Method for detecting viable microorganisms which are potentially present in a sample of cellular products

Abstract

The present invention relates to a method for isolating viable microorganisms which are potentially present in a sample comprising 106 to 108 immune cells or progenitor cells thereof, the method comprising the following steps: - bringing the sample into contact with: • a lysing composition comprising o a lysis buffer comprising a non-ionic detergent at a concentration of between 0.004% and 0.050% of the reaction volume o and / or a lysis solution comprising a saponin at a concentration of between 0.03% and 4% of the reaction volume; • an endonuclease for digesting the nucleic acids released by the action of the lysing composition; • an endopeptidase acting at a pH of between 7 and 8; - separating the viable microorganisms which are potentially present in the sample by filtering the sample through a filter, the pores of which have a diameter of between 0.30 and 0.50 μm. The present invention also relates to the associated solid-phase cytometry detection method.

Description

[0001] Method for detecting viable microorganisms potentially present in a sample of cellular products.

[0002] The present invention relates to the technical field of microbiological control in the pharmaceutical industry. In particular, the invention relates to the preparation of a sample of cellular products for the detection of viable microorganisms, in particular by solid phase cytometry.

[0003] Advanced Therapy Medicinal Products (ATMPs) represent a rapidly expanding class of pharmaceutical products. Although still limited to a few targeted applications (hemopathies, rare diseases), having already demonstrated their effectiveness, they are destined to take an increasingly important place in our healthcare system (more than a thousand clinical trials were underway in 2020, including nearly a hundred in phase III).

[0004] These drugs are based on the use of cellular products (autologous, allogeneic or xenogeneic) that have been subjected to substantial ex vivo manipulations, modifying their biological characteristics, physiological functions or structural properties, before administering them to humans.

[0005] However, the large-scale production and use of these drugs faces a number of difficulties, in particular:

[0006] - Production costs much higher than for chemical drugs (new complex production processes, low volumes produced, etc.)

[0007] - Very short stability of the finished product when used fresh (of the order of a few hours) and therefore the need to be able to have quality controls within a very short timeframe

[0008] The impossibility of sterilizing them and therefore an increased risk of microbial contamination.

[0009] Microbiological controls represent a critical step in the ATMP production cycle, particularly sterility tests that condition release. They are described in the European Pharmacopoeia, section 2.6.27 “Microbiological examination of cell-based preparations”. The time and cost constraints associated with these microbiological controls are currently one of the main obstacles to the development of the ATMP industry.

[0010] Sterility testing is mandatory for final products by the European Pharmacopoeia, as a condition for their release. The traditional method, namely cell culture in broths and visual inspection, requires a minimum of 14 days to ensure non-contamination. Alternative methods, also based on cell culture, but with automated reading, have been approved by the Pharmacopoeia, and require a minimum of 7 days of incubation, most often 10 days, to produce a result. bioMérieux's BACT / ALERT® solution, which detects the growth of microorganisms through CO2 production, is now the standard for sterility testing of ATMPs / MTIs.

[0011] However, these methods have particularly long response times, which can impact patients' lives. Furthermore, they often require sampling that is not compatible with the volumes produced in the context of MTI manufacturing activities, especially in autologous situations.

[0012] In order to reduce delays but also to improve detection capabilities, other technologies are currently being studied to carry out sterility tests on MTIs:

[0013] - Determination of ATP (Adenosine triphosphate)

[0014] - Molecular biology tests.

[0015] Another technology that has not been explored for sterility testing of ATMPs is solid-phase cytometry.

[0016] A solid-phase cytometer allows for the rapid quantification of microorganisms such as bacteria, yeasts, or molds in a sample. The sample is filtered through a membrane so that any microorganisms present in the sample are recovered on the membrane. The living microorganisms are then specifically labeled by a molecule that penetrates the viable microorganisms and is then cleaved by esterases, releasing a fluorochrome, fluorescein, into the microorganism, emitting at a specific wavelength. A laser beam scans the entire surface of the membrane to excite the fluorochrome, which makes it possible to detect and count any viable microorganisms present in the sample.

[0017] The use of solid-phase cytometry, applied to sterility testing on ATMPs, raises many challenges. The main limitations of this technology lie in the high cell concentration equal to or greater than 10 5 cells / ml and in the nature of the cells to be lysed, in particular immune cells and their progenitor cells.

[0018] This problem of high cell concentration is encountered in the detection of bacteria in blood products, i.e. generally whole blood. Thus, document W003025207 describes a method for concentrating bacteria from blood products which consists of reducing the concentration of blood cell populations by means of selective aggregation thereof. A filtration step makes it possible to collect non-aggregated pathogenic germs in the filtrate and to retain the blood cell aggregates on the filter, then possibly selective lysis of the residual cells takes place before a new passage on the filter. The step of reducing the concentration of blood cell populations by aggregation allows a reduction of the order of 4 log (from 10 9 at 10 5 cells / ml) with regard to the concentration of platelets, and of the order of 5 log (since 10 10 at 10 5cells / ml) with respect to the concentration of red blood cells. Although microorganisms are smaller than aggregated blood cells, there is a risk that they may be retained, thus generating a loss of microorganisms and false negatives by the detection method.

[0019] The problem of high cell concentration is also encountered in blood samples for other detection methods such as in molecular biology. Patents EP2718713 and EP2510123 describe the selective lysis of blood cells in a sample likely to contain microorganisms using controlled lysis buffers to recover intact, dead or live microorganisms. The DNA of the microorganisms is then extracted for PCR detection. The viability of the microorganisms is not, for detection in molecular biology, an essential criterion. Thus, the disadvantage of these methods is that the lysis buffers do not ensure the preservation of the viability of the microorganisms necessary for detection by solid-phase cytometry with good sensitivity.

[0020] Zelenin (Biotechnol Lett 2015 37: 825-830) describes a protocol for isolating viable bacteria from a blood sample using saponin at a concentration between 0.5% and 2.5% to destroy erythrocytes followed by osmotic shock to lyse leukocytes. The disadvantage of this method is that it is not applicable to a sample with a high concentration of immune cells such as leukocytes, which are much more difficult to lyse than erythrocytes. This isolation method is followed by molecular biology detection and antibiotic susceptibility testing.

[0021] Document EP3063290 describes a method for isolating microorganisms from a sample with high cell density comprising the use of a lysis buffer containing 0.05 to 0.5% SDS and Benzonase® nuclease. The samples contain 10 6 5x10 cells 8cells in a sample of at least 5 ml. The microorganisms are then lysed and detected by molecular biology. This method has the disadvantage of not preserving the viability of the microorganisms by using an anionic detergent such as SDS.

[0022] On the other hand, document EP2601304 describes a preparation of a blood sample for identification by mass spectrometry. This requires the lysis of human particles by saponin and centrifugation or microfiltration to recover only the living microorganisms. These microorganisms are then cultured, separated from the culture medium and then identified by mass spectrometry. After the centrifugation step, a 1% saponin solution is added to the precipitate which contains the potential microorganisms but also 5 x 10 9Erythrocytes 7000 leukocytes and 50,000 platelets per milliliter of blood. This mixture is then centrifuged again to recover the precipitate. This process is not suitable for a sample with a high concentration of immune cells such as leukocytes, which are much more difficult to lyse than erythrocytes.

[0023] Another challenge for application in ATMPs lies in the filterability of cell matrices. One solution then consists of a preparation process to make the sample filterable. However, this process can irreversibly alter the microbial load either by completely eliminating microbes or by degrading them in such a way that viability labeling no longer detects them. Thus, document WO2019051272 describes the preparation of a viscous, non-filterable sample for detection by solid-phase cytometry. The use of an isopropyl myristate-based solvent does not resolve the problems encountered with a sample with a high cell concentration.

[0024] Similarly, the Journal of Microbiological Methods 64 (2006) 420-423 describes the cytometric detection of Aspergillus fumigatus in respiratory secretions, a difficult-to-filter sample. The proposed pretreatment includes the addition of rhDNAsel, trypsin, HEPES buffer, and DTT / Triton X-100. Using this pretreatment, adapted to a particularly viscous sample, does not resolve the problems encountered with a sample with a high cell concentration.

[0025] There is therefore a need to develop a method for detecting viable microorganisms from samples containing a high concentration of immune cells or their progenitor cells, which are difficult to filter.

[0026] SUMMARY OF THE INVENTION

[0027] An objective of the present invention is to provide an isolation of viable microorganisms potentially present in very small quantities in a sample while eliminating immune cells or their progenitor cells present in very large quantities.

[0028] Thus, a first object of the invention relates to a method for isolating viable microorganisms potentially present in a sample comprising 10 6 at 10 8 immune cells or progenitor cells thereof, comprising the following steps:

[0029] - Bring the sample into contact with

[0030] • a lysing composition comprising o a lysis buffer comprising a non-ionic detergent at a concentration of between 0.004% and 0.050% o and / or a lysis solution comprising a saponin at a concentration of between 0.03% and 4% • an endonuclease for digesting the nucleic acids released by the action of the lysing composition

[0031] • an endopeptidase acting at a pH between 7 and 8,

[0032] - Filter potentially present viable microorganisms through a filter with a pore diameter between 0.30 and 0.50 pm or in other words separate potentially present viable microorganisms from the sample by filtering the sample through a filter with a pore diameter between 0.30 and 0.50 pm.

[0033] Preferably, the invention relates to a method for isolating viable microorganisms potentially present in a sample comprising 106 at 10 8 immune cells or progenitor cells thereof, comprising the following steps:

[0034] - Bring the sample into contact with

[0035] • a lysing composition, the lysing composition being a lysis buffer comprising a non-ionic detergent at a concentration of between 0.004% and 0.050% of the reaction volume and a saponin at a concentration of between 0.03% and 4% of the reaction volume

[0036] • an endonuclease for digesting the nucleic acids released by the action of the lysing composition

[0037] • an endopeptidase acting at a pH between 7 and 8,

[0038] - Filter potentially present viable microorganisms through a filter with a pore diameter between 0.30 and 0.50 pm or in other words separate potentially present viable microorganisms from the sample by filtering the sample through a filter with a pore diameter between 0.30 and 0.50 pm.

[0039] The present invention therefore consists in efficiently lysing the immune cells or their progenitor cells present in very high concentration while keeping the microorganisms viable and thus allowing their detection by methods which require keeping the microorganisms viable. Preferably, the immune cells are CAR-T. An advantage of the present invention is that it allows efficient and rapid sample preparation. The time required to bring the sample into contact with the lysing composition, the endonuclease and the endopeptidase and then the filtration is less than 30 minutes, preferably less than 20 minutes. The method according to the invention allows the viability of the microorganisms to be maintained for detection, in particular by solid-phase cytometry, which is a particularly rapid detection method.

[0040] The effectiveness of the lysis according to the invention is notably due to the synergy between the lysis composition, the endopeptidase and the endonuclease. Indeed, the lysis step by the lysing composition induces the loss of viability of the immune cells or their progenitor cells. It acts by destructuring and permeabilizing the plasma membranes of the cells, by forming complexes with the lipids of the membranes. This mechanism then allows the endopeptidase to hydrolyze the membrane proteins and burst the cells. The action of the endopeptidase also continues in order to degrade the nuclear membranes and release the intracellular material. The endonuclease, by degrading the nucleic acids, also improves the lysis.

[0041] Preferably, the endopeptidase is a trypsin.

[0042] Preferably, the non-ionic detergent is a polyoxyethylene detergent such as BRIJ or NP40.

[0043] In the presence of a high cell concentration, the sample is difficult to filter and the efficiency of lysis is then a key element. The small debris obtained by this lysis prevents the filter from clogging, which would prevent the detection of microorganisms. In the case of detection by solid-phase cytometry, effective lysis avoids having a significant fluorescence background noise that would mask the presence of microorganisms.

[0044] Another objective of the present invention is to enable rapid detection by solid phase cytometry. Detection is carried out between 4 and 6 hours. Thus, another object of the invention relates to a method for the detection of microorganisms potentially present in a sample comprising 10 6 at 10 8 immune cells or progenitor cells thereof, comprising the following steps:

[0045] - Carry out the isolation process according to the invention - Mark the viable microorganisms retained on the porous membrane with a viability marker

[0046] - Perform a scan of the porous membrane using a laser beam or camera

[0047] - Determine the presence of viable microorganisms captured by the membrane

[0048] Since immune cells or their progenitor cells are marked like microorganisms by a viability marker, complete lysis of immune cells prevents the marking of these cells and therefore reduces false positives. In addition, since lysis allows for very small cellular debris to be obtained, this prevents the adhesion of microorganisms to this debris and thus prevents the microorganisms from being masked. This method therefore reduces false negatives.

[0049] Another object of the invention relates to a lysis kit comprising:

[0050] - a lysis buffer comprising a non-ionic detergent at a concentration of between 0.004% and 0.050% of the reaction volume

[0051] - a solution comprising a saponin at a concentration of between 0.03% and 4% of the reaction volume

[0052] - an endonuclease

[0053] - an endopeptidase acting at a pH between 7 and 8.

[0054] The lysis kit according to the invention allows efficient lysis of immune cells or their progenitor cells while being non-toxic to microorganisms.

[0055] DETAILED DESCRIPTION OF THE INVENTION

[0056] The present invention relates to a method for isolating viable microorganisms potentially present in a sample comprising a high quantity of immune cells or their progenitor cells, and the detection of said microorganisms by solid phase cytometry.

[0057] A first subject of the invention relates to a method for isolating viable microorganisms potentially present in a sample comprising 10 6 at 10 8 immune cells or progenitor cells thereof, comprising the following steps: - Contacting the sample with o a lysing composition comprising

[0058] • a lysis buffer comprising a non-ionic detergent at a concentration of between 0.004% and 0.050% of the reaction volume

[0059] • and / or a lysis solution comprising a saponin at a concentration of between 0.03% and 4% of the reaction volume o an endonuclease for digesting the nucleic acids released by the action of the lysing composition o an endopeptidase acting at a pH of between 7 and 8.

[0060] - Separate potentially present viable microorganisms from the sample by filtering the sample through a filter with a pore diameter between 0.30 and 0.50 pm.

[0061] Preferably, the lysing composition is a lysis buffer comprising a non-ionic detergent at a concentration of between 0.004% and 0.050% of the reaction volume and a saponin at a concentration of between 0.03% and 4% of the reaction volume.

[0062] The sample according to the present invention is a clinical sample comprising 10 6 10 cells 8 immune cells or their progenitor cells. In a preferred embodiment, the amount of cells in the sample is 10 6 at 5x10 7 immune cells or their progenitor cells and even more preferably 5x10 6 and 10 7cells. The amount of sample used varies depending on the sample source. In a preferred embodiment, the sample volume is from 100 µl to 2 ml, even more preferably from 500 µl to 1.5 ml.

[0063] Immune cells or progenitor cells are part of advanced therapy medicinal products or cell therapy products. Immune cells can be chosen from NK cells, monocytes, B lymphocytes, T lymphocytes, natural or genetically modified, such as regulatory T lymphocytes, tumor-infiltrating T lymphocytes, cytotoxic T lymphocytes, helper T lymphocytes and T lymphocytes with a chimeric antigen receptor (CAR). NK cells (or NK lymphocytes) are cells of innate immunity. They are non-T (CD3-), non-B (CD19-) lymphocytes, characterized in humans by the markers CD56, CD16 and NK.

[0064] Monocytes are leukocytes that evolve into macrophages, dendritic cells or osteoclasts.

[0065] B lymphocytes are the immune cells responsible for producing antibodies.

[0066] Regulatory T cells are a subpopulation of CD4+ T cells, which inhibit the proliferation of other effector T cells.

[0067] Cytotoxic T lymphocytes are a subpopulation of CD8+ T lymphocytes, which destroy infected cells.

[0068] Helper T lymphocytes are a subpopulation of CD4+ T lymphocytes that act as intermediaries in the immune response.

[0069] Finally, T lymphocytes with a chimeric antigen receptor (CAR), also called CAR-T cells, correspond to a particular cellular engineering technology. These are T lymphocytes that express a chimeric antigen receptor. CAR-T cells are capable of killing cancer cells, by recognizing and binding to the tumor antigen present on said cancer cells. Preferably, the sample according to the invention comprises CAR-T cells.

[0070] Progenitor cells of immune cells can be mesenchymal stem cells.

[0071] The cell sample can come from the patient to be treated, in which case the patient and the donor are the same person, by biopsy or blood sample. In this case, the composition obtained and stored will be administered to the same patient: it is an autologous product.

[0072] Alternatively, the cell sample may come from another source, such as another individual or through cellular engineering, such as a biopsy or blood sample. In this case, the resulting and stored composition will be administered to a patient to be treated other than the donor: it is an allogeneic product.

[0073] The sample can be packaged fresh in a liquid medium with storage limited to a few hours or in the form of a frozen solution. The sample can include cryopreservatives. Preferably, the method according to the invention uses a fresh sample. The sample can be preserved with BSA (bovine serum albumin).

[0074] The sample comprises or is likely to contain a microorganism. The microorganisms that can be isolated and / or detected according to the present invention may be bacteria, yeasts, fungi. The microorganisms may be aerobic or anaerobic. The microorganisms detected can be for example Corynebacterium tuberculosteariticum, Moraxella catarrhalis, Moraxella osloensis, Bacteroides fragilis Pichia carsonii Bretanomyces bruxellensis Paracoccus yeei, Sacharomyces cerevisiae, Streptococcus pyogenes, Penicillium expensum, Sphingomonas paucimobilis, Kocuria rhizophila, Staphylococcus epidermidis, Aspergillus brasiliensis, Candida albicans, Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Cutibacterium acnes, Clostridium sporogenes.

[0075] In order to isolate microorganisms, the present invention provides a method for isolating microorganisms directly from the sample. The sample of immune cells or their progenitor cells is directly contacted with a lysis composition. The sample of immune cells or their progenitor cells does not first undergo centrifugation, filtration or selective aggregation to reduce the concentration of said cells.

[0076] Indeed, these preliminary steps can lead to a loss of microorganisms and generate false negatives. High-speed centrifugation, for example, can promote adhesion between immune cells and certain bacteria, preventing their separation.

[0077] According to the present invention, it is therefore necessary to carry out a selective lysis destroying the immune cells or their progenitor cells in order to recover the viable microorganisms. Thus, the addition of a chemical detergent or surfactant is commonly practiced in order to solubilize the membrane proteins of mammalian cells. Detergents are amphiphilic compounds capable of forming micelles in aqueous solution, inside which are the hydrophobic heads and outside which are the hydrophilic heads. These micelles disorganize the phospholipid membranes. Detergents can be ionic such as Sodium dodecyl sulfate (SDS), non-ionic and milder such as Triton X or zwitterionic.

[0078] The bacterial cell wall is solid and consists mainly of peptidoglycan. In the case of Gram-positive bacteria, teichoic acid is also present. This network of covalent bonds resists the dissolving effect of surfactants for several minutes. However, depending on the quantity and nature of the surfactant, they can penetrate the microorganisms and destroy the protein structure, impacting their ability to reproduce and therefore their viability.

[0079] The difficulty with selective lysis lies in choosing a lysis composition that is both effective in lysing immune cells or their progenitor cells and non-toxic to microorganisms. In the present invention, it is essential that the microorganisms be viable. Thus, it is necessary to use a weak, non-toxic surfactant.

[0080] On the other hand, the effectiveness of the lysis composition depends on the quantity of cells present in the sample. However, the sample of the present invention is composed of a very high cell concentration. It also depends on the nature of the sample. A lysis composition that is effective for erythrocytes is not necessarily effective for immune cells such as leukocytes. Indeed, blood is composed mainly of erythrocytes and of approximately 4000 to 7000 leukocytes per ml of blood. However, erythrocytes are small cells that lyse more easily than leukocytes.

[0081] Thus, according to the present invention, the sample is brought into contact with

[0082] - a lysing composition comprising o a lysis buffer comprising a non-ionic detergent at a concentration of between 0.004% and 0.050% of the reaction volume o and / or a lysis solution comprising a saponin at a concentration of between 0.03% and 4% of the reaction volume

[0083] - an endonuclease for digesting the nucleic acids released by the action of the lysing composition

[0084] - an endopeptidase acting at a pH between 7 and 8, The lysis composition is used to break cell membranes by acting on their physicochemical properties.

[0085] Preferably, the lysis composition comprises saponin. Quillaja Saponin will be mentioned. In the case of leukocytes, the membrane is more robust than that of erythrocytes. In addition, immune cells or progenitor cells also have a nucleus and intracellular organelles which make them more resistant to lysis. Preferably, the final saponin concentration is between 0.03% and 2%, even more preferably between 0.05% and 1%, even more preferably between 0.05% and 0.8%, even more preferably between 0.05% and 0.2% of the reaction volume.

[0086] Preferably, the lysis composition contains a non-ionic detergent. Preferably the non-ionic detergent is a polyoxyethylene detergent. Preferably, said detergent is NP40. More preferably, said detergent is BRIJ. Mention may be made of BrijOI 0. Preferably, the final concentration of non-ionic detergent is between 0.004% and 0.050%, more preferably between 0.004% and 0.030% of the reaction volume.

[0087] When saponin is not present, the final concentration of nonionic detergent is between 0.025% and 0.05% of the reaction volume.

[0088] When there is no detergent, the final saponin concentration is between 0.10% and 4% of the reaction volume. Preferably, when there is no detergent, the buffer is very concentrated in salt, for example, greater than 50g / l of TRIZMA buffer.

[0089] Immune cells or their progenitor cells, present in very high concentrations, are partially lysed with low concentration saponin and / or low concentration non-ionic detergent, without destroying the microorganisms present in very small quantities. Advantageously, in order not to degrade the microorganisms, the lysis composition does not contain ionic detergents such as SDS.

[0090] The preparation of the lysis composition and in particular of the lysis buffer comprising a non-ionic detergent is known to those skilled in the art. The ingredients are dissolved and mixed.

[0091] The sample is also brought into contact with an endopeptidase that acts effectively at a pH between 7 and 8.5, preferably at a pH between 7 and 8. The endonuclease allows for complete lysis of immune cells. Indeed, the non-ionic polyoxyethylene detergent such as BRIJ and / or saponin deconstructs and permeabilizes the plasma membranes of immune cells and their progenitor cells, by forming complexes with the membrane lipids. This mechanism then allows the endopeptidase to hydrolyze the membrane proteins and burst the cells.

[0092] The endopeptidase used according to the invention does not impair the survival of microorganisms. Examples of endopeptidases include trypsin or an endopeptidase mixture such as the composition accutase® from biowest. Preferably, the endopeptidase is a trypsin. It has optimal activity at pH 8. The trypsin can be in liquid form or in powder form which is redissolved. Preferably, the final concentration of trypsin is in excess. It can be, for example, greater than 0.1%.

[0093] According to the present invention, detection can be carried out using a viability marker. However, since immune cells or their progenitor cells can be marked like microorganisms by a viability marker, complete lysis of immune cells reduces false positives. In addition, since lysis allows very small cellular debris to be obtained, this prevents the adhesion of microorganisms to this debris and thus prevents the microorganisms from being masked. This method therefore reduces false negatives.

[0094] In one embodiment, the sample is contacted with an endopeptidase prior to contacting with the lysis composition. This embodiment is particularly advantageous when the sample comprises BSA.

[0095] pH is also a factor to consider to optimize the effectiveness of lysis agents. Typically, lysis agents have a pH between 5 and 9. Preferably, lysis agents act at a pH between 7 and 8.5, more preferably between 7 and 8.

[0096] Thus, at a basic pH between 7 and 8, saponin and trypsin have very good efficiency. Moreover, at this pH, the viability of the target microorganisms is maintained, which is not the case for certain target microorganisms above a pH of 8.5. After the addition of the endopeptidase, a rinsing step with an appropriate fluid is carried out in order to stop the enzymatic and chemical reactions. Preferably, fluid D is used.

[0097] The lysis composition and endopeptidase are added to the sample for an appropriate time and in a quantity to lyse immune cells or their progenitor cells while preserving potentially present, living microorganisms.

[0098] The sample is also brought into contact with one or more enzymes for lysing nucleic acids. These enzymes are known and conventionally used by those skilled in the art. Endonuclease non-specifically degrades all forms of DNA and RNA. Examples include Benzonase® Nuclease, commercially available from Merck Mi II ipore. Preferably, the final concentration of endonuclease is in excess. The final concentration may be between 50 IU / ml and 100 IU / ml.

[0099] The endonuclease may be separate from the lysing composition or be part of the lysing composition. It may be added before, alongside, or after the lysing composition and the endopeptidase. The endopeptidase may optionally be inactivated before the addition of the endonuclease.

[0100] By degrading nucleic acids, the endonuclease prevents the filter from clogging. Filtration is therefore improved, avoiding false positives.

[0101] The endonuclease can be in dried form.

[0102] After the step of bringing the sample into contact with the lysis composition and the endopeptidase, the whole is homogenized or mixed, i.e. by vortexing or stirring. In addition, an incubation step with stirring can take place for a few minutes.

[0103] Potentially present viable microorganisms are then separated, i.e. isolated, from the sample by filtration. The separation is carried out on a porous membrane with pores smaller than the size of the bacteria.

[0104] Preferably, the porous membrane has pores between 0.30 and 0.50 pm, preferably between 0.32 and 0.45 pm. Bacteria have a smaller diameter than yeasts and molds. They are nevertheless retained by the filter. Due to the very small diameter of the membrane, sample lysis must be very efficient to avoid clogging the filter and causing a very high fluorescence background that can mask the specific fluorescence of microorganisms. This method therefore reduces false negatives.

[0105] The microorganism isolation step is a rapid step of less than 30 minutes, preferably less than 20 minutes.

[0106] After the isolation of the microorganisms, their detection can take place by any type of method such as molecular biology or by cytometry. The detection according to the invention is carried out by solid phase cytometry allowing detection of viable microorganisms potentially present.

[0107] Thus, another object of the invention relates to a method for the detection of microorganisms potentially present in a sample comprising 10 6 at 10 8 immune cells or their progenitor cells, comprising the following steps: carrying out the isolation method according to the invention carrying out a marking of the viable microorganisms retained on the porous membrane with a viability marker carrying out a scanning by a laser beam or by camera of the porous membrane determining the presence of viable microorganisms captured by the membrane

[0108] The detection according to the present invention is particularly rapid and is carried out between 4 and 6 hours, the detection of aerobic microorganisms being faster than that of anaerobic microorganisms which requires longer incubation times. Solid phase cytometry is a promising technology for sterility testing on ATMPs. It allows very sensitive detection of microorganisms. Thus, the SCANRDI® technology developed by the applicant makes it possible to detect any presence of microorganisms in a filterable sample with very rapid results. The protocol according to the invention thus allows an ultra-rapid sterility test of a sample of immune cells or their progenitor cells in less than 6 hours. This solid phase cytometer makes it possible to very quickly quantify microorganisms (bacteria, yeasts or molds) in a sample.The sample is filtered through a membrane so that any microorganisms present in the sample are recovered on the membrane. Live microorganisms are then specifically labeled with a viability marker that penetrates the microorganisms and is then cleaved by esterases, releasing a fluorochrome, fluorescein, into the microorganism, emitting at a specific wavelength. Immune cells or their progenitor cells also contain esterases and can absorb the substrate in the same way as microorganisms. Labeling them with fluorescein can then lead to false positives. Cell lysis is therefore essential to distinguish microorganisms present in the sample from immune cells or their progenitor cells.After the labeling step, a laser beam scans the entire surface of the membrane to excite the fluorochrome, which makes it possible to detect and count any viable microorganisms present in the sample. A microscope coupled with the Scan makes it possible to visually validate the presence of microorganisms. The present invention makes it possible to achieve a detection limit of 1 CFU per sample.

[0109] On the other hand, a challenge for the use of this technology for application to sterility testing on innovative therapy drugs lies in the presence of a high concentration of cells equal to or greater than 10 5 cells / ml, preferably between 10 6 at 5x10 7cells / ml. Indeed, this non-filterable matrix causes a very significant fluorescence background on the membrane which can mask the specific fluorescence of microorganisms whose quantity is very low. It is therefore necessary to have a very effective lysis to obtain very small debris. The porous membrane of the solid phase cytometer allows the microorganisms to be retained while allowing the fluid and cellular debris to pass through it. The membrane corresponds to a plurality of pores having an average diameter of less than 0.50 pm, preferably between 0.32 and 0.45 pm.

[0110] Another object of the invention relates to a lysis kit comprising:

[0111] - a lysis buffer comprising a non-ionic detergent at a concentration of between 0.004% and 0.050% of the reaction volume

[0112] - a solution comprising a saponin at a concentration of between 0.03% and 4% of the reaction volume

[0113] - an endonuclease

[0114] - an endopeptidase acting at a pH between 7 and 8 In an alternative method, detection can be carried out by molecular biology. This is then a method for the detection of microorganisms potentially present in a sample comprising 10 6 at 10 8 immune cells or their progenitor cells, comprising the following steps: carrying out the isolation method according to the invention incubating the sample in an enrichment medium allowing the growth of the microorganisms eliminating or inactivating the endonuclease lysing the microorganisms to recover the nucleic acids bringing the nucleic acid solution into contact with primers making it possible to amplify at least one gene or a gene fragment of said microorganism detecting said microorganism

[0115] The step of isolating viable microorganisms allows residual DNA to be eliminated.

[0116] Endonuclease inactivation can be achieved by dilution in a large volume. It can take place before or after incubation in the enrichment medium.

[0117] The invention is illustrated with the non-limiting examples presented below.

[0118] Examples

[0119] Example 1: Preparation of lysing agents

[0120] Lysis buffer A is prepared with:

[0121] - Trizma: 138 g

[0122] - H2O: 900 ml

[0123] The pH of the buffer is adjusted to 8. Then 4 μl of benzonase (Benzonase E1014-25KU, Sigma) are added to 7 ml of the previously prepared solution. 2 ml of trypsin, (Trypsin EDTA: ref 25200-072- 500 ml, Sigma) will be added during the lysis protocol.

[0124] The following lysing compositions are prepared and then 4pl of benzonase (Benzonase E1014-25KU, Sigma) are added to 7ml of the previously prepared solution. 2ml of trypsin, (Trypsin EDTA: ref 25200-072-500ml, Sigma) will be added during the lysis protocol.

[0125] A lysis composition B comprising:

[0126] - Trizma (SIGMA base / T6066-5KG): 138 g

[0127] - H2O: 900 ml

[0128] The pH of the buffer is adjusted to 8.

[0129] In this buffer, 1.5 g (1.5 g / l of lysing composition) of saponin (Saponine Quillaja S4521 -10G, Sigma) are added.

[0130] A lysis composition C comprising:

[0131] - Trizma: 6 g

[0132] - BrijOlO (SIGMA / P6136-100G): 0.1296g

[0133] - H2O: 900 ml

[0134] The pH of the buffer is adjusted to 8.

[0135] In this buffer, 1.5 g (1.5 g / l of lysing composition) of saponin (Saponine Quillaja S4521 -10G, Sigma) are added.

[0136] A lysis composition D comprising:

[0137] - Trizma: 6 g

[0138] - BrijOlO: 0.1296g

[0139] - H2O: 900 ml

[0140] - Sodium thiosulfate: add 1 ml of the stock solution. (0.0499 g / L)

[0141] The pH of the buffer is adjusted to 8.

[0142] In this buffer, 1.5 g (1.5 g / l of lysing composition) of saponin (Saponine Quillaja S4521 -10G, Sigma) are added.

[0143] A lysis composition E comprising:

[0144] - Trizma: 6 g

[0145] - NP40 (Nonidet P 40 Sigma ref 74385-1 L): 0.75g

[0146] - H2O: 900 ml

[0147] The pH of the buffer is adjusted to 8.

[0148] In this buffer, 1.5 g (1.5 g / l of lysing composition) of saponin (Saponin Quillaja S4521 -10G, Sigma) are added. A lysis composition F comprising:

[0149] - Trizma: 6 g

[0150] - NP40: 0.75g

[0151] - H2O: 900 ml

[0152] The pH of the buffer is adjusted to 8.

[0153] A lysis composition G comprising:

[0154] - HEPES: mass of HEPES Acid: 3.12g, mass of HEPES Base: 9.61g

[0155] - BrijOlO: 0.1296g

[0156] - H2O: 900 ml

[0157] The pH of the buffer is adjusted to 8.

[0158] In this buffer, 1.5 g (1.5 g / l of lysing composition) of saponin (Saponine Quillaja S4521 -10G, Sigma) are added.

[0159] An H lysis composition comprising:

[0160] - HEPES: mass of HEPES Acid: 3.12g, mass of HEPES Base: 9.61g

[0161] - NP40: 0.75g

[0162] - H2O: 900 ml

[0163] The pH of the buffer is adjusted to 8.

[0164] In this buffer, 1.5 g (1.5 g / l of lysing composition) of saponin (Saponine Quillaja S4521 -10G, Sigma) are added.

[0165] Example 2: Preparation of the sample of immune cells or their progenitor cells

[0166] 1 ml of cells in RPMI 1640 medium (ref MS0A6Y100A biowest) is placed in contact with 5 CFU of microorganisms (1 OOpl).

[0167] The immune cells used are JURKAT cells or CAR-T cells: JURKAT cells (clone E6-1, ATCC TIB-152 or ECACC 88042803), 1 ml, at 10 7 cells / ml, CAR-T cells (ProMab PM-CAR2003-2M), 0.5ml, at 2x10 6 cells / ml, CAR-T cells (ProMab PM-CAR2003-2M), 1 ml, at 4x10 6 cells / ml, T cells (patient cells French Blood Establishment), 1 ml, at 2x10 7 cells / ml.

[0168] Progenitor cells are: umbilical cord mesenchymal stem cells (patient cells), 1 ml with 1x10 7 cells, lymphocytes, 1 ml with 2x10 7 cells, bone marrow mesenchymal stem cells, 1.5 ml with 1.5x10 6 cells.

[0169] Example 3: Lysis protocol

[0170] The contaminated sample is brought into contact with 1 ml of each of the lysis compositions and the benzonase according to example 1.

[0171] This solution is incubated at 37°C for 5 minutes with agitation at 1000 RPM in a ThermoMixer C Ependorf SN (37°C). After the 5 minutes of incubation, 2 ml of trypsin are added and this mixture is again agitated at 1000 RPM and 37°C for 5 minutes. The solution is then diluted by adding a few ml of fluiD (ref 42624 BioMérieux) to stop the chemical and enzymatic reactions.

[0172] Example 4: Detection of microorganisms in the lysed sample

[0173] This solution is then filtered through a membrane with a pore size between 0.32 and 0.38 pm on the SCANRDI® device (CB04 membrane and ScanFilter 415701). Several washes are possible with FluiD.

[0174] 1 ml of counterstain solution named CSE / CSM ((CSE ref 205-R4070- 01 bioMérieux / CSM 205-R4109-01) dilution of 1 Z100 of CSE in the CSM) is added and left on the SCANRDI® membrane for 10 seconds then the CSE / CSM is then passed through the membrane.

[0175] The membrane is then recovered and placed on the activation PAD on which the activation medium is placed (CELL BURST ACTIVATE kit bioMérieux ref 424512: CELL BURST ACTIVATE AER equivalent to a TSB broth for aerobic germs, CELL BURST ACTIVATE ANAER equivalent to a thioglycolate broth for anaerobic germs). The PAD is placed in an incubator at 30°C for 2 hours for aerobic germs and 2.5 hours for anaerobic germs under anaerobic conditions (GEN bag Anaer bimérieux 455534). The PADs are then changed and a new PAD with the marker (ChemChrome V6 ref 201 -R 1007-03 diluted 1 / 100 in ChemSol B16 205-R2023-02) is incubated for 45 minutes for aerobic germs and 1 hour 30 minutes for anaerobic germs under anaerobic conditions.

[0176] The marker used in SCANRDI® technology is a viability marker. SCANRDI® only detects viable germs.

[0177] At the end of the incubation, the membranes are recovered and analyzed using SCANRDI®.

[0178] Example 5: Efficiency of cell lysis

[0179] The sample preparation (Jurkat cells without the presence of microorganisms), the lysis protocol with lysis buffer A and lysis compositions B to H are described in Examples 1 to 4.

[0180] The RDI SCAN reading shows that the lysis protocol with buffer A results in too high a presence of unfiltered debris. This lysis protocol therefore does not allow for a process of isolating microorganisms from the sample. Conversely, the lysis protocol with buffers B to H allows for effective lysis of Jurkat cells, which allows for isolation of germs.

[0181] Example 6: Efficiency of cell lysis as a function of the concentration of saponin, non-ionic detergent and endopeptidase

[0182] The sample preparation (JURKAT cells without the presence of microorganisms), the lysis protocol and the reading by SCAN RDI are described in examples 1, 2, 3 and 4.

[0183] The results show that immune cells (Jurkat cells) can be effectively lysed. RDI SCAN reading of the membrane shows that lysis of cells and debris does not interfere with RDI SCAN reading for germ enumeration.

[0184] Example 7: Results obtained with the method of detecting aerobic microorganisms according to the invention

[0185] The sample preparation (Jurkat cell), the lysis protocol with lysis compositions B, D, E as well as the detection are described in examples 1, 2, 3 and 4. The microorganisms used are: Staphylococcus aureus, Pseudomonas aeruginosa, bacillus subtilis, Streptococcus pyogenes, kocuria rhyzophila. Each microorganism is tested 5 times (5 replicates) and in parallel with 5 Petri dishes to verify the accuracy of the inoculum (5CFU) on the dishes.

[0186] The results show that the microorganisms inoculated with the cells to undergo lysis remained viable and were detected by SCANRDI®. The results obtained show a sensitivity of less than 50 CFU for the microorganisms tested. All these results are in accordance with the recommendations of the pharmacopoeias which require a recovery rate between 50 and 200% between the dish technique and the SCAN RDI detection technique.

[0187] Example 8: Results obtained with the method of detecting anaerobic microorganisms according to the invention

[0188] The sample preparation (Jurkat cell), the lysis protocol with lysis compositions B, D, E as well as the detection are described in examples 1, 2, 3 and 4 specific for anaerobes. The microorganisms used are: Bacteroides fragilis, Clostridium sporogenes, Cutibacterium acnes

[0189] The results show that the anaerobic microorganisms that were inoculated with the cells to undergo lysis remained viable and were detected by SCANRDI®. The presence of thiosulfate in the detection protocol allowed for very good recovery.

[0190] The results obtained show a sensitivity of less than 30 CFU for the microorganisms tested.

[0191] Example 9: Results obtained with the method of detection according to the invention of the anaerobic microorganisms Candida albicans and Aspergillus brasiliensis

[0192] Sample preparation (Jurkat cells), lysis protocol with lysis compositions B, D, E and detection are described in example 1, 2, 3 and 4. The microorganisms used are: Candida albicans, Aspergillus brasiliensis and Penicillium chrysogenum The results show that the anaerobic microorganisms inoculated with the cells to undergo lysis remained viable and were detected by SCANRDI®. The results obtained show a sensitivity of less than 50 CFU for the germs tested.

[0193] Example 10: Influence of the concentration of Saponin and non-ionic detergent on the viability of microorganisms.

[0194] According to Example 1 (composition D), Example 2 (varying the cell volume) and Example 3, the toxicity of the protocol was tested with different microorganisms. The sample consists of JURKAT cells.

[0195] Following example 3, the solution is filtered through a NALGENE filter with a membrane with a pore diameter of 0.45 μm to recover the microorganisms and grow them in Petri dish media adapted to the types of microorganisms.

[0196] The results obtained in the table above show that at Saponin concentrations of 0.075 to 0.1364% of the reaction volume and BRIJ concentrations between 0.007 and 0.0127% of the reaction volume there is no toxicity for the germs tested while allowing the lysis of T lymphocytes. Example 11: Lysis of Jurkat cells (T lymphocytes) by a lysis composition and / or an endopeptidase

[0197] 1) Jurkat cell samples tested

[0198] Jurkat bioMérieux cell culture, harvested on 09 / 15 / 2022, stored in 5% DMSO + 10% BSA, thawed on 09 / 16 / 2022.

[0199] The cells are diluted to 1 / 10, i.e. 10 7 cells / ml (1 ml of cells in 9 ml of RPMI).

[0200] 2) Cell lysis

[0201] Lysis is carried out according to example 1. 1 ml of cells and 1 ml of lysis composition C and endonuclease are added for 5 min at 37 °C with stirring at 1000 rpm (triplicate). After the lysis step, 200 μl of the lysates are collected in a first tube in order to carry out the measurement on the Gallios cytometry system (beckman Coulter, Gallios 10 colors).

[0202] In the remaining tube, 2 ml of 0.25% endopeptidase solution is added. The tube is incubated for 5 minutes at 37°C, then, as before, 200 μl of the lysates are taken in order to carry out the measurement on the Gallios system.

[0203] 3) Flow cytometry

[0204] The Gallios flow cytometer is used to observe the effect of lysis on cells at both stages.

[0205] The table below shows the cytometric profile of cells that did not undergo lysis, of cells that underwent lysis by the lysing composition with endonuclease, and of cells that underwent lysis by the lysing composition, endonuclease and endoptidase.

[0206]

[0207] We observe 3 groups of cells:

[0208] - a group of living Jurkat cells

[0209] - a group of dead Jurkat cells

[0210] -a group of cellular debris

[0211] The lysis step by the lysing composition and the endonuclease (column 3) induces the loss of viability of Jurkat cells. Indeed, a shift of almost all cells from the viable state to the dead cell window is observed on the flow cytometer. A reduction in cell size is observed corresponding to the action of the lysis composition on the cell membranes. The saponin and BRIJ present in the lysis composition act by destructuring and permeabilizing the plasma membranes of the cells, by forming complexes with the membrane lipids.

[0212] This mechanism then allows the endopeptidase to hydrolyze the membrane proteins and burst the cells (column 4). Thus, the flow cytometer observes a displacement of almost all the dead cells towards the cell debris window. The action of trypsin also continues to degrade the nuclear membranes and release the intracellular material.

[0213] In conclusion, the lysis composition and the endopeptidase act in synergy allowing a better lysis of immune cells.

Claims

Claims 1 - Method for isolating viable microorganisms potentially present in a sample comprising 10 6 at 10 8 immune cells or progenitor cells thereof, comprising the following steps: - Bring the sample into contact with • a lysing composition comprising o a lysis buffer comprising a non-ionic detergent at a concentration of between 0.004% and 0.050% of the reaction volume o and / or a lysis solution comprising a saponin at a concentration of between 0.03% and 4% of the reaction volume • an endonuclease for digesting the nucleic acids released by the action of the lysing composition • an endopeptidase acting at a pH between 7 and 8, - Separate potentially present viable microorganisms from the sample by filtering the sample through a filter with a pore diameter between 0.30 and 0.50 pm. 2- Method according to claim 1 in which the sample is brought into contact with • a lysing composition, the lysing composition being a lysis buffer comprising a non-ionic detergent at a concentration of between 0.004% and 0.050% of the reaction volume and a saponin at a concentration of between 0.03% and 4% of the reaction volume • an endonuclease for digesting the nucleic acids released by the action of the lysing composition • an endopeptidase acting at a pH between 7 and 8. 3- Method according to any one of the preceding claims, characterized in that the sample comprises 10 6 at 5x10 7immune cells or progenitor cells of the latter, preferably between 5x10 6 and 10 7 cells. 4- Method according to any one of the preceding claims, characterized in that the immune cells are CAR-T. 5- Method according to any one of the preceding claims, characterized in that the endopeptidase is a trypsin. 6- Method according to any one of the preceding claims, characterized in that the non-ionic detergent is a polyoxyethylene detergent. 7- Method according to claim 5 characterized in that the polyoxyethylene detergent is NP40. 8- Method according to claim 5 characterized in that the polyoxyethylene detergent is BRIJ. 9- Method according to any one of claims 1, 3 to 8 characterized in that the final concentration of non-ionic detergent is between 0.025% and 0.05% in the absence of saponin. 10- Method according to any one of claims 1, 3 to 8 characterized in that the final concentration of saponin is between 0.10% and 4% of the reaction volume in the absence of non-ionic detergent. 11- Method according to any one of the preceding claims, characterized in that the final concentration of endonuclease is greater than 0.1% of the reaction volume. 12- Method according to any one of the preceding claims, characterized in that the endonuclease is brought into contact with the sample before the endopeptidase or after inactivation of the endopeptidase. 13- Method according to any one of the preceding claims, characterized in that the final concentration of endopeptidase is in excess. 14- Method according to any one of the preceding claims, characterized in that the porous membrane has pores between 0.32 and 0.45 μm. 15- Method according to any one of the preceding claims, characterized in that the sample is fresh. 16- Method for the detection of microorganisms potentially present in a sample comprising 10 6 at 10 8 immune cells or progenitor cells thereof, comprising the following steps: - Carrying out the isolation process according to any one of claims 1 to 15 - Mark the viable microorganisms retained on the porous membrane with a viability marker - Perform a scan of the porous membrane using a laser beam or camera - Determine the presence of viable microorganisms captured by the membrane 17- Lysis kit including: - a lysis buffer comprising a non-ionic detergent at a concentration of between 0.004% and 0.050% of the reaction volume - a solution comprising a saponin at a concentration of between 0.03% and 4% of the reaction volume - an endonuclease - an endopeptidase acting at a pH between 7 and 8.

Images