Method for producing device for treating fluid and device for treating fluid

Abstract

A method for manufacturing a device (1) for treating a fluid, comprising a binding step in which a substrate comprising a hollow fiber membrane is bound to a compound having one or more peptides of the sequence KKIRVRLSA (SEQ ID NO. 1) such that a chemical bond is formed between each other; the hollow fiber membrane comprises polysulfone, polyethersulfone, polysulfone and polyvinylpyrrolidone, polyethersulfone and polyvinylpyrrolidone and derivatives thereof; the device (1) is surprisingly able to remove viable bacteria and endotoxins from biological fluids, such as plasma.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of Italian Patent Application No. 102022000025365, filed on December 12, 2022, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to a method for manufacturing a device for treating a fluid, a device for treating a fluid and a use thereof. The present invention also relates to a treated hollow fiber membrane. Background Art

[0004] Sepsis is a clinical syndrome caused by disorders of the body's coagulation and immune systems.

[0005] Septic shock is characterized by alterations in blood circulation and cellular metabolism, resulting in a decrease in oxygen and nutrient supply to tissues and organs, which may lead to acute functional insufficiency.

[0006] Sepsis is a leading cause of death among people of all ages, with a mortality rate significantly higher than other conditions such as stroke and heart attack.

[0007] Patients with sepsis are typically treated with intravenous antibiotics, oxygen, fluids, and medications to stimulate the heart and maintain acceptable blood pressure levels.

[0008] Thanks to earlier diagnosis and continued implementation of best practices, sepsis mortality is no longer an immediately fatal condition, but rather a long-term, chronic illness often associated with long-term inflammation, immunosuppression, organ damage, and loss of lean body mass. Furthermore, patients who survive sepsis remain at risk of death and long-term cognitive and functional deficits.

[0009] Lipopolysaccharide (LPS) is the main component of the outer membrane of Gram-negative bacteria and is one of the main causes of the clinical syndrome of sepsis.

[0010] LPS is able to bind to specific host receptors and trigger an inflammatory response characterized by the release of large amounts of inflammatory mediators that enable the host to respond to pathogens. However, the production of this inflammatory mediator can become uncontrolled and excessive, leading to the development of septic shock.

[0011] Furthermore, lipoteichoic acid (LTA), a major cell wall component of Gram-positive bacteria, has been implicated in a variety of inflammatory diseases ranging from mild skin disorders to severe sepsis.

[0012] WO20100338220 discloses the peptide sequence KKIRVRLSA, which exists in monomeric, dendrimeric and multimeric forms, in particular in the form of compounds of formula (III) (see below), and describes the ability of M33 to neutralize LPS.

[0013] The peptide M33 has been identified and characterized for its ability to act as an effective antibacterial agent. Its mechanism of action is based on the peptide's chemical and physical properties. In particular, M33 exhibits an excess of positive charge due to its constituent charged amino acids, which allows it to bind to bacterial surfaces with considerable affinity through interactions with LPS and LTA (which, of course, are negatively charged). The peptide is produced in a tetrapod-like form, with four identical peptide sequences attached to a lysine core.

[0014] The most recent patent application WO2018193011 proposes attaching M33 to an agarose substrate via a procedure that requires several relatively complex and time-consuming steps.

[0015] Therefore, there remains a need for alternatives compared to the prior art, which can potentially be used to treat and / or prevent sepsis.

[0016] In particular, the need is also felt to identify methods that would allow the peptide M33 to exert and improve its endotoxin-removing action in a simple and inexpensive manner and, at the same time (in synergy with other ingredients), to remove bacteria.

[0017] The object of the present application is to provide a method for producing a device for treating fluids, a treated hollow fiber membrane, a device for treating fluids and the use thereof, which allow at least partially to overcome the disadvantages of the prior art while being easy and economical to implement. Summary of the Invention

[0018] According to the present application, there are provided a method for manufacturing a device for treating fluids, a treated hollow fiber membrane, a device for treating fluids and the use of such a device according to the following independent claim and preferably according to any claim directly or indirectly dependent on the independent claim. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The present application will be described below with reference to the accompanying drawings, which illustrate some non-limiting implementation examples of the present application, in which:

[0020] Figure 1 Schematically illustrates an apparatus according to the present application;

[0021] Figure 2 is a photograph of substrate particles (untreated hollow fibers) that can be used in the method according to the present application;

[0022] Figure 3 A, 3B and 3C are SEM images of substrates (untreated hollow fibers) that can be used in the method according to the present application;

[0023] Figure 4 Show Figure 2SEM images of the cross-section of the material;

[0024] Figure 5 shows Formula (III) in expanded form; and

[0025] Figure 6 shows chromatograms produced by the washing liquid passing through the device according to the present application (chromatogram with dashed line) and the device comprising a hollow fiber membrane simply bound to the compound of formula (III) (without beta radiation) (the abscissa represents time in minutes; the ordinate represents arbitrary units in mV);

[0026] Figure 7 is a graph showing the results of a comparative test between the device according to the present application and another device (the abscissa represents time in minutes; the ordinate represents CFUs-colony forming units / ml);

[0027] Figure 8 is a graph showing the results of a comparative test between the device according to the present application and another device (the abscissa represents time in minutes; the ordinate represents EU / ml of endotoxin - EU: endotoxin unit); and

[0028] Figure 9 Sampling locations are shown for some of the experiments performed. DETAILED DESCRIPTION

[0029] exist Figure 1 1 represents as a whole a device for treating a fluid, and the device 1 includes a treated hollow fiber membrane 7 .

[0030] In particular, the device 1 comprises a barrel 2, which in turn is provided with a side wall 3 (generally cylindrical) adapted to define an adsorption chamber 4. In particular, the barrel 2 further comprises a pair of circular closure elements 5 arranged to close the axial ends of the side wall 3. An opening is formed in each of the circular closure elements 5 to allow the inlet and outlet of the liquid to be treated. Each of the circular closure elements 5 comprises a fitting 6 at the corresponding opening for connecting a treatment tube. Typically, the barrel 2 is made of a plastic material, such as polycarbonate.

[0031] Additionally or alternatively, openings for the inlet and outlet of the liquid to be treated can also be formed on the side of the cartridge.

[0032] According to a first aspect of the present application, a method of manufacturing a device 1 is provided.

[0033] The method comprises a binding step in which a substrate comprising a hollow fiber membrane (particularly consisting of a hollow fiber membrane) is bound to at least one compound (having a formula) containing at least one peptide having the sequence KKIRVRLSA (SEQ ID NO. 1), such that the at least one compound is chemically (particularly covalently) linked to the substrate (and the hollow fiber membrane), and (thereby) a treated hollow fiber membrane 7 is obtained. The hollow fiber membrane comprises (or consists of) a substrate (particularly a polymer) selected from the group consisting of polysulfone, polyethersulfone, polysulfone and polyvinylpyrrolidone, polyethersulfone and polyvinylpyrrolidone (and derivatives thereof). In particular, the hollow fiber membrane comprises (or consists of) a substrate (particularly a polymer) selected from the group consisting of polyethersulfone, polyethersulfone and polyvinylpyrrolidone (and derivatives thereof). In some specific and non-limiting cases, the substrate is polyethersulfone (particularly with polyvinylpyrrolidone).

[0034] Each amino acid in the above peptides has an L or D configuration independently of the other amino acids.

[0035] The above method allows to obtain treated hollow fiber membranes 7 (and devices for treating fluids containing them) simply and quickly in an industrial manner (and in quantities). According to experimental observations, the devices thus obtained are surprisingly able to reduce (in particular remove) endotoxins (derived from Gram-negative bacteria), lipoteichoic acid (derived from Gram-positive bacteria) and bacteria from liquids (in particular organic liquids such as plasma and blood).

[0036] In the bonding step, the substrate is contacted with the compound to obtain a mixture of substrate and compound; in the bonding step, the mixture is treated with (subjected to) radiation (in particular ionizing radiation; in particular particulate radiation), in particular so that at least one compound is covalently attached to the hollow fiber membrane (in particular to the substrate), and a treated hollow fiber membrane 7 is obtained. For example, such radiation is beta and / or gamma radiation (in particular beta radiation).

[0037] According to experimental observations, by treating the mixture according to the present application, it is surprisingly possible to obtain a chemical bond (more precisely, a covalent bond) between the compound and the substrate (between the hollow fiber membrane and the compound; in particular between the substrate and the compound) in a simple and rapid manner. In this regard, it should be noted that in this way, laborious and lengthy steps of protecting functional groups, and / or reacting under controlled conditions, and / or using linkers and / or other special reagents and / or stresses (such as thermal stress, ultraviolet light pH, etc.) are unnecessary. It should also be noted that the production time is particularly reduced, given that the coupling between the substrate and the compound occurs simultaneously with the sterilization of the material (beta radiation, in addition to causing the bond between the substrate and the compound, also allows the sterilization of the medical material).

[0038] According to some non-limiting embodiments, during the bonding step, the substrate and the compound are brought into contact with each other within a cartridge 2 (particularly sealed; more particularly, the opening of which is sealed). In particular, during the bonding step, the cartridge containing the mixture is treated with radiation as described above (particularly ionizing radiation; particularly particulate radiation), such as beta and / or gamma radiation (particularly beta radiation).

[0039] This speeds up and simplifies the handling of materials which are all processed (and therefore also sterilized) at the same time.

[0040] Advantageously, but not necessarily, the mixture is treated with beta radiation in an amount of less than 150 kGy (particularly less than 50 kGy). More advantageously, but not necessarily, the mixture is treated with beta radiation in an amount of less than 30 kGy (particularly equal to or less than 28 kGy; more particularly equal to or less than 25 kGy) during the combining step. In particular, the mixture is treated with beta radiation in an amount of greater than 5 kGy (particularly greater than 10 kGy).

[0041] The radiation dose is measured (during the radiation dose mapping test) in accordance with ISO 11137-1:2020, in particular by a thin film dosimeter (FWT, Far West Technology). A reference dosimeter is conventionally used.

[0042] By keeping the amount of radiation provided at a relatively low level, the risk of damage to the compound, the substrate and, if present, the cartridge 2 can be reduced.

[0043] According to some non-limiting embodiments, in the combining step, a liquid (particularly a solution) containing the aforementioned compound is contacted with the substrate (so as to obtain a mixture). Particularly, the liquid is loaded into the cartridge 2, and more particularly the substrate is located in the cartridge 2.

[0044] Advantageously, but not necessarily, the liquid is an aqueous solution (eg, 0.9 wt% NaCl saline solution or PBS-phosphate buffered saline).According to some non-limiting embodiments, the solution consists of at least about 95 wt% water.

[0045] Advantageously, but not necessarily, the concentration of the compound in the liquid (solution) is up to about 5 mg / mL (particularly to about 3 mg / mL; preferably less than about 2 mg / mL).

[0046] According to some non-limiting embodiments, the concentration of the compound in the liquid (solution) is at least about 0.3 mg / mL (preferably, at least about 0.5 mg / mL).

[0047] For example, the concentration of the compound in the liquid (solution) ranges from about 1 mg / mL to about 2 mg / mL (particularly from about 0.5 mg / mL to about 1.5 mg / mL).

[0048] Advantageously, but not necessarily, the compound contains at least two (especially four) of the aforementioned peptides having the sequence KKIRVRLSA (SEQ ID NO. 1).

[0049] According to some preferred but non-limiting embodiments, the compound has a formula selected from the group consisting of (I) and (II).

[0050]

[0051] Among them, each R 1 represents the aforementioned peptide; X 1 、X 2 and X 3 are identical or different from each other and each independently represents at least a bifunctional group (particularly having at least two amino groups; particularly amino acids; more particularly lysine); Y 1 For example, it is selected from -H, another amino acid (particularly -β-alanine or -cysteine, more particularly -β-alanine) and another peptide.

[0052]

[0053] Among them, each R 1 represents the aforementioned peptide; X 1 、X 2 、X 3 、X 4 、X 5 、X 6 、X 7 、X 8 、X 9 and X 10 are identical or different from each other and each independently represents at least a bifunctional group (particularly having at least two amino groups; particularly amino acids; more particularly lysine); Y 1 For example, it is selected from -H, another amino acid (particularly -β-alanine or -cysteine; more particularly -β-alanine) and another peptide (particularly -β-alanine and -cysteine).

[0054] Alternatively or additionally, Y 1 Selected from -H and organic groups (with or without functional groups).

[0055] According to some specific and non-limiting embodiments, the compound has formula (III).

[0056]

[0057] Here, according to common standardization, K represents lysine; I represents isoleucine; R represents arginine; V represents valine; L represents leucine; S represents serine; and A represents alanine. Figure 5 Formula (III) is shown in expanded form.

[0058] The compound can be synthesized according to methods known in the art, more specifically, as described in WO20100338220 and European Patents EP2344178B1 (see in particular paragraph

[0020] ) and EP2595496B1 (see in particular paragraphs

[0017] and

[0018] ) and in the following articles: Pini, A., Falciani, C., Mantengoli, E., Bindi, S., Brunetti, J., Iozzi, S., Rossolini, G.M. & Bracci L. A novel tetrabranched antimicrobial peptide that neutralizes bacterial lipopolysaccharide and prevents septic shock in vivo. FASEB J.24,1–8(2010); and Brunetti, J., Falciani, C., Roscia, G., Pollini, S., Bindi, S., Scali, S., Arrieta, UC,Gómez-Vallejo,V.,Quercini,L.,Ibba,E.,Prato,M.,Rossolini,GM,Llop,J.,Bracci,L.&Pini,A.In vitroand in vivo efficacy,toxicity,bio-distribution and resistance selection of anovel antibacterial drug candidate.Sci.Rep.6,26077(2016);and Cresti L,Falciani C,Cappello G,Brunetti J,Vailati S,Melloni E,Bracci L,Pini A.Safetyevaluations of a synthetic antimicrobial peptide administered intravenously in rats and dogs.Sci Rep. 2022Nov 11;12(1):19294.doi:10.1038 / s41598-022-23841-2.

[0059] According to some non-limiting embodiments, the hollow fiber membrane is porous, particularly for filtration (more particularly for microfiltration or ultrafiltration).

[0060] In some non-limiting cases, the hollow fiber membrane has multiple conjugated (delocalized) π systems. In particular, the hollow fiber membrane has multiple aromatic groups (in other words, in these cases, the conjugated π systems are aromatic groups).

[0061] In particular, each of the hollow fiber membranes has a side wall comprising (composed of) a substrate and an inner cavity defined by the side wall.

[0062] According to some non-limiting embodiments, the sidewall has a thickness ranging from about 25 μm (particularly from about 40 μm) to about 400 μm (particularly to about 150 μm). In some cases, the sidewall has a thickness of up to about 90 μm (particularly up to about 85 μm).

[0063] In particular, the sidewall has an outer surface provided with pores having diameters ranging from about 0.1 μm to about 15 μm and an inner surface provided with pores having diameters ranging from about 5 nm to about 200 nm.

[0064] According to some non-limiting embodiments, the hollow fiber membrane has a length ranging from about 0.1 mm to about 50 mm (particularly to about 30 mm; more particularly to about 5 mm).

[0065] The length of the hollow fiber membrane is measured by successive sieving with sieves having pores of decreasing size. The diameter of the pores of the first sieve that does not allow the particle to pass represents the size (ie, diameter) of the particle.

[0066] In particular, the average length of the hollow fiber membranes ranges from about 0.1 mm to about 50 mm (in particular, to about 30 mm; more particularly, to about 5 mm).

[0067] Unless otherwise expressly stated, in this document, the average length is determined by measuring (e.g. with the aid of an optical microscope) the length of 50 randomly selected hollow fiber membranes. More generally, in this document, the average measurement value is obtained by averaging 50 random measurements.

[0068] The diameter of the pores is measured by SEM. More precisely, the size is measured along one direction (random sampling).

[0069] According to some non-limiting embodiments, the pores of the outer surface have an average diameter ranging from about 0.1 μm to about 15 μm. Additionally or alternatively, the pores of the inner surface have an average diameter ranging from about 5 nm to about 200 nm.

[0070] Unless otherwise specified, the average diameter of the pores herein is measured by SEM. More precisely, the average diameter is calculated by measuring the dimensions of 50 pores (randomly sampled) in one direction (randomly sampled) and averaging the measured dimensions. In particular, a SEM instrument is used.

[0071] According to some non-limiting embodiments, the cross-sectional area of ​​the lumen is in the range of about 5000 μm 2 to about 200,000 μm 2 .

[0072] In particular, the lumen has an inner diameter (more particularly an average inner diameter) of up to about 1000 μm (more particularly up to about 900 μm; even more particularly up to about 400 μm). According to some non-limiting embodiments, the lumen has an inner diameter (more particularly an average inner diameter) of at least 10 μm (particularly at least 100 μm).

[0073] Advantageously, but not necessarily, the hollow fiber membrane has a molecular weight cut-off of up to about 1500 kDalton, in particular up to 1000 kDalton.

[0074] According to some embodiments, the hollow fiber membrane has a molecular weight cut-off ranging from (at least) about 10 kDalton (more precisely, from 14 kDalton). The molecular weight cut-off refers to the molecular weight of a substance (polymer, bacteria, virus, etc.) that is retained by the membrane at 90%.

[0075] The molecular weight cut-off is determined (especially indirectly) by performing cut-off (filtration) experiments on molecules of known (and decreasing) molecular weight.

[0076] In particular, the molecules in question are proteins or dextran conjugated to fluorescent (fluorine-labeled dextran) or dye markers. Typically, fluorine-labeled dextran is used. In short, a solution containing one or more molecules with a known molecular weight (MW) is produced and filtered through a membrane. If the detection technology allows, solutions containing several molecules with a known MW (molecular weight) can be filtered simultaneously. The concentration may vary depending on the size and number of fibers in the device. When albumin is used, the concentration is 35-40 mg / ml, which is similar to physiological concentrations. If fluorine-labeled dextran is used, the concentration used is about 20 mg / ml. The MWs of fluorine-labeled dextran are 4, 70, 150 and 2000 kDa.

[0077] The concentration of molecules with known MW is measured at the inlet, outlet and in the filtrate (for tangential filtration). Depending on the protein / dextran and any conjugates, various techniques can be used to measure the concentration. For example, it can be detected by spectrophotometry, immunoenzymatic methods, fluorescence, electrophoresis, or chromatography. The molecular weight cut-off of the filter corresponds to the lowest molecular weight of a molecule retained by a membrane having a sieving coefficient of at least 9.0. For tangential filtration, the sieving coefficient is calculated as follows: [2 x filtrate concentration] / [input concentration + output concentration].

[0078] Similarly, the treated hollow fiber membrane 7 also includes sidewalls and an inner cavity, the inner cavity being defined by the sidewalls and having the same characteristics and dimensions as the hollow fiber membrane described above. In other words, the treated hollow fiber membrane 7 is essentially the same as the hollow fiber membrane, differing only in that it has (attached to) the above-described compound (on its surface). Therefore, the properties described for the hollow fiber membrane also apply to the treated hollow fiber membrane 7.

[0079] In particular, hollow fiber membranes are similar to those described in patent applications with publication numbers EP 2 316 560 A1 and / or EP 3 208 241 A1 and / or can be produced as described therein (with appropriate modifications). Hollow fiber membranes of this type are also manufactured by Medica spa (Via Degli Artigiani, 7, 41036 Medola MO, Italy) under the name and Nominal sales.

[0080] It is important to note that it has been experimentally observed that hollow fiber membranes (whether treated or not) clog surprisingly very little.

[0081] According to a second aspect of the present application, a treated hollow fiber membrane 7 is provided as described above (with reference to the first aspect of the present application).

[0082] According to a third aspect of the present application, there is provided a treated hollow fiber membrane 7 (as described above) for treating (and / or preventing) sepsis.

[0083] According to a fourth aspect of the present application, a method for treating (and / or preventing) sepsis using the treated hollow fiber membrane 7 (as described above) is provided. In some specific non-limiting cases, the method uses the device 1 (as described above).

[0084] According to a fifth aspect of the present application, there is provided a device 1 for treating a fluid. In particular, the device 1 is as defined above. More particularly, the compound and the treated hollow fiber membrane 7 are as described and / or defined above.

[0085] According to some non-limiting embodiments, the device 1 is obtained (obtainable) by the method mentioned in the first aspect of the present application.

[0086] According to a sixth aspect of the present application, there is provided an (in vitro) use of an apparatus 1 for (at least partially) removing viable bacteria and endotoxins from a (particularly biological) fluid. In particular, the fluid is a liquid, such as blood or plasma. In some specific and non-limiting cases, it is plasma.

[0087] In particular, the use provides for feeding a fluid through the device 1 .

[0088] A method for (at least partially) (in vitro) removal of viable bacteria and endotoxins from a (particularly biological) fluid is also provided. In particular, the fluid is a liquid, such as blood or plasma. In some specific and non-limiting cases, it is plasma.

[0089] In particular, the use provides for feeding a fluid through the device 1 .

[0090] Unless expressly stated otherwise, the contents of the references (articles, books, patent applications, etc.) cited herein are hereby incorporated by reference in their entirety.

[0091] Other features of the present application will become apparent from the following description which is given by way of illustrative and non-limiting examples only.

[0092] Example 1

[0093] This example describes the realization of an embodiment of the device 1 according to the present application.

[0094] The compound of formula (III) was used without modification (eg cysteylation or insertion of other linking groups).

[0095] The compound of formula (III) was dissolved in saline solution (0.9% NaCl or PBS) at a concentration of 1 mg / ml.

[0096] Porous particles containing a matrix / substrate (PES -Versatile- ) cartridge 2 is produced. The cartridge is washed with a disposable saline solution (note that PBS can also be used).

[0097] In a cleanroom production environment, a saline solution (0.9% NaCl-PBS can also be used) containing the compound of formula (III) (for some tests, the concentration was 1 mg / ml, for other tests, the concentration was 2 mg / ml - see Table 1 below) was loaded into the cartridge 2 by a pump (a syringe can also be used). The volume loaded was equal to the fill volume of the cartridge (approximately 5 mL) so that the device was completely filled with the solution containing the compound of formula (III). More precisely, in this way, each cartridge 2 contained 0.8 g of substrate and 5 ml of solution.

[0098] The device is sealed with a sealing cap and sealed in its packaging. The device is then transported to the sterilization facility, where it is exposed to beta radiation. The time from compound loading to sterilization can range from a few days to approximately 15 days. Returning the sterile product from sterilization can take another 1 to 10 days. At this point, the device 1 has been shipped at room temperature (uncontrolled) and stored in its carton.

[0099] A 13.3 kGy irradiation test and a 25 kGy irradiation test were performed, and in both cases there were two turns on the conveyor belt (with the cartons in different directions from one turn to the other).

[0100] In both cases, the device 1 was washed with saline solution to remove excess unattached peptide. During the initial wash (priming), some of the unattached excess compound (III) was removed, after which the device remained stable and no release of compound (III) was observed in subsequent steps.

[0101] These observations were confirmed by experiments carried out in HPLC reverse phase chromatography and mass spectrometry. In particular, the HPLC tests highlighted a significant difference in the presence of compound (III-M33) in the wash liquid between the two devices, one of which was subjected to beta irradiation and the other not, and therefore not attached to the substrate. In the non-irradiated device (black curve in the figure below), a clear peak appears at around 32 minutes (light blue arrow), corresponding to the retention time of compound (III-M33), which is even beyond the measurement range of the instrument. The same peak at 32 minutes is present in the (dashed) curve of the device subjected to irradiation, but with a much smaller size (see Figure 6 , where the abscissa represents retention time in the column in minutes and the ordinate represents arbitrary units in mV). Other peaks are not associated with the peptide or its truncated or aggregated forms but may be attributed to products of the substrate present in the column.

[0102] The results obtained indicate that compound (III) has been covalently linked to the hollow fiber membranes in the devices subjected to beta radiation irradiation.

[0103] Example 2

[0104] This example describes experiments carried out in a device 1 obtained as described in Example 1.

[0105] The tests were performed in dynamic mode (recirculation) and static mode (static contact) and evaluated the removal of bacteria and / or endotoxins and / or lipoteichoic acid from specially generated solutions (challenge solutions).

[0106] Dynamic recirculation conditions were used with a flow rate of 10 mL / min, and samples were collected at different time intervals over a 2-h period.

[0107] Table 1 below summarizes the devices tested during the project.

[0108] Table 1

[0109]

[0110] Concentration M33 represents the concentration of compound (III) in the solution that has been loaded into cartridge 2 (see Example 1 described above).

[0111] Device 1 (PES-M33) was compared with a control (CTRL-PES) without compound (III) to demonstrate the activity of device 1. The bacterial challenge solution consisted of a 10 5 -10 6 CFU / ml of Bacillus diminuta in saline lactose broth is expressed. The number of CFU was detected in samples collected from the challenge solution (batch) and the post-drum recirculation line (post). The samples were analyzed by diluting them and culturing them in TSA (tryptone soy agar) at 32.5° for 48 hours, followed by colony counts.

[0112] The results are shown in Table 2 and Figure 7 shown in the chart.

[0113] The number of CFU is the mean of counts performed on plates containing samples at different initial dilutions.

[0114] Table 2

[0115]

[0116] In fact, in Figure 7Middle: the concentration of Bacillus difficile in the recirculating solution of the control cartridge at different treatment times, in CFU / ml; CTRL-PES post represents the determined post-cartridge solution of the control cartridge at different treatment times; PES-M33 batch represents the recirculating solution of the cartridge containing peptide M33 at different treatment times; PES-M33 represents the determined post-cartridge solution of the cartridge containing peptide M33 at different treatment times; and PES-M33 batch represents the solution at the post-cartridge collection point of the cartridge containing peptide M33 at different treatment times.

[0117] Figure 9 The sample locations of the solution are shown. The solution sample marked as "batch" was collected at B. The sample marked as "post" was collected at P.

[0118] The number of CFUs of Bacillus diminuta was significantly reduced with both the PES-M33 and CTRL-PES devices. This suggests that the PES itself has the activity to retain bacteria, as demonstrated by the performance of the CTRL-PES. This activity appears to be quite non-specific, as the CTRL-PES device showed a reduction with each pass through the cartridge (after sampling), but this reduction over time was not reflected in the recirculated batch solution, suggesting the possibility of non-specific adsorption and release with different kinetics. However, the most significant results were obtained with the PES-M33 device, where the bactericidal activity was both significant and specific, as a reduction in the number of CFU / ml was achieved during the 2-hour treatment period, which was reflected in the entire recirculated solution (batch) and corresponded to a 98.8% reduction in the initial CFU concentration of Bacillus diminuta.

[0119] Subsequently, the endotoxin removal efficacy of the device containing PES-M33 (cartridge) and the control device (CTRL-PES) was also tested.

[0120] Endotoxin removal was quantified by preparing a spiked solution of LPS at a concentration of 5000 EU / ml in water. A volume of 200 ml of the spiked solution was recirculated through the prototype device PES-M33 and the control CTRL-PES. Samples were collected from the recirculated solution (batch) and post-drum production line (post), and their endotoxin concentrations were measured using a chromogenic kinetic LAL test using an MCS Nexgen instrument (Charles River).

[0121] Quantification allowed calculation of the logarithmic decay (LRV) of endotoxin concentration, which was equal to 0 for CTRL-PES and 3 for PES-M33.

[0122] The results obtained are shown in Table 3 and Figure 8 In the chart mentioned in .

[0123] Table 3

[0124]

[0125] In fact, in Figure 8 Middle: CTRL-PES batch represents the concentration of Bacillus diffusus in the recirculating solution of the control cartridge at different treatment times, in CFU / ml; CTRL-PES post represents the post-cartridge solution of the control cartridge at different treatment times; PES-M33 batch represents the recirculating solution of the cartridge containing peptide M33 at different treatment times; PES-M33 batch represents the solution at the post-cartridge collection point of the cartridge containing peptide M33 at different treatment times.

[0126] Figure 9 The sample locations of the solution are shown. The solution sample marked as "batch" was collected at B. The sample marked as "post" was collected at P.

Claims

1. A method of manufacturing a device (1) for treating a fluid; The device (1) comprises a treated hollow fiber membrane (7); The method comprises: a binding step, in which the substrate containing the hollow fiber membrane is bound to at least one compound having at least one peptide having the sequence KKIRVRLSA (SEQ ID NO. 1) so that the at least one compound is covalently linked to the substrate, and the treated hollow fiber membrane (7) is obtained; The hollow fiber membrane comprises a substrate selected from the group consisting of polyethersulfone, polyethersulfone and polyvinylpyrrolidone and derivatives thereof; In the combining step, the substrate is contacted with the compound to obtain a mixture of the substrate and the compound; In the binding step, the mixture is treated with ionizing radiation to cause the at least one compound to be covalently linked to the hollow fiber membrane, and the treated hollow fiber membrane (7) is obtained; Each amino acid in the peptide, independent of the other amino acids, has either the L or D configuration.

2. The method according to claim 1, wherein During the combining step, the mixture is treated with beta and / or gamma radiation.

3. The method according to claim 2, wherein: In the combining step, the mixture is treated with beta and / or gamma radiation (particularly beta radiation) of less than 50 kGy (particularly equal to or less than 30 kGy; more particularly equal to or less than 28 kGy).

4. The method according to claim 2 or 3, wherein: During the bonding step, the substrate and the compound are brought into contact with each other within a cartridge; during the bonding step, the cartridge containing the mixture is treated with beta and / or gamma radiation, in particular beta radiation.

5. The method according to any one of the preceding claims, wherein During the binding step, a liquid (particularly a solution) containing the compound is brought into contact with the substrate; in particular, the liquid is introduced into the cartridge, and more particularly, the substrate is located in the cartridge.

6. The method according to any one of the preceding claims, wherein The compound has at least two (especially four) peptides having the sequence KKIRVRLSA (SEQ ID NO. 1).

7. The method according to any one of the preceding claims, wherein The compound has a formula selected from the group consisting of (I) and (II): Among them, each R 1 represents the peptide; X 1 、X 2 and X 3 are identical or different from each other and each independently represents at least a bifunctional group (especially an amino acid); Y 1 is selected from -H, another amino acid (particularly -β-alanine or -cysteine) and another peptide; Among them, each R 1 represents the peptide; X 1 、X 2 、X 3 、X 4 、X 5 、X 6 、X 7 、X 8 、X 9 and X 10 are identical or different from each other and each independently represents at least a bifunctional group (particularly an amino acid; more particularly lysine); Y 1 is selected from -H, another amino acid (particularly -β-alanine or -cysteine) and another peptide.

8. A method according to any one of the preceding claims, wherein Each of the hollow fiber membranes has a side wall comprising a substrate and an inner cavity defined by the side wall; the side wall has a thickness in the range of about 25 μm (particularly about 40 μm) to about 150 μm (particularly about 80 μm), an outer surface provided with pores having a diameter in the range of about 0.1 μm to about 15 μm, and an inner surface provided with pores having a diameter in the range of about 5 nm to about 200 nm; the inner cavity has an area of ​​about 5000 μm 2 to about 200,000 μm 2 A cross-section within a range, particularly an average inner diameter within a range of about 100 μm to about 400 μm; The hollow fiber membranes are particles having a length ranging from about 0.1 mm to about 50 mm (particularly to about 30 mm; more particularly to about 5 mm).

9. The method according to any one of the preceding claims, wherein The compound has the formula (III):

10. The method according to any one of the preceding claims, wherein In the combining step, the mixture is treated with beta radiation.

11. A device for treating a fluid; the device (1) comprises a treated hollow fiber membrane (7), the hollow fiber membrane (7) comprising (in particular consisting of) a substrate selected from the group consisting of polyethersulfone, polyethersulfone and polyvinylpyrrolidone and derivatives thereof; The treated hollow fiber membrane (7) further comprises a compound having at least one peptide having the sequence KKIRVRLSA (SEQ ID NO. 1), wherein the peptide is covalently linked to the substrate; Each amino acid in the peptide, independently of the other amino acids, has an L or D configuration; In particular, the device (1) is obtained according to any one of claims 1 to 9.

12. The device according to claim 11, comprising a cartridge provided with at least one inlet and outlet and housing the treated hollow fiber membranes (7); The treated hollow fiber membranes are particles having a length ranging from about 0.1 mm to about 50 mm (particularly to about 30 mm; more particularly to about 5 mm); The compound has at least two (particularly four) peptides having the sequence KKIRVRLSA (SEQ ID NO. 1).

13. The device according to claim 11 or 12, wherein: The compound has a formula selected from the group consisting of (I) and (II): Among them, each R 1 represents the peptide; X 1 、X 2 and X 3 are identical or different from each other and each independently represents at least a bifunctional group (particularly having at least two amino groups; particularly amino acids; more particularly lysine); Y 1 is selected from -H, another amino acid (particularly -β-alanine or -cysteine) and another peptide (particularly -β-alanine or -cysteine); and Among them, each R 1 represents the peptide; X 1 、X 2 、X 3 、X 4 、X 5 、X 6 、X 7 、X 8 、X 9 and X 10 are identical or different from each other and each independently represents at least a bifunctional group (particularly having at least two amino groups; particularly amino acids; more particularly lysine); Y 1 is selected from -H, another amino acid (particularly -β-alanine or -cysteine) and another peptide (particularly -β-alanine or -cysteine).

14. The device according to any one of claims 11 to 13, wherein: Each of the hollow fiber membranes has a side wall comprising a substrate, and an inner cavity defined by the side wall; in particular, the thickness of the side wall is in the range of from about 25 μm (in particular from about 40 μm) to about 150 μm (in particular to about 80 μm), in particular, the compound has formula (III):

15. Use of a device (1) according to any one of claims 10 to 14 for at least partially removing live bacteria and endotoxins from a fluid, in particular a biological fluid; the use provides for feeding the fluid through the device (1).

16. A treated hollow fiber membrane; the treated hollow fiber membrane (7) comprises a substrate (in particular consists of a substrate), the substrate being selected from the group consisting of polyethersulfone, polyethersulfone and polyvinylpyrrolidone and derivatives thereof; The treated hollow fiber membrane (7) further comprises a compound having at least one peptide having the sequence KKIRVRLSA (SEQ ID NO. 1), wherein the peptide is covalently linked to the substrate; Each amino acid in the peptide, independent of the other amino acids, has either the L or D configuration.

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