Improved mononuclear cell activation testing

Abstract

The present invention is in the field of in vitro assays, in particular for the detection of pyrogens. The present invention provides an improved method for an animal-free pathway by providing an improved mononuclear cell activation test. The improved test allows for reduced reagent consumption and improved flux.

Description

Technical Field

[0001] The present invention belongs to the field of in vitro assays, particularly in the field of pyrogen and endotoxin detection. The present invention provides an improved monocyte activation test to provide an improved method for animal-free approaches. The improved test allows for reduced reagent consumption and improved throughput. Background Art

[0002] Since pyrogens and endotoxins cause severe adverse reactions in patients, the detection of the presence of pyrogens and endotoxins is highly relevant to the quality and safety testing of pharmaceutical compositions and medical devices. Conventionally used detection assays include the rabbit pyrogen test (RPT) and the Limulus amebocyte lysate assay (LAL) (also known as the bacterial endotoxin test (BET)). These tests are expensive, time-consuming, and require the use of experimental animals. Additionally, the Limulus amebocyte lysate assay is generally limited to the detection of Gram-negative bacteria and is prone to false positives.

[0003] An alternative to the above assays is the monocyte activation test (MAT), which does not require the use of animals and is more representative of the human immune response. MAT is a test subject to specific regulatory guidelines for sample preparation, testing, and result analysis established in the European Pharmacopoeia (Ph.Eur.; European Pharmacopoeia, 10th Edition, 2019, Monograph 2.6.30, Council of Europe). Subject to these guidelines, currently used protocols have low throughput, are relatively expensive, and require the use of large amounts of reagents and sample volumes. Therefore, there is still a need for improved animal-free pyrogen and endotoxin detection assays. Additionally, there is still a need for an improved monocyte activation test. There is a need to improve the throughput of the monocyte activation test. There is a need for further miniaturization of the monocyte activation test. There is a need to reduce the cost of the monocyte activation test. There is a need to reduce the material consumption of the monocyte activation test. Summary of the Invention

[0004] In a first aspect, there is provided a method for detecting pyrogens in a sample, the method comprising the steps of:

[0005] i) providing one or more samples;

[0006] ii) contacting the samples with peripheral blood mononuclear cells (PBMCs) in an incubation medium; and

[0007] iii) determining the response of the PBMCs,

[0008] wherein the volume of the incubation medium is at most 175 μL per sample.

[0009] In some embodiments, the method is a monocyte activation test. 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 contact in step ii) is carried out in a standardized 384-well plate. In some embodiments, the incubation medium contains 1 to 4 volume % (such as about 2%) of a human medium supplement. In some embodiments, the determined PBMC response is the secretion of inflammatory cytokines (such as IL-6, IL-1β, IL-8, TNF-α, MCP-1, IL-10, IFN-α, IFN-β, IFN-γ, IFN-λ), prostaglandins, or high mobility group proteins. In some embodiments, the response of PBMC is determined by ELISA assay. In some embodiments, PBMC is present at a density of at most 500×1000 cells / cm 2 , preferably at most 250×1000 cells / cm 2 . In some embodiments, PBMC is present at a density of about 10×1000 cells / cm 2 to about 250×1000 cells / cm 2 , preferably about 50×1000 cells / cm 2 to about 150×1000 cells / cm 2 , more preferably about 90×1000 cells / cm 2 to about 130×1000 cells / cm 2 . In some embodiments, for multiple identical samples, the determined PBMC response has a coefficient of variation of at most 20%.

[0010] In some embodiments, in step i), an additional control sample, preferably a lipopolysaccharide sample, is provided, and the control sample preferably contains about 0.005 to about 15 endotoxin units / mL.

[0011] In some embodiments, at least 50, preferably at least 97 samples are provided. In some embodiments, the volume of the incubation medium is 80 to 120 μL, and PBMC is present at a density of about 90 to about 130×1000 cells / cm 2 .

[0012] In another aspect, a method for releasing a pharmaceutical composition or a medical device for use is provided, the method comprising subjecting the pharmaceutical composition or a sample derived from the medical device to the method of the first aspect.

[0013] In another aspect, a kit is provided, the kit comprising a pyrogen or endotoxin standard, PBMC, and one or more 384-well plates. Detailed Description

[0014] In one aspect, a method for detecting pyrogens in a sample is provided, the method comprising the steps of:

[0015] i) providing one or more samples;

[0016] ii) contacting the samples with peripheral blood mononuclear cells (PBMCs) in an incubation medium; and iii) determining the response of the PBMCs,

[0017] wherein the volume of the incubation medium is at most 175 μL per sample. The method is attractive because it does not require the use of experimental animals. The method has high predictive power.

[0018] Pyrogen

[0019] Pyrogens are substances that can trigger an immune response in a subject by activating a series of immune processes, typically characterized by a rise in body temperature above normal levels (fever). The biological activity of a pyrogen is its ability to cause fever in a subject, or its pyrogenicity as referred to herein. A pyrogen can be an exogenous pyrogen. An "exogenous" or "external" pyrogen refers to a pyrogen that originates outside the subject. A pyrogen can be an endotoxin. Endotoxins (such as lipopolysaccharide (LPS)) are cell components of bacteria such as Gram-negative bacteria and are the main components that make up their cell outer walls. The presence of endotoxins in a 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 intravascular coagulation (DIC), endotoxin shock, and adult respiratory distress syndrome (ARDS).

[0020] 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 being flagellin, peptidoglycan, lipoproteins, lipoteichoic acid, fibroblast-stimulating lipopeptide 1, macrophage-activating lipopeptide 2, viral pyrogens, yeast pyrogens, and fungal pyrogens (e.g., yeast or fungal polysaccharides).

[0021] A pyrogen can be a product- or process-related impurity present in a pharmaceutical composition or on the surface (e.g., of a medical device). Examples of pyrogenic impurities are chemical reagents 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), and any other impurity that exhibits pyrogenicity.

[0022] A pyrogen can be a damage-associated molecular pattern (DAMP), which refers to biomolecules typically released by dead or damaged cells. Examples of DAMP pyrogens include biglycan, decorin, versican, hyaluronic acid, fibronectin, tenascin, uric acid, S100 proteins, ATP, GTP, F-actin, cyclophilin A, histones, HMGB1, HMGN1, IL-1α, IL-33, SAP130, DNA, RNA, mtDNA, TFAM, formyl peptides, mROS, calreticulin, defensins, heat shock proteins, and any other biomolecules released by cells that exhibit pyrogenicity.

[0023] A pyrogen can be a medium component of a pharmaceutical composition. Examples of such components include excipients, solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (such as antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, etc.

[0024] A pyrogen can be an endogenous pyrogen. "Endogenous" or "internal" pyrogens refer to pyrogens produced by a subject's body after contact with exogenous pathogens. Endogenous pyrogens can be associated with an inflammatory response. Endogenous pyrogens can be DAMPs. Examples of endogenous pyrogens include cytokines and chemokines.

[0025] In some embodiments, the pyrogen is an exogenous pyrogen. In some embodiments, the pyrogen is an endotoxin. In some embodiments, the endotoxin is a cell component (preferably lipopolysaccharide) of Gram-negative bacteria. "Gram-negative" bacteria refer to bacteria that do not retain the crystal violet dye used in the standard Gram staining method. In some embodiments, the Gram-negative bacteria are pathogenic bacteria. Examples of 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 product- or process-related impurity present in or on a pharmaceutical composition. In some embodiments, the pyrogen is a damage-associated molecular pattern (DAMP). In some embodiments, the pyrogen is a medium component of a pharmaceutical composition.

[0026] 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.

[0027] Step i) Provide a sample

[0028] In step i) of the method, one or more samples are provided. The sample(s) can be taken from (derived from) an original source, such as from a product like 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 generated by dilution or concentration of the original sample.

[0029] The sample can also be a replica of the original sample or subsample. The replicate samples are preferably 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 replicas can be prepared individually, or can be generated by obtaining equivalent subsamples from the original sample or subsample. The sample is preferably a liquid sample, more preferably an aqueous sample.

[0030] Samples can be obtained from a pharmaceutical composition (e.g., a therapeutic composition, a diagnostic composition, or a composition for preventing a disease or disorder or alleviating its symptoms (such as a vaccine)) to be tested for the presence of pyrogens or endotoxins. The pharmaceutical composition can be in any form, e.g., it can be a pharmaceutical composition suitable for topical, transdermal, intravenous, intramuscular, intraperitoneal, intracerebral, subcutaneous, intra-articular, intra-adipose, oral, intrahepatic, intra-visceral, intra-otic, intrathoracic, intracardiac, intraocular, or intratracheal administration or administration via inhalation. Intraocular administration is preferred. In some embodiments, the pharmaceutical composition is a vaccine.

[0031] Samples can be obtained from a surface (e.g., the surface of a medical device or instrument) to be tested for the presence of pyrogens or endotoxins. Examples of medical devices and instruments include bedpans, catheters, cardioverters, defibrillators, catheters, dialyzers, electrocardiographs, enema devices, endoscopes, gas cylinders, gauze sponges, surgical scissors, hypodermic needles, syringes, infection control devices (such as masks, surgical gowns, face shields, and goggles), instrument sterilizers, kidney dishes, nasogastric tubes, scalpels, nebulizers, ophthalmoscopes, otoscopes, pipettes, proctoscopes, radiologic technologists, sphygmomanometers, thermometers, tongue depressors, blood transfusion packs, tuning forks, ventilators, watches, etc. For example, such a sample can be obtained by rinsing the surface to be tested with a solution (e.g., water or buffer), collecting the rinse fluid, and using the rinse fluid for sample preparation.

[0032] In some embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, at least 99, at least 100, at least 132, at least 164, at least 196, at least 228, at least 260, at least 292, at least 324, at least 356, or at least 384 samples are provided. In some embodiments, at least 50, preferably at least 97 samples are provided. In some embodiments, at least 96 samples are provided. In some embodiments, at least 384 samples are provided. Advantageously, the method according to the invention can be practiced using a 384-well plate.

[0033] In some embodiments, in step i), additional control samples are provided. The control sample may contain a pyrogen (positive control) or may not contain a pyrogen (negative control). A pyrogen positive control sample containing a known pyrogen concentration can improve the quantification accuracy. For example, by using multiple samples containing different pyrogen concentrations, a standard curve can be made. The control sample can be a sample from a (reference) standard. Such a standard is described later herein.

[0034] In some embodiments, the control sample is an endotoxin sample, preferably a lipopolysaccharide (LPS) sample. Endotoxins are typically measured in endotoxin units per mL (EU / mL). One EU / mL is equal to approximately 0.1 - 0.2 ng endotoxin / mL (preferably 0.15 ng / mL) solution. In embodiments where the control sample is an endotoxin (preferably lipopolysaccharide) sample, the control sample preferably contains from about 0.005 to about 15 endotoxin units / mL. In some embodiments, the control sample contains from about 0.005 to about 1 endotoxin unit / mL. In some embodiments, the control sample contains from about 0.008 to about 0.5 endotoxin units / mL, or from about 0.01 to about 0.4, preferably from about 0.05 to about 0.3, more preferably from about 0.1 to about 0.2 EU / mL.

[0035] Step ii) Contact with PBMC

[0036] In step ii), one or more samples are contacted with peripheral blood mononuclear cells (PBMCs) in an incubation medium. In some embodiments, contact is made with whole peripheral blood or a fraction thereof containing PBMCs. In some embodiments, contact is made with isolated PBMCs. Blood fractions containing PBMCs and isolated PBMCs can be obtained by standard methods (e.g., using density gradient centrifugation).

[0037] In some embodiments, contact is made with a PBMC cell line. In some embodiments, contact is made with PBMCs obtained from a single donor. In some embodiments, contact is made with PBMCs obtained from pooled whole peripheral blood from multiple donors. In cases where PBMCs are obtained from a single or multiple donors, these donors are preferably qualified according to standardized guidelines, more preferably as described in sections 5 - 3, 5 - 4, 5 - 5, 6 - 3 of the European Pharmacopoeia (Ph.Eur.; European Pharmacopoeia, 10th Edition, 2019, Council of Europe) and / or monograph 2.6.30.

[0038] In some embodiments, contact is made with fresh PBMCs. In preferred embodiments, contact is made with cryopreserved PBMCs. Cryopreservation of PBMCs can be carried out according to standard procedures, e.g., as described in a standard manual (e.g., Hubel, A., 2018: Preservation of Cells: A Practical Manual, 1st Edition, Wiley - Blackwell, New Jersey, USA).

[0039] In some embodiments, the PBMCs are mammalian, preferably human. In some embodiments, the PBMCs are white blood cells. In some embodiments, the PBMCs are macrophages. In preferred embodiments, the PBMCs comprise or are monocytes, preferably mammalian monocytes, more preferably human monocytes. In some embodiments, the macrophages or monocytes are derived from pluripotent stem cells. PBMCs are well known and well characterized and typically contain 10 - 20% monocytes.

[0040] The method is preferably a monocyte activation test. More preferably, the monocyte activation test is performed according to the guidelines set forth in the European Pharmacopoeia monograph 2.6.30.

[0041] Contact with the PBMCs in step (ii) can be adding the sample to a culture medium containing the PBMCs. It can also be adding the culture medium containing the PBMCs to the sample. It can be carried out in any suitable container (e.g., a microplate (with one or more wells), a test tube (such as an Eppendorf tube), a flask (such as a conical flask), a bottle (such as a Schott bottle), a fermenter, etc.). In some embodiments, the contact is carried out in a standardized 96 - well plate. In preferred embodiments, the contact is carried out in a standardized 384 - well plate. Standardized well plates are widely available from commercial suppliers. Suitable standards are all five of the ANSI / SLAS standards, preferably 1 - 2004 (R2012), 2 - 2004 (R2012), 3 - 2004 (R2012), 4 - 2004 (R2012) and 6 - 2012 (R2012).

[0042] In the case of using a 96 - well plate or a 384 - well plate, a single sample can be placed in each well. The present invention uniquely allows the use of a 384 - well plate. Using a 384 - well plate has the advantages of enabling a higher detection throughput (since more samples can be tested simultaneously) and reducing reagent requirements and total costs. In the standardized use of well plates in MAT, a given number of wells are required for control or reference samples. This limits the number of wells available for actual test samples. A 384 - well plate has a better test - to - control ratio because after allocating wells to the required control samples, there are still more wells available for test samples. In this context, the following can be a conventional well allocation for a 96 - well plate, using 4 replicates for each data point:

[0043]

[0044]

[0045] Alternatively, in the case of 2 dilutions of each test sample and omitting the NEP control, the following may apply:

[0046] Sample Quadruplicate wells LPS curve 8 32 Two concentrations of Test Sample 1 2 8 Test Sample 1 LPS spike 2 8 Two concentrations of Test Sample 2 2 8 Test Sample 2 LPS spike 2 8 Two concentrations of Test Sample 3 2 8 Test Sample 3 LPS spike 2 8 Two concentrations of Test Sample 4 2 8 Test Sample 4 LPS spike 2 8 Total wells 96

[0047] It should be noted that the 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 above table, when each well is considered to contain the provided sample, a single test sample 1 results in multiple samples. When not explicitly stated, this difference will be clear from the context.

[0048] PBMC can be present at a ratio density during the contact step, and this ratio density is preferably in terms of the number of cells / cm 2 Measured (cm 2 refers to the growth area, which is preferably the area of the well cross-section; the preferred well is a flat-bottom well). In the context of the present disclosure, the density of PBMC refers to the density of PBMC used for each contacted sample. Considering the growth area (cm 2 ), cell concentration (number of cells / mL), and volume (mL), a person skilled in the art can easily calculate, for example, by using a cell counter or by using microscopy techniques, the density in terms of the number of cells / cm 2 in a container (preferably a well of a standardized 384-well plate). This density is a commonly used parameter, and a person skilled in the art should understand that there may be dead cells in the population. In some embodiments, PBMC (preferably containing monocytes) is present at a density of at most 500×1000 cells / cm 2 , preferably at most 250×1000 cells / cm 2 .

[0049] In some embodiments, PBMC (preferably containing monocytes) is present at about 10×1000 cells / cm 2 to about 250×1000 cells / cm 2 , preferably about 50×1000 cells / cm 2 to about 150×1000 cells / cm 2 , preferably about 60×1000 cells / cm 2 to about 140×1000 cells / cm 2 , preferably about 70×1000 cells / cm 2 to about 130×1000 cells / cm 2 , preferably about 80×1000 cells / cm 2 to about 120×1000 cells / cm 2 , more preferably about 90×1000 cells / cm 2 to about 130×1000 cells / cm 2 ; in some embodiments about 105×1000 cells / cm 2 to about 115×1000 cells / cm 2The density exists. In some embodiments, PBMC (preferably containing monocytes) is present at a density of 110×1000 cells / cm 2 or approximately 110×1000 cells / cm 2 .

[0050] In some embodiments, PBMC (preferably containing monocytes) is present at a density of from about 560 cells / well to about 14,000 cells / well, preferably from about 2,800 cells / well to about 8,400 cells / well, preferably from about 3,360 cells / well to about 7,840 cells / well, preferably from about 3,920 cells / well to about 7,280 cells / well, preferably from about 4,480 cells / well to about 6,720 cells / well, more preferably from about 5,040 cells / well to about 7,280 cells / well. In some embodiments, PBMC (preferably containing monocytes) is present at a density of 6,160 cells / well or approximately 6,160 cells / well. The growth area of a conventional 384-well plate is about 0.056 cm per well 2 . 6,160 cells / well can be considered as approximately 110×1000 cells / cm 2 .

[0051] The incubation medium can have a specific volume (preferably measured in mL or μL) during the contacting step. In the context of the present disclosure, the volume of the incubation medium refers to the volume per sample. In the provided method, the volume of the incubation medium is at most 175 μL per sample. In some embodiments, it has a volume of at most 150 μL per sample

[0052] In some embodiments, the volume of the incubation medium is from 20 to 150 μL, preferably from 30 to 140 μL, more preferably from 50 to 110 μL. In some embodiments, the volume of the incubation medium is from 80 to 120 μL. In some embodiments, the volume of the incubation medium is from 40 to 130 μL, preferably from 60 to 120 μL, more preferably from 70 to 115 μL, more preferably from 75 to 105 μL, more preferably from 85 to 105 μL

[0053] In some embodiments, the volume of the incubation medium is from 20 to 100 μL, preferably from 30 to 100 μL, more preferably from 50 to 100 μL. In some embodiments, the volume of the incubation medium is from 80 to 100 μL. In some embodiments, the volume of the incubation medium is 100 μL or approximately 100 μL

[0054] In some embodiments, the volume of the incubation medium is from 20 to 150 μL, and PBMC (preferably monocytes) is present at a density of from about 10×1000 cells / cm 2 to about 250×1000 cells / cm2 The density exists. In some embodiments, the volume of the incubation medium is 20 to 150 μL, and PBMCs (preferably monocytes) are present at a density of about 50 × 1000 cells / cm 2 to about 150 × 1000 cells / cm 2 The density exists. In some embodiments, the volume of the incubation medium is 20 to 150 μL, and PBMCs (preferably monocytes) are present at a density of about 90 × 1000 cells / cm 2 to about 130 × 1000 cells / cm 2 The density exists.

[0055] In some embodiments, the volume of the incubation medium is 30 to 140 μL, and PBMCs (preferably monocytes) are present at a density of about 10 × 1000 cells / cm 2 to about 250 × 1000 cells / cm 2 The density exists. In some embodiments, the volume of the incubation medium is 30 to 140 μL, and PBMCs (preferably monocytes) are present at a density of about 50 × 1000 cells / cm 2 to about 150 × 1000 cells / cm 2 The density exists. In some embodiments, the volume of the incubation medium is 30 to 140 μL, and PBMCs (preferably monocytes) are present at a density of about 90 × 1000 cells / cm 2 to about 130 × 1000 cells / cm 2 The density exists.

[0056] In some embodiments, the volume of the incubation medium is 80 to 120 μL, and PBMCs (preferably monocytes) are present at a density of about 10 × 1000 cells / cm 2 to about 250 × 1000 cells / cm 2 The density exists. In some embodiments, the volume of the incubation medium is 80 to 120 μL, and PBMCs (preferably monocytes) are present at a density of about 50 × 1000 cells / cm 2 to about 150 × 1000 cells / cm 2 The density exists. In some embodiments, the volume of the incubation medium is 80 to 120 μL, and PBMCs (preferably monocytes) are present at a density of about 90 × 1000 cells / cm 2 to about 130 × 1000 cells / cm 2 The density exists.

[0057] In some embodiments, the volume of the incubation medium is 50 to 110 μL, and PBMCs (preferably monocytes) are present at a density of about 10 × 1000 cells / cm 2to about 250 × 1000 cells / cm 2 are present at a density. In some embodiments, the volume of the incubation medium is 50 to 110 μL, and PBMCs (preferably monocytes) are present at a density of about 50 × 1000 cells / cm 2 to about 150 × 1000 cells / cm 2 are present at a density. In some embodiments, the volume of the incubation medium is 50 to 110 μL, and PBMCs (preferably monocytes) are present at a density of about 90 × 1000 cells / cm 2 to about 130 × 1000 cells / cm 2 are present at a density.

[0058] In some embodiments, the volume of the incubation medium is 20 to 100 μL, and PBMCs (preferably monocytes) are present at a density of about 10 × 1000 cells / cm 2 to about 250 × 1000 cells / cm 2 are present at a density. In some embodiments, the volume of the incubation medium is 20 to 100 μL, and PBMCs (preferably monocytes) are present at a density of about 50 × 1000 cells / cm 2 to about 150 × 1000 cells / cm 2 are present at a density. In some embodiments, the volume of the incubation medium is 20 to 100 μL, and PBMCs (preferably monocytes) are present at a density of about 90 × 1000 cells / cm 2 to about 130 × 1000 cells / cm 2 are present at a density.

[0059] In some embodiments, the volume of the incubation medium is 30 to 100 μL, and PBMCs (preferably monocytes) are present at a density of about 10 × 1000 cells / cm 2 to about 250 × 1000 cells / cm 2 are present at a density. In some embodiments, the volume of the incubation medium is 30 to 100 μL, and PBMCs (preferably monocytes) are present at a density of about 50 × 1000 cells / cm 2 to about 150 × 1000 cells / cm 2 are present at a density. In some embodiments, the volume of the incubation medium is 30 to 100 μL, and PBMCs (preferably monocytes) are present at a density of about 90 × 1000 cells / cm 2 to about 130 × 1000 cells / cm 2 are present at a density.

[0060] In some embodiments, the volume of the incubation medium is 50 to 100 μL, and PBMCs (preferably monocytes) are present at a density of about 10 × 1000 cells / cm2 at a density of from about 250×1000 cells / cm 2 In some embodiments, the volume of the incubation medium is 50 to 100 μL, and PBMCs (preferably monocytes) are present at a density of from about 50×1000 cells / cm 2 to about 150×1000 cells / cm 2 In some embodiments, the volume of the incubation medium is 50 to 100 μL, and PBMCs (preferably monocytes) are present at a density of from about 90×1000 cells / cm 2 to about 130×1000 cells / cm 2 are present.

[0061] In some embodiments, the volume of the incubation medium is 80 to 100 μL, and PBMCs (preferably monocytes) are present at a density of from about 10×1000 cells / cm 2 to about 250×1000 cells / cm 2 In some embodiments, the volume of the incubation medium is 80 to 100 μL, and PBMCs (preferably monocytes) are present at a density of from about 50×1000 cells / cm 2 to about 150×1000 cells / cm 2 In some embodiments, the volume of the incubation medium is 80 to 100 μL, and PBMCs (preferably monocytes) are present at a density of from about 90×1000 cells / cm 2 to about 130×1000 cells / cm 2 are present.

[0062] In some embodiments, the volume of the incubation medium is 100 μL or about 100 μL, and PBMCs (preferably monocytes) are present at a density of 110×1000 cells / cm 2 or about 110×1000 cells / cm 2 are present.

[0063] The incubation medium can be any medium suitable for the culture of PBMCs, preferably mammalian PBMCs, more preferably human PBMCs, and most preferably human monocytes. Preferably, the incubation medium follows the guidelines set forth in the monograph 2.6.30 of the European Pharmacopoeia (ibid.). A preferred incubation medium is RPMI (Roswell Park Memorial Institute) medium, more preferably RPMI 1640 medium, which is commercially available from, for example, Thermo Fisher Scientific (Waltham, MA, USA). However, other suitable media can also be considered, such as DMEM (Dulbecco’s Modified Eagle’s Medium), EMEM (Eagle’s Minimum Essential Medium), Ham F-10 or F-12 medium, Iscove’s Modified Dulbecco’s Medium (IMDM), etc., all of which are commercially available. Additional examples of suitable media are given in the Examples section later in this document.

[0064] The incubation medium can be supplemented to optimize incubation and the subsequent PBMC response determined in step (iii). Thus, in some embodiments, the incubation medium contains 0.1 to 15% by volume, preferably 0.1 to 10% by volume, more preferably 1 to 4% by volume of a medium supplement. A preferred medium supplement is a human medium supplement. In some embodiments, the incubation medium contains 1% by volume or approximately 1% by volume of the medium supplement. In some embodiments, the incubation medium contains 2% by volume or approximately 2% by volume of the medium supplement. In some embodiments, the incubation medium contains 3% by volume or approximately 3% by volume of the medium supplement. In some embodiments, the incubation medium contains 4% by volume or approximately 4% by volume of the medium supplement. As used herein, "medium supplement" refers to additional medium components that can enhance the activity (such as cytokine production) of PBMCs (preferably human PBMCs). In some embodiments, the medium supplement is fetal bovine serum (FBS). In some embodiments, the medium supplement is human AB serum (AB).

[0065] Incubate one or more samples in contact with peripheral blood mononuclear cells (PBMCs) for a duration sufficient to induce a response in the PBMCs (preferably monocytes). In some embodiments, the duration of incubation is at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, or at least 24 hours. Preferably, the duration of incubation is at least 16 hours. In some embodiments, the contact is carried out for at most 72 hours, preferably at most 36 hours, preferably at most 30 hours, more preferably at most 24 hours, still more preferably at most 18 hours, and most preferably at most 16 hours.

[0066] The 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 (such as about 37°C) and a CO2 level of 0 - 8% (such as about 5%). In a preferred embodiment, the incubation is carried out at 37°C and a CO2 level of 5% for at least 16 hours. After incubation, the incubated sample can be used directly for step iii), or it can be frozen and step iii) can be carried out at a later time point.

[0067] Step iii) Determine the response of PBMC

[0068] In step (iii), the response of PBMC (preferably mammalian PBMC, more preferably human PBMC, most preferably human monocytes) is determined. 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 inflammatory cytokines. Examples of inflammatory cytokines are IL-6, IL-1β, I-6, IL-8, TNF-α, MCP-1, IL-10, IFN-α, IFN-β, IFN-γ, and IFN-λ, preferably IL-6, IL-1β, I-6, IL-8, TNF-α, MCP-1, IL-10, more preferably IL-6. The response of PBMC can be the production and / or secretion (preferably secretion) of prostaglandins (an example of which is PGE2). The response of PBMC can be the production and / or secretion (preferably secretion) of high-mobility group proteins (an example of which is HMGB1). The response can be the production and / or secretion of neopterin. In some embodiments, the determined PBMC response is the expression of surface activation markers.

[0069] In some embodiments, the determined PBMC response is the production and / or secretion (preferably secretion) of inflammatory cytokines (such as IL-6, IL-1β, I-6, IL-8, TNF-α, MCP-1, IL-10, IFN-α, IFN-β, IFN-γ, IFN-λ), prostaglandins, or high-mobility group proteins. Those skilled in the art will understand that the determination of the PBMC response can also involve the combined determination of the production and / or secretion (preferably secretion) of multiple inflammatory cytokines, prostaglandins, and / or high-mobility group proteins.

[0070] In preferred embodiments, the determined PBMC response is the production and / or secretion (preferably secretion) of IL-6.

[0071] The determination of the PBMC response can be carried out directly in the same or different containers after the contact step, or the incubation mixture can be stored, optionally frozen, and used for response determination at different time points. Generally, the response of PBMC is related to the detection of pyrogens. It is possible that no response is detected, in which case no pyrogen is detected.

[0072] The determination of the PBMC response can be carried out, for example, by quantitative PCR, flow cytometry techniques (such as FACS analysis), or by immunoassays (preferably ELISA assays). A person skilled in the art knows how to perform such immunoassays, the description of which can be found in standard manuals such as The Immunoassay Handbook: Theory and Applications of Ligand Binding, ELISA and Related Techniques, 2013, 4th edition, edited by Wild, D., Elsevier Science, Netherlands (incorporated herein by reference in its entirety). Commercial ELISA kits (such as the MabTech ELISAbasic IL-6 kit (HRP, MabTech AB, Nack Strand, SE, Stockholm, Sweden)) are also available. When used in the methods of the present invention, ELISA assays are particularly advantageous because they enable high-throughput testing of multiple samples.

[0073] Thus, in some embodiments, the response of PBMCs is determined by ELISA assay. In some embodiments, the ELISA assay is performed using antibodies against IL-6, IL-1β, I-6, IL-8, TNF-α, MCP-1, IL-10, IFN-α, IFN-β, IFN-γ, IFN-λ, prostaglandin, or high-mobility group proteins. In a preferred embodiment, the ELISA assay is performed using an antibody against IL-6 (anti-IL-6). Such antibodies are commercially available, for example, clone 13A5 from MabTech AB, Stockholm, Sweden. In some embodiments, the ELISA assay is performed in a standardized 384-well plate. Examples of ELISA applied in the context of the present disclosure are provided in the Examples section later in this document.

[0074] In particular, when testing multiple identical samples, the detection method of the present disclosure demonstrates low variability between measurements. Thus, in some embodiments, for multiple identical samples, the determined PBMC response has a coefficient of variation of at most 30%. In some embodiments, for multiple identical samples, the determined PBMC response has a coefficient of variation of at most 25%, or 24%, 23%, 22%, or 21%. Preferably, for multiple identical samples, the determined PBMC response has a coefficient of variation of at most 20%. More preferably, for multiple identical samples, the determined PBMC response has a coefficient of variation of at most 15%. Even more preferably, for multiple identical samples, the determined PBMC response has a coefficient of variation of at most 10%, and most preferably at most 9.5%.

[0075] The detection method of the present disclosure is capable of detecting pyrogens or endotoxins in samples obtained from various products intended for therapeutic use or therapy (preferably for human therapeutic use or human therapy). Examples of such products include pharmaceutical compositions, medical devices, and medical instruments as previously described herein. If no pyrogens or endotoxins are detected in or on the product, they can be safely released (approved) for use. In this context, release can be considered as making available to the public while certifying that the product meets certain (relevant) standards.

[0076] Accordingly, in one aspect, there is provided a method for releasing a product (preferably a pharmaceutical product) for use, the method comprising subjecting a sample derived from the product to the method for detecting pyrogens or endotoxins as previously described herein. Preferably, the product is released only if no pyrogens are detected or a low pyrogen level is detected. This can depend on the standards met.

[0077] In some embodiments, there is provided 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 the method for detecting pyrogens or endotoxins as previously described herein.

[0078] The present disclosure further provides a kit. Preferably, the kit is suitable for carrying out the methods of the present invention. The kit may comprise a pyrogen or endotoxin standard of known concentration. A preferred standard is a lipopolysaccharide (LPS) standard. The pyrogen or endotoxin standard may be prepared according to the Ph.Eur. guidelines (monograph 2.6.30, ibid.), or ready-made standards may be obtained from commercial suppliers, for example from the EDQM (European Directorate for the Quality of Medicines & Healthcare; see, for example, the European Pharmacopoeia reference standards provided by the EDQM: order and catalogue). Examples of pyrogen standards are provided in the examples section later in this document.

[0079] The pyrogen or endotoxin standard may correspond to one or more samples. Preferably, a plurality of samples each containing a different concentration of pyrogen or endotoxin are used to generate a standard curve. In the case of endotoxin (preferably LPS), exemplary standard concentrations are 0.5 EU / ml, 0.25 EU / ml, 0.125 EU / ml, 0.06 EU / ml, 0.03 EU / ml, 0.016 EU / ml and 0.008 EU / ml. A plurality of replicate (identical) samples may be provided, preferably at least two, more preferably at least three, and most preferably at least four.

[0080] The kit may comprise PBMCs, preferably monocytes, more preferably mammalian monocytes, and most preferably human monocytes. Suitable PBMCs have been described previously herein.

[0081] The kit may comprise one or more 384-well plates. Compared to standard pyrogen detection methods (e.g., compared to the monocyte activation test using 96-well plates), 384-well plates can increase the test throughput while minimizing reagent and overall costs.

[0082] Accordingly, in one aspect, there is provided a kit comprising a pyrogen standard, PBMCs and one or more 384-well plates. In some embodiments, the kit is a monocyte activation test (MAT) kit. In some embodiments, the kit further comprises an incubation medium optionally in combination with a (human) medium supplement as described previously herein. In some embodiments, the concentration of the (human) medium supplement is 1 to 4 volume % (about 2%).

[0083] In some embodiments, the kit is suitable for simultaneously testing at least three different products (e.g., different pharmaceutical compositions, medical devices, or medical instruments) for the presence of pyrogens. In some embodiments, the kit is suitable for simultaneously testing at least four different products. In some embodiments, the kit is suitable for simultaneously testing at least five different products. In some embodiments, the kit is suitable for simultaneously testing at least six different products. In some embodiments, the kit is suitable for simultaneously testing at least seven different products. In some embodiments, the kit is suitable for simultaneously testing at least eight different products. In some embodiments, the kit is suitable for simultaneously testing at least nine different products. In some embodiments, the kit is suitable for simultaneously testing at least ten different products. The testing is preferably carried out according to the Ph.Eur. guidelines on the detection of pyrogens and endotoxins (Monograph 2.6.30, ibid).

[0084] "Simultaneous" testing means testing samples corresponding to different products in a single plate, which allows for increased test throughput and minimization of reagents and overall cost.

[0085] In some embodiments, the volume of the incubation medium in each sample tested in each well is at most 175 μL. In some embodiments, it has a volume of at most 170, 165, 160, 155, or 150 μL.

[0086] In some embodiments, the volume of the incubation medium in each sample tested in each well is from 20 to 150 μL, preferably from 30 to 140 μL, more preferably from 40 to 130 μL, more preferably from 50 to 120 μL, more preferably from 60 to 115 μL, more preferably from 70 to 110 μL, more preferably from 80 to 105 μL, more preferably from 90 to 100 μL. In some embodiments, the volume of the incubation medium in each sample tested in each well is from 20 to 100 μL, preferably from 30 to 100 μL, more preferably from 50 to 100 μL. In some embodiments, the volume of the incubation medium in each sample tested in each well is from 80 to 120 μL. In some embodiments, the volume of the incubation medium in each sample tested in each well is from 80 to 100 μL. In some embodiments, the volume of the incubation medium in each sample tested in each well is 100 μL or about 100 μL.

[0087] In some embodiments, PBMCs are present at a density of at most 500×1000 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 to about 250×1000 cells / cm 2, preferably at a density of about 50×1000 cells / cm 2 to about 150×1000 cells / cm 2 , more preferably at a density of about 90×1000 cells / cm 2 to about 130×1000 cells / cm 2 . In some embodiments, PBMCs are present at a density of 110×1000 cells / cm 2 or about 110×1000 cells / cm 2 .

[0088] In some embodiments, the volume of incubation medium in each sample tested in each well is 80 to 120 μL, and PBMCs are present at a density of about 90 to about 130×1000 cells / cm 2 . In some embodiments, the volume of incubation medium in each sample tested in each well is 100 or about 100 μL, and PBMCs are present at a density of 110×1000 cells / cm 2 or about 110×1000 cells / cm 2 .

[0089] Generally, the above volumes can be considered 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.

[0090] General definition

[0091] In this document and its claims, the verb "to comprise" and its conjugations are used in their non - restrictive sense to mean including the item following this word, but not excluding items not specifically mentioned. Additionally, the verb "consisting of" may be replaced by "consisting essentially of", meaning that the methods, and correspondingly the components, as defined herein may include additional steps, and correspondingly components, other than the specifically specified steps, and correspondingly components, provided that the additional steps, and correspondingly components, do not change the unique characteristics. Additionally, the mention of an element by the indefinite article "a / an" does not exclude the possibility of there being more than one such element, unless the context clearly requires that there be one and only one such element. Thus, the indefinite article "a / an" generally means "at least one / at least one kind".

[0092] As used herein, a specific value with "at least" means that specific 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.

[0093] The term "about" or "approximately" when used in connection with a numerical value (e.g., about 10) is preferably intended to mean that this value can 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 in combination with all of the stated circumstances. Various embodiments are described herein. Unless otherwise stated, each embodiment described herein can be combined together.

[0094] All patent applications, patents, and publications mentioned herein are hereby incorporated 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

[0095] Figure 1A —Effect of cell density on IL-6 production in the monocyte activation test. PBMCs were seeded into 384-well plates at the indicated cell densities (×1000 cells / cm 2 ), with a total volume of 33 μL. The x-axis indicates the cell density (×1000 cells / cm 2 ). The y-axis indicates IL-6 per 1000 cells. Four replicates (symbols) and the mean (horizontal line) are plotted. The figure caption shows the total MAT volume.

[0096] Figure 1B —As in 1A, but with a total MAT volume of 50 μL.

[0097] Figure 1C —As in 1A, but with a total MAT volume of 66 μL.

[0098] Figure 1D —As in 1A, but with a total MAT volume of 100 μL.

[0099] Figure 2 —Graph of absorbance values (in optical density (OD)) (y-axis) at each density (×1000 cells / cm 2 , increasing from left to right) at each LPS concentration (in EU / ml) (x-axis). The dashed line indicates 0.1 OD. The mean of three experiments, each with four replicates, is depicted.

[0100] Figure 3 —Graph of signal-to-noise ratio (bars) versus density (1000 cells / cm 2 ). The signal-to-noise ratio is calculated by dividing the OD at 0.016 EU / ml by the OD of the blank.

[0101] Figure 4—Coefficient of Variation (CV) at different cell densities. The average CV% of four replicates was calculated for each concentration of LPS (EU / ml), and then the average was taken for each density (1000 cells / cm 2 ). The figure shows the average (bars) and standard deviation (error bars) of three different experiments.

[0102] Figure 5 —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 at a 100-μl assay volume. The x-axis represents the cell density in cells / cm 2 counted as 1000. Error bars represent the standard deviation of three experiments.

[0103] Figure 6 —Average CV% at different assay volumes. The average CV% of four replicates was calculated for each LPS concentration (EU / ml), and then the average was taken for each assay volume (μl) and density. The figure shows the average (bars) and standard deviation (error bars) of three different experiments (from left to right, 55 / 110 / 220). The patterns represent the cell density in cells / cm 2 counted as 1000.

[0104] Figure 7 —Graph of absorbance (OD) versus LPS concentration (EU / ml). The x-axis is on a logarithmic scale. The gray line represents the standard curve at a density of 110 (×1000 cells / cm 2 ) and an assay volume of 100 μl. The black line represents the standard curve at a density of 220 (×1000 cells / cm 2 ) and an assay volume of 66 μl. Error bars indicate the standard deviation of four replicates.

[0105] Figure 8 —Curve slope. The bars indicate the curve slope of the four-parameter logistic curve. Left: LPS standard curve at a density of 110 (×1000 cells / cm 2 ) and an assay volume of 100 μl. Right: LPS standard curve at a density of 220 (×1000 cells / cm 2 ).

[0106] Figure 9 —Average CV%. The average CV% of four replicates was calculated for each LPS concentration (EU / ml), and then the average was taken for each volume / density combination. Left: Average CV% of the LPS standard curve at a density of 110 (×1000 cells / cm 2 ) and an assay volume of 100 μl. Right: Average CV% of the LPS standard curve at a density of 220 (×1000 cells / cm 2) and the average CV% of the LPS standard curve at a measurement volume of 66 μL.

[0107] Example

[0108] Materials and Methods

[0109] Control Preparation

[0110] Lipopolysaccharide (LPS) was obtained from the EDQM (Batch 5.1) and processed as per the instructions of the EDQM. LPS was rehydrated by vortexing for 30 minutes in 5 mL of LAL reagent water (LRW, Lonza Bioscience, Basel, CH, Switzerland) and diluted to a stock concentration of 10 endotoxin units / mL (EU / ml) by vortexing for 3 minutes in LRW. Subsequently, a LPS reference standard endotoxin curve (RSE) was created via serial dilution and mixed by resuspension in RPMI 1640 (Thermo Fisher Scientific, Waltham, MA, USA).

[0111] Cell Thawing

[0112] Vials of PBMC (10 million PBMC / ml) were rapidly thawed in a water bath set at 37 °C and resuspended by slowly adding pre-warmed (37 °C) RPMI medium containing 4% human media supplement (Mediatech, Manassas, VA, USA).

[0113] Cell Density

[0114] Samples of 0.2 EU / ml LPS were plated at 50% of the final volume onto 384-well microplates (Thermo Fisher Scientific, Waltham, MA, USA) to obtain various different final volumes as indicated (per experiment). Subsequently, cell suspensions were added at a ratio of 1:1 at different cell concentrations as indicated (per experiment) to obtain a final concentration of 2% (v / v) human media supplement (HMS). The final concentration of LPS corresponded to a two-fold dilution series starting at 0.1 EU / ml and was obtained by resuspension in RPMI in the plate. Cells were incubated with LPS in an incubator (Binder (CB60), Tuttlingen, Germany) set at 37 °C and 5% CO2 for 20 hours + / - 1 hour, after which the IL-6 concentration was measured by ELISA as explained below. The IL-6 per 1000 cells was calculated by interpolating the measured optical density (OD) on the linear regression model of the IL-6 standard curve and dividing the resulting total IL-6 by the total number of cells in the well.

[0115] Plate the LPS standard curve (33 μl, at concentrations of 0.064 EU / ml, 0.032 EU / ml, 0.016 EU / ml, 0.008 EU / ml, 0.004 EU / ml) onto a 384-well microplate. Thaw the cryopreserved peripheral blood mononuclear cells (PBMCs) and resuspend them in RPMI medium containing 4% human medium supplement. Serially dilute the cell suspension at concentrations of 1514 cells / μl, 757 cells / μl, 378 cells / μl, 189 cells / μl, 94.7 cells / μl, and 47.4 cells / μl (dilution factor of 2). Add 33 μl of each cell suspension 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 of final cell density. At each density, the final concentration of LPS is 0.032 EU / ml, 0.016 EU / ml, 0.008 EU / ml, 0.004 EU / ml, 0.002 EU / ml. The final concentration of HMS in each well is 2%.

[0116] MAT incubation volume

[0117] Plate the 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) onto a 384-well microplate in three different volumes (16.7 μl, 33 μl, and 50 μl). Thaw the cryopreserved PBMCs and reconstitute them in RPMI medium containing 4% human medium supplement. Dilute the cell suspension at different cell concentrations and add it to the plate at a 1:1 ratio, resulting in 55,000 cells / cm 2 、110,000 cells / cm 2 and 220,000 cells / cm 2 of final cell density at each assay volume (33 μl, 66 μl, and 100 μl) at a final HMS concentration of 2%.

[0118] MAT

[0119] Samples for the LPS standard curve were added to the culture plates in different volumes (at a 1:1 ratio with the resuspended PBMCs) and incubated for 16 hours in an incubator at 37 °C 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%.

[0120] ELISA

[0121] The ELISA plates were coated with the capture antibody for IL-6 (clone 13A5, MabTech AB, Stockholm, Sweden) at a concentration diluted 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 plates.

[0122] Statistics

[0123] Statistical analysis (including four-parameter / five-parameter logistic regression) was performed using Graphpad Prism 8 (GraphPad Software, San Diego, CA, USA).

[0124] Results

[0125] LPS responses at different densities and volumes at 0.1 EU / ml

[0126] At a concentration of 0.1 EU / ml and different 384MAT volumes, increasing the cell density showed an improvement in the IL-6 response per 1000 cells, with a sharp increase observed at a density of 303 (×1000 cells / cm 2 ) in 50 μL, 66 μL, and 100 μL. Using a volume of 33 μL in the MAT, a 3-fold increase in signal was observed at a density of 227 (×1000 cells / cm 2 ) compared to a density of 152 (×1000 cells / cm 2 ), while there was a slight decrease in IL-6 per cell at a density of 303 (×1000 cells / cm 2 )( Figure 1A -D).

[0127] Cell density and LPS response

[0128] Higher density means a stronger LPS signal (OD) at each LPS concentration, but also increases the background signal (at 0.00 EU / ml) - up to 0.12 OD at 440 density (×1000 cells / cm 2 ). Figure 2 )

[0129] In addition, the signal-to-noise ratio (calculated as the signal at 0.032 EU / ml divided by the background signal) increases with increasing density, reaching an optimum value (signal-to-noise ratio of 6.4) at 110 density (×1000 cells / cm 2 ).

[0130] In addition, although the LPS signal and the signal-to-noise ratio increase with increasing density ( Figure 3 ), the coefficient of variation also increases (as Figure 4 shown), reaching an average CV% of up to 28.5% at 440 density (×1000 cells / cm 2 ). Between 55 and 220 density (×1000 cells / cm 2 ), the signal-to-noise ratio is the highest among the tested densities, while the average CV% is the lowest.

[0131] Effect of volume on the LPS signal

[0132] A 33-μL MAT volume shows a significant increase in the LPS response, as Figure 5 depicted (showing a relative gain at 0.032 EU / ml normalized to 100 μL). When plating higher cell densities, the relative gain also increases, with an average relative gain of 219% at 55 density (×1000 cells / cm 2 ), 304% at 110 density (×1000 cells / cm 2 ), and 387% at 220 density (×1000 cells / cm 2 ). However, smaller volumes show an increase in the variation between replicates ( Figure 6 ). At each density, the 100-μL MAT volume shows the lowest CV% (11.8% at 55 density (×1000 cells / cm 2 ), 13.3% at 110 density (×1000 cells / cm 2 ), and 18.6% at 220 density (×1000 cells / cm 2 )). A pattern of increasing CV% with increasing density is observed for each assay volume, but at 110 density (×1000 cells / cm 2)Except that it showed a lower CV% than 220 density (×1000 cells / cm 2 ) in all tested assay volumes.

[0133] LPS standard curve

[0134] For both configurations: at a MAT volume of 66 μL and a cell density of 220 (×1000 cells / cm 2 ) and at a MAT volume of 100 μL and a cell density of 110 (×1000 cells / cm 2 ), an LPS standard curve ( Figure 7 ) was created. Four-parameter logistic regression showed R 2 values of 0.99 (66 μL / 220 density (×1000 cells / cm 2 )) and 0.98 (100 μL / 100 density (×1000 cells / cm 2 ). Figure 8 The curve slopes of the two plots are shown, and the slope between the two configurations differed by approximately a factor of two. Additionally, the average CV% under 66 μL / 220 density (×1000 cells / cm 2 ) MAT was significantly higher than that under 100 μL / 110 density (×1000 cells / cm 2 ) (23.4% and 9.4% respectively) ( Figure 9 ).

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; And iii) Determining the response of the PBMCs, wherein the volume of the incubation medium is at most 175 μL per sample.

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 20 to 150 μL, preferably 30 to 140 μL, more preferably 50 to 110 μL.

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

5. The method according to any one of claims 1-4, wherein the incubation medium contains 1 to 4% by volume, such as about 2%, of a medium supplement.

6. The method according to any one of claims 1-5, wherein the determined response of the PBMCs is the secretion of inflammatory cytokines such as IL-6, IL-1β, IL-8, TNF-α, MCP-1, IL-10, IFN-α, IFN-β, IFN-γ, IFN-λ, prostaglandins, or high-mobility group proteins.

7. The method according to any one of claims 1-6, wherein the response of the PBMCs is determined by ELISA assay.

8. The method according to any one of claims 1-7, wherein the PBMCs are present at a density of at most 500×1000 cells / cm 2 , preferably at most 250×1000 cells / cm 2 .

9. The method according to any one of claims 1 - 8, wherein the PBMCs are present at a density of about 10×1000 cells / cm 2 to about 250×1000 cells / cm 2 , preferably about 50×1000 cells / cm 2 to about 150×1000 cells / cm 2 , more preferably about 90×1000 cells / cm 2 to about 130×1000 cells / cm 2 .

10. The method according to any one of claims 1-9, wherein for a plurality of identical samples, the determined response of the PBMCs has a coefficient of variation of at most 20%.

11. The method according to any one of claims 1-10, wherein in step i), an additional control sample is provided, wherein the additional control sample is preferably a lipopolysaccharide sample, and wherein the additional control sample preferably contains about 0.005 to about 15 endotoxin units / mL.

12. The method according to any one of claims 1-11, wherein at least 50 samples, preferably at least 97 samples, are provided.

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

14. 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 the method according to any one of claims 1-13.

15. A kit comprising a pyrogen or endotoxin standard, PBMCs, and one or more 384-well plates.

Images