, such as Graphpad Prism (GraphPad Software, San Diego, CA, USA). A higher quality detection method is characterized by a lower standard deviation of the measurements. Furthermore, it also exhibits a higher "goodness of fit" (R ) of the 4PL or 5PL model fit to the PBMC response / pyrogen concentration curve. 2 ). Furthermore, higher quality methods are characterized by an increased (dynamic) measurement range, which may be defined as the absolute difference between the lowest and highest values ​​of the fit. An increased (dynamic) range may enhance the accuracy of measurements within curves with similar slopes, i.e., provide a larger concentration range over which pyrogens can be accurately detected.

[0066] In some embodiments where multiple samples of different concentrations are provided, the goodness of fit of the method (R) for a model fit of 4PL or 5PL, preferably 4PL, is measured. 2 ) is higher than for a comparable method that differs only in that human platelet serum is replaced with a different media supplement, preferably hAB or FBS (or in that no media supplement is used). A higher goodness of fit can be at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, or at least 50% higher. In some embodiments, the R of a method in a 4PL model fit 2 is at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.9, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, or at least 0.98, preferably at least 0.95.

[0067] In some embodiments in which multiple samples of different concentrations are provided, the (dynamic) range of the method is increased relative to a comparable method that differs only in that human platelet serum is replaced with a different media supplement, preferably hAB or FBS (or in that no media supplement is used). The increase in (dynamic) range may be at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 250%, or at least 300%.

[0068] In some embodiments in which multiple samples of different concentrations are provided, the standard deviation of measurements obtained in the method is lower than that of a comparable method that differs only in that human platelet serum is replaced with a different media supplement, preferably hAB or FBS (or in that no media supplement is used). A lower standard deviation can be at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least 200% lower. In some embodiments, the standard deviation of the method has a value of at most about 0.3, preferably at most about 0.2, more preferably at most about 0.1, and most preferably at most about 0.08. The standard deviation of the replicates is preferably calculated by summing the squares of the distances of each replicate from the mean of that set of replicates, then dividing that sum by the degrees of freedom, and then calculating the square root.

[0069] The detection method of the present disclosure allows for the detection of pyrogens in samples taken from various products intended for therapeutic use or treatment, preferably for human therapeutic use or treatment. Examples of such products include pharmaceutical compositions, medical devices, and medical equipment, as already described herein. If no pyrogens are detected in / on the product, they can be safely released (removed) for use. In this context, release can be recognized as approval that the product complies with certain (relevant) standards while being made publicly available.

[0070] Thus, in one aspect, there is provided a method for releasing a product, preferably a pharmaceutical product, for use, comprising subjecting a sample from the product to a method for detecting pyrogens as previously described herein. The product is preferably released only if no or low pyrogen levels are detected. This may depend on which standard is being adhered to. In some embodiments, there is provided a method for releasing a pharmaceutical composition or medical device for use, comprising subjecting a sample from the pharmaceutical composition or medical device to a method for detecting pyrogens as previously described herein.

[0071] 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 standard of known concentration. A preferred standard is a lipopolysaccharide (LPS) standard. The standard may be a non-endotoxin pyrogen standard, preferably a standard comprising a triacylated lipopeptide or a bacterial cell component, more preferably a standard comprising a Toll-like receptor 1 / 2 (TLR1 / 2) agonist (e.g., PAM3CSK4) or a bacterial protein (e.g., flagellin). The pyrogen standard may be prepared according to the Ph.Eur. Guidelines (Monograph 2.6.30, supra) or may be obtained from a commercial source, e.g., EDQM (European Directorate for the Quality of Medicines & Healthcare; see, e.g., Ph.Eur. Reference Standards: Orders and Catalogs provided by EDQM). Examples of pyrogen standards are provided in the Examples section below. A preferred kit of parts includes a pyrogen or endotoxin standard and a vial containing human platelet lysate, and optionally, PBMCs. Preferably, the kit further includes an incubation medium, optionally in combination with the human platelet lysate.

[0072] The pyrogen standard may correspond to one or more samples. Preferably, multiple samples, each containing different concentrations of pyrogen, allow for the preparation 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. Preferably, at least two, more preferably at least three, and most preferably at least four replicate (identical) samples may be provided. The kit may also include PBMCs, preferably monocytes, more preferably mammalian monocytes, most preferably human monocytes. Suitable PBMCs have been described previously herein. The kit may also include one or more 96-well or 384-well plates. 384-well plates allow for increased testing throughput while minimizing reagent and overall costs compared to standard pyrogen detection methods, e.g., monocyte activation tests using 96-well plates.

[0073] Thus, in one aspect, a kit of parts is provided that includes a pyrogen standard, PBMCs, human platelet lysate, and one or more 96-well or 384-well plates. In some embodiments, the kit is a monocyte activation test (MAT) kit. In some embodiments, the kit optionally further includes an incubation medium in combination with the human platelet lysate as previously described herein. In some embodiments, the human platelet lysate is present at a concentration of 0.05-20 vol%, preferably 0.2-10 vol%, more preferably 0.5-5 vol%, even more preferably 1-4 vol%, and most preferably 0.5-2.5 vol%, e.g., about 2 vol%. In some embodiments, the human platelet lysate is present at a concentration of 0.1-15 vol%, preferably 0.1-10 vol%, more preferably 1-4 vol%, and most preferably 0.5-2.5 vol%, e.g., about 2 vol%.

[0074] In some embodiments, the kit is suitable for simultaneous testing of at least three different products, e.g., different pharmaceutical compositions, medical devices, or medical equipment, for the presence of pyrogens. In some embodiments, the kit is suitable for simultaneous testing of at least four different products. In some embodiments, the kit is suitable for simultaneous testing of at least five different products. In some embodiments, the kit is suitable for simultaneous testing of at least six different products. In some embodiments, the kit is suitable for simultaneous testing of at least seven different products. In some embodiments, the kit is suitable for simultaneous testing of at least eight different products. In some embodiments, the kit is suitable for simultaneous testing of at least nine different products. In some embodiments, the kit is suitable for simultaneous testing of at least ten different products. Testing is preferably performed in accordance with the Ph.Eur.Guidelines for the Detection of Pyrogens and Endotoxins (Monograph 2.6.30, supra).

[0075] "Parallel" testing refers to the testing of samples corresponding to different products in a single plate, allowing for increased testing throughput and minimization of reagent and overall costs.

[0076] In some embodiments, the incubation medium in each sample tested per well has a volume of up to 300 μL, preferably up to 250 μL. In some embodiments, it has a volume of about 175 μL. In some embodiments, it has a volume of up to 170, 165, 160, 155, or 150 μL. In some embodiments, the incubation medium in each sample tested per well has a volume of 20 to 250 μL, preferably 30 to 175 μL, and more preferably 50 to 110 μL. In some embodiments, the incubation medium in each sample tested per well has a volume of 20 to 150 μL, preferably 30 to 140 μL, more preferably 40 to 130 μL, more preferably 50 to 120 μL, more preferably 60 to 115 μL, more preferably 70 to 110 μL, more preferably 80 to 105 μL, and more preferably 90 to 100 μL. In some embodiments, the incubation medium in each sample tested per well 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 in each sample tested per well has a volume of 80 to 120 μL. In some embodiments, the incubation medium in each sample tested per well has a volume of 80 to 100 μL. In some embodiments, the incubation medium in each sample tested per well has a volume of 100 μL or about 100 μL.

[0077] In some embodiments, PBMCs are present at up to 500 x 1000 cells / cm 2 , preferably up to 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 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

[0078] In some embodiments, the incubation medium in each sample tested per well has a volume of 80-120 μL, and the PBMCs are present at a density of about 90 to about 130×1000 cells / cm. 2 In some embodiments, the incubation medium in each sample tested per well has a volume of at or 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

[0079] Generally, the above volume can be considered the total volume present in the well. In some embodiments, incubation medium is added in an amount such that the above total volume is achieved.

[0080] [General definition] In this document and its claims, the verb "to comprise" and its conjugations are used in their open-ended sense to mean that the items following the term are included, but not that items not specifically mentioned are excluded. Furthermore, the verb "to consist" may be substituted by "to consist essentially of," which means that a method, individually, a component as defined herein, may include additional steps, individually, components, which do not alter its inherent characteristics beyond those specifically identified. Furthermore, the reference to an element by the article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be only one. Thus, the article "a" or "an" typically means "at least one."

[0081] As used herein, a particular value followed by "at least" means the particular value or more. For example, "at least 2" is understood to be the same as "2 or more," i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more.

[0082] The term "about" or "approximately," when used in connection with a numerical value (e.g., about 10), preferably means that the value is the given value or may be 5%, preferably 1%, higher or lower than the given value. As used herein, the term "and / or" indicates that one or more of the stated examples may be present alone or in combination with at least one of the stated examples and up to all of the stated examples. Various embodiments are described herein. Each embodiment, when specified herein, may be combined together unless otherwise indicated.

[0083] 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 present invention is further described by the following examples, which are presented for illustrative purposes only and should not be construed as limiting the scope of the invention. [Brief explanation of the drawings]

[0084] [Figure 1A] IL-6 production in response to endotoxin (LPS) in the presence of different media supplements. PBMCs were incubated with a range of LPS concentrations for 20 hours in the presence of either FBS, hPL, or hAB in an incubator. IL-6 production in the presence of 1% vol. of media supplement was then assessed by ELISA. Results are shown as the mean OD of four replicates + / - standard deviation. [Figure 1B]IL-6 production in response to endotoxin (LPS) in the presence of different media supplements. PBMCs were incubated with a range of LPS concentrations for 20 hours in the presence of either FBS, hPL, or hAB in an incubator. IL-6 production in the presence of 2% vol. of media supplement was then assessed by ELISA. Results are shown as the mean OD of four replicates + / - standard deviation. [Figure 1C] IL-6 production in response to endotoxin (LPS) in the presence of different media supplements. PBMCs were incubated with a range of LPS concentrations for 20 hours in the presence of either FBS, hPL, or hAB in an incubator. IL-6 production in the presence of 4% vol. of media supplements was then assessed by ELISA. Results are shown as the mean OD of four replicates + / - standard deviation. [Figure 1D] IL-6 production in response to endotoxin (LPS) in the presence of different media supplements. PBMCs were incubated with a range of LPS concentrations for 20 hours in the presence of either FBS, hPL, or hAB in an incubator. The area under the curve for different concentrations of media supplements is shown. [Figure 2A] Comparison of curve statistics for the effects of media supplements FBS, hPL, and hAB on IL-6 production by PBMCs in response to endotoxin (LPS). A four-parameter logistic (4PL) nonlinear regression model was fitted to the data in Figures 1A-1D. Standard deviations (SD, Figure 2A) are shown. [Figure 2B] Comparison of curve statistics for the effects of media supplements FBS, hPL, and hAB on IL-6 production by PBMCs in response to endotoxin (LPS). A four-parameter logistic (4PL) nonlinear regression model was fitted to the data in Figures 1A-1D. The dynamic range is indicated. [Figure 2C]Comparison of curve statistics for the effects of media supplements FBS, hPL, and hAB on IL-6 production by PBMCs in response to endotoxin (LPS). A four-parameter logistic (4PL) nonlinear regression model was fitted to the data in Figures 1A-1D. The goodness of fit (R2, Figure 2C) is shown. [Figure 3A] IL-6 production in response to PAM3CSK4 in the presence of different media supplements. PBMCs were incubated with a range of PAM3CSK4 concentrations in the presence of either FBS, hPL, or hAB in an incubator for 20 hours. IL-6 production in the presence of 1% vol. of media supplements was then assessed by ELISA. Results are shown as the mean OD + / - standard deviation of four replicates. [Figure 3B] IL-6 production in response to PAM3CSK4 in the presence of different media supplements. PBMCs were incubated with a range of PAM3CSK4 concentrations in the presence of either FBS, hPL, or hAB in an incubator for 20 hours. IL-6 production in the presence of 2% vol. of media supplements was then assessed by ELISA. Results are shown as the mean OD + / - standard deviation of four replicates. [Figure 3C] IL-6 production in response to PAM3CSK4 in the presence of different media supplements. PBMCs were incubated with a range of PAM3CSK4 concentrations in the presence of either FBS, hPL, or hAB in an incubator for 20 hours. IL-6 production in the presence of 4% vol. of media supplements was then assessed by ELISA. Results are shown as the mean OD + / - standard deviation of four replicates. [Figure 3D] IL-6 production in response to PAM3CSK4 in the presence of different media supplements. PBMCs were incubated with a range of concentrations of PAM3CSK4 for 20 hours in the presence of either FBS, hPL, or hAB in an incubator. The area under the curve for different concentrations of media supplements is shown. [Figure 4A]Comparison of curve statistics for the effects of media supplements FBS, hPL, and hAB on IL-6 production by PBMCs in response to PAM3CSK4. A four-parameter logistic (4PL) nonlinear regression model was fitted to the data in Figures 3A-3D. Standard deviations (SD, Figure 4A) are shown. [Figure 4B] Comparison of curve statistics for the effects of media supplements FBS, hPL, and hAB on IL-6 production by PBMCs in response to PAM3CSK4. A four-parameter logistic (4PL) nonlinear regression model was fitted to the data in Figures 3A-3D. The dynamic range is indicated. [Figure 4C] Comparison of curve statistics for the effects of media supplements FBS, hPL, and hAB on IL-6 production by PBMCs in response to PAM3CSK4. A four-parameter logistic (4PL) nonlinear regression model was fitted to the data in Figures 3A-3D. The goodness of fit (R2, Figure 4C) is shown. [Figure 5A] IL-6 production in response to flagellin-BS in the presence of different media supplements. PBMCs were incubated with a range of concentrations of flagellin-BS in the presence of either FBS, hPL, or hAB in an incubator for 20 hours. IL-6 production in the presence of 1% vol. of media supplements was then assessed by ELISA. Results are shown as the mean OD of four replicates + / - standard deviation. [Figure 5B] IL-6 production in response to flagellin-BS in the presence of different media supplements. PBMCs were incubated with a range of concentrations of flagellin-BS in the presence of either FBS, hPL, or hAB in an incubator for 20 hours. IL-6 production in the presence of 2% vol. of media supplements was then assessed by ELISA. Results are shown as the mean OD + / - standard deviation of four replicates. [Figure 5C]IL-6 production in response to flagellin-BS in the presence of different media supplements. PBMCs were incubated with a range of concentrations of flagellin-BS in the presence of either FBS, hPL, or hAB in an incubator for 20 hours. IL-6 production in the presence of 4% vol. of media supplements was then assessed by ELISA. Results are shown as the mean OD + / - standard deviation of four replicates. [Figure 5D] IL-6 production in response to flagellin BS in the presence of different media supplements. PBMCs were incubated with a range of concentrations of flagellin BS for 20 hours in the presence of either FBS, hPL, or hAB in an incubator. The area under the curve for different concentrations of media supplements is shown. [Figure 6A] Comparison of curve statistics for the effects of media supplements FBS, hPL, and hAB on IL-6 production by PBMCs in response to flagellin B. A four-parameter logistic (4PL) nonlinear regression model was fitted to the data in Figures 5A-5D. Standard deviations (SD, Figure 6A) are shown. [Figure 6B] Comparison of curve statistics for the effects of media supplements FBS, hPL, and hAB on IL-6 production by PBMCs in response to flagellin B. A four-parameter logistic (4PL) nonlinear regression model was fitted to the data in Figures 5A-5D. The dynamic range is shown. [Figure 6C] Comparison of curve statistics for the effects of media supplements FBS, hPL, and hAB on IL-6 production by PBMCs in response to flagellin B. A four-parameter logistic (4PL) nonlinear regression model was fitted to the data in Figures 5A-5D. The goodness of fit (R2, Figure 6C) is shown. [Figure 7A] FBS induces IL-6 responses to NEP at lower percentages. PBMCs were incubated with a range of endotoxin (LPS, Figure 7A) concentrations for 20 hours in an incubator in the presence of different concentrations of FBS. [Figure 7B]FBS induces IL-6 responses to NEP at lower percentages. PBMCs were incubated with a range of concentrations of PAM3CSK4 for 20 hours in an incubator in the presence of different concentrations of FBS. [Figure 7C] FBS induces IL-6 responses to NEP at lower percentages. PBMCs were incubated with a range of concentrations of flagellin BS for 20 hours in an incubator in the presence of different concentrations of FBS. [Figure 7D] The areas under the curve for different pyrogens and concentrations are shown. [Figure 8A] hPL induces IL-6 responses to pyrogens. PBMCs were incubated with a range of endotoxin concentrations in the presence of different concentrations of hPL in an incubator for 20 hours. IL-6 production was then assessed by ELISA. Results are shown as the mean OD + / - standard deviation of four replicates. [Figure 8B] hPL induces IL-6 responses to pyrogens. PBMCs were incubated with a range of PAM3CSK4 concentrations in the presence of different concentrations of hPL for 20 hours in an incubator. IL-6 production was then assessed by ELISA. Results are shown as the mean OD + / - standard deviation of four replicates. [Figure 8C] hPL induces IL-6 responses to pyrogens. PBMCs were incubated with a range of flagellin BS concentrations in the presence of different concentrations of hPL in an incubator for 20 hours. IL-6 production was then assessed by ELISA. Results are shown as the mean OD + / - standard deviation of four replicates. [Figure 8D] The areas under the curve for different pyrogens and concentrations are shown. [Figure 9A]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 indicates cell density (×1000 cells / cm2). The y-axis indicates IL-6 / 1000 cells. Four replicates (symbols) and the mean (horizontal line) are plotted. The figure title indicates the total MAT volume. [Figure 9B] 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 50 μL. The x-axis indicates cell density (×1000 cells / cm2). The y-axis indicates IL-6 / 1000 cells. Four replicates (symbols) and the mean (horizontal line) are plotted. The figure title indicates the total MAT volume. [Figure 9C] 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 66 μL. The x-axis indicates cell density (×1000 cells / cm2). The y-axis indicates IL-6 / 1000 cells. Four replicates (symbols) and the mean (horizontal line) are plotted. The figure title indicates the total MAT volume. [Figure 9D] 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 100 μL. The x-axis indicates cell density (×1000 cells / cm2). The y-axis indicates IL-6 / 1000 cells. Four replicates (symbols) and the mean (horizontal line) are plotted. The figure title indicates the total MAT volume. [Figure 10] Plot of absorbance values ​​(y-axis) at optical density (OD) at each concentration of LPS (x-axis) in EU / ml for each density (×1000 cells / cm, increasing from left to right). The dotted line indicates 0.1 OD. Each represents the average of three experiments with four replicates. [Figure 11]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 at the blank. [Figure 12] Coefficient of variation (CV) at various cell densities. The average CV% of four replicates was calculated for each concentration of LPS (EU / ml) and then averaged per density (1000 cells / cm). Plots show the mean (bars) and standard deviation (error bars) of three different experiments. [Figure 13] Relative gain (y-axis) plotted as percentage of optical density (OD) at 0.032 EU / ml LPS normalized to the optical density at 100 microliter assay volume. The x-axis shows cell density in 1000 cells / cm. Error bars represent standard deviation of three experiments. [Figure 14] Average CV% at different assay volumes. The average CV% of four replicates was calculated for each concentration of LPS (EU / ml) and then averaged per assay volume (microliters) and density. Plots show the mean (bars) and standard deviation (error bars) of three different experiments (55 / 110 / 220 from left to right). Patterns show cell density in 1000 cells / cm2. [Figure 15] Absorbance (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 an assay volume of 100 microliters. The black line represents the standard curve at a density of 220 (×1000 cells / cm2) and an assay volume of 66 microliters. Error bars indicate the standard deviation of four replicates. [Figure 16] Curve slope. Bars indicate 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 17]Average CV%. The average CV% of four replicates was calculated for each concentration of LPS (EU / ml) and then averaged per volume / density combination. Left: Average CV% of the LPS standard curve at 110 density (×1000 cells / cm2) and 100 microliter assay volume. Right: Average CV% of the LPS standard curve at 220 density (×1000 cells / cm2) and 66 microliter assay volume.

[0085] [Example] Example 1. Human plate lysates provide enhanced reactivity to endotoxin and non-endotoxin pyrogens. [PBMC processing] PBMCs (10 million PBMCs / ml) were rapidly thawed in a water bath (Grant JB Nova, Cambridge, UK) set to 37°C and resuspended by the slow addition (approximately 1 mL per 5 seconds) of preheated (37°C) RPMI 1640 + Glutamax + HEPES (Gibco, Grand Island, NY, USA) medium. Immediately, the cells were added to plates containing the following media supplements: human platelet lysate (hPL, PLTMax human platelet lysate, available from Millipore (product number: SCM141; catalog number: 6D0352)), fetal bovine serum (FBS, Avantor by VWR, USA), or human AB (hAB, Merck, West Point, PA, USA). Each sample contained approximately 300 × 1000 cells / cm. 2 The cells contained approximately 1 million PBMCs / ml, corresponding to a density of 100 μg / ml.

[0086] [LPS, PAM3CSK4, and flagellin BS preparation] Lipopolysaccharide (LPS) was obtained from the European Directorate for the Quality of Medicines & Healthcare (EDQM, batch 5.1) and processed as directed by EDQM. Briefly, LPS was rehydrated in 5 mL of LAL reagent water (LRW, Lonza Bioscience, Basel, Switzerland) by vortexing for 30 minutes and diluted to a stock concentration of 100 (EEU / mL) by vortexing for 3 minutes in LRW. LPS reference endotoxin (RSE) samples were prepared in RPMI 1640 + Glutamax + HEPES (Gibco, Grand Island, NY, USA) and mixed by resuspension 20 times.

[0087] PAM3CSK4 (Toll-like receptor 1 / 2 (TLR1 / 2) agonist) (Invivogen, Toulouse, France) stock solution (10 μg / mL) was made by adding 950 μL of RPMI 1640 + Glutamax (Gibco, Grand Island, NY, USA) to 50 μL of a 10 μg / mL aliquot and mixing by brief vortexing. The stock solution was diluted to a sample concentration of 50 ng / mL, and then two-fold serial dilutions (2 mL + 2 mL) were made by resuspension in RPMI. 50 μL of each dilution mastermix was added to the plate, maintaining equal concentrations for each media supplement tested.

[0088] Flagellin-BS (Invivogen) stock solution (500 ng / mL) was made by adding 950 μL of RPMI to 50 microliters of a 10 μg / mL aliquot and mixing by resuspension. The stock solution was diluted to a sample concentration of 125 ng / mL, and then two-fold serial dilutions (2 mL + 2 mL) were made by resuspension in RPMI. 50 μL of each dilution master mix was added to the plate to maintain equal concentrations for each media supplement tested.

[0089] [ELISA] ELISA plates (MaxiSorp, NUNC, Amsterdam, The Netherlands) were coated with IL-6 capture antibody (clone 13A5, Mabtec) diluted in PBS (VWR, Solon, OH, USA) at a concentration of 1:2000 and incubated overnight at 4°C. ELISA was performed according to the manufacturer's protocol (MabTech AB, Nacka Strand, Sweden). Optical density (OD) was measured using an absorbance microplate reader, Multiscan Ascent (Thermo Scientific, Vantaa, Finland), at a wavelength of 450 nm. The background OD at 630 nm was subtracted from the signal at 450 nm before further analysis. Supernatant (50 μL) was diluted 1:4 with ELISA diluent (PBST (ELISA wash buffer, Biolegend, Amsterdam, The Netherlands) + 0.1% BSA) and added to the ELISA microplate.

[0090] [Statistics] Statistical analyses, including 4PL logistic regression, were performed using Graphpad Prism 8.

[0091] [result] To investigate whether hPL could be used to improve pyrogen detection using MAT, we incubated pools of PBMCs from four single donors with a range of endotoxin concentrations and then tested IL-6 production by measuring ELISA absorbance at optical density (OD) after 20 hours using hPL, FBS, and hAB as media supplements (Figures 1A-1D). Results show dose-dependent IL-6 production for all media supplements, increasing with increasing vol.% of media supplement (Figures 1A-1C). Of all media supplements, hPL consistently demonstrated a higher LPS response compared to the other media supplements (Figure 1D), while LPS incubation in the presence of FBS and hAB induced similar IL-6 production across the percentages tested. To further investigate the effect of medium supplements on the IL-6 response of PBMCs to LPS, we fitted a four-parameter logistic (4PL) nonlinear regression model to the curves in Figures 1A–1C, and used the standard deviation (Figure 2A), (dynamic) range (Figure 2B), and goodness of fit (R 2 , Figure 2C). The 4PL logistic regression model is a type of nonlinear regression commonly used in dose-response curves, for example, in MAT and / or receptor binding assays, which require four parameters to fit an S-shaped curve. From this model, interpolation of the sample data can be performed to determine the concentration of the contaminant, expressed in EEU / mL, in the sample to be tested in MAT. Therefore, the quality of this 4PL logistic regression model fitted to the curve of the reference endotoxin standard (RSE), is essential for accurately determining the contaminant level of the sample. A high-quality 4PL regression model is characterized by low variability (standard deviation), increased range, and a high goodness of fit (R) of the model to the RSE data. 2) The 4PL regression results showed a consistently lower standard deviation (SD) for hPL compared to the other media supplements (Figure 2A), indicating reduced variability across all percentages tested. Furthermore, the range for hPL was consistently increased when using hPL compared to the other supplements (Figure 2B), indicating an increased dynamic range for determining IL-6 production. The goodness of fit (R 2 ) was higher for hPL compared to the other supplements (Figure 2C). These results indicate that hPL exhibits the strongest response in detecting LPS, with lower variability, a larger range, and increased fidelity compared to the other supplements.

[0092] Extending the results for endotoxin, we examined the effects of the media supplements hPL, FBS, and hAB on the ability of MAT to determine the presence of Toll-like receptor (TLR)-1 / 2 ligands. To this end, pools of PBMCs from four single donors were incubated with a range of PAM3CSK4 concentrations and tested for IL-6 production by ELISA (Figures 3A–3D). Both human-based media supplements induced strongly enhanced IL-6 responses to PAM3CSK4 compared with FBS (Figures 3A–3D), demonstrating an increased ability of the human-based supplements to determine TLR-1 / 2 ligands over the animal-based supplements. Similar to LPS, hPL induced the highest IL-6 response to PAM3CSK4 across all percentages tested, with the area under the curve increasing compared to the other supplements. Comparing the statistics of the (4PL) nonlinear regression models fitted to the data in Figures 3A-3C, FBS showed a lower SD compared to the other supplements (Figure 4A), likely highlighted by the lower overall OD values ​​for FBS compared to the human-based supplements. Among the human-based supplements, hPL consistently showed a lower SD compared to hAB, again demonstrating reduced variability across the vol.% tested (Figure 4A). Furthermore, hPL showed an increased range (Figure 4B) and improved fit (Figure 4C) compared to the other supplements. These results indicate that human-based supplements are superior to FBS for the detection of TLR-1 / 2 ligands, and among them, hPL exhibits reduced variability, improved fit, and a higher range compared to hAB.

[0093] In addition to endotoxin and PAM3CSK4, we examined the effects of the media supplements hPL, FBS, and hAB on the IL-6 response to the common NEP flagellin (flagellin BS) from Bacillus subtilis. Again, pools of PBMCs from four different donors were incubated with a range of flagellin BS concentrations, and IL-6 production was then determined by ELISA. Across all vol.% values ​​tested, hPL induced the strongest IL-6 production in response to flagellin BS, with increased area under the curve compared to all other media supplements (Figures 5A–5D), indicating an increased ability to detect flagellin BS in the case of hPL. Comparison of the (4PL) nonlinear regression models fitted to the data in Figures 5A–5C showed a consistently reduced SD (Figure 6A), increased range (Figure 6B), and improved fit (Figure 6C) for hPL over the other media supplements. These results indicate that hPL is a superior medium supplement compared to hAB and FBS for the detection of flagellin BS.

[0094] The effect of media supplements hPL, FBS, and hAB on the limit of quantitation (LoQ), a stringent measure of sensitivity of MAT to endotoxin, PAM3CSK4, and flagellin BS, was compared. LoQ was calculated as the mean OD of the blank + 10 of the blank. *The LoQ was determined by identifying the first mean signal (OD) above a cutoff value defined as the standard deviation. Among all media supplements tested, the LoQ of hPL was consistently lower than that of other media supplements for all pyrogens included in the study compared to FBS (Table 1). Specifically, the LoQ of hPL for PAM3CSK4 was 0.9 ng / mL and 0.04 ng / mL lower than that of FBS and hAB, respectively. Furthermore, the LoQ of hPL for flagellin BS was 0.4 and 0.8 ng / mL lower than that of FBS and hAB, respectively. Finally, the LoQ of the human-based supplements for LPS was 0.008 EEU / mL lower than that of FBS but was equivalent when compared to each other. These observations indicate that hPL is the most consistent media supplement for detecting samples containing multiple pyrogens, with relatively high sensitivity across all pyrogens included in the study.

[0095] [Table 3]

[0096] Example 2. Determining the optimal %vol. of medium supplements for MAT Combining the data from Figures 1A-1C, 3A-3C, and 5A-5C for FBS (Figures 7A-7D), we compared the responses between different vol.% of LPS, PAM3CSK4, and flagellin BS. From the data, we can see a dose-response relationship between FBS and IL-6 production in response to LPS (Figure 7A). However, while lower vol.% FBS (1% and 2%) induced an increased IL-6 response compared to 4% FBS in response to NEP (Figures 7B and C), LPS was still detectable at these lower percentages (Figure 7A). For this reason, the use of 1% and 2% FBS is superior to 4% FBS. Furthermore, combining the data from Figures 1A-1C, 3A-3C, and 5A-5C for hPL (Figures 8A-8D), we compared the responses between different vol.% of LPS, PAM3CSK4, and flagellin BS. Similar to FBS, a dose-response relationship between hPL and IL-6 production can be observed for LPS (Fig. 8A). Furthermore, 2% vol.% induced an increased IL-6 response to NEP compared with 4% (Fig. 8D).

[0097] Example 3. Optimization of cell density and assay volume [Control formulation] Lipopolysaccharide (LPS) was obtained from EDQM (batch 5.1) and processed as directed by EDQM. LPS was rehydrated in 5 mL of LAL reagent water (LRW, Lonza Bioscience, Basel, CH) by vortexing for 30 minutes and diluted to a stock concentration of 10 endotoxin units per milliliter (EU / ml) by vortexing for 3 minutes in LRW. An LPS reference endotoxin curve (RSE) was then generated via serial dilutions and resuspension in RPMI 1640 (Thermo-Fisher Scientific, Waltham, MA, USA).

[0098] [PBMC processing] A vial of PBMCs (10 million PBMCs / ml) was rapidly thawed in a water bath set at 37°C and resuspended by the gradual addition of prewarmed (37°C) RPMI medium containing 4% human media supplement (Mediatech, Manassas, VA, USA).

[0099] [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 for various different final volumes, as specified (per experiment). Cell suspensions were then added at different cell concentrations, as specified (per experiment), in a 1:1 ratio to obtain a final concentration of 2% (vol. / vol.) human medium supplement (HMS). Final LPS concentrations corresponded to a two-fold dilution series starting at 0.1 EU / mL, obtained by resuspension in RPMI within the plate. Cells were incubated 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 / 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.

[0100] An LPS standard curve (33 microliters at concentrations of 0.064 EU / ml, 0.032 EU / ml, 0.016 EU / ml, 0.008 EU / ml, and 0.004 EU / ml) was 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 suspension was 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. 33 microliters of each cell suspension was added to the plate, resulting in 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.

[0101] [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, resulting in a final HMS concentration of 2% and a cell density of 55,000 cells / cm in each assay volume (33 microliters, 66 microliters, and 100 microliters). 2 , 110.000 cells / cm 2 and 220,000 cells / cm 2 A final cell density of 0.1% was obtained.

[0102] [MAT] Samples for the LPS standard curve were added to the culture plate in different volumes (at a 1:1 ratio with the resuspended PBMCs) and incubated for 16 hours in an incubator at 37°C under 5% CO. The final concentrations of LPS were 0.5, 0.25, 0.125, 0.06, 0.03, 0.016, and 0.008 EU / ml. The final concentration of HMS was 2%.

[0103] [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 using a Thermo-Scientific absorbance plate reader at a wavelength of 450 nanometers. Background at 630 nanometers was subtracted. Supernatant (16.7 microliters) was diluted 1 + 1 with incubation buffer and added to the ELISA plate.

[0104] [Statistics] Statistical analyses, including four-parameter / five-parameter logistic regression, were performed using Graphpad Prism 8 (GraphPad Software, San Diego, CA, USA).

[0105] [result] Increasing cell density showed improved IL-6 response at a concentration of 0.1 EU / ml and different MAT volumes (×1000 cells / cm) at densities of 50 μL, 66 μL, and 100 μL. 2 A rapid increase was observed at a density of 227 (×1000 cells / cm) using a volume of 33 μL in MAT. 2) and a density of 152 (×1000 cells / cm 2 ), whereas a three-fold increase in signal was observed compared to 303 density (×1000 cells / cm 2 ), a slight decrease in IL-6 was observed per cell (Figures 9A to 9D).

[0106] A stronger LPS signal (OD) was inferred at each LPS concentration from the higher density, but the background signal (at 0.00 EU / ml) was also reduced to a density of 440 (×1000 cells / cm 2 ) to 0.12 OD (Figure 10). 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 ) (signal to noise 6.4) was reached as an optimum.

[0107] Furthermore, while the LPS signal and signal-to-noise ratio increased with density (Figure 11), the variability of replicates also increased, with an average CV% of 440 densities (×1000 cells / cm), as shown in Figure 12. 2 ) to 28.5%. 55-220 density (×1000 cells / cm 2 ), the signal-to-noise ratio was the highest among the densities tested, while the average CV% was the lowest.

[0108] A MAT volume of 33 microliters showed a significant increase in LPS response, with a relative gain of 0.032 EU / ml normalized to 100 μL, as shown in Figure 13. The relative gain also increased with plating at higher cell densities, with the average relative gain at a MAT volume of 33 μL reaching a density of 55 (×1000 cells / cm). 2 ) and 219%, and the density (×1000 cells / cm 2 ) and 304% at a density of 220 (×1000 cells / cm 2 ) was 387%. However, smaller volumes showed increased variability between replicates (Figure 14). At each density, a MAT volume of 100 μL showed the lowest CV% (55 densities (×1000 cells / cm)). 2) at 11.8%, 110 density (×1000 cells / cm 2 ) at 13.3% and 220 densities (×1000 cells / cm 2 The pattern of increasing CV% with increasing density was observed at 220 densities (×1000 cells / cm) in all assay volumes tested. 2 ) showed a lower CV% than the 110 density (×1000 cells / cm 2 ) were allowed for each assay volume.

[0109] 220(×1000 cells / cm 2 MAT volume of 66 μL and a cell density of 110 (× 1000 cells / cm 2 LPS standard curves were generated for two configurations: 100 μL MAT volume at a cell density of 100 μL (Figure 15). Four-parameter logistic regression yielded an R of 0.99. 2 Value (66 μL / 220 density (×1000 cells / cm 2 )) and an R of 0.98 2 Value (100 μL / 100) Density (×1000 cells / cm 2 )). Figure 16 shows the curve slopes for both graphs, with an approximately two-fold difference in slope between the two configurations. Furthermore, the average CV% was 66 μL / 220 density (×1000 cells / cm 2 ) in MAT at a density of 100 μL / 110 (×1000 cells / cm 2 ) were significantly higher (23.4% and 9.4%, respectively) (Figure 17).

Claims

1. 1. A method for detecting pyrogens in a sample, comprising: i) providing one or more samples; ii) contacting the sample with peripheral blood mononuclear cells (PBMCs) in an incubation medium containing human platelet lysate (hPL); iii) determining the response of the PBMCs; A method comprising:

2. The method of claim 1, which is a monocyte activation test.

3. 3. The method of claim 1 or 2, wherein the incubation medium has a volume of up to 300 μL or up to 250 μL per sample, preferably the incubation medium has a volume of 20-250 μL, more preferably 30-175 μL, most preferably 50-110 μL.

4. The method according to any one of claims 1 to 3, wherein said contacting in step ii) is carried out in a standardized 96-well or 384-well plate, preferably in a 96-well plate.

5. The method according to any one of claims 1 to 4, wherein the incubation medium comprises 0.05 to 20 vol.%, preferably 0.5 to 4 vol.%, more preferably 0.8 to 3 vol.%, even more preferably 1 to 2.5 vol.%, and most preferably 1.2 to 2.2 vol.%, for example about 2 vol.% human platelet lysate.

6. 6. The method of any one of claims 1 to 5, wherein the response of the PBMCs that is determined is the excretion of inflammatory cytokines such as IL-6, IL-1β, IL-8, TNF-α, MCP-1, IFN-α, IFN-β, IFN-γ, IFN-λ, prostaglandins, or high mobility group proteins.

7. 7. The method of claim 6, wherein the response of the PBMCs is higher than the response of the PBMCs in a comparative method that differs only in the substitution of human platelet serum with human AB serum or with fetal bovine serum or in the absence of human platelet serum.

8. The method of any one of claims 1 to 7, wherein the response of the PBMCs is determined by an ELISA assay.

9. The PBMCs have a density of up to 500 x 1000 cells / cm ２ , preferably up to 250 x 1000 cells / cm ２ The method of any one of claims 1 to 8, wherein the cellulose is present at a density of

10. The PBMCs are at a density of about 10 x 1000 cells / cm ２ ~Approx. 350×1000 cells / cm ２ , preferably about 50 x 1000 cells / cm ２ ~Approx. 150 x 1000 cells / cm ２ , more preferably about 90 x 1000 cells / cm ２ ~Approx. 130×1000 cells / cm ２ The method of any one of claims 1 to 9, wherein the cellulose is present at a density of

11. 11. The method according to claim 1, wherein the limit of quantification for lipopolysaccharides is less than 0.01 EEU / mL, and / or the limit of quantification for triacylated lipopeptides is less than 0.1 ng / mL, and / or the limit of quantification for bacterial proteins is less than 1 ng / mL.

12. 12. The method according to any one of claims 1 to 11, wherein in step iii) the response is determined for a plurality of samples, and the response of the PBMCs for the same plurality of samples has a coefficient of variation of up to 30%.

13. The incubation medium has a volume of about 80 to about 120 μL, and the PBMCs have a density of about 90 to about 130×1000 cells / cm. ２ The method of any one of claims 1 to 12, wherein the granules are present at a density of

14. The method according to any one of claims 1 to 13, wherein the incubation medium has a volume of 30 to 175 μL, most preferably 50 to 110 μL.

15. The PBMCs have a density of about 50 x 1000 cells / cm ２ ~Approx. 150 x 1000 cells / cm ２ , more preferably about 90 x 1000 cells / cm ２ ~Approx. 130×1000 cells / cm ２ The method of any one of claims 1 to 14, wherein the granules are present at a density of

16. The method of any one of claims 1 to 15, wherein the PBMCs are non-immortalized PBMCs.

17. The incubation medium has a volume of 30-175 μL, and the PBMCs are cultured at a density of about 50×1000 cells / cm. ２ ~Approx. 150 x 1000 cells / cm ２ The method of any one of claims 1 to 16, wherein the granules are present at a density of

18. 18. The method according to any one of claims 1 to 17, wherein the incubation medium containing hPL is RPMI (Roswell Park Memorial Institute) medium, DMEM (Dulbecco's Modified Eagle's Medium), EMEM (Eagle's Minimum Essential Medium), Ham's F-10 or F-12 medium, or Iscove's Modified Dulbecco's Medium (IMDM).

19. A method for releasing a pharmaceutical composition or a medical device for use, comprising subjecting a sample from said pharmaceutical composition or said medical device to the method of any one of claims 1 to 18,

20. A kit of parts comprising a pyrogen or endotoxin standard and a vial containing human platelet lysate and optionally containing PBMCs.

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