Improved mononuclear cell activation testing using human platelet serum

Abstract

The invention belongs to the field of in vitro assay, in particular to the field of pyrogen and endotoxin detection. The present invention provides improved compositions suitable for use in animal-free testing, such as for mononuclear cell activation testing. The improved composition allows the test response to have improved sensitivity, improved dynamic range, and improved goodness of fit.

Description

Technical Field

[0001] The present invention relates to the field of in vitro assays, particularly pyrogen and endotoxin detection. The present invention provides improved compositions suitable for animal-free testing, such as monocyte activation assays. The improved compositions allow for test responses with increased sensitivity, reduced variability, improved dynamic range, and improved goodness of fit. Background Art

[0002] Because pyrogens and endotoxins cause serious adverse reactions in patients, the presence of pyrogens and endotoxins is highly relevant to the quality and safety testing of pharmaceutical compositions and medical devices. Traditional detection assays include rabbit pyrogen test (RPT) and Limulus amebocyte lysate assay (LAL) (also referred to as bacterial endotoxin test (BET)). These tests are expensive, time consuming, and require the use of experimental animals. In addition, Limulus amebocyte lysate assays are usually limited to detecting gram-negative bacteria and are 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 European Commission has chosen to replace the rabbit pyrogen test (RPT) with the MAT by 2025. It is a test that is subject to specific regulatory guidelines for sample preparation, testing, and result analysis as set out in the European Pharmacopoeia (Ph. Eur.; European Pharmacopoeia [European Pharmacopoeia] 10th edition, 2019, monograph 2.6.30, European Commission). Due to these guidelines, the currently used protocols have low throughput, are relatively expensive, and require the use of large amounts of reagents and sample volumes.

[0004] To date, classical MAT has typically been performed using the culture medium supplements fetal bovine serum (FBS) or human AB (hAB) serum (Ph. Eur.; European Pharmacopoeia, supra). However, while FBS offers good reactivity for endotoxin detection, it has difficulty detecting low concentrations of non-endotoxin pyrogens. In contrast, hAB serum has been shown to offer good sensitivity for non-endotoxin pyrogens, but has a reduced ability to detect endotoxins, for example, compared to FBS. Therefore, there is a need for MAT assays using fetal bovine serum and MAT assays using hAB serum that can detect the full spectrum of pyrogens with sufficiently high sensitivity (Molenaar-de Backer 2021, ALTEX-Alternatives to animal experimentation, 38(2); 307–315).

[0005] Therefore, there remains a need for improved animal-free pyrogen and endotoxin detection assays. Additionally, there remains a need for improved monocyte activation tests. There is 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. Summary of the Invention

[0006] The inventors have discovered that human platelet lysate (hPL), a human-based culture medium supplement, results in assays that unexpectedly exhibit higher sensitivity to both endotoxins and a variety of non-endotoxin pyrogens, thereby demonstrating advantages over other culture medium supplements. For example, as shown in the Examples, assays using either of the commonly used supplements, hAB serum and FBS, were found to be unable to simultaneously and effectively detect low concentrations of endotoxins and non-endotoxin pyrogens. However, assays using hPL consistently demonstrated greater sensitivity to both endotoxins and non-endotoxin pyrogens, particularly compared to FBS and hAB.

[0007] Therefore, in a first aspect, there is provided a method for detecting a pyrogen in a sample, the method comprising the steps of:

[0008] i) providing one or more samples;

[0009] ii) contacting the sample with peripheral blood mononuclear cells (PBMCs) in an incubation medium comprising human platelet lysate (hPL); and

[0010] iii) Determination of PBMC responses.

[0011] In certain embodiments, the method is a monocyte activation test.In certain embodiments, the volume of incubation medium is 300 μ L or 250 μ L at the most for each sample, preferably 20 to 250 μ L, more preferably 30 to 175 μ L, most preferably 50 to 110 μ L.In certain embodiments, step ii) contact is carried out in standardization 96 orifice plates or 384 orifice plates (preferably 96 orifice plates).In certain embodiments, incubation medium comprises 0.05 to 20 volume %, preferably 0.5 to 4 volume %, more preferably 0.8 to 3 volume %, even more preferably 1 to 2.5 volume %, most preferably 1.2 to 2.2 volume %, such as the human platelet lysate of about 2 volume %.In certain embodiments, the response of determined PBMC is the secretion of inflammatory cytokines (such as IL-6, IL-1 β, 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 greater than the response of PBMCs in a comparative assay that differs only in that the human platelet serum is replaced with human AB serum or fetal bovine serum, or in the absence of human platelet serum. In some embodiments, the response of PBMCs is determined by ELISA. In some embodiments, PBMCs are expressed at a population of at most 500 x 1000 cells / cm 2 , preferably at most 250×1000 cells / cm 2 In some embodiments, PBMCs are present at a density of about 10×1000 cells / cm 2 About 350 × 1000 cells / cm 2 (optionally to about 300 x 1000 cells / cm 2 ), preferably about 50×1000 cells / cm 2 About 150 × 1000 cells / cm 2 , more preferably about 90×1000 cells / cm 2 About 130 × 1000 cells / cm 2 In some embodiments, the limit of quantification of lipopolysaccharide 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 protein is less than 1, preferably less than 0.5 ng / mL. In some embodiments, in step iii), the PBMC response determined for multiple identical samples has a coefficient of variation of at most 30%. In some embodiments, the volume of the incubation medium is about 80 to about 120 μL, and the PBMCs are incubated at a density of about 90 to about 130×1000 cells / cm 2In another aspect, a method for releasing a pharmaceutical composition or medical device for use is provided, the method comprising subjecting the pharmaceutical composition or a sample derived from a medical device to the method of the first aspect. In another aspect, a kit is provided, the kit comprising a pyrogen or endotoxin standard and a vial comprising human platelet lysate, optionally further comprising PBMCs. DETAILED DESCRIPTION

[0012] In one aspect, a method for detecting a pyrogen in a sample is provided, the method comprising the steps of:

[0013] i) providing one or more samples;

[0014] ii) contacting the sample with peripheral blood mononuclear cells (PBMCs) in an incubation medium comprising human platelet lysate (hPL); and

[0015] iii) Determination of PBMC responses.

[0016] This method is attractive because it offers the following advantages over currently available methods in the art: it does not require the use of experimental animals, it provides improved sensitivity to endotoxins and / or non-endotoxin pyrogens, it provides an improved range of responses to endotoxins and / or non-endotoxin pyrogens, it provides improved reliability in determining PBMC responses, it provides lower variability between measurements, and it results in lower costs. The steps of the method are preferably performed in the order in which they are numbered.

[0017] Pyrogens

[0018] Pyrogens are well known in the art. They are substances that can trigger an immune response in a subject by activating a series of immune processes, typically characterized by an increase in body temperature beyond normal levels (fever). The biological activity of a pyrogen is its ability to cause fever in a subject, or referred to herein as its pyrogenicity. A pyrogen can be an exogenous pyrogen. "Exogenous" or "external" pyrogen refers to a pyrogen derived from outside the subject's body. A pyrogen can be an endotoxin, preferably lipopolysaccharide (LPS). Endotoxins (such as lipopolysaccharides) are cellular components of bacteria such as Gram-negative bacteria and are the main components of their outer cell walls. The presence of endotoxins in the subject's bloodstream is associated with a variety of adverse symptoms (including fever, hypotension, nausea, chills and shock) and can lead to complications such as disseminated vascular coagulation (DIC), endotoxic shock and acute respiratory distress syndrome (ARDS). A pyrogen can be a non-endotoxin pyrogen (NEP). Non-endotoxin pyrogens include microbe-associated molecular patterns (MAMPs) and pathogen-associated molecular patterns (PAMPs), examples of which are bacterial cell components such as bacterial proteins (e.g., flagellin), peptidoglycans, 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, flagellin may be derived from gram-positive bacteria, such as Bacillus subtilis.

[0019] Pyrogens may be product or process related impurities present in a pharmaceutical composition or on the surface of, for example, a medical device. Examples of pyrogenic impurities are chemical agents such as polyadenylic acid, polyuridylic acid, polyriboinosinic acid, dinitrophenol, trinitrophenol, 4,6-dinitro-o-cresol, N-phenyl-p-naphthylamine, aldehyde-α-naphthylamine, metals and nanoparticles (typically <1 nm), as well as any other impurities that exhibit pyrogenic properties.

[0020] Pyrogens can be damage-associated molecular patterns (DAMPs), which refer to biomolecules typically released by dead or damaged cells. Examples of DAMP pyrogens include biglycan, decorin, versican, hyaluronic acid, fibronectin, tenascin, uric acid, S100 protein, 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 cells exhibiting pyrogenicity. Pyrogens can be toll-like receptor 1 / 2 (TLR1 / 2) agonists. Pyrogens can be the vehicle component of a pharmaceutical composition. Examples of such components include excipients, solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, and the like. The pyrogen may be a triacylated lipopeptide. The pyrogen may be an endogenous pyrogen. "Endogenous" or "internal" pyrogens refer to pyrogens produced by the subject's body after contact with an exogenous pathogen. Endogenous pyrogens may be associated with an inflammatory response. An endogenous pyrogen may be a DAMP. An endogenous pyrogen may be a toll-like receptor 1 / 2 (TLR1 / 2) agonist. Examples of endogenous pyrogens include cytokines and chemokines.

[0021] 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 do not usually retain the crystal violet dye used in standard Gram staining, in contrast to "Gram-positive" bacteria that usually retain the dye. In some embodiments, Gram-negative bacteria are pathogenic or potentially pathogenic bacteria. Examples of pathogenic or potentially pathogenic Gram-negative bacteria are bacteria of the genera Escherichia, Salmonella, Shigella, Pseudomonas, Neisseria, Haemophilus, Bordetella, Vibrio, etc. In some embodiments, the pyrogen is a non-endotoxin pyrogen (NEP). In some embodiments, the pyrogen is a cellular component of Gram-positive bacteria (preferably a bacterial protein (e.g., flagellin)). In some embodiments, Gram-positive bacteria are pathogenic or potentially pathogenic. Examples of pathogenic or potentially pathogenic Gram-positive bacteria are bacteria of the genera Streptococcus, Staphylococcus, Corynebacterium, Listeria, Bacillus (e.g., Bacillus subtilis), Clostridium, and the like. In some embodiments, the pyrogen is a product- or process-related impurity present in or on the surface of the pharmaceutical composition. In some embodiments, the pyrogen is a damage-associated molecular pattern (DAMP). In some embodiments, the pyrogen is a component of the medium of the 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.

[0022] Step i) Providing a sample

[0023] In step i) of the method, one or more samples are provided. The sample can be taken from (or derived from) an original source, for example, from a product such as a pharmaceutical composition or a medical device. The sample can also be a subsample taken from an original sample (or another subsample). The sample can also be or be taken from a subsample produced by dilution or concentration of an original sample.

[0024] The sample can also be a replica of the original sample or subsample. Replicas are preferably intended to be identical, and more preferably they are identical. In the case where the sample is a replica of the original sample or subsample, at least two, at least three, or at least four (preferably at least four) replicas of the original sample or subsample are provided. For example, the replica can be prepared separately, or can be produced by obtaining an equivalent subsample from the original sample or subsample. The sample is preferably a liquid sample, more preferably an aqueous sample.

[0025] Samples can be obtained from pharmaceutical compositions to be tested for the presence of pyrogens (e.g., therapeutic compositions, diagnostic compositions, or compositions for preventing a disease or condition or alleviating its symptoms, such as vaccines). The pharmaceutical composition can be in any form. In some embodiments, the pharmaceutical composition is a vaccine.

[0026] Samples can be obtained from surfaces to be tested for the presence of pyrogens (e.g., surfaces of medical devices or instruments). Examples of medical devices and instruments include bedpans, cannulas, cardioverters, defibrillators, catheters, dialyzers, electrocardiographs, enema devices, endoscopes, gas cylinders, gauze sponges, surgical scissors, hypodermic needles, syringes, infection control equipment (e.g., masks, surgical gowns, face shields, and goggles), instrument sterilizers, kidney trays, nasogastric tubes, scalpels, nebulizers, ophthalmoscopes, otoscopes, pipettes, proctoscopes, radiographers, sphygmomanometers, thermometers, tongue depressors, transfusion bags, tuning forks, ventilators, watches, and the like. For example, such samples can be obtained by rinsing the surface to be tested with a solution (e.g., water or a buffer), collecting the rinsing fluid, and using the rinsing fluid for sample preparation.

[0027] 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, 8, 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 87, at least 88, at least 89, at least 90, at least 91, at least 92, at least 93, at least 94, In certain embodiments, 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 certain embodiments, at least 50, preferably at least 97 samples are provided. In certain embodiments, at least 96 samples are provided. In certain embodiments, at least 384 samples are provided. Advantageously, 384 orifice plates can be used to put into practice according to method of the present invention.

[0028] In some embodiments, in step i), an additional control sample is provided. The control sample may comprise a pyrogen standard (positive control) or may be pyrogen-free (negative control). A pyrogen positive control sample comprising a known pyrogen concentration can improve quantitative accuracy. For example, a standard curve can be produced using multiple samples comprising different pyrogen concentrations. The control sample may be a sample from a (reference) standard. Such standards are described later herein.

[0029] 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, wherein EEU is "equivalent endotoxin unit"). One EEU / mL (EU / mL) is equal to approximately 0.1-0.2 ng endotoxin / mL (preferably 0.15 ng / mL) of solution. In embodiments where the control sample is an endotoxin (preferably lipopolysaccharide) sample, the control sample preferably comprises about 0.005 to about 15 endotoxin units / mL. In some embodiments, the control sample comprises about 0.005 to about 1 endotoxin units / mL. In some embodiments, the control sample comprises 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.

[0030] In some embodiments, the control sample is a non-endotoxin pyrogen (NEP) sample, such as a sample of 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, an example being PAM3CSK4 (CAS No. 112208-00-1). In some embodiments where the control sample is a non-endotoxin pyrogen sample, the control sample preferably comprises from about 0.01 to about 125 ng / mL of the non-endotoxin pyrogen. In some embodiments, the control sample comprises from about 0.1 to about 20 ng / mL, preferably from about 0.1 to about 15 ng / mL, and more preferably from about 1 to about 10 ng / mL of the non-endotoxin pyrogen.

[0031] Step ii) Contact with PBMC

[0032] 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 certain embodiments, the sample is contacted with whole peripheral blood or a fraction containing PBMCs therefrom. In certain embodiments, the sample is contacted with isolated PBMCs. Blood fractions containing PBMCs and isolated PBMCs can be obtained using standard methods (e.g., using leukocyte apheresis and / or density gradient centrifugation).

[0033] In certain embodiments, contact is made with a PBMC cell line. In certain embodiments, contact is made with immortal or non-immortal cells (preferably non-immortal cells). In certain embodiments, contact is made with PBMC obtained from a single donor. In certain embodiments, contact is made with PBMC obtained from the pooled whole peripheral blood of multiple donors. In the case of obtaining PBMC from a single or multiple donors, these donors are preferably according to standardized guidelines, more preferably as described in European Pharmacopoeia (Ph.Eur.; European Pharmacopoeia [European Pharmacopoeia] 10th edition, 2019, European Commission) sections 5-3, 5-4, 5-5, 6-3 and / or monograph 2.6.30 for qualification certification.

[0034] In some embodiments, contact is made with fresh PBMC. In a preferred embodiment, contact is made with cryopreserved PBMC. Cryopreservation of PBMC can be carried out according to standard procedures, for example, as described in a standard manual (e.g., Hubel, A., 2018: Preservation of Cells: A Practical Manual [Cell Cryopreservation: A Practical Manual], 1st edition, Wiley-Blackwell [Wiley-Blackwell Publishing], New Jersey, USA). In some embodiments, PBMC is mammalian, preferably human. In some embodiments, PBMC is a leukocyte. In some embodiments, PBMC is a macrophage. In a preferred embodiment, PBMC comprises or is a monocyte, preferably a mammalian monocyte, more preferably a human monocyte. In some embodiments, macrophages or monocytes are derived from pluripotent stem cells. PBMC is well known and well characterized and typically contains 10-20% monocytes. Preferred PBMCs are non-immortalized PBMCs, preferably non-immortalized monocytes.

[0035] The method is preferably a monocyte activation test, or part of a monocyte activation test. More preferably, the monocyte activation test is performed according to the guidelines set out in European Pharmacopoeia Monograph 2.6.30 (supra).

[0036] In step ii), contacting with PBMC can be adding one or more samples to a culture medium comprising PBMC. It can also be adding a culture medium comprising PBMC to one or more samples. It can be carried out in any suitable container (e.g., microplate (with one or more holes), test tube (e.g., Eppendorf tube), flask (e.g., Erlenmeyer flask), bottle (e.g., Schott flask), fermentor, etc.). In a preferred embodiment, contact is carried out in a standardized 96-well plate. In certain embodiments, contact is carried out in a standardized 384-well plate. Standardized well plates are widely available from commercial suppliers. Suitable standards are ANSI / SLAS standards, preferably all five of 1-2004 (R2012), 2-2004 (R2012), 3-2004 (R2012), 4-2004 (R2012), and 6-2012 (R2012).

[0037] When using 96-well plates or 384-well plates, a single sample can be placed in each well. The present invention advantageously allows the use of 384-well plates. Using 384-well plates has the further advantage of being able to achieve higher detection throughput (because 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 that can be used for actual test samples. 384-well plates have a better test-to-control ratio because after the wells are assigned to the desired control samples, more wells are still available for test samples. In this context, the following can be the conventional well distribution for 96-well plates, with 4 repetitions used for each data point:

[0038] sample Quadruplicate wells LPS curve 8 32 Three concentrations of test sample 1 3 12 Test sample 1 LPS spiked 3 12 Test Sample 1 Test Sample 1 NEP Control 2 8 Three concentrations of test sample 2 3 12 Test sample 2LPS spiked 3 12 Test sample 2 Test sample 1 NEP control 2 8 Total hole 96

[0039] Alternatively, with 2 dilutions per test sample and omitting the NEP control, the following may apply:

[0040]

[0041]

[0042] It should be noted that a sample of the product to be analyzed can result in multiple samples, which are provided in the method for detecting pyrogens. For example, in the table above, a single test sample 1 results in multiple samples when each well is considered to contain the provided sample. This difference will be clear from the context unless explicitly stated.

[0043] PBMCs (preferably comprising monocytes) may be present during the contacting step at a specific density, preferably expressed as cells / cm 2 Measurement (cm 2= refers to the growth area, which is preferably the area of ​​the well cross section; preferred wells are flat bottom wells). In the context of this disclosure, the density of PBMCs refers to the density of PBMCs used per sample contacted. Considering the growth area (cm 2 ), cell concentration (cells / mL) and volume (mL), a skilled person can easily calculate the number of cells / cm in a container (preferably the wells of a standardized 96-well or 384-well plate), for example, by using a cell counter or by using microscopy techniques. 2 This density is a commonly used parameter, and the skilled person will understand that there may be some dead cells in the population. In some embodiments, PBMCs (preferably comprising monocytes) are present at a density of at most 500 x 1000 cells / cm 2 , preferably at most 250×1000 cells / cm 2 The density exists.

[0044] In some embodiments, PBMCs (preferably comprising monocytes) are cultured at a density of about 10 x 1000 cells / cm 2 About 350 × 1000 cells / cm 2 , preferably about 50×1000 cells / cm 2 About 150 × 1000 cells / cm 2 , preferably about 60×1000 cells / cm 2 About 140 × 1000 cells / cm 2 , preferably about 70×1000 cells / cm 2 About 130 × 1000 cells / cm 2 , preferably about 80×1000 cells / cm 2 About 120 × 1000 cells / cm 2 , more preferably about 90×1000 cells / cm 2 About 130 × 1000 cells / cm 2 In some embodiments, PBMCs (preferably comprising monocytes) are present at a density of 110×1000 cells / cm 2 or approximately 110 × 1000 cells / cm 2 The density exists.

[0045] Incubation medium can have a specific volume (preferably measured in mL or μL) during the contact step. In the context of the present disclosure, the volume of incubation medium refers to the volume of each sample. In certain embodiments, the volume of incubation medium is 300 μL per sample at the most or 250 μL per sample at the most, preferably 250 μL per sample at the most. In a preferred embodiment, its volume is 200 μL per sample at the most, 175 μL per sample at the most, or 150 μL per sample at the most, preferably 250 μL per sample at the most or about 250 μL. In certain embodiments, the volume of incubation medium is 20 to 250 μL, preferably 30 to 175 μL, more preferably 50 to 110 μL.

[0046] In some embodiments, the volume of the incubation medium is 20 to 150 μL, preferably 30 to 140 μL, more preferably 50 to 110 μL. In some embodiments, the volume of the incubation medium is 80 to 120 μL. In some embodiments, the volume of the incubation medium is 40 to 130 μL, preferably 60 to 120 μL, more preferably 70 to 115 μL, more preferably 75 to 105 μL, more preferably 85 to 105 μL.

[0047] In some embodiments, the volume of the incubation medium is 20 to 100 μL, preferably 30 to 100 μL, more preferably 50 to 100 μL. In some embodiments, the volume of the incubation medium is 80 to 100 μL. In some embodiments, the volume of the incubation medium is 100 μL or about 100 μL. In some embodiments, the volume of the incubation medium is 1 to 99 μL, preferably 30 to 70 μL, such as 33 μL or 66 μL.

[0048] In some embodiments, the volume of incubation medium is 20 to 150 μL, and PBMCs (preferably monocytes) are cultured at a density of approximately 10×1000 cells / cm 2 About 250 × 1000 cells / cm 2 In some embodiments, the volume of the incubation medium is 20 to 150 μL, and the PBMCs (preferably monocytes) are present at a density of about 50×1000 cells / cm 2 About 150 × 1000 cells / cm 2 In some embodiments, the volume of the incubation medium is 20 to 150 μL, and the PBMCs (preferably monocytes) are present at a density of about 90×1000 cells / cm 2 About 130 × 1000 cells / cm 2 The density exists.

[0049] In some embodiments, the volume of incubation medium is 30 to 140 μL, and PBMCs (preferably monocytes) are cultured at a population of approximately 10×1000 cells / cm 2 About 250 × 1000 cells / cm 2 In some embodiments, the volume of the incubation medium is 30 to 140 μL, and the PBMCs (preferably monocytes) are present at a density of about 50×1000 cells / cm 2 About 150 × 1000 cells / cm 2 In some embodiments, the volume of the incubation medium is 30 to 140 μL, and the PBMCs (preferably monocytes) are present at a density of about 90×1000 cells / cm 2 About 130 × 1000 cells / cm 2 The density exists.

[0050] In some embodiments, the volume of incubation medium is 80 to 120 μL, and PBMCs (preferably monocytes) are cultured at a density of approximately 10×1000 cells / cm 2 About 250 × 1000 cells / cm 2 In some embodiments, the volume of the incubation medium is 80 to 120 μL, and the PBMCs (preferably monocytes) are present at a density of about 50×1000 cells / cm 2 About 150 × 1000 cells / cm 2 In some embodiments, the volume of the incubation medium is 80 to 120 μL, and the PBMCs (preferably monocytes) are present at a density of about 90×1000 cells / cm 2 About 130 × 1000 cells / cm 2 The density exists.

[0051] In some embodiments, the volume of incubation medium is 50 to 110 μL, and PBMCs (preferably monocytes) are cultured at a density of approximately 10×1000 cells / cm 2 About 250 × 1000 cells / cm 2 In some embodiments, the volume of the incubation medium is 50 to 110 μL, and the PBMCs (preferably monocytes) are present at a density of about 50×1000 cells / cm 2 About 150 × 1000 cells / cm 2 In some embodiments, the volume of the incubation medium is 50 to 110 μL, and the PBMCs (preferably monocytes) are present at a density of about 90×1000 cells / cm 2 About 130 × 1000 cells / cm 2 The density exists.

[0052] In some embodiments, the volume of incubation medium is 20 to 100 μL, and PBMCs (preferably monocytes) are cultured at a density of approximately 10×1000 cells / cm 2 About 250 × 1000 cells / cm 2 In some embodiments, the volume of the incubation medium is 20 to 100 μL, and the PBMCs (preferably monocytes) are present at a density of about 50×1000 cells / cm 2 About 150 × 1000 cells / cm 2 In some embodiments, the volume of the incubation medium is 20 to 100 μL, and the PBMCs (preferably monocytes) are present at a density of about 90×1000 cells / cm 2 About 130 × 1000 cells / cm 2 The density exists.

[0053] In some embodiments, the volume of incubation medium is 30 to 100 μL, and PBMCs (preferably monocytes) are cultured at a density of approximately 10×1000 cells / cm 2 About 250 × 1000 cells / cm 2 In some embodiments, the volume of the incubation medium is 30 to 100 μL, and the PBMCs (preferably monocytes) are present at a density of about 50×1000 cells / cm 2 About 150 × 1000 cells / cm 2 In some embodiments, the volume of the incubation medium is 30 to 100 μL, and the PBMCs (preferably monocytes) are present at a density of about 90×1000 cells / cm 2 About 130 × 1000 cells / cm 2 The density exists.

[0054] In some embodiments, the volume of incubation medium is 50 to 100 μL, and PBMCs (preferably monocytes) are cultured at a density of approximately 10×1000 cells / cm 2 About 250 × 1000 cells / cm 2 In some embodiments, the volume of the incubation medium is 50 to 100 μL, and the PBMCs (preferably monocytes) are present at a density of about 50×1000 cells / cm 2 About 150 × 1000 cells / cm 2 In some embodiments, the volume of the incubation medium is 50 to 100 μL, and the PBMCs (preferably monocytes) are present at a density of about 90×1000 cells / cm 2 About 130 × 1000 cells / cm 2 The density exists.

[0055] In some embodiments, the volume of incubation medium is 80 to 100 μL, and PBMCs (preferably monocytes) are cultured at a density of approximately 10×1000 cells / cm 2 About 250 × 1000 cells / cm 2 In some embodiments, the volume of the incubation medium is 80 to 100 μL, and the PBMCs (preferably monocytes) are present at a density of about 50×1000 cells / cm 2 About 150 × 1000 cells / cm 2 In some embodiments, the volume of the incubation medium is 80 to 100 μL, and the PBMCs (preferably monocytes) are present at a density of about 90×1000 cells / cm 2 About 130 × 1000 cells / cm 2 The density exists.

[0056] In some embodiments, the volume of incubation medium is 100 μL or about 100 μL, and PBMCs (preferably monocytes) are cultured at a density of 110×1000 cells / cm 2 or approximately 110 × 1000 cells / cm 2 The density exists in the general surface area of ​​0.32cm 2 110 × 1000 cells / cm in a 96-well plate 2 This is equivalent to approximately 35,200 cells / well.

[0057] The incubation medium comprising human platelet lysate (hPL) can be any culture medium suitable for PBMC, preferably mammalian PBMC, more preferably human PBMC, most preferably human monocyte culture. Preferably, the incubation medium is according to the guidelines set forth in the monograph 2.6.30 (ibid.) of the European Pharmacopoeia. Preferred incubation medium is RPMI (Roswell Park Memorial Institute (Roswell Park Memorial Institute)) culture medium, more preferably RPMI 1640 culture medium, which is widely commercially available. However, other suitable culture media can also be considered, such as DMEM (Dulbecco's Modified Eagle's Medium (Dulbecco's Modified Eagle's Medium)), EMEM (Eagle's Minimum Essential Medium (Eagle's Minimum Essential Medium)), Ham F-10 or F-12 culture medium, Iscove's Modified Dulbecco's Medium (Iscove's Modified Dulbecco's Medium, IMDM), α-MEM (Minimum Essential Medium α), etc., all of which are commercially available. Further examples of suitable culture media are given in the Examples section later herein.In some embodiments, the incubation medium is not α-MEM.

[0058] As used herein, human platelet lysate (hPL) has its common meaning in the art. It refers to a culture medium supplement obtained by lysing human platelets. In certain embodiments, hPL is autologous relative to PBMC. In certain embodiments, hPL is allogeneic relative to PBMC. Human platelet lysate can be prepared using standard methods available in the art (e.g., as described in Burnouf et al., 2016, Biomaterials [Biological Materials] 76; 371-387, Mohamed et al., 2020, Blood Res [Blood Research] 55; 35-43, and Guiotto et al., 2020, J Transl Med [Journal of Translational Medicine] 18: 351). Human platelet lysate can be prepared from platelet concentrates, for example, 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 the PRP (platelet rich plasma) method, or it can be prepared using platelet apheresis. Typically, the buffy coat method involves centrifuging whole blood and then pooling four buffy coat units from four donors plus one plasma unit. After the second centrifugation step, the platelet concentrate is filtered through a leukocyte filter and stored. Typically, the PRP method involves pooling four or five blood units, which are then centrifuged to separate blood cells from the upper layer consisting of platelets mixed with plasma. Typically, platelet apheresis involves processing blood via an apheresis machine that uses centrifugation to remove platelets.

[0059] hPL can be prepared from platelet concentrate by repeated freeze / thaw cycles (e.g., freezing at a temperature of about -30°C to about 80°C, then one to five cycles of thawing at 37°C). As another example, it can be prepared by ultrasonicating the platelet concentrate (e.g., performing up to 30 minutes at a frequency of about 20kHz). Ultrasonication can optionally be combined with repeated freeze / thaw cycles. As another example, it can be prepared by direct platelet activation in platelet concentrate, which involves adding a calcium salt (e.g., CaCl ), activating the platelet thrombin cascade and causing platelet lysis. Alternatively, platelet lysis can be induced by solvent / detergent treatment. In some cases, human platelet lysate preparation involves adding an anticoagulant, such as heparin, to whole blood or platelet concentrate. In some cases, once human platelet lysis is performed, additional centrifugation steps are performed to remove platelet fragments. Human platelet lysate can be used fresh (ie, immediately following preparation), or alternatively, stored lysate can be used, such as frozen human platelet lysate after thawing.

[0060] Human platelet lysates can also be obtained from commercial suppliers, for example, such as those from Mediatech (Manassas, VA, USA), StemCell Technologies GmbH (Cologne, Germany), and others. 06960), Sigma-Aldrich (St Louis, MO, USA; SCM141 and SCM142), etc. In some embodiments, the hPL comprises about 7 to about 90 mg / mL total protein.

[0061] Preferably, the human platelet lysate comprises one or more growth factors. In some embodiments, it comprises one or more growth factors selected from PDGF, PDGF-AA, PDGF-AB, PDGF-BB, TGF-β, VEGF, bFGF, EGF, BDNF, IGF-1, and HGF, preferably selected from PDGF-AA, PDGF-AB, PDGF-BB, TGF-β, VEGF, and IGF-1. In some embodiments, the PDGF concentration in the hPL is between about 0 and 70 ng / mL, preferably between about 10 and about 70 ng / mL. In some embodiments, the PDGF-AA concentration in the hPL is between about 0 and about 240 ng / mL, preferably between about 0.01 and about 240 ng / mL. In some embodiments, the PDGF-AB concentration in the hPL is between about 0 and about 580 ng / mL, preferably between about 0.01 and about 580 ng / mL. In some embodiments, the PDGF-BB concentration in the hPL is between about 0 and about 23 ng / mL, preferably between about 0.01 and about 23 ng / mL. In some embodiments, the concentration of TGF-β in hPL is between about 0 and about 250 ng / mL, preferably between about 0.04 and about 250 ng / mL. In some embodiments, the concentration of VEGF in hPL is between about 0 and about 0.8 ng / mL, preferably between about 0.15 and about 0.8 ng / mL. In some embodiments, the concentration of bFGF in hPL is between about 0 and about 5.5 ng / mL, preferably between about 0.04 and about 5.5 ng / mL. In some embodiments, the concentration of EGF in hPL is between about 0 and about 20 ng / mL, preferably between about 0.01 and about 20 ng / mL. In some embodiments, the concentration of BDNF in hPL is between about 0 and about 100 ng / mL, preferably between about 0.03 and about 100 ng / mL. In some embodiments, the concentration of IGF-1 in hPL is between about 0 and about 500 ng / mL, preferably between about 0.1 and about 500 ng / mL. In some embodiments, the concentration of HGF in hPL is between about 0 and about 2.6 ng / mL, preferably between 0.1 and about 2.6 ng / mL.

[0062] 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, total protein of 4.0-6.5 g / dL, endotoxins USP of <10 EU / mL, mycoplasma not detected, and sterility test negative for growth. Preferably, the hPL has been sterile filtered, such as 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.

[0063] In some embodiments, the incubation medium comprises 0.05 to 20% by volume, preferably 0.2 to 10% by volume, more preferably 0.5 to 5% by volume, even more preferably 1 to 4% by volume, most preferably 0.5 to 2.5% by volume, such as about 2% by volume of human platelet lysate. In some embodiments, it comprises 0.1 to 15% by volume, preferably 0.1 to 10% by volume, more preferably 1 to 4% by volume, most preferably 0.5 to 2.5% by volume, such as about 2% by volume of human platelet lysate. In a preferred embodiment, the incubation medium comprises 0.05 to 20% by volume, preferably 0.5 to 4% by volume, more preferably 0.8 to 3% by volume, even more preferably 1 to 2.5% by volume, most preferably 1.2 to 2.2% by volume, such as about 2% by volume of human platelet lysate. In some embodiments, the incubation medium comprises 1 or about 1% by volume of human platelet lysate. In a preferred embodiment, the incubation medium comprises 2 or about 2% by volume of human platelet lysate. In some embodiments, the incubation medium comprises 3 or about 3% by volume of human platelet lysate. In some embodiments, the incubation medium comprises 4 or about 4% by volume of human platelet lysate. In preferred embodiments, the incubation medium does not comprise 1.5-2.5% or 4.5-5.5% by volume of hPL, and in particular, the incubation medium does not comprise or consist of α-MEM containing 1.5-2.5% or 4.5-5.5% by volume of hPL.

[0064] One or more samples contacted with peripheral blood mononuclear cells (PBMC) are hatched for a duration sufficient to induce PBMC (preferably monocytes) to respond. In certain embodiments, the duration of hatching 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 hatching is at least 16 hours. In certain embodiments, contact is carried out for up to 72 hours, preferably up to 36 hours, preferably up to 30 hours, more preferably up to 24 hours, also more preferably up to 18 hours, most preferably up to 16 hours.

[0065] Incubation is preferably carried out under cell culture (preferably human cell culture) conditions. Preferred conditions are a temperature in the range of 30°C-42°C (e.g., about 37°C) and a CO2 level of 0-8% (e.g., about 5%). In a preferred embodiment, incubation is carried out at 37°C and a CO2 level of 5% for at least 16 hours. After incubation, the incubated sample can be used directly in step iii), or can be frozen and step iii) can be carried out at a later time point.

[0066] Step iii) Determining the response of PBMCs

[0067] In step iii), determine the response of PBMC (preferably mammalian PBMC, more preferably human PBMC, most preferably human monocytes). The response of PBMC can be the activation of PBMC. The response of PBMC can be the expression of surface activation markers. Examples of surface activation markers include CD80, CD86, CD11c, CD38, CD282 and CD64. The response of PBMC can be the production and / or secretion (preferably secretion) of cytokines (such as inflammatory cytokines or anti-inflammatory cytokines, most preferably inflammatory cytokines). Examples of inflammatory cytokines are IL-6, IL-1 β, 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-1 β, IL-8, TNF-α, MCP-1, more preferably IL-6. In some embodiments, the response of PBMC is the generation and / or secretion of prostaglandin (PGE2). In some embodiments, the response of PBMC is the generation and / or secretion of neopterin. In some embodiments, the response of PBMC is the expression of surface activation markers.

[0068] In certain embodiments, the response of determined PBMC is the generation and / or secretion (preferably secretion) of inflammatory cytokines (such as IL-6, IL-1 β, IL-8, TNF-α, MCP-1, IFN-α, IFN-β, IFN-γ, IFN-λ), prostaglandins or high mobility group proteins.Technical staff will appreciate that the determination that PBMC responds can also relate to the generation and / or secretion (preferably secretion) combination of multiple inflammatory cytokines, prostaglandins and / or high mobility group proteins to determine.In a preferred embodiment, the response of determined PBMC is the generation and / or secretion (preferably secretion) of IL-6.

[0069] The determination of the PBMC response can be carried out directly in the same or different container after the contacting step, or the incubation mixture can be stored, optionally frozen, and used for response determination at different time points. In general, the response of PBMC is related to the detection of pyrogens. It is possible that no response is detected, in which case no pyrogens are detected. The determination of the PBMC response can be carried out by, for example, quantitative PCR, flow cytometry techniques (such as FACS analysis), or by immunoassay (preferably ELISA assay). The skilled person knows how to perform such immunoassays, and descriptions of the immunoassays can be found in standard manuals such as The Immunoassay Handbook: Theory and Applications of Ligand Binding, ELISA and Related Techniques [Immunoassay Handbook: Theory and Applications of Ligand Binding, ELISA and Related Techniques], 2013, 4th edition, Wild, D. ed., Elsevier Science [Elsevier Science Publishing Group], The Netherlands (incorporated herein in its entirety by reference). Commercial ELISA kits (eg, MabTech ELISA basic IL-6 kit (HRP, MabTech AB, Nack Strand, SE, Stockholm, Sweden) are also available. ELISA assays are particularly advantageous when used in the methods of the present invention because they enable high-throughput testing of multiple samples.

[0070] In certain embodiments, the response of PBMC is determined by ELISA. In certain embodiments, the antibody for IL-6, IL-1 β, IL-8, TNF-α, MCP-1, IFN-α, IFN-β, IFN-γ, IFN-λ, prostaglandin or high mobility group protein is used to carry out ELISA. In a preferred embodiment, the antibody for IL-6 (anti-IL-6) is used to carry out ELISA. Such antibody is commercially available, for example, from the clone 13A5 of MabTech AB company in Stockholm, Sweden. In certain embodiments, ELISA is carried out in standardization 96 orifice plates or 384 orifice plates (preferably 96 orifice plates). The example of the ELISA used in the context of the present disclosure is provided in the examples section later herein.

[0071] In certain embodiments, the response of PBMC is higher than the response of PBMC in comparative method (determination), and the difference of this comparative method is only that human platelet serum is replaced by different culture medium supplements (or does not use culture medium supplements).As used herein, " culture medium supplements " refers to the additional culture medium components that can promote the response (such as activation, expression of surface activation markers, cytokine production, prostaglandin production or high mobility group protein production) of PBMC (preferably human PBMC, more preferably human monocyte).Preferably, the difference of comparative method is only that human platelet serum is replaced by human AB serum (hAB) or fetal bovine serum (FBS), or there is no human platelet serum.Human AB serum and fetal bovine serum are used in this article with their common meaning, and can be, for example, obtained from commercial suppliers, such as VWR Avantor (Radnor, PA, USA) FBS catalogue provided and Merck (Merck) (West Point, PA, USA) Pennsylvania, USA hAB provided.

[0072] In some embodiments where the pyrogen is an endotoxin (preferably lipopolysaccharide), the response of PBMCs is higher than the response of PBMCs in a comparative method where the only difference is that the human platelet serum is replaced by 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 where the only difference is that the human platelet serum is replaced by human AB serum (hAB) or fetal bovine serum (FBS) (preferably fetal bovine serum). A "higher" PBMC response 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%, at least 200%, at least 250%, or at least 300% higher than a comparable method, the comparative method differing only in that the human platelet serum is replaced with a different medium supplement (preferably hAB or FBS) (or no medium supplement is used). PBMC responses can be determined as previously described herein. For example, in embodiments where the PBMC response determined is IL-6 production and / or secretion (preferably secretion), a higher PBMC response means that the PBMC produces and / or secretes more IL-6.

[0073] In some embodiments, the pyrogen detection sensitivity of the method is higher than that of a comparative method, the difference of which is that human platelet serum is replaced by different culture medium supplements (preferably hAB or FBS) (or no culture medium supplement is used). Sensitivity can preferably be expressed using a limit of quantitation (LoQ), which defines the lowest pyrogen concentration in a sample that can be detected by a method. A lower LoQ means that the method has improved sensitivity. It is understood by the skilled person that the exact calculation used in the LoQ determination may vary depending on the assay used to determine the PBMC response. For example, in the case of a spectrophotometric immunoassay (such as ELISA), the LoQ can be determined (preferably determined at about 450nm) by identifying the first average signal (OD) of the sample that exceeds the critical value, which can be defined as blank average OD+10*blank standard deviation. Optionally, the background OD measured at 630nm can be subtracted from the signal before analysis. Another example of LoQ determination is provided in the examples.

[0074] In some embodiments, the limit of quantification for endotoxins (preferably lipopolysaccharides) 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 quantification 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 quantification 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 quantification is determined in a spectrophotometric immunoassay, more preferably in an ELISA, and even more preferably in an ELISA at about 450 nm.

[0075] In some embodiments, the LoQ of an endotoxin (preferably lipopolysaccharide) is lower than the LoQ exhibited by a comparative method, the only difference being that the human platelet serum is replaced by 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 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)) is lower than the LoQ exhibited by a comparative method, the only difference being that the human platelet serum is replaced by human AB serum (hAB) or fetal bovine serum (FBS), preferably fetal bovine serum. A "lower" LoQ 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%, at least 200%, at least 250%, or at least 300% lower than the LoQ exhibited by a comparative method that differs only in that the human platelet serum is replaced with a different medium supplement (preferably hAB or FBS) (or no medium supplement is used).

[0076] In particular, when testing multiple identical samples, the detection methods of the present disclosure demonstrate low variability between measurements. Thus, in some embodiments, for multiple identical samples, the PBMCs determined respond with a coefficient of variation (ratio of standard deviation to mean measurement) of at most 30%. In some embodiments, for multiple identical samples, the PBMCs determined respond with a coefficient of variation of at most 25%, or 24%, 23%, 22%, or 21%. Preferably, for multiple identical samples, the PBMCs determined respond with a coefficient of variation of at most 20%. More preferably, for multiple identical samples, the PBMCs determined respond with a coefficient of variation of at most 15%. Still more preferably, for multiple identical samples, the PBMCs determined respond with a coefficient of variation of at most 10%, most preferably at most 9.5%.

[0077] The detection method of the present disclosure further demonstrates low variability between measurements of samples of multiple different concentrations. For example, this variability can be determined by generating a PBMC response / pyrogen concentration curve (for example, using a pyrogen standard as described later in this article) and applying nonlinear regression analysis. Therefore, in some cases, the concentration curve is a standard curve. Typically, a four-parameter or five-parameter logistic (4PL or 5PL) regression model is applied in bioassay analysis (preferably in ELISA). Such analysis is well known to the technician and can be performed using commercial software such as Graphpad Prism (GraphPad software, San Diego, CA, USA). The higher quality detection method is characterized in that the standard deviation of the measured values ​​is lower. It is further characterized in that the "goodness of fit" (R 2 ) is higher. In addition, higher quality methods are characterized by an increased (dynamic) measurement range, which can be defined as the absolute difference between the lowest and highest values ​​of the fit. An increased (dynamic) range can improve measurement accuracy within curves of similar slope, i.e., provide a larger concentration range over which pyrogens can be accurately detected.

[0078] In some embodiments where multiple samples of different concentrations are provided, the method is tested for goodness of fit (R 2 ) than a comparative method that differs only in that the human platelet serum is replaced by a different medium supplement (preferably hAB or FBS) (or no medium supplement is used). The 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%. In some embodiments, the method has an R 2 It 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.

[0079] In some embodiments where a plurality of samples of varying concentrations are provided, the (dynamic) range of the method is increased relative to a comparable method that differs only in that the human platelet serum is replaced with a different medium supplement (preferably hAB or FBS) (or no medium supplement is used). The increase in (dynamic) range 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%, at least 200%, at least 250%, or at least 300%.

[0080] In some embodiments where a plurality of samples of varying concentrations are provided, the standard deviation of the measurements obtained in the method is lower than that of a comparative method where the only difference is that the human platelet serum is replaced with a different culture medium supplement (preferably hAB or FBS) (or no culture medium supplement is used). The 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%. In some embodiments, the standard deviation of the method is 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 the set of replicates, then dividing this sum by its degrees of freedom, and then calculating the square root.

[0081] The detection method of the present disclosure is capable of detecting pyrogens in samples obtained from various products intended for therapeutic use or therapy (preferably for therapeutic use or human therapy for humans). Examples of such products include pharmaceutical compositions, medical devices and medical instruments as previously described herein. If no pyrogens are detected in / on the products, they can be safely released (approved) for use. In this context, release can be considered as being provided to the public while certifying that the product meets certain (relevant) standards.

[0082] Thus, in one aspect, a method for releasing a product (preferably a pharmaceutical product) for use is provided, comprising subjecting a sample derived from the product to a method for detecting pyrogens as previously described herein. Preferably, the product is released only when no pyrogen levels are detected or low pyrogen levels are detected. This may depend on the criteria met. In some embodiments, a method for releasing a pharmaceutical composition or medical device for use is provided, comprising subjecting a pharmaceutical composition or a sample derived from a medical device to a method for detecting pyrogens as previously described herein.

[0083] The present disclosure further provides kits. Preferably, the kits are suitable for practicing the methods of the present invention. The kits 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 comprising a triacylated lipopeptide or a bacterial cell component, more preferably comprising a toll-like receptor 1 / 2 (TLR1 / 2) agonist (e.g., PAM3CSK4) or a bacterial protein (e.g., flagellin). Pyrogen standards can be prepared according to Ph. Eur. guidelines (monograph 2.6.30, supra), or readily available from commercial suppliers, such as the EDQM (European Directorate for the Quality of Medicines & Healthcare; see, for example, the European Pharmacopoeia Reference Standards: Order and List provided by the EDQM). Examples of pyrogen standards are provided in the Examples section later herein. Preferred kits include a pyrogen or endotoxin standard and a vial containing human platelet lysate, and optionally further contain PBMCs. Preferably, the kit further comprises an incubation medium, optionally in combination with human platelet lysate.

[0084] The pyrogen standard can correspond to one or more samples. Preferably, a plurality of samples each containing different concentrations of pyrogens are used to produce a standard curve. In the case of endotoxin (preferably LPS), exemplary standard concentrations are 0.5EU / ml, 0.25EU / ml, 0.125EU / ml, 0.06EU / ml, 0.03EU / ml, 0.016EU / ml and 0.008EU / ml. Multiple repeated (identical) samples can be provided, preferably at least two, more preferably at least three, most preferably at least four. The test kit can include PBMC, preferably monocytes, more preferably mammalian monocytes, most preferably human monocytes. Suitable PBMCs are described herein before. The test kit can include one or more 96-well or 384-well plates. Compared to standard pyrogen detection methods (for example, compared to monocyte activation tests using 96-well plates), 384-well plates can improve test throughput while minimizing reagents and total cost.

[0085] Therefore, in one aspect, a kind of complete kit is provided, this complete kit comprises pyrogen standard, PBMC, human platelet lysate and one or more 96-well or 384-well plates.In certain embodiments, this kit is monocyte activation test (MAT) test kit.In certain embodiments, this kit further comprises the incubation culture medium optionally combined with human platelet lysate as described herein before.In certain embodiments, the concentration of human platelet lysate is 0.05 to 20 volume %, preferably 0.2 to 10 volume %, more preferably 0.5 to 5 volume %, even more preferably 1 to 4 volume %, most preferably 0.5 to 2.5 volume %, such as about 2 volume %.In certain embodiments, the concentration of human platelet lysate is 0.1 to 15 volume %, preferably 0.1 to 10 volume %, more preferably 1 to 4 volume %, most preferably 0.5 to 2.5 volume %, such as about 2 volume %.

[0086] In certain embodiments, the test kit is applicable to testing simultaneously at least three different products (e.g., different pharmaceutical compositions, medical devices, or medical instruments) for the presence of pyrogens. In certain embodiments, the test kit is applicable to testing simultaneously at least four different products. In certain embodiments, the test kit is applicable to testing simultaneously at least five different products. In certain embodiments, the test kit is applicable to testing simultaneously at least six different products. In certain embodiments, the test kit is applicable to testing simultaneously at least seven different products. In certain embodiments, the test kit is applicable to testing simultaneously at least eight different products. In certain embodiments, the test kit is applicable to testing simultaneously at least nine different products. In certain embodiments, the test kit is applicable to testing simultaneously at least ten different products. Test is preferably carried out according to the guidance of Ph.Eur. about pyrogen and endotoxin detection (monograph 2.6.30, ibid.).

[0087] "Simultaneous" testing refers to testing samples corresponding to different products in a single plate, which allows for increased testing throughput and minimizes reagent and overall costs.

[0088] In some embodiments, the volume of incubation medium in each sample tested in each well is at most 300 μL, preferably at most 250 μL. In some embodiments, its volume is about 175 μL. In some embodiments, it has a volume of at most 170, 165, 160, 155, or 150 μL. In some embodiments, the volume of incubation medium in each sample tested in each well is 20 to 250 μL, preferably 30 to 175 μL, more preferably 50 to 110 μL. In some embodiments, the volume of incubation medium in each sample tested in each well is 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, more preferably 90 to 100 μL. In some embodiments, the volume of incubation medium in each sample tested per well is 20 to 100 μL, preferably 30 to 100 μL, more preferably 50 to 100 μL. In some embodiments, the volume of incubation medium in each sample tested per well is 80 to 120 μL. In some embodiments, the volume of incubation medium in each sample tested per well is 80 to 100 μL. In some embodiments, the volume of incubation medium in each sample tested per well is 100 μL or about 100 μL.

[0089] In some embodiments, PBMCs are cultured at a density of at most 500 x 1000 cells / cm 2 , preferably at most 250×1000 cells / cm 2 In some embodiments, PBMCs are present at a density of about 10×1000 cells / cm 2 About 250 × 1000 cells / cm 2 , preferably about 50×1000 cells / cm 2 About 150 × 1000 cells / cm 2 , more preferably about 90×1000 cells / cm 2 About 130 × 1000 cells / cm 2 In some embodiments, PBMCs are present at a density of 110×1000 cells / cm 2 or approximately 110 × 1000 cells / cm 2 The density exists.

[0090] In some embodiments, the volume of incubation medium in each sample tested per well is 80 to 120 μL, and the PBMCs are cultured at a population of about 90 to about 130×1000 cells / cm 2 In some embodiments, the volume of incubation medium in each sample tested per well is 100 or about 100 μL, and the PBMCs are present at a density of 110×1000 cells / cm 2 or approximately 110 × 1000 cells / cm 2 The density exists.

[0091] Generally, the above volumes can be considered to be the total volume present in the well.In some embodiments, the incubation medium is added in an amount such that the total volume as described above is achieved.

[0092] General Definition

[0093] In this document and its claims, the verb "comprise" and its conjugations are used in its non-restrictive sense, meaning that the items following the word are included, but items not specifically mentioned are not excluded. In addition, the verb "compose" can be replaced by "essentially consisting of...", meaning that the method, respectively, component as defined herein may include additional steps, respectively, components in addition to the steps, respectively, components specifically indicated, and the additional steps, respectively, components do not change the unique characteristics. In addition, referring to an element by the indefinite article "a / an" does not exclude the possibility of the presence of more than one such element, unless the context clearly requires the presence of one and only one such element. Therefore, the indefinite article "a / an" generally means "at least one".

[0094] As used herein, having "at least" a particular value means that 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 ... and so on.

[0095] The word "about" or "approximately" when used in conjunction with a numerical value (e.g., about 10) preferably means that the value may be the given value or the given value plus or minus 5%, preferably 1%. As used herein, the term "and / or" means that one or more of the stated circumstances may occur alone or in combination with at least one of the stated circumstances, up to and including all of the stated circumstances. Various embodiments are described herein. Unless otherwise stated, each embodiment described herein can be combined together.

[0096] All patent applications, patents, and publications mentioned herein are incorporated herein by reference in their entirety. The present invention is in no way limited to the methods and materials specifically described. The present invention is further illustrated by the following examples, which are provided for illustrative purposes only and should not be construed as limiting the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] Figures 1A-1D . IL-6 production in response to endotoxin (LPS) in the presence of different media supplements. PBMC were incubated with a range of LPS concentrations in the presence of FBS, hPL, or hAB for 20 h in an incubator. IL-6 production in the presence of 1% (v / v) LPS was then assessed by ELISA. Figure 1A ), 2% volume ( Figure 1B ), or 4% by volume ( Figure 1C ) medium supplements. The results are shown as the mean OD + / - standard deviation of 4 replicates. Figure 1D The areas under the curves for different concentrations of media supplements are shown.

[0098] Figures 2A-2C Statistical comparison of the effects of culture medium supplements FBS, hPL, and hAB on IL-6 production by PBMC in response to endotoxin (LPS). Figures 1A-1D The data were fitted with a 4-parameter logistic (4PL) nonlinear regression model. The standard deviation (SD, Figure 2A ), (dynamic) range( Figure 2B ), and goodness of fit (R 2 , Figure 2C ).

[0099] Figures 3A-3D . 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 FBS, hPL, or hAB for 20 h in an incubator. IL-6 production in the presence of 1% (v / v) PAM3CSK4 was then assessed by ELISA. Figure 3A ), 2% volume ( Figure 3B ), or 4% by volume ( Figure 3C ) medium supplements. The results are shown as the mean OD + / - standard deviation of 4 replicates. Figure 3D The areas under the curves for different concentrations of media supplements are shown.

[0100] Figures 4A-4C Statistical comparison of the effects of culture medium supplements FBS, hPL, and hAB on IL-6 production by PBMC in response to PAM3CSK4. Figures 3A-3DThe data were fitted with a 4-parameter logistic (4PL) nonlinear regression model. The standard deviation (SD, Figure 4A ), (dynamic) range( Figure 4B ), and goodness of fit (R 2 , Figure 4C ).

[0101] Figures 5A-5D . IL-6 production in response to flagellin BS in the presence of different medium supplements. PBMC were incubated with a range of concentrations of flagellin BS in the presence of FBS, hPL, or hAB for 20 hours in an incubator. IL-6 production in the presence of 1% (v / v) flagellin BS was then assessed by ELISA. Figure 5A ), 2% volume ( Figure 5B ), or 4% by volume ( Figure 5C ) medium supplements. The results are shown as the mean OD + / - standard deviation of 4 replicates. Figure 5D The areas under the curves for different concentrations of media supplements are shown.

[0102] Figures 6A-6C Statistical comparison of the effects of culture medium supplements FBS, hPL, and hAB on IL-6 production by PBMC in response to flagellin BS. Figures 5A-5D The data were fitted with a 4-parameter logistic (4PL) nonlinear regression model. The standard deviation (SD, Figure 6A ), (dynamic) range( Figure 6B ), and goodness of fit (R 2 , Figure 6C ).

[0103] Figures 7A-7D .FBS induces IL-6 responses against NEP at lower percentages. In the presence of different concentrations of FBS, PBMCs were treated with a range of endotoxins (LPS, Figure 7A )、PAM3CSK4( Figure 7B ) or flagellin BS ( Figure 7C ) were incubated together in an incubator for 20 hours. Figure 7D The areas under the curves for different pyrogens and concentrations are shown.

[0104] Figures 8A-8D hPL induces IL-6 responses against pyrogens. In the presence of different concentrations of hPL, PBMCs were treated with a range of endotoxin concentrations ( Figure 8A )、PAM3CSK4( Figure 8B ) or flagellin BS ( Figure 8C) were incubated in an incubator for 20 hours. IL-6 production was then assessed by ELISA. Results are presented as the mean OD + / - standard deviation of four replicates. Figure 8D The areas under the curves for different pyrogens and concentrations are shown.

[0105] Figures 9A-9D Effect of cell density on IL-6 production in monocyte activation assay. PBMCs were cultured at the indicated cell densities (×1000 cells / cm 2 ) were inoculated into 384-well plates with a total volume of 33 μL ( Figure 9A )、50μL( Figure 9B )、66μL( Figure 9C ), or 100 μL ( Figure 9D ), the X-axis indicates the cell density (×1000 cells / cm 2 ), the y-axis indicates IL-6 per 1000 cells. 4 replicates (symbols) and the mean (horizontal line) are plotted. The figure title indicates the total MAT volume.

[0106] Figure 10 Each density (×1000 cells / cm 2 Figure 2 is a graph of absorbance values ​​(in optical density (OD)) (y-axis) at various LPS concentrations (in EU / ml) (increasing from left to right). The dashed line indicates 0.1 OD. The average of three experiments, each with four replicates, is depicted.

[0107] Figure 11 Signal-to-noise ratio (bars) versus density (1000 cells / cm 2 The signal-to-noise ratio was calculated by dividing the OD at 0.016 EU / ml by the OD at the blank.

[0108] Figure 12 The coefficient of variation (CV) at different cell densities was calculated for each concentration of LPS (EU / ml) and the average CV% of four replicates was then calculated for each density (1000 cells / cm 2 ) were averaged. The graph shows the mean (bars) and standard deviation (error bars) of three different experiments.

[0109] Figure 13 The relative gain (y-axis) is plotted as a percentage of the optical density (OD) at 0.032 EU / ml LPS normalized to the optical density in a 100 μl assay volume. The x-axis represents the number of cells per cm2 at 1000 cells / cm2. 2 The error bars represent the standard deviation of three experiments.

[0110] Figure 14Average CV% at different assay volumes The average CV% of four replicates was calculated for each LPS concentration (EU / ml) and then averaged for each assay volume (μl) and density. The graph shows the average (bars) and standard deviation (error bars) of three different experiments (from left to right, 55 / 110 / 220). The pattern represents 1000 cells / cm 2 Calculate the cell density.

[0111] Figure 15 . Relationship between absorbance (OD) and LPS concentration (EU / ml). The X-axis is a logarithmic scale. The gray line represents the density (×1000 cells / cm 2 ) and a 100 μl assay volume. The black line represents the 220 density (×1000 cells / cm 2 ) and a standard curve with an assay volume of 66 μl. Error bars indicate the standard deviation of four replicates.

[0112] Figure 16 . Curve slope. The bar indicates the curve slope of the four-parameter logistic curve. Left: At a density of 110 (×1000 cells / cm 2 ) and LPS standard curve at 100 μl assay volume. Right: LPS standard curve at 220 cells / cm 2 ) and an LPS standard curve in a 66 μl assay volume.

[0113] Figure 17 Average CV% The average CV% of four replicates was calculated for each LPS concentration (EU / ml) and then averaged for each volume / density combination. Left: At a density of 110 (×1000 cells / cm 2 ) and an assay volume of 100 μl. Right: Average CV% of LPS standard curve at a density of 220 (×1000 cells / cm 2 ) and the average CV% of the LPS standard curve in an assay volume of 66 μl.

[0114] Examples

[0115] Example 1. Human platelet lysate results in increased reactivity to endotoxin and non-endotoxin pyrogens

[0116] PBMC treatment

[0117] PBMCs (10 million PBMCs / ml) were rapidly thawed in a water bath (Grant JB Nova, Cambridge, UK) set at 37°C and resuspended by slowly adding (approximately 1 mL every 5 seconds) pre-warmed (37°C) RPMI 1640+Glutamax+HEPES (Gibco, Grand Island, NY, USA) medium. The cells were immediately added to plates containing the following medium supplements: human platelet lysate (hPL, PLTMax human platelet lysate, available from Millipore (Product No.: SCM141; Catalog No.: 6D0352)), fetal bovine serum (FBS, Avantovida, USA), or human AB serum (hAB, Merck, West Point, PA, USA). Each sample well contained approximately 1 million PBMCs / ml, corresponding to approximately 300 × 1000 cells / cm 2 density.

[0118] LPS, PAM3CSK4, and flagellin BS preparations

[0119] Lipopolysaccharide (LPS) was obtained from the European Directorate for the Quality of Medicines & Healthcare (EDQM, batch 5.1) and processed according to EDQM instructions. Briefly, LPS was rehydrated by vortexing in 5 mL of LAL reagent water (LRW, Lonza Bioscience, Basel, Switzerland) for 30 minutes and diluted to a stock concentration of 100 (EEU / mL) by vortexing in LRW for 3 minutes. LPS reference endotoxin (RSE) samples were prepared in RPMI 1640 + Glutamax + HEPES (Gibco, Grand Island, NY, USA) and resuspended and mixed 20 times.

[0120] A stock solution (10 μg / mL) of PAM3CSK4 (a toll-like receptor 1 / 2 (TLR1 / 2) agonist) (Invivogen, Toulouse, France) was prepared by adding 950 μL of RPMI 1640 + Glutamax (Gibco, Grand Island, NY, USA) to a 50 μL aliquot of 10 μg / mL and mixing by brief vortexing. This stock solution was diluted to a sample concentration of 50 ng / mL, after which two-fold serial dilutions (2 mL + 2 mL) were prepared by resuspending in RPMI. 50 μL of each dilution mix was added to the plate, maintaining the same concentration of each test medium supplement.

[0121] A stock solution (500 ng / mL) of flagellin-BS (Anolun Biotech) was prepared by adding 950 μL of RPMI to a 50 μL aliquot of 10 μg / mL and mixing by resuspending. This stock solution was diluted to a sample concentration of 125 ng / mL, after which two-fold serial dilutions (2 mL + 2 mL) were created by resuspending in RPMI. 50 μL of each dilution mix was added to the plate, maintaining the same concentration of each test medium supplement.

[0122] ELISA

[0123] ELISA plates (MaxiSorp, NUNC, Amsterdam, the Netherlands) were coated with IL-6 capture antibody (clone 13A5, Mabtec) at a dilution of 1:2000 in PBS (VWR, Solon, OH, USA) and incubated overnight at 4°C. ELISA was performed according to the manufacturer's protocol (MabTech AB, Stockholm, Sweden). Optical density (OD) was measured at a wavelength of 450 nm using an absorbance microplate reader Multiscan Ascent (Thermo Scientific, Vantaa, Finland). Background OD at 630 nm was subtracted from the 450 nm signal before further analysis. The supernatant (50 μL) was diluted 1:4 in ELISA diluent (PBST (ELISA wash buffer, Biolegend, Amsterdam, The Netherlands) + 0.1% BSA) and added to an ELISA microplate.

[0124] statistics

[0125] Statistical analyses (including 4PL logistic regression) were performed using Graphpad Prism 8.

[0126] result

[0127] To investigate whether hPL could be used to improve pyrogen detection using MAT, we incubated PBMC pools from four single donors with a range of endotoxin concentrations using hPL, FBS, and hAB as media supplements, followed by measurement of ELISA absorbance (in optical density (OD)) after 20 h ( Figures 1A-1D ) to test IL-6 production. The results showed that all medium supplements showed dose-dependent IL-6 production, which increased with increasing volume percentage of medium supplements ( Figures 1A-1C Across all media supplements, hPL consistently displayed a higher LPS response compared to other media supplements ( Figure 1D ), whereas LPS incubation in the presence of FBS and hAB induced similar IL-6 production at each tested percentage. To further investigate the effect of medium supplementation on the IL-6 response of PBMC to LPS, we performed Figures 1A-1C The curves were fitted with a 4-parameter logistic (4PL) nonlinear regression model, and the statistical parameters standard deviation ( Figure 2A ), (dynamic) range( Figure 2B ), and goodness of fit (R 2 , Figure 2C ). The 4PL logistic regression model is a nonlinear regression commonly used in dose-response curves (e.g., in MAT and / or receptor binding assays) that requires four parameters to fit a sigmoidal curve. Based on this model, sample data can be interpolated to determine the concentration of contaminants (expressed in EEU / mL) in the sample to be tested in the MAT. Therefore, the quality of this 4PL logistic regression model that is fitted to the reference endotoxin standard (RSE) curve is crucial for accurately determining the sample contaminant level. High-quality 4PL regression models are characterized by low variation (standard deviation), increased range, and goodness of fit of the model to the RSE data (R 2 ) was higher. The results of 4PL regression showed that the standard deviation (SD) of hPL was consistently lower than that of other culture medium supplements ( Figure 2A ), indicating that the variation in all tested percentages was reduced. In addition, the range of hPL was consistently increased when hPL was used compared to other supplements ( Figure 2B ), indicating an improved dynamic range for determining IL-6 production. The goodness of fit (R 2 ) is also higher ( Figure 2C These results indicate that hPL showed the strongest response in detecting LPS, with less variation, a wider range, and improved goodness of fit compared to the other supplements.

[0128] Expanding on the results with endotoxin, the effects of the culture medium supplements hPL, FBS, and hAB on the ability of MAT to identify the presence of toll-like receptor (TLR)-1 / 2 ligands were investigated. To this end, PBMC pools from four single donors were incubated with a range of concentrations of PAM3CSK4 and assayed for IL-6 production by ELISA ( Figures 3A-3D Compared to FBS, both human-based media supplements induced a strongly enhanced IL-6 response to PAM3CSK4 ( Figures 3A-3D), indicating that human-based supplements have an increased ability to identify TLR-1 / 2 ligands compared to animal-based supplements. Similar to LPS, hPL demonstrated the highest IL-6 response to PAM3CSK4 in all tested percentages, with increased area under the curve compared to other media supplements. Figures 3A-3C The statistical fitting of the (4PL) nonlinear regression model to the data revealed that the SD of FBS was lower than that of the other supplements ( Figure 4A ), which may be due to the overall lower OD values ​​of FBS compared to human-based supplements. Among the human-based supplements, hPL showed consistently lower SD compared to hAB, again indicating reduced variation across the volume percentages tested ( Figure 4A Furthermore, hPL exhibited an increased range ( Figure 4B ) and improved goodness of fit ( Figure 4C These results indicate that human-based supplements are superior to FBS in detecting TLR-1 / 2 ligands, with hPL demonstrating reduced variation, improved goodness of fit, and higher range compared to hAB.

[0129] In addition to endotoxin and PAM3CSK4, the effects of the culture medium supplements hPL, FBS, and hAB on the IL-6 response to the common NEP flagellin protein (flagellin BS) of Bacillus subtilis were also investigated. Similarly, PBMC pools from four different donors were incubated with a range of concentrations of flagellin BS, after which IL-6 production was measured by ELISA. At all volume percentages tested, hPL induced the strongest IL-6 production in response to flagellin BS, with an increased area under the curve (AUC) compared to all other culture medium supplements. Figures 5A-5D ), indicating that the ability of hPL to detect flagellin BS was increased. Figures 5A-5C Comparison of the (4PL) nonlinear regression models fitted to the data showed that hPL was consistently smaller in SD than other media supplements ( Figure 6A ), Range Increase ( Figure 6B ) and the goodness of fit improved ( Figure 6C These results indicate that hPL is a superior medium supplement for detecting flagellin BS compared to hAB and FBS.

[0130] The effects of the culture medium supplements hPL, FBS, and hAB on the limit of quantification (LoQ), a stringent measure of sensitivity, of MAT for endotoxin, PAM3CSK4, and flagellin BS were compared. The LoQ was determined by identifying the first mean signal (OD) exceeding a critical value, defined as the blank mean OD + 10*blank standard deviation. Among all culture medium supplements tested, hPL consistently had lower LoQs compared to FBS for all pyrogens included in the study (Table 1). Specifically, hPL had a LoQ for PAM3CSK4 that was 0.9 and 0.04 ng / mL lower than FBS and hAB, respectively. Furthermore, hPL had a LoQ for flagellin BS that was 0.4 and 0.8 lower than FBS and hAB, respectively. Finally, the human-based supplement had a LoQ for LPS that was 0.008 EEU / mL lower than FBS, but the human-based supplements were equivalent when compared to each other. These observations indicate that hPL is the most desirable medium supplement for testing samples containing multiple types of pyrogens, with relatively high sensitivity for all pyrogens included in the study.

[0131] Table 1. Limits of quantitation (LoQ) of MAT using 2% FBS, 2% hPL, or 2% hAB as media supplements.

[0132]

[0133] Example 2. Determination of the Optimal Medium Supplement Volume % for MAT

[0134] Will Figures 1A-1C , 3A-3C and 5A-5C FBS data were combined ( Figures 7A-7D ) to compare the BS responses of LPS, PAM3CSK4, and flagellin at different volume percentages. From the data, it can be observed that for LPS, there is a dose-response relationship between FBS and IL-6 production ( Figure 7A However, lower volume percentages of 1% and 2% FBS induced increased IL-6 responses against NEP compared to 4% ( Figure 7B and C), but LPS could still be detected at those lower percentages ( Figure 7A For this reason, 1% and 2% FBS are preferred over 4% FBS. Figures 1A-1C , 3A-3C and 5A-5C hPL data were combined ( Figures 8A-8D ) to compare the responses of LPS, PAM3CSK4, and flagellin BS at different volume percentages. Similar to FBS, a dose-response relationship between hPL and IL-6 production was observed for LPS ( Figure 8A In addition, 2% by volume induced an increased IL-6 response to NEP compared to 4% ( Figure 8D ).

[0135] Example 3. Optimization of cell density and assay volume

[0136] Control preparation

[0137] Lipopolysaccharide (LPS) was obtained from EDQM (batch 5.1) and processed according to the instructions of EDQM. LPS was rehydrated by vortexing in 5 mL of LAL reagent water (LRW, Lonza Bioscience, Basel, CH) for 30 minutes and diluted to a stock concentration of 10 endotoxin units / ml (EU / ml) by vortexing in LRW for 3 minutes. Subsequently, an LPS reference endotoxin curve (RSE) was created via serial dilutions by mixing by resuspending in RPMI 1640 (Thermo Fisher Scientific, Waltham, MA, USA).

[0138] PBMC treatment

[0139] A vial of PBMCs (10 million PBMCs / ml) was rapidly thawed in a water bath set at 37°C and resuspended by slowly adding pre-warmed (37°C) RPMI medium containing 4% human culture medium supplement (Mediatech, Manassas, VA, USA).

[0140] Cell density

[0141] The sample of 0.2EU / ml LPS is plated on 384-well microplates (Thermo Fisher Scientific, Waltham, Massachusetts, USA) with 50% of the final volume, to obtain various different final volumes (each experiment) as indicated. Subsequently, cell suspensions are added at a ratio of 1: 1 by different cell concentrations (each experiment) as indicated, to obtain a final concentration of 2% (volume / volume) of human culture medium supplement (HMS). The final concentration of LPS corresponds to a two-fold dilution series starting with 0.1EU / mL, obtained by being resuspended in the RPMI in the plate. Cells are incubated together with LPS at 37°C and 5% CO2 in an incubator (Binder (CB60), Tuttlingen, Germany (Tuttlingen, Germany)) for 20 hours + / - 1 hour, followed by measurement of IL-6 concentration by ELISA as explained below. IL-6 per 1000 cells was calculated by interpolating the measured optical density (OD) on 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.

[0142] An LPS standard curve (33 microliters, concentrations of 0.064 EU / ml, 0.032 EU / ml, 0.016 EU / ml, 0.008 EU / ml, 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 of 2) at 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 At each cell density, the final LPS concentration was 0.032 EU / ml, 0.016 EU / ml, 0.008 EU / ml, 0.004 EU / ml, and 0.002 EU / ml. The final HMS concentration in each well was 2%.

[0143] MAT incubation volume

[0144] Samples of the LPS standard curve (concentrations: 0.064 EU / ml, 0.032 EU / ml, 0.016 EU / ml, 0.008 EU / ml, 0.004 EU / ml) were plated onto 384-well microplates in three different volumes (16.7 microliters, 33 microliters, and 50 microliters). Cryopreserved PBMCs were thawed and reconstituted in RPMI culture medium containing 4% human culture medium supplement. Cell suspensions were diluted with different cell concentrations and added to the plate at a 1:1 ratio, resulting in 55,000 cells / cm at each assay volume (33 microliters, 66 microliters, and 100 microliters) at a final HMS concentration of 2%. 2 , 110,000 cells / cm 2 and 220,000 cells / cm 2 final cell density.

[0145] MAT

[0146] Samples for the LPS standard curve were added to the culture plate at different volumes (at a 1:1 ratio with resuspended PBMCs) and incubated for 16 hours at 37°C in an incubator with 5% CO2. The final concentrations of LPS were 0.5 EU / ml, 0.25 EU / ml, 0.125 EU / ml, 0.06 EU / ml, 0.03 EU / ml, 0.016 EU / ml, and 0.008 EU / ml. The final concentration of HMS was 2%.

[0147] ELISA

[0148] ELISA plates were coated with IL-6 capture antibody (clone 13A5, MabTech AB, Stockholm, Sweden) at a dilution of 1:2000 in phosphate-buffered saline and incubated overnight at 4°C. ELISA was performed using the MabTech ELISAbasic IL-6 kit (HRP) according to the manufacturer's protocol (MabTech). Absorbance was measured at a wavelength of 450 nm using a Thermo-Scientific absorbance microplate reader. The background at 630 nm was subtracted. The supernatant (16.7 μl) was diluted 1+1 in incubation buffer and added to the ELISA plate.

[0149] statistics

[0150] Statistical analyses (including four-parameter / five-parameter logistic regression) were performed using Graphpad Prism 8 (GraphPad Software, San Diego, CA, USA).

[0151] result

[0152] Increasing cell density at different 384MAT volumes at a concentration of 0.1 EU / ml showed improved IL-6 responses per 1000 cells, with 303 densities (×1000 cells / cm2) in 50 μL, 66 μL, and 100 μL showing an improvement in IL-6 responses per 1000 cells. 2 A sharp increase was observed at 33 μL volume in MAT with a density of 152 (×1000 cells / cm 2 ) compared to 227 cells per cm 2 ) was observed to increase the signal by 3 times, while at 303 density (×1000 cells / cm 2 ) showed a slight decrease in IL-6 per cell ( Figures 9A-9D ).

[0153] Higher density means stronger LPS signal (OD) at each LPS concentration, but also increased background signal (at 0.00 EU / ml) - at a density of 440 (×1000 cells / cm 2 ) up to 0.12OD( Figure 10 Furthermore, the signal-to-noise ratio (as calculated by dividing the signal at 0.032 EU / ml by the background signal) increased with increasing density, reaching 0.032 at a density of 110 (×1000 cells / cm 2 ) reaches the optimal value (SNR 6.4) under .

[0154] In addition, although the LPS signal and signal-to-noise ratio increase with density ( Figure 11 ), but repeated variation also increases (e.g. Figure 12 As shown), at a density of 440 (×1000 cells / cm 2 ) with an average CV% as high as 28.5%. 2 ), the signal-to-noise ratio was the highest and the average CV% was the lowest among the densities tested.

[0155] The 33 μl MAT volume showed a significant increase in the LPS response, as Figure 13 Depicted (relative gain normalized to 0.032 EU / ml in 100 μL is shown). Relative gain also increased when higher cell densities were plated, with the relative gain increasing at a density of 55 (×1000 cells / cm) at a MAT volume of 33 μL. 2 ) was 219% at a density of 110 (×1000 cells / cm 2 ) was 304% and at a density of 220 (×1000 cells / cm 2 ) was 387%. However, smaller volumes showed increased variation between replicates ( Figure 14 At each density, 100 μL MAT volume showed the lowest CV% (55 density (×1000 cells / cm 2 ) was 11.8% and the density was 110 (×1000 cells / cm 2 ) was 13.3%, and the density (×1000 cells / cm 2 ) was 18.6%. The pattern of increasing CV% with increasing density was observed for each assay volume, but the CV% at 110 density (×1000 cells / cm 2 ), which showed a density greater than 220 (×1000 cells / cm ) in all assay volumes tested. 2 )Lower CV%.

[0156] For both configurations: at a MAT volume of 66 μL and a cell density of 220 (×1000 cells / cm 2 ) and a MAT volume of 100 μL and a cell density of 110 (×1000 cells / cm 2 ), an LPS standard curve was created ( Figure 15 Four-parameter logistic regression showed that 0.99 (66 μL / 220 density (×1000 cells / cm 2 )) and 0.98 (100 μL / 100 density (× 1000 cells / cm 2 )) of R 2 value. Figure 16 The slopes of the curves for both plots are shown, and the slopes differ by a factor of about two between the two configurations. 2 ) The average CV% under MAT was higher than that of 100 μL / 110 cells / cm 2 ) were significantly higher (23.4% and 9.4%, respectively) ( Figure 17 ).

Claims

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

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

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

4. The method according to any one of claims 1 to 3, wherein the contacting of step ii) is performed in a standardized 96-well plate or a 384-well plate, preferably 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% by volume, preferably 0.5 to 4% by volume, more preferably 0.8 to 3% by volume, even more preferably 1 to 2.5% by volume, most preferably 1.2 to 2.2% by volume, such as about 2% by volume of human platelet lysate.

6. The method according to any one of claims 1 to 5, wherein the response of the PBMCs determined is the secretion 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 box proteins.

7. The method of claim 6, wherein the response of the PBMCs is higher than the response of PBMCs in a comparator method that differs only in that the human platelet serum is replaced by human AB serum or fetal calf serum, or in the absence of human platelet serum.

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

9. The method according to any one of claims 1 to 8, wherein the PBMCs are present at a density of at most 500 x 1000 cells / cm 2 , preferably at most 250×1000 cells / cm 2 The density exists.

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

11. The method according to any one of claims 1 to 10, wherein the limit of quantification of lipopolysaccharide is lower than 0.01 EEU / mL, and / or the limit of quantification of triacylated lipopeptides is lower than 0.1 ng / mL, and / or the limit of quantification of bacterial protein is lower than 1 ng / mL.

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

13. The method according to any one of claims 1-12, wherein the volume of the incubation medium is about 80 to about 120 μL, and wherein the PBMCs are cultured at a population of about 90 to about 130 x 1000 cells / cm 2 The density exists.

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

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

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

17. The method according to any one of claims 1-16, wherein the volume of the incubation medium is 30 to 175 μL, and wherein the PBMCs are cultured at a density of about 50×1000 cells / cm 2 About 150 × 1000 cells / cm 2 The density exists.

18. The method according to any one of claims 1 to 17, wherein the incubation medium containing hPLs 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, the method comprising subjecting the pharmaceutical composition or a sample derived from the medical device to a method according to any one of claims 1 to 18.

20. A kit comprising a pyrogen or endotoxin standard and a vial comprising human platelet lysate, further optionally comprising PBMCs.

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