Methods and apparatus for producing nuclease-free surfaces

Abstract

The invention relates to a method (1000) for generating a nuclease-free surface, wherein the method comprises: generating (1100) a first non-thermal plasma (10) at atmospheric pressure, wherein the first non-thermal plasma (10) is generated at a first power on a discharge surface; generating (1200) a second non-thermal plasma (20) at atmospheric pressure, wherein the second non-thermal plasma (20) is generated at a second power on the discharge surface, wherein the second power is higher than the first power; generating (1300) a plasma stream (26) from the first non-thermal plasma and the second non-thermal plasma; conducting (1400) the plasma stream (26) to a surface (102, 104) to be cleaned; and inactivating (1500) nucleases on the surface (102, 104, 106) to be cleaned.

Description

Technical Field

[0001] The present invention relates to a method and apparatus for producing nuclease-free surfaces, particularly nuclease-free polymer surfaces. Background Technology

[0002] RNA (ribonucleic acid) and DNA (deoxyribonucleic acid) are two important nucleic acids that perform different functions in living organisms. As is well known, DNA carries genetic information and significantly influences an organism's development, growth, function, and reproduction. DNA is passed on to the next generation. RNA plays a crucial role in protein biosynthesis. So-called messenger RNA (mRNA) transmits genetic information from DNA in the cell nucleus to ribosomes in the cytoplasm, where proteins are produced.

[0003] Novel vaccines and medical treatments based on RNA or DNA are currently being developed. For example, so-called mRNA vaccines have been successfully used as a COVID-19 vaccine. Other mRNA vaccines are currently being developed for diseases such as Lyme disease, chlamydia, cytomegalovirus, dengue fever, herpes, influenza, HIV, and malaria (to name just a few).

[0004] Furthermore, RNA- or DNA-based active agents are being investigated in cancer treatment (particularly cancer immunotherapy) and for treating genetic defects. These RNA- and / or DNA-based technologies have great potential to successfully treat currently untreatable or poorly treated diseases, or at least limit the symptoms of these diseases.

[0005] However, even small impurities containing nucleases (ribonuclease, or RNase for short, and deoxyribonuclease, or DNase for short) can damage or even render novel vaccines or drugs unusable. This is because RNases are enzymes that catalyze the hydrolysis of phosphodiester bonds in the ribonucleic acid (RNA) chain. Deoxyribonucleases are enzymes that catalyze the hydrolysis of the deoxyribonucleic acid (DNA) molecule into shorter chains or individual building blocks. If this catalysis occurs, the vaccine or active agent may be damaged, inactivated, or even rendered unusable.

[0006] Therefore, all containers and other objects that come into contact with the vaccine or active agent (e.g., during production, storage, or administration) must be thoroughly cleaned and have active nucleases removed.

[0007] The cleaning process includes, for example, autoclaving, treatment with hydrogen peroxide, diethyl pyrocarbonate or ribonuclease inhibitors, treatment with heat, UV radiation, or other methods.

[0008] These cleaning methods are often expensive and time-consuming, and can cause lasting damage to the treated surfaces or objects. For example, RNases and DNases are known to be destroyed by autoclaving at at least 121°C for 15 minutes or by heating to 180°C for at least eight hours. High temperatures and / or the chemicals used cause lasting damage to many materials, particularly polymers, rendering them unusable or only suitable for limited use in the production and storage of new vaccines and active agents.

[0009] In conventional bioreactors, vaccines or active agents to be produced are typically stored in stainless steel tanks lined with polymer membranes or polymer membrane bags and / or connected via polymer tubing.

[0010] Typical polymers used in bioreactors include: • ETFE—ethylene tetrafluoroethylene copolymer, • FEP—Tetrafluoroethylene-hexafluoropropylene copolymer, • HDPE—High-density polyethylene • LDPE—Low-density polyethylene • PC—Polycarbonate, polyethylene terephthalate • PFA—a perfluoroalkoxy polymer, • PP / PCO — Polypropylene / Polycyclooctene • PMP—Polymethylpentene, • Barex—acrylonitrile plastics, • HIPS—High-impact polystyrene • PVC—Polyvinyl chloride • TPE—Thermoplastic elastomer, • etc.

[0011] However, these methods are unsuitable or only suitable to a limited extent for removing active DNA and RNA enzymes using conventional methods.

[0012] JW Lackmann et al. "A dielectric barrier discharge terminally inactivates RNase A by oxidizing sulfur-containing amino acids and breaking structural disulfide bonds , 2015, J .

[0013] Phys. D: Appl. Phys. 48 494003 describes the inactivation of RNase A by means of dielectric barrier discharge. Summary of the Invention

[0014] Therefore, the object of the present invention is to provide a method and apparatus for producing nuclease-free surfaces, particularly suitable for polymer surfaces. This object is achieved by the method according to claim 1 and the apparatus according to claim 14. Other aspects of the invention are set forth in the dependent claims and the following description.

[0015] Specifically, this objective is achieved by a method for producing nuclease-free surfaces—particularly nuclease-free polymer surfaces. The terms nuclease-free, DNase-free, or RNase-free are used synonymously below. The term nuclease-free surface refers to a surface that is substantially free of active RNases and DNases. A surface substantially free of RNases and DNases is one whose RNase and DNase contamination is so low that products containing RNA or DNA (such as active agents or vaccines) that come into contact with this surface are no longer damaged or inactivated.

[0016] This method is applicable to all types of surfaces, especially polymer surfaces. However, it should be understood that other surfaces (such as metal surfaces, ceramic surfaces, glass surfaces, etc.) can also be made free of active nucleases by the method according to the invention.

[0017] To check if a surface is nuclease-free (i.e., RNase-free or DNase-free), a sample of the surface can be extracted for 1 hour in RNase-free water (ultrapure DNase-free / RNase-free distilled water) or DNase-free water. If the surface is the inner surface of a volume (e.g., a polymer membrane bag), the volume can be filled with RNase-free (or DNase-free) water and extracted for 1 hour. An RNase test kit (e.g., RNaseAlert) can be used. ™ Treat 45 μL of this RNase-free water with the laboratory test kit (catalog number: AM1964) or the DNase test kit and examine it using a fluorometer (Mithras LB 940, Berthold Technologies). If the amount of RNase (or DNase) in the sample water is less than 1.5 relative to the amount of RNase (or DNase) in the RNase-free / DNase-free water (blank), the surface can be described as RNase-free (or DNase-free). A value between 1.5 and 2.5 indicates slight contamination of the sample. A value of 2.5 or higher indicates severe contamination of the sample.

[0018] The method according to the present invention includes the following steps: • A first nonthermal plasma is generated at atmospheric pressure, wherein the first nonthermal plasma is generated at a first power on a discharge surface; • A second nonthermal plasma is generated at atmospheric pressure, wherein the second nonthermal plasma is generated at a second power on the discharge surface; The second power is higher than the first power. • A plasma flow is generated by a first nonthermal plasma and a second nonthermal plasma; • Conducting the plasma flow to the surface to be cleaned, and • Inactivate nucleases on the surface to be cleaned.

[0019] The inventors' tests showed that by using two different nonthermal plasmas with different power levels, nucleases could be removed from surfaces more effectively than by using only one type of nonthermal plasma. The first nonthermal plasma (hereinafter sometimes referred to as "ozone-dominant nonthermal plasma") had a higher ozone content, and the second nonthermal plasma (hereinafter occasionally referred to as "nitrogen-dominant nonthermal plasma") had a higher nitrogen oxide content. The inventors attributed this to the low solubility of O3, NO, and NO2 in water (in humid conditions). This was particularly effective in forming polyvalent NO using two types of plasma. x Such a level of N2O5, not observed under other conditions, was observed. It was found that N2O5 can be generated efficiently and stably by mixing the effluent gases from the first and second air plasmas. Using a surface treated with the mixed gas resulted in a reduction of nucleases to 1 / 10. 6 .

[0020] Optionally, inactivated nucleases can be removed from the surface to be cleaned—for example, by rinsing with RNase-free water. However, inactivation of RNases and / or DNases is usually sufficient, as the inactivated enzymes are no longer capable of cleaving RNA / DNA. Optionally, after removing inactivated RNases and / or DNases from the surface to be cleaned, a plasma stream can be passed to the cleaned surface again. This reprocessing with a plasma stream allows the cleaning medium (e.g., RNase-free water) to be removed from the surface and allows the surface to dry.

[0021] Nonthermal plasmas (NTPs) are plasmas that are not in thermal equilibrium. Therefore, the temperatures of the types of particles they contain (neutral particles, ions, electrons) vary, sometimes significantly. Ozone-dominant NTPs are those that primarily lead to the formation of ozone (O3) in the plasma gas. Nitrogen-dominant NTPs are those that primarily lead to the formation of nitrogen compounds (such as NO, NO2, NO3-) in the plasma gas. x Non-thermal plasmas containing N2O, HNO3, HNO2 and / or N2O5.

[0022] The first nonthermal plasma, primarily composed of ozone, can be generated, for example, by means of dielectric barrier discharge or corona discharge. Dielectric barrier discharge is an alternating voltage gas discharge in which at least one electrode is electrically isolated from the gas space by means of current isolation using a dielectric material.

[0023] A gaseous space filled with process gas (or through which process gas flows) (e.g., air, particularly (filtered) ambient air) can be ionized by electrically isolating electrodes to generate plasma. For this purpose, an alternating voltage is applied to the electrodes, causing a discharge. The displacement current sustains the discharge even through insulation, allowing for continuous electrical transfer into the plasma.

[0024] The frequency of the AC voltage is preferably selected such that it corresponds to the resonant frequency of the plasma generator. The resonant frequency of the plasma generator depends particularly on the size and materials of the plasma generator and the process gas used. In one example, the frequency of the AC voltage is approximately 40 kHz (±10%). The higher the selected frequency, the higher the proportion of reactive nitrogen substances (RNS, such as NO). The lower the selected frequency, the higher the proportion of reactive oxygen species (ROS, such as ozone). Furthermore, the plasma density increases as the frequency decreases.

[0025] Electrodes can be designed as, for example, parallel plates, metal meshes on dielectric materials, or tips opposite to the plates.

[0026] Corona discharge is a discharge in a non-conductive medium, such as in a process gas (e.g., air, especially (filtered) ambient air). The discharge requires ions as charge carriers. These charge carriers may already be present (in plasma), or they may form in the medium due to field ionization if the electric field strength is sufficiently high (e.g., >100 kV / m).

[0027] The first type of nonthermal plasma (nonthermal plasma dominated by ozone) is, for example, discharged at a rate of less than 0.5 W / cm² on the discharge surface. 2 Especially less than 0.4 W / cm 2 or less than 0.3 W / cm 2 or less than 0.2 W / cm 2 The power generation. The preferred range for generating ozone-dominant nonthermal plasma is between 0.1 W / cm². 2 and 0.2W / cm 2 Between. In particular, the surface temperature of the electrodes used to generate ozone-dominant non-thermal plasma can be less than 90°C, especially less than 75°C or less than 60°C.

[0028] On the other hand, a second nonthermal plasma (nitrogen-dominant nonthermal plasma) is generated by dielectric barrier discharge or corona discharge. For example, the nitrogen-dominant nonthermal plasma discharges at a rate greater than 0.1 W / cm² on the discharge surface. 2 Especially greater than 0.2 W / cm 2 or greater than 0.5 W / cm 2 or greater than 1.5 W / cm2 or greater than 2W / cm 2 The power generated is preferably within the range of 0.5 W / cm². 2 and 2.2W / cm 2 Between. In particular, the surface temperature of the electrodes used to generate nitrogen-based non-thermal plasma can be greater than 90°C.

[0029] However, it is preferably specified that the first non-thermal plasma discharges at a rate of 0.2 W / cm² on the discharge surface. 2 Up to <0.45W / cm 2 Preferably 0.3W / cm 2 Up to <0.45W / cm 2 The power generated, wherein the second nonthermal plasma (20) is discharged at a rate of >0.45 W / cm² on the discharge surface. 2 Up to 0.7W / cm 2 Preferably >0.45W / cm 2 Up to 0.6W / cm 2 The power generation. Preferably, in this case, dielectric barrier discharge or corona discharge is provided.

[0030] The process gas can be, for example, air, particularly dried and / or sterilized air and / or filtered ambient air, argon, etc. Alternatively, a mixture of at least two different process gases can be used.

[0031] The process gases can be purified—for example, by passing through at least one filter—before the generation of the plasma (primarily ozone and / or nitrogen). The same process gases (e.g., air, particularly (filtered) ambient air) can be used to generate the first and second nonthermal plasmas, or different process gases or mixtures can be used.

[0032] Specifically, the generation of a first nonthermal plasma dominated by ozone and a second nonthermal plasma dominated by nitrogen can occur simultaneously, preferably in a common plasma generator. For this purpose, the electrodes can be designed such that different powers exist at their surfaces. For example, the first nonthermal plasma can be primarily generated in regions with lower power density, while the second nonthermal plasma can be primarily generated in regions with higher power density.

[0033] It is also possible that the first and second nonthermal plasmas are generated one after the other (i.e., in time) in successive segments. For this purpose, the power of the plasma generator can be adjusted (periodically).

[0034] In another aspect of the invention, a first non-thermal plasma is generated in a first plasma generator, and a second non-thermal plasma is generated in a second plasma generator different from the first plasma generator.

[0035] The resulting plasma (primarily ozone and nitrogen) is then used to generate a plasma flow. For this purpose, ionized process gases are exhausted from the plasma generator. Exhaust can be achieved, for example, by means of at least one fan and / or at least one pump. Alternatively, the plasma generator can be designed so that the process gases are exhausted directly.

[0036] The plasma flow is then directed to the surface to be cleaned. Thus, the plasma flow comes into contact with the surface. To enhance the effectiveness of the plasma flow, it can be selectively blown onto the surface to be cleaned. This can be achieved using plasma gas nozzles, fans, and / or pumps. This allows for particularly rapid inactivation of nucleases located on the surface.

[0037] In another preferred embodiment, the plasma stream is mixed with the aerosol and conducted to the surface to be cleaned. The aerosol is generated and may include water, particularly distilled water, RNase-free / DNase-free water, and / or plasma-activated water. The aerosol can be generated, for example, by means of a sprayer or atomizer.

[0038] In particular, the addition of fine water droplets, preferably smaller than 100 μm and especially preferably smaller than 80 μm, results in a high inactivation rate of nucleases. In this case, an ultrasonic nebulizer is preferably used to atomize distilled water (and / or RNase-free and / or DNase-free water), and the aerosol mixture is mixed with a plasma stream (generated by ozone-based and / or nitrogen-based non-thermal plasma) and conducted to the surface to be cleaned.

[0039] In another particularly preferred embodiment, plasma-activated water (PAW) is generated and applied as an aerosol mixture to the surface to be cleaned. Particularly preferably, an ultrasonic atomizer is used to generate the aerosol mixture, and the finest PAW aerosol is applied to the surface to be cleaned.

[0040] The surface to be cleaned can be, for example, a polymer surface, such as a polymer film, polymer sheet, or a polymer surface formed in another manner (injection molding, extrusion, etc.). The use of non-thermal plasma or plasma streams will not damage the polymer. The use of ozone-based and nitrogen-based plasmas allows for rapid and reliable inactivation of RNases and / or DNases.

[0041] It has been shown that a ratio of 70:30 (based on the discharge surface) of the first nonthermal plasma to the second nonthermal plasma results in a good deactivation rate. xPreferably, it includes NO, NO2, and / or higher levels of N. x O y (x≥1; y≥3), in which the NO component is dominant.

[0042] The ratio of the components of the plasma flow can be determined and / or changed by the plasma generator power, particularly by the AC voltage (frequency, amplitude, signal shape, etc.) applied to the generator and / or the generator geometry.

[0043] The surface to be cleaned can be located inside the hollow body. This hollow body can be formed, for example, from at least one polymer membrane bag or a decontamination container. To inactivate any nucleases, a plasma flow is conducted into the interior of the hollow body. Specifically, the plasma flow can be conducted into the hollow body at at least one inlet. Furthermore, the plasma flow can be conducted into the hollow body at multiple inlets. This improves cleaning performance.

[0044] If the surface to be cleaned comprises multiple interiors of multiple hollow bodies, these hollow bodies can be interconnected via at least one plasma gas line, allowing plasma flow to be introduced into the respective interiors. For example, several polymer membrane bags can be interconnected, or several polymer membrane bags can be filled / flowed through in parallel using plasma flow via a suitable dispenser. Furthermore, the inner surfaces of so-called disposable components can be cleaned. For this purpose, plasma flow is introduced into the disposable component and the inner surfaces are cleaned. Disposable components typically comprise one or more polymer membrane bags and at least one hose line. Additionally, valves and / or the like can be part of the disposable component. These disposable components are particularly used in the production of active agents or vaccines, or their precursors or intermediates.

[0045] Besides the internal inner surface, the objects within the hollow body may also be devoid of active nucleases. For example, a filter can be retained inside the hollow body through which the plasma flow is directed. Similarly, other items to be cleaned can be provided in the decontamination container, for example. These items may include, for example, pipettes, pipette tips, cannulas, vials, disposable medical or pharmaceutical plastic products, disposable bioprocess containers, laboratory equipment, centrifuges, PCR units, etc. The incoming or outgoing plasma flow then inactivates the nucleases.

[0046] The method may also include a leak test on at least the hollow body, wherein the plasma gas of the generated plasma stream is used as the test gas. If the plasma gas is introduced into the hollow body and the pressure inside the hollow body is measured, the pressure drop can determine whether the hollow body (e.g., a polymer membrane bag) is sealed or leaking. Therefore, the additional leak test can be omitted. Thus, after inactivating RNase / DNase, it can be immediately determined whether the hollow body (e.g., a polymer membrane bag) is sealed and free of RNase / DNase. A hollow body that meets both criteria can be used, for example, in a bioreactor in a bioprocess pipeline for the production of vaccines or active agents, for the transport and / or storage of vaccines or active agents. Similarly, precursors and / or intermediates of vaccines or active agents can be produced, transported, and / or stored in the polymer membrane bag.

[0047] The method may further include unfolding and / or extruding a polymer membrane, wherein a plasma flow is conducted to at least one side of the extruded and / or unfolded polymer membrane. Therefore, the method can be directly integrated into the membrane production process or into the production process of membrane products (such as polymer membrane bags). Thus, a separate deactivation step can be omitted, since the produced membrane or membrane product is directly RNase-free or DNase-free. Surfaces can also be treated multiple times with the plasma flow. For example, after the membrane has been unfolded, extruded, and / or stretched (unidirectional or bidirectional), the plasma flow can be immediately conducted to at least one surface of the membrane (particularly the subsequent interior of the polymer membrane bag). For this purpose, a plasma gas nozzle preferably spanning the entire width of the membrane can be provided, or multiple plasma gas nozzles arranged adjacent to or offset from each other (in the direction of membrane pull-out) can be provided such that the plasma flow preferably flows across the surface of the membrane across the entire width of the membrane. Similarly, at least one plasma gas nozzle can be arranged to be movable and mobile on the surface of the membrane to conduct the plasma flow to the surface to be cleaned.

[0048] In one aspect, the membrane is guided through a decontamination zone where a plasma flow is introduced. This decontamination zone may have a longitudinal extension. The treatment time of the membrane surface with the plasma flow can then be determined based on the membrane removal rate. Within the decontamination zone, the membrane can be reoriented (multiple times) (e.g., using rollers for reorientation).

[0049] Specifically, the plasma flow can be conducted to the surface to be cleaned for a period of at least 10 minutes, at least 15 minutes, or at least 20 minutes.

[0050] Furthermore, the method may include generating at least one volume, particularly at least one polymer film bag. Preferably, the volume is generated by at least one polymer film. For this purpose, the polymer film may be folded, and the edge regions of the folded polymer film may be interconnected (preferably integrally interconnected), or multiple polymer films (at least two) may be placed one on top of the other and joined together to form a volume with an inner surface (e.g., a polymer film bag). The generation of at least one volume may involve polymer bonding and / or polymer welding. For example, polymer welding may be thermal welding, high-frequency welding, pulse welding, ultrasonic welding, friction welding, laser welding, etc.

[0051] Nuclease-free inner surfaces of volumes can be created before and / or after polymer bonding and / or polymer welding. For example, nuclease-free membranes can be welded together, or volumes can be created first and then plasma flow can be introduced into those volumes.

[0052] This objective is also achieved by an apparatus for generating a nuclease-free surface. The apparatus is designed to perform the methods described above. The apparatus includes at least one plasma reactor having at least one discharge surface on a discharge device, wherein a first non-thermal plasma can be generated on the discharge surface at atmospheric pressure with a first power using the plasma reactor. Furthermore, a second non-thermal plasma can be generated on the discharge surface at atmospheric pressure with a second power, wherein the second power is higher than the first power. Additionally, the apparatus includes at least one plasma gas line connected to the at least one plasma reactor and configured to conduct the plasma flow generated by the first and second non-thermal plasmas to the surface to be cleaned, thereby inactivating nucleases on the surface to be cleaned.

[0053] Preferably, the plasma reactor having a discharge surface on the discharge device includes a dielectric discharge device or a corona discharge device.

[0054] Regarding the generation of non-thermal plasma, it is preferably specified that the discharge device used to generate the first non-thermal plasma is capable of discharging at a rate of 0.2 W / cm² on the discharge surface. 2 Up to <0.45W / cm 2 Preferably 0.3W / cm 2 Up to <0.45W / cm 2 The power operation is such that the discharge device used to generate the second non-thermal plasma is capable of operating at >0.45 W / cm² on the discharge surface. 2 Up to 0.7W / cm 2 Preferably >0.45W / cm 2 Up to 0.6W / cm 2Power operation. For this purpose, for example, a control device can be provided that operates at 0.2 W / cm² in the discharge region. 2 Up to <0.45W / cm 2 Preferably 0.3W / cm 2 Up to <0.45W / cm 2 The power is used to operate the discharge device for generating the first nonthermal plasma, and at the discharge surface at a power of >0.45 W / cm². 2 Up to 0.7W / cm 2 Preferably >0.45W / cm 2 Up to 0.6W / cm 2 The power is used to operate the discharge device for generating the second non-thermal plasma. In this case, two separate discharge devices can be set, one of which is set to a first power level by the control device, and the other is set to a second power level by the control device.

[0055] Surprisingly, it was found that the discharge surface of the discharge device was 1.0 cm. 2 Up to 6.0cm 2 Preferably 2.5cm 2 Up to 3.5cm 2 In the case of operation under both power modes, preferably at low ozone concentrations, RNS (reactive nitrogen species) are generated by the plasma reactor in the plasma generation unit. RNS (especially nitrogen oxides such as N₂O₅ and NO) react with nucleases. This results in a reduction in the amount of nucleases on the entire treated surface. Therefore, it is preferably specified that the plasma reactor is designed such that the first nonthermal plasma and the second nonthermal plasma can be generated simultaneously using the plasma reactor. This is accomplished via the discharge surface of the discharge device. In this case, the control device can control the plasma reactor in this manner.

[0056] The plasma reactor is suitable for atmospheric dielectric barrier discharge and includes, for example, a glass tube and an inner electrode formed of bundled stainless steel fibers, wherein the bundled stainless steel fibers are helically arranged within the glass tube, providing an outer electrode designed as a sieve cylinder. Preferably, the inner electrode is made of bundled stainless steel fibers with a fiber diameter of about 1.0 μm to 1.2 μm, preferably about 1.0 μm, wherein the fiber length is about 3 cm to 6 cm. When installed, the fibers largely overlap. The sieve cylinder is preferably made of wire mesh, wherein the wire diameter is preferably between about 0.18 mm and 0.36 mm, and the spacing between the longitudinal wires is preferably between about 0.4 mm and 1.25 mm.

[0057] The glass tube and the sieve cylinder, which serves as the external electrode, together form a dielectric material. When the glass tube has a material thickness of 0.18 mm to 0.36 mm, the spacing between the longitudinal metal wires is 0.4 mm to 1.25 mm, and the internal electrode is made of bundled stainless steel wire with a thickness of 1.0 μm to 1.2 μm, a particularly uniform dielectric discharge occurs.

[0058] During operation of the device, oxygen in the air is converted into hydroxyl radicals through a discharge tube. This effect is optimally achieved by high-energy voltage pulses of alternating current, preferably at a frequency of 25 kHz to 44 kHz.

[0059] For example, nitrogen-dominant nonthermal plasmas at discharge surfaces with a velocity greater than 0.1 W / cm² 2 Especially greater than 0.2 W / cm 2 or greater than 0.5 W / cm 2 or greater than 1.5 W / cm 2 or greater than 2W / cm 2 The power generated is preferably within the range of 0.5 W / cm². 2 and 2.2W / cm 2 Between. In particular, the surface temperature of the electrodes used to generate nitrogen-based non-thermal plasma can be greater than 90°C.

[0060] The plasma reactors for generating a first nonthermal plasma at atmospheric pressure and the plasma reactors for generating a second nonthermal plasma at atmospheric pressure can be designed as a single unit. The generation of the first and second nonthermal plasmas can occur at electrodes with different power densities, at different electrodes, or continuously. Two or more different plasma generators can also be used.

[0061] Furthermore, the plasma gas pipeline can be configured to connect the plasma reactor to at least one decontamination container, and / or to at least one hollow body having an inner surface, particularly at least one polymer membrane bag, and / or to at least one plasma gas nozzle. The plasma gas nozzle can conduct the plasma flow into the hollow body having an inner surface, or directly to the surface to be cleaned (e.g., to an unfolded polymer membrane).

[0062] In particular, the plasma gas nozzle may be directed at at least one polymer film, wherein the polymer film is an extruded film, an expanded film, and / or a film to be welded, and wherein the plasma gas nozzle is optionally designed to be movable.

[0063] On the other hand, the device forms a closed loop for the plasma flow. Therefore, the generated plasma flow is guided in a circular pattern. After generation, the plasma flow is directed to the surface to be cleaned and then, after optional cleaning (e.g., filtration), returns to the plasma generator. This allows the plasma flow to be generated in an energy-efficient manner. Furthermore, additional process gas (e.g., air, particularly (filtered) ambient air) can be supplied to the plasma flow guided in the loop. Preferably, the process gas is supplied upstream of the plasma generator.

[0064] The device may also include a polymer welding component (e.g., a thermal welding component, an ultrasonic welding component, a friction welding component, a laser welding component, a high-frequency welding component, a pulse welding component, etc.) configured to weld a polymer membrane to form a polymer membrane bag. Deactivation can be performed before or after welding.

[0065] The apparatus may also include an unfolding device for unfolding the polymer film. Deactivation can be performed immediately after unfolding. Alternatively and / or alternatively, deactivation can also be performed immediately after the film has been produced, for example, after extrusion and / or stretching, i.e., before the film is wound or further processed.

[0066] Furthermore, the device may include a decontamination container connected to at least one plasma gas line. Therefore, the decontamination container and its contents may be free of active RNA enzymes / DNA enzymes.

[0067] Furthermore, this objective is achieved through polymer membrane bags used in bioreactors, wherein the polymer membrane bags have been produced from RNase-free polymer membranes, and / or wherein the polymer membrane bags have had active RNase / DNase removed after their production. Therefore, the inner surface of the polymer membrane bag is free of RNase and / or DNase. Attached Figure Description

[0068] The accompanying drawings illustrate specific embodiments of the invention. These embodiments are helpful in understanding the invention. In particular, Figure 1 This is a schematic diagram of a method for producing surfaces free of RNase and / or DNase; Figure 2 This is a schematic diagram of a first device for producing an RNAse-free and / or DNAse-free surface; Figure 3 This is a schematic diagram of a second device for producing an RNAse-free and / or DNAse-free surface; Figure 4 This is a schematic diagram of a third device for producing surfaces free of RNase and / or DNase; Figure 5This is a schematic diagram of a fourth device for producing an RNAse-free and / or DNAse-free surface, and Figure 6 This is a schematic diagram of a fifth device for producing surfaces free of RNase and / or DNase; Figure 7 This is a schematic diagram of a sixth device for producing surfaces free of RNase and / or DNase; Figure 8 The illustration schematically shows the testing of five plastic surfaces (B1 to B5) contaminated with RNase before and after the method according to the invention. Detailed Implementation

[0069] Figure 1 This is a schematic diagram of a method 1000 for producing an RNase-free and / or DNase-free surface. Method 1000 includes the following steps: • Generates 1100 ozone-dominant first nonthermal plasma at atmospheric pressure; • Generates 1200 nitrogen-dominant second nonthermal plasmas 20 at atmospheric pressure; • A 1300 plasma flow is generated from ozone-dominant nonthermal plasma and nitrogen-dominant nonthermal plasma; • Conduct the plasma flow 1400° to the surface to be cleaned; and • Inactivate RNase and / or DNase on the surface to be cleaned for 1500 minutes.

[0070] Optionally, 1600 inactivated RNases and / or DNases can also be removed from the surface to be cleaned.

[0071] Method 1000 was applied to a 1-liter polymer membrane bag. First, the polymer membrane bag was filled with 50 mL of RNase-free water (ultrapure distilled water, DNase-free / RNase-free, Thermo Fisher Scientific) and extracted for at least 1 hour. Subsequently, 45 μL of sample was treated with an RNase assay kit and examined using a fluorometer (Mithras LB 940, Berthold Technologies). The RNase assay kit used (RNaseAlert) ™ The laboratory test kit (catalog number: AM1964, from Thermo Fisher Scientific) has a detection limit of 3.5 × 10⁻⁶. -7 One unit (approximately 0.5 pg) of RNase A. A threshold for standardizing the terms RNase-free / DNase-free has not yet been established.

[0072] Longer extraction times were found to lead to an increase in measured RNase levels. Nearly twice the RNase levels were observed at 12 hours of extraction compared to one hour, and nearly 7.5 times the RNase levels were observed at 24 hours. Treating the inner surface of the polymer membrane bag for 20 minutes according to Method 1000 reduced the relative RNase levels from an initial 11.5 to below 1.5—specifically, to 1.17—compared to a pure, RNase-free water sample. Therefore, the treated surface was again free of RNase.

[0073] The effect of the method according to the present invention is also Figure 8 As illustrated in the figure, this test is based on five plastic surfaces. RNaseAlert from Thermo Fisher Scientific was used. ™ The laboratory test kit (catalog number: AM1964) was used to measure the presence of RNase. The ordinate represents relative fluorescence units. Values ​​of 51 were obtained in the control (=RNase-free) before and after treatment. Five samples were coated with different amounts of RNase. After treatment, all samples were below the threshold of 100, which was considered RNase-free. After treatment, most samples (B1, B2, B4, and B5) were at control levels.

[0074] Figure 2 This is a schematic diagram of a first apparatus 100 for generating RNase-free and / or DNase-free surfaces 102, 104, 106. Apparatus 100 includes at least one plasma reactor 112 for generating ozone-dominant nonthermal plasma 12 at atmospheric pressure, and at least one plasma reactor 114 for generating nitrogen-dominant nonthermal plasma 14 at atmospheric pressure.

[0075] To generate the first nonthermal plasma 12 and the second nonthermal plasma 14, two different plasma reactors 112 and 114 can be used, or a common plasma reactor 110 can be used. Therefore, the plasma reactors for generating ozone-dominant nonthermal plasma 12 and nitrogen-dominant nonthermal plasma 14 can be integrally formed in a single housing.

[0076] The plasma flow 26 generated by ozone-based nonthermal plasma 12 and nitrogen-based nonthermal plasma 14 can be conducted via plasma gas line 126, which is connected to plasma reactors 110, 112, 114 and to surfaces 102, 104, 106 to be cleaned, so as to inactivate RNase and / or DNase on surfaces 102, 104, 106 to be cleaned.

[0077] exist Figure 2 In the illustrated embodiment, device 100 includes a decontamination container 50 connected to a plasma gas line 126. The generated plasma flow 26 is thus introduced into the decontamination container 50 and conducted to the surfaces 102, 104, 106 to be cleaned. Therefore, not only can the inner surface 102 of the decontamination container 50 be free of active RNase / DNase, but the surfaces 104, 106 of objects disposed inside the decontamination container 50 can also be free of active RNase / DNase. These objects may include, for example, pipettes, pipette tips, cannulas, vials, etc.

[0078] Plasma stream 26 can be guided in the loop via plasma gas line 127. After passing through decontamination container 50, plasma stream 26 is cleaned, for example, in filter 210, and then transferred back to plasma reactors 110; 112, 114. Additionally, other process gases (e.g., air, particularly (filtered) ambient air) can be introduced into plasma reactors 110; 112, 114 via gas inlet 212. The process gases can be cleaned, particularly filtered, before entering plasma reactors 110; 112, 114.

[0079] Figure 3 This is a schematic diagram of a second device 100' for generating an RNase-free and / or DNase-free surface. The generation of the plasma flow is essentially as described in the reference. Figure 1 The process is performed as described above. However, as shown, the plasma gas line 126 is not connected to the decontamination container; instead, the plasma flow 26 is introduced directly into the polymer membrane bag 42 to remove active RNase / DNase from its inner surface 102. The polymer membrane bag 42 can be connected to other polymer membrane bags 44, 46 via additional plasma gas lines (particularly hoses), such that the inner surfaces 104, 106 of these bags, as well as the components connected to them (e.g., hoses, valves, connectors, etc.), are also free of active RNase / DNase. The polymer membrane bags 42, 44, 46 can be connected in series as shown, or filled with the plasma flow 26, or the plasma flow can flow through them in parallel via a suitable distributor. Here, the plasma flow 26 can also be guided in a loop.

[0080] Similarly, the device 100' can be used to clean the inner surface of a so-called disposable component. For this purpose, a plasma flow 26 is introduced into the disposable component and cleans the inner surface. A disposable component typically includes one or more polymer membrane bags 42, 44, 46 and at least one hose connecting these polymer membrane bags. Furthermore, valves and / or the like may be part of the disposable component. By introducing the plasma flow 26, the inner surface of a substantially fully assembled disposable component can be cleaned, or individual parts (or sub-components) of the disposable component can be cleaned.

[0081] The devices 100 and 100' can also be configured to perform a leak test on at least one polymer membrane bag 42, 44, 46 or decontamination container 50, wherein the plasma gas of the generated plasma stream 26 is used as the test gas. For example, if the pressure inside the polymer membrane bag 42, 44, 46 or decontamination container 50 remains constant during a predetermined test period, it can be determined that the polymer membrane bag / decontamination container is leak-proof.

[0082] Figure 4 This is a schematic diagram of a third device 100'' for generating an RNase-free and / or DNase-free surface. The generation of the plasma flow is essentially as described in the reference. Figure 1 The process is carried out as described above. However, as shown, the plasma gas line 126 is not connected to the decontamination container, but rather to the plasma gas nozzle 226. The plasma gas nozzle 226 is directed towards the polymer membrane 60, which is present in a folded state. The folded membrane can be welded into a polymer membrane bag using the polymer welding member 240 of the device 100''. The plasma gas nozzle 226 conducts the plasma flow 26 to the subsequent inner surface of the polymer membrane bag before and / or during welding. Thus, RNase-free / DNase-free polymer membrane bags can be produced, which can be used, for example, as reaction chambers in bioreactors or for storing / transporting RNA / DNA active agents or vaccines and / or their precursors and / or intermediates.

[0083] Figure 5 This is a schematic diagram of a fourth device 100''' for generating an RNase-free and / or DNase-free surface. The generation of the plasma flow is essentially as described in the reference. Figure 1The process is performed as described above. However, as shown, the plasma gas line 126 is not connected to the decontamination container, but rather to two plasma gas nozzles 222, 224. The first plasma gas nozzle 222 is directed towards the polymer film 60, which is deployed by the deployment device 260 of the apparatus 100'''. The second plasma gas nozzle 224 is directed towards the polymer film 62, which is deployed by the deployment device 262. It should be understood that the apparatus 100''' is not limited to two plasma gas nozzles 222, 224 and two deployment devices 260, 262. For example, multiple films (at least three, four, five, etc.) can also be used for polymer film bag production—for example, to produce three-dimensional polymer film bags. Depending on the number of films, a corresponding number of deployment devices can be provided. Each of these deployment devices can be assigned at least one plasma gas nozzle.

[0084] The first polymer film 60 and the second polymer film 62 can be welded together using the polymer welding member 240 of the apparatus 100''' to form a polymer film bag. The polymer welding member 240 can weld the polymer films 60 and 62 together by means of weld seam 47 at a suitable interval (depending on the required bag size). Alternatively, extruded membrane tubes can be used to produce the polymer film bag instead of two separate films 60 and 62.

[0085] Plasma gas nozzle 222 conducts plasma flow 26 to the surface of membrane 60 after deployment device 260, which will later become the inner surface of the polymer membrane bag. Correspondingly, plasma gas nozzle 224 conducts plasma flow 26 to the surface of membrane 62 after deployment device 262, which will later become the inner surface of the polymer membrane bag. Thus, RNase-free / DNase-free polymer membrane bags can be produced, which can be used, for example, as reaction chambers in bioreactors or for storing / transporting RNA / DNA active agents or vaccines and / or their precursors and / or intermediates.

[0086] Figure 6 This is a schematic diagram of a fifth device 100'''' for producing an RNase-free and / or DNase-free surface. The device 100'''' is essentially like... Figure 3The device 100' is constructed as described above. Device 100''' is connected downstream of the polymer bag production apparatus 250. The polymer film bags 42 produced by means of the polymer bag production apparatus 250 can then be connected to device 100''. This allows the inner surface of the polymer film bags 42 to be free of active RNase / DNase. Device 100'''' can be integrated into a system for producing polymer film bags, preferably inline, wherein the produced polymer film bags are preferably automatically connected to device 100''''. Device 100'''' can also be configured to perform leak tests on the produced polymer film bags 42, wherein the plasma gas of the generated plasma stream 26 is used as the test gas.

[0087] The polymer bag production apparatus 250 may include at least one polymer welding component 240, such as Figure 4 and Figure 5 As shown schematically in the diagram.

[0088] Figure 7 This is a schematic diagram of a sixth device 100 for generating nuclease-free surfaces. The device 100 includes a gas inlet 212, a pressure control 214, a downstream flow control 216, a first plasma generator 112, and a downstream second plasma generator 114. A plasma gas line 126 (with a plasma flow 26) leads from the first plasma generator and the downstream second plasma generator to a decontamination container 50, in which the surface to be treated is arranged (not shown). Downstream of the decontamination container 50 are an on / off switch 228 and a flow sensor 229. This is followed by an ozone filter 230 to prevent ozone from escaping from the device 100.

[0089] Referring to the accompanying drawings, some embodiments are described in more detail than others. However, other embodiments are also included in this disclosure. This disclosure should not be construed as limited to the embodiments explicitly set forth herein; rather, these embodiments are given by way of example to convey to those skilled in the art the scope of the subject matter according to the invention. Of course, the invention may also be practiced in ways other than those set forth herein without prejudice to the essential characteristics of the invention. Embodiments of the invention are considered illustrative rather than restrictive in every respect, and therefore variations of the described embodiments may also fall within the scope of protection.

[0090] List of reference numerals 12 (Ozone-dominant) First nonthermal plasma 14. (Nitrogen-dominant) Second nonthermal plasma 26 Plasma Flow 42 Polymer film bags 44 Polymer film bags 46 Polymer film bags 47 Weld 50 Decontamination Containers 60 Polymer Film 62 Polymer film 100 devices 102 Surface to be cleaned 104 Surfaces to be cleaned 106 Surfaces to be cleaned 110 Plasma Generator 112 Plasma Generator 114 Plasma Generator 126 Plasma Gas Line 127 Plasma Gas Line 210 Filter 214 Pressure Control 216 Flow Control 212 Gas Inlet 222 Plasma Gas Nozzle 224 Plasma Gas Nozzle 226 Plasma Gas Nozzle 228 On / Off Switch 229 Flow Sensor 230 Ozone Filter 240 Polymer welded components 250 Polymer Bag Production Unit 260 Deployment Device 262 Deployment Device 1000 methods 1100 generates the first nonthermal plasma at atmospheric pressure. 1200 generates a second nonthermal plasma at atmospheric pressure. 1300 The plasma flow is generated by the first nonthermal plasma and the second nonthermal plasma. 1400 Conducts the plasma flow to the surface to be cleaned. 1500 inactivates RNase and / or DNase. 1600 Removes inactivated RNases and / or DNases 1700 The plasma flow is then redirected back to the surface to be cleaned. This invention also relates to the following points: Point 1. A method (1000) for producing an RNase-free and / or DNase-free surface, particularly an RNase-free and / or DNase-free polymer surface, wherein the method comprises: (10) Non-thermal plasma (1100) mainly composed of ozone is generated under atmospheric pressure. A nitrogen-dominant nonthermal plasma (20) is generated at atmospheric pressure (1200); A plasma flow (26) is generated (1300) by the ozone-dominant nonthermal plasma and the nitrogen-dominant nonthermal plasma. The plasma flow (26) is conducted (1400) to the surface to be cleaned (102, 104), and RNase and / or DNase are inactivated on the surface to be cleaned (102, 104, 106) (1500).

[0091] Point 2. The method (1000) according to claim 1, the method further comprising removing (1600) the inactivated RNase and / or DNase from the surfaces (102, 104, 106) to be cleaned, and optionally, after removing (1600) the inactivated RNase and / or DNase from the surfaces (102, 104, 106) to be cleaned, conducting (1700) the plasma flow (26) to the cleaned surfaces (102, 104).

[0092] Point 3. According to the method (1000) of point 1 or 2, the ozone-dominant nonthermal plasma (10) is generated by means of dielectric barrier discharge or corona discharge, and the ozone-dominant nonthermal plasma optionally has a discharge rate of less than 0.5 W / cm² on the discharge surface. 2 Especially less than 0.4 W / cm 2 or less than 0.3 W / cm 2 or less than 0.2 W / cm 2 The power generated, and / or the nitrogen-dominant nonthermal plasma (20) is generated by means of dielectric barrier discharge or corona discharge, and wherein the nitrogen-dominant nonthermal plasma (20) optionally generates a power greater than 0.1 W / cm² on the discharge surface. 2 Especially greater than 0.2 W / cm 2 or greater than 0.5 W / cm 2 or greater than 1.5 W / cm 2 or greater than 2W / cm 2 The power generation, and / or the generation (1100, 1200) of the ozone-dominant nonthermal plasma and the nitrogen-dominant nonthermal plasma, are carried out simultaneously, preferably simultaneously in a common plasma generator (110).

[0093] Point 4. The method (1000) according to one of points 1 to 3, wherein the plasma flow (26) is mixed with the aerosol and then conducted to the surface to be cleaned (102, 104).

[0094] Point 5. The method (1000) according to any one of points 1 to 4, wherein the surface to be cleaned comprises an inner surface of at least one volume, and wherein the at least one volume is particularly formed by a polymer membrane bag (42, 44, 46) or by a decontamination container (50), wherein the plasma flow (26) is conducted into the volume at at least one inlet.

[0095] Point 6. The method (1000) according to any one of points 1 to 5, wherein the method further includes a leak test of the at least one volume, wherein the plasma gas of the generated plasma flow (26) is used as the test gas.

[0096] Point 7. The method (1000) according to any one of points 1 to 6, wherein the method further comprises unfolding the polymer film (60, 62) and / or extruding the polymer film (60, 62), and wherein the plasma flow (26) is conducted to at least one side of the extruded and / or unfolded polymer film (60, 62).

[0097] Point 8. According to the method (1000) of any one of points 1 to 7, wherein the plasma flow (26) is conducted to the surface (102, 104, 106) to be cleaned for a period of time of at least 10 minutes, or at least 15 minutes, or at least 20 minutes.

[0098] Point 9. The method (1000) according to any one of points 1 to 8, wherein the method further comprises generating at least one volume, in particular at least one polymer membrane bag (42, 44, 46), wherein the volume is generated by at least one polymer membrane (60, 62), and generating the at least one volume comprises polymer bonding and / or polymer welding, wherein the RNase-free and / or DNase-free inner surface of the volume is generated before and / or after the polymer bonding and / or polymer welding.

[0099] Point 10. An apparatus (100) for generating RNase-free and / or DNase-free surfaces—particularly RNase-free and / or DNase-free polymer surfaces, wherein the apparatus (100) is configured to perform the method (1000) according to any one of points 1 to 9, and comprises: at least one plasma reactor (110, 112) for generating ozone-dominant nonthermal plasma (12) at atmospheric pressure; at least one plasma reactor (110, 114) for generating nitrogen-dominant nonthermal plasma (14) at atmospheric pressure; at least one plasma gas line (126) connected to the plasma reactor (100, 112, 114) and configured to conduct plasma flows (26) generated by the ozone-dominant nonthermal plasma and the nitrogen-dominant nonthermal plasma to the surfaces to be cleaned (102, 104, 106) to inactivate RNases and / or DNases on the surfaces to be cleaned (102, 104, 106).

[0100] Point 11. According to the apparatus (100) described in point 10, the plasma reactor (110, 112) for generating ozone-dominant nonthermal plasma (12) at atmospheric pressure and the plasma reactor (110, 114) for generating nitrogen-dominant nonthermal plasma at atmospheric pressure are integrally formed.

[0101] Point 12. In the apparatus (100) according to point 10 or 11, the plasma gas line (126) is configured to connect the plasma reactor (100, 112, 114) to at least one decontamination container (50), and / or to at least one volume, particularly at least one polymer membrane bag (42, 44, 46), and / or to at least one plasma gas nozzle (222, 224, 226), wherein the plasma gas nozzle (222, 224, 226) optionally points to at least one polymer membrane (60, 62), wherein the polymer membrane (60, 62) is an extruded membrane, a wound membrane and / or a membrane to be welded, and wherein the plasma gas nozzle (222, 224, 226) is further optionally configured to be movable.

[0102] Point 13. The device (100) according to any one of points 10 to 12, wherein the device (100) forms a closed loop for the plasma flow (26).

[0103] Point 14. The apparatus (100) according to any one of points 10 to 13, wherein the apparatus (100) includes a polymer welding member (240) configured to weld polymer films (60, 62) to form a polymer film bag (42), and / or wherein the apparatus (100) includes a deployment device (260, 262) for deploying the polymer films (60, 62), and / or wherein the apparatus (100) includes a decontamination container (50) connected to the at least one plasma gas line, and / or wherein the apparatus (100) further includes an aerosol generator configured to generate aerosol, and wherein the apparatus (100) is configured to mix the generated aerosol with the plasma stream (26) and conduct it to the surface to be cleaned (102, 104, 106).

[0104] Point 15. A polymer membrane bag (42, 44, 46) for a bioreactor, wherein the polymer membrane bag (42, 44, 46) is produced according to the method (1000) described in point 9, and its inner surface is free of RNase and / or DNase.

Claims

1. A method (1000) for producing a nuclease-free surface, characterized by the following steps: A first nonthermal plasma (10) is generated at atmospheric pressure (1100), wherein the first nonthermal plasma (10) is generated at a first power on a discharge surface. A second nonthermal plasma (20) is generated at atmospheric pressure (1200), wherein the second nonthermal plasma (20) is generated at a second power on the discharge surface. The second power is higher than the first power. A plasma flow (26) is generated by the first nonthermal plasma and the second nonthermal plasma (1300); The plasma flow (26) is conducted (1400) to the surface (102, 104) to be cleaned, and Nucleases are inactivated (1500) on the surfaces to be cleaned (102, 104, 106).

2. The method (1000) according to claim 1, the method further comprising removing (1600) inactivated nucleases from the surfaces (102, 104, 106) to be cleaned, and optionally, after removing (1600) the inactivated nucleases from the surfaces (102, 104, 106) to be cleaned, conducting (1700) the plasma flow (26) to the cleaned surfaces (102, 104).

3. The method (1000) according to claim 1 or 2, characterized in that, The non-thermal plasma (10), which is mainly composed of ozone, is generated by dielectric barrier discharge or corona discharge.

4. The method (1000) according to any one of claims 1 to 3, characterized in that, The first non-thermal plasma at the discharge surface is 0.2 W / cm 2 Up to <0.45W / cm 2 Preferably 0.3W / cm 2 Up to <0.45W / cm 2 The power generated, wherein the second non-thermal plasma (20) is generated on the discharge surface at a rate >0.45 W / cm 2 Up to 0.7W / cm 2 Preferably >0.45W / cm 2 Up to 0.6W / cm 2 The power generated.

5. The method according to any one of claims 1 to 4, characterized in that, The generation (1100, 1200) of the first non-thermal plasma and the second non-thermal plasma occurs simultaneously.

6. The method (1000) according to any one of claims 1 to 5, characterized in that, Aerosols are also generated, wherein the plasma flow (26) mixes with the aerosols and is then conducted to the surfaces to be cleaned (102, 104).

7. The method (1000) according to any one of claims 1 to 6, characterized in that, The surface to be cleaned is located inside the hollow body.

8. The method (1000) according to claim 7, characterized in that, The hollow body is a polymer film bag (42, 44, 46) or a decontamination container (50).

9. The method (1000) according to claim 7 or claim 8, wherein the method further comprises a leak test of the at least one hollow body, wherein the plasma gas of the generated plasma flow (26) is used as the test gas.

10. The method (1000) according to any one of claims 1 to 9, wherein the method further comprises unfolding a polymer film (60, 62) or extruding a polymer film (60, 62), and wherein the plasma flow (26) is conducted to at least one side of the extruded polymer film or the unfolded polymer film (60, 62).

11. The method (1000) according to any one of claims 1 to 10, wherein the plasma flow (26) is conducted to the surface to be cleaned (102, 104, 106) for a period of time of at least 10 minutes, at least 15 minutes, or at least 20 minutes.

12. The method (1000) according to any one of claims 7 to 11, wherein the method comprises: The hollow body is produced by at least one polymer film (60, 62), and the production of the at least one hollow body includes polymer bonding or polymer welding, wherein... Nuclease-free inner surfaces of volume are generated before and after polymer bonding or polymer welding.

13. The method (1000) according to any one of claims 1 to 7, characterized in that, The surface is part of a pipette tip, a disposable plastic product for medical or pharmaceutical use, a disposable biological process container, laboratory equipment, a centrifuge, or a PCR unit.

14. An apparatus (100) for generating a nuclease-free surface, wherein the apparatus (100) is configured to perform the method (1000) according to any one of claims 1 to 13, wherein the apparatus comprises: At least one plasma reactor (110, 112, 114), said at least one plasma reactor having at least one discharge surface on the discharge device. The plasma reactor (110, 112, 114) can be used to generate a first non-thermal plasma (12) at atmospheric pressure and a first power on the discharge surface. The feature is that the plasma reactor (110, 112, 114) can generate a second non-thermal plasma (14) at atmospheric pressure with a second power on the discharge surface, wherein the second power is higher than the first power; At least one plasma gas line (126) is connected to the at least one plasma reactor (100, 112, 114) and is configured to conduct a plasma flow (26) generated by the first nonthermal plasma and the second nonthermal plasma to the surface to be cleaned (102, 104, 106) to inactivate nucleases on the surface to be cleaned (102, 104, 106).

15. The apparatus (100) according to claim 14, characterized in that, The plasma reactor (110, 112, 114) having the discharge surface on the discharge device includes a dielectric discharge device or a corona discharge device.

16. The apparatus (100) according to claim 14 or claim 15, wherein the plasma reactor (110, 112, 114) is configured such that the first nonthermal plasma and the second nonthermal plasma can be generated simultaneously using a single plasma reactor.

17. The apparatus (100) according to any one of claims 14 to 16, characterized in that, Two plasma reactors (110, 112, 114) are provided, wherein the first plasma reactor (110, 112) can be used to generate the first nonthermal plasma and the second plasma reactor (110, 114) can be used to generate the second nonthermal plasma.

18. The apparatus (100) according to claim 14 or 17, wherein the plasma gas line (126) has plasma gas nozzles (222, 224, 226) that can be connected to at least one decontamination container (50) or to at least one hollow body, particularly at least one polymer film bag (42, 44, 46).

19. The apparatus (100) according to any one of claims 14 to 18, the apparatus further comprising an unfolding device (260, 262) for unfolding the polymer film (60, 62) and a polymer welding device (240).

20. The apparatus according to any one of claims 14 to 19, wherein The device (100) includes a decontamination container (50) connected to the at least one plasma gas line.

21. The apparatus according to any one of claims 14 to 20, wherein The device (100) further includes an aerosol generator configured to generate aerosols, and wherein the device (100) is configured to mix the generated aerosols with the plasma flow (26) and conduct them to the surfaces to be cleaned (102, 104, 106).

22. A polymer membrane bag (42, 44, 46) for a bioreactor, wherein the polymer membrane bag (42, 44, 46) is produced by the method (1000) according to claim 12, and the inner surface of the polymer membrane bag is free of nucleases.

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