Bioreactors configured to be UV sterilized and methods of using UV sterilization in bioprocesses - Patents.com

Abstract

In some variations, the UV sterilizable bioreactor system comprises a chamber configured to carry out a reaction, a component configured to introduce a gas into the chamber, and one or more UV light sources configured to irradiate surfaces within the chamber with ultraviolet light. Some variations provide a method for cleaning and sterilizing a bioreactor, comprising providing a bioreactor system comprising a chamber configured to carry out a reaction, a component for introducing a gas into the chamber, and one or more UV light sources for irradiating surfaces of the chamber with ultraviolet light, cleaning the chamber, and exposing the chamber to UV radiation to sterilize the chamber. The disclosed technology is a fundamental advance in the field of spray bioreactors for many types of commercial fermentation. The bioreactor can incorporate materials that are not explicitly compatible with steam sterilization, allowing the use of lower cost materials, among many other benefits from UV sterilization.

Description

[Technical field]

[0001] Priority Data This international patent application claims priority to U.S. Provisional Patent Application No. 63 / 327,258, entitled "UV BIOREACTOR," filed April 4, 2022, and U.S. Patent Application No. 18 / 121,153, filed March 14, 2023, each of which is incorporated herein by reference.

[0002] Field The present invention relates generally to sterilization of bioreactors, bioreactor designs that allow for efficient sterilization, and methods for sterilizing bioreactors and other bioprocess components. [Background technology]

[0003] background Fermentation uses biological systems to convert feedstocks into more valuable products. Advanced fermentation processes can be carried out in bioreactors, which are physical systems that create an optimal environment for biological systems by combining solid, liquid or gaseous feedstocks in a controlled environment. In modern implementations of bioreactors, this often involves the combination of at least one liquid feedstock with at least one gaseous feedstock.

[0004] For biological production to be effective, it is important that the presence of undesirable biological agents (known as adventitious organisms) is limited to the greatest extent possible. This is important to ensure that the highest possible percentage of the feedstock is converted to the desired product or products, rather than alternative undesirable products. In modern bioreactor systems, the presence of adventitious organisms is reduced by thoroughly cleaning the reactor system, crucially using steam. This process of reducing adventitious populations is often considered sterilization, although in some cases traces of adventitious organisms may remain in the system. Summary of the Invention [Problem to be solved by the invention]

[0005] It is well understood that radiation inactivates microorganisms. In the prior art, adventitious agents have been removed from bioreactor systems by using high energy gamma radiation in a controlled environment. The ionizing nature of gamma radiation allows for uniform distribution of sterilizing radiation throughout the bioreactor volume. However, the ability of gamma radiation to penetrate structural features substantially complicates the sterilization process, as this radiation poses a substantial threat to human health. For that reason, sterilization of bioreactors by gamma irradiation is performed in centralized manufacturing facilities, and these containers are traditionally discarded after a single use. [Means for solving the problem]

[0006] overview In some variations, the present invention provides a method for producing a composition comprising: (a) a chamber configured to carry out a reaction; (b) a component configured to introduce a gas into the chamber; and (c) one or more UV light sources configured to irradiate surfaces within the chamber with UV light; The present invention provides a UV sterilizable bioreactor system comprising:

[0007] In a preferred embodiment, the chamber is configured to maintain a sterile boundary with the environment.

[0008] In some embodiments, the component for introducing the gas is a gas sparger. In some embodiments, the component for introducing the gas is a membrane. The gas can be air, oxygen, syngas, hydrogen, carbon monoxide, methane, natural gas, or combinations thereof. In typical embodiments, the gas is oxygen, air, oxygen-enriched air, or oxygen-depleted air.

[0009] In a preferred embodiment, the component for introducing gas is UV sterilizable. In some embodiments, the component for introducing gas comprises a UV transparent material. The UV transparent material can comprise a UV transparent polymer, a UV transparent ceramic, a UV transparent glass, or a combination thereof. The UV transparent polymer can be selected from the group consisting of polyacrylate, silicone, fluoropolymer, and combinations thereof. The fluoropolymer can be selected from the group consisting of polyvinylidene fluoride, polytetrafluoroethylene, perfluoroalkoxyalkane, and combinations thereof. The UV transparent ceramic can be selected from the group consisting of quartz, fused silica, borosilicate, and combinations thereof.

[0010] In some embodiments, the UV sterilizable bioreactor system further comprises one or more conduits (e.g., valves) configured to supply input materials into the chamber and / or withdraw output materials from the chamber. Preferably, at least one of the one or more conduits is UV sterilizable. In some embodiments, there are two, three, or more conduits, and each of the conduits is UV sterilizable.

[0011] In some embodiments, one or more UV light sources are located within the chamber. The one or more UV light sources may be permanently located within the chamber. Alternatively or additionally, the one or more UV light sources may be reversibly located within the chamber.

[0012] In some embodiments, the one or more UV light sources are located in a UV-transparent well disposed in the chamber. The UV-transparent well can be made of a UV-transparent material including a UV-transparent polymer, a UV-transparent ceramic, a UV-transparent glass, or a combination thereof. The UV-transparent polymer can be selected from the group consisting of polyacrylate, silicone, fluoropolymer, and a combination thereof. The fluoropolymer can be selected from the group consisting of polyvinylidene fluoride, polytetrafluoroethylene, perfluoroalkoxyalkane, and a combination thereof. The UV-transparent ceramic can be quartz. The UV-transparent glass can be fused silica, borosilicate, or a combination thereof.

[0013] In some embodiments, at least some (including at least one) of the one or more UV light sources are external to the chamber, hi some embodiments, all of the one or more UV light sources are external to the chamber.

[0014] In some embodiments, the chamber has walls made of metal, metal alloy, polymer, ceramic, composite, glass, concrete, or combinations thereof. In some embodiments, the metal is aluminum, copper, nickel, silver, or combinations thereof. In some embodiments, the metal alloy is carbon steel or stainless steel. In some embodiments, the polymer is selected from the group consisting of polyolefins, polyacrylates, polycarbonates, fluoropolymers, silicones, and combinations thereof. In some embodiments, the composite is a polymer reinforced with glass fibers. In some embodiments, the polymer is selected from high density polyethylene, polypropylene, polycarbonate, or combinations thereof. The polymer can be selected from the group consisting of poly(methyl methacrylate), polyvinylidene fluoride, hexafluoropropylene-tetrafluoroethylene copolymer, perfluoroether-tetrafluoroethylene copolymer, or combinations thereof. The chamber walls can also be coated with an antimicrobial surface, such as, for example, copper, silver, or a nanostructured coating.

[0015] In some embodiments, the chamber has a UV-transparent chamber wall comprising a UV-transparent material. The UV-transparent material can include a UV-transparent polymer, a UV-transparent ceramic (e.g., quartz), a UV-transparent glass (e.g., fused silica and / or borosilicate), or a combination thereof. The UV-transparent polymer can be selected from the group consisting of a polyacrylate (e.g., poly(methyl methacrylate)), a silicone, a fluoropolymer, and a combination thereof. The fluoropolymer can be selected from the group consisting of polyvinylidene fluoride, hexafluoropropylene-tetrafluoroethylene copolymer, perfluoroether-tetrafluoroethylene copolymer, poly(ethylene-co-tetrafluoroethylene), and a combination thereof.

[0016] In some embodiments, the chamber has UV-reflective chamber walls that include or are internally coated with a UV-reflective material, which may be selected from, for example, aluminum, stainless steel, polytetrafluoroethylene, or combinations thereof.

[0017] In some embodiments, the chamber is configured with a chamber top that is not sealed from the environment, but the chamber top is UV sterilizable to form a sterile barrier with the environment.

[0018] The one or more UV light sources can be configured to irradiate at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the total surface area within the chamber, including its internal components, with ultraviolet light.

[0019] The one or more UV light sources may be configured to irradiate at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the total volume of the chamber with UV light.

[0020] In some embodiments, the one or more UV light sources each have a UV wavelength selected from about 100 nm to about 400 nm, hi some embodiments, the UV wavelength is selected from about 220 nm to about 300 nm for at least one of the one or more UV light sources, such as for all of the UV light.

[0021] The one or more UV light sources may each be selected from the group consisting of, for example, a UV light emitting diode, a UV mercury lamp, a UV xenon lamp, and a UV krypton lamp.

[0022] In some embodiments, the bioreactor system includes an impeller located within the chamber, and the impeller is preferably UV sterilizable, hi other embodiments, the chamber does not include an impeller.

[0023] In some embodiments, the UV sterilizable bioreactor system further comprises a filtration unit configured to filter the input material before it is provided into the chamber, hi some embodiments, the filtration unit is UV sterilizable.

[0024] In some embodiments, the UV sterilizable bioreactor system includes a bioreactor sensor located within the chamber. The bioreactor sensor can be configured to detect or measure a bioreactor parameter selected from the group consisting of, for example, pH, temperature, oxygen, carbon dioxide, foaming, mixing, cell density, feed substrate concentration, reaction intermediate concentration, and product concentration. There can be multiple bioreactor sensors located within the chamber. In some embodiments, the bioreactor sensor is configured to transmit a wireless signal.

[0025] In some embodiments, the bioreactor sensor is UV sterilizable. The bioreactor sensor can be configured with a UV light guide for sterilizing the bioreactor sensor. The UV light guide can be, for example, a UV optical fiber.

[0026] In some embodiments, the bioreactor sensor is disposed in or through a probe port, which is UV sterilizable. In some embodiments, the bioreactor sensor is contained within a UV sterilizable housing located within the chamber. The UV sterilizable housing can include a UV transparent material, such as a UV transparent polymer, a UV transparent ceramic, a UV transparent glass, or a combination thereof. The UV transparent polymer can be selected from the group consisting of polyacrylates, silicones, fluoropolymers, and combinations thereof. An exemplary polyacrylate is poly(methyl methacrylate). The fluoropolymer can be selected from the group consisting of polyvinylidene fluoride, hexafluoropropylene-tetrafluoroethylene copolymer, perfluoroether-tetrafluoroethylene copolymer, poly(ethylene-co-tetrafluoroethylene), and combinations thereof. An exemplary UV transparent ceramic is quartz. An exemplary UV transparent glass includes fused silica, borosilicate, or a combination thereof.

[0027] In some embodiments, the bioreactor system includes a wash-in-place arm located within the chamber. The wash-in-place arm is preferably UV sterilizable.

[0028] Some variations are generally: (a) a chamber configured to carry out a reaction; (b) a component configured to introduce a gas into the chamber; and (c) one or more light sources configured to irradiate a surface within the chamber with radiation, the radiation having a wavelength greater than 1 nm and less than 1 mm; The present invention provides a radiation sterilizable bioreactor system comprising:

[0029] In a preferred embodiment, the one or more light sources are configured to irradiate surfaces within the chamber with UV radiation, visible light, IR radiation, or a combination thereof.

[0030] Some variations provide a method for cleaning and sterilizing a bioreactor, the method comprising: (i) providing a UV sterilizable bioreactor system comprising: (a) a chamber configured to carry out a reaction; (b) a component configured to introduce a gas into the chamber; and (c) one or more UV light sources configured to irradiate surfaces within the chamber with ultraviolet light; (ii) cleaning the chamber; and (iii) exposing the chamber to UV radiation to sterilize the chamber; Includes.

[0031] In some methods, the UV sterilizable bioreactor system further comprises one or more conduits (e.g., valves) configured to provide input materials into the chamber and / or withdraw output materials from the chamber, hi some methods, at least one of the conduits is UV sterilizable.

[0032] In some methods, one or more UV light sources, such as all of the UV light sources, are located within the chamber. In some methods, one or more UV light sources, such as all of the UV light sources, are permanently located within the chamber. In some methods, one or more UV light sources, such as all of the UV light sources, are reversibly located within the chamber.

[0033] In some methods, one or more of the UV light sources, such as all of the UV light sources, are located in a UV-transparent well that is disposed within the chamber.

[0034] In some methods, at least some of the one or more UV light sources are external to the chamber. In some methods, all of the one or more UV light sources are external to the chamber.

[0035] In some methods, the chamber has walls made from a metal, a metal alloy, a polymer, a ceramic, a composite material, glass, concrete, or a combination thereof.

[0036] In some methods, the chamber has UV-transparent chamber walls that include a UV-transparent material.

[0037] In some methods, the chamber has UV-reflective chamber walls that include or are internally coated with a UV-reflective material.

[0038] In some methods, the chamber is configured with a chamber top that is not sealed from the environment, and the chamber top is UV sterilizable to form a sterile barrier with the environment.

[0039] In some methods, an impeller is located within the chamber, and the impeller is UV sterilizable.

[0040] In some methods, the UV sterilizable bioreactor system further comprises a filtration unit configured to filter the input material before it is provided into the chamber. In some methods, the filtration unit is UV sterilizable.

[0041] In some methods, a bioreactor sensor is located in the chamber. The bioreactor sensor can be configured to detect or measure a bioreactor parameter selected from the group consisting of pH, temperature, oxygen, carbon dioxide, foaming, mixing, cell density, feed substrate concentration, reaction intermediate concentration, and product concentration. During operation, the bioreactor sensor can actually detect or measure the bioreactor parameter. The bioreactor sensor can be configured to transmit a wireless signal during or after the sensor measurement.

[0042] In some methods, the bioreactor sensor is UV sterilizable. The bioreactor sensor can be configured with a UV light guide for sterilizing the bioreactor sensor. Alternatively or additionally, the bioreactor sensor is contained within a UV sterilizable housing located within the chamber. The UV sterilizable housing can include a UV transparent material. Alternatively or additionally, the bioreactor sensor may be disposed within or through a probe port, and the probe port is UV sterilizable.

[0043] In some methods, the bioreactor includes a wash-in-place arm located within the chamber, and the wash-in-place arm is UV sterilizable.

[0044] In some methods, step (ii) utilizes a cleaning agent selected from the group consisting of hot water, alkaline detergent, sodium hydroxide, sodium percarbonate, acidic detergent, phosphoric acid, peracetic acid, isopropanol, ethanol, sodium hypochlorite, and combinations thereof. Other cleaning agents may be utilized instead of or in addition to these chemicals.

[0045] In some methods, step (iii) is about 50 mJ / cm 2 The sterilization energy per unit total area within the chamber is utilized.

[0046] In some methods, step (iii) is about 0.5 mW / cm 2 ~About 10000mW / cm 2 Utilize the total UV power output of

[0047] In some methods, step (iii) is about 0.1 W / m 3 ~about 10W / m 3 Utilizes the total UV power capacity of

[0048] In some methods, step (iii) utilizes a UV sterilization time of from about 60 seconds to about 2 hours.

[0049] In some methods, step (iii) exposes at least 80% of the total surface area of ​​the chamber, including its internal components, to UV radiation. Step (iii) may expose at least 85%, at least 90%, at least 95%, or at least 99% (including 100%) of the total surface area of ​​the chamber, including its internal components, to UV radiation.

[0050] In some methods, step (iii) exposes at least 80% of the total volume of the chamber to UV radiation. Step (iii) may expose at least 85%, at least 90%, at least 95% or at least 99% (including 100%) of the total volume of the chamber to UV radiation.

[0051] In some methods, step (iii) utilizes a UV wavelength selected from about 100 nm to about 400 nm, such as a UV wavelength selected from about 220 nm to about 300 nm.

[0052] In some methods, the one or more UV light sources are each selected from the group consisting of a UV light emitting diode, a UV mercury lamp, a UV xenon lamp, and a UV krypton lamp.

[0053] In various methods, step (iii) is effective to achieve a 4-log reduction in adventitious microorganisms present prior to step (iii). In some methods, step (iii) is effective to achieve a 6-log reduction in adventitious microorganisms present prior to step (iii). In some methods, step (iii) is effective to achieve an 8-log reduction in adventitious microorganisms present prior to step (iii). In some methods, step (iii) is effective to achieve a 10-log reduction in adventitious microorganisms present prior to step (iii).

[0054] The method may further include introducing a gas into the chamber after step (iii). The gas may be, for example, air, oxygen, syngas, hydrogen, carbon monoxide, methane, natural gas, or combinations thereof. The gas may be a mixture of O2 and N2, with various concentrations of O2 above or below 21 vol% O2.

[0055] In a preferred method, the chamber maintains a sterile boundary with the environment.

[0056] The various variations generally provide a method for cleaning and sterilizing a bioreactor, the method comprising: (i) providing a radiation sterilizable bioreactor system comprising: (a) a chamber configured to carry out a reaction; (b) a component configured to introduce a gas into the chamber; and (c) one or more light sources configured to irradiate a surface within the chamber; (ii) cleaning the chamber; and (iii) sterilizing the chamber by exposing the chamber to radiation, the radiation having a wavelength longer than 1 nm and less than 1 mm; Includes. [Brief description of the drawings]

[0057] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] FIG. 1 is a schematic diagram (side view) of a UV sterilizable bioreactor system according to some embodiments of the present invention. [Diagram 2] FIG. 1 is a schematic (isometric) view of a UV sterilizable bioreactor system according to some embodiments of the present invention. [Diagram 3] FIG. 1 is a schematic diagram (side view) of a UV sterilizable bioreactor top with a stirrer seal in some embodiments of the invention. [Figure 4] FIG. 2 is a schematic diagram (top view) of a UV sterilizable sparger in some embodiments of the present invention. [Diagram 5]FIG. 1 is a schematic (isometric view) of a UV sterilizable sparger in some embodiments of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0058] Detailed Description of the Embodiments The devices, structures, methods, systems and articles of manufacture of the present invention are described in detail with reference to various non-limiting embodiments.

[0059] This specification is intended to enable a person skilled in the art to make and use the invention and describes several embodiments, adaptations, variations, alternatives and uses of the invention. These and other embodiments, features and advantages of the invention will become more apparent to those skilled in the art upon review of the following detailed description of the invention in conjunction with any accompanying figures.

[0060] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0061] Unless otherwise indicated, all numerical values ​​expressing conditions, concentrations, dimensions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending, at least on routine analytical techniques.

[0062] The term "comprising" is synonymous with "including," "containing," or "characterized by" and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. "Comprising" is a term used in claim language to mean that the specified claim element is essential, but that other claim elements may be added, which may even form part of the claim's scope.

[0063] The phrase "consisting of" as used herein excludes any element, step, or ingredient not specified in the claim. When the phrase "consisting of" (or variations thereof) appears in a section of the body of a claim rather than immediately following the preamble, it limits only the elements set forth in that section and does not exclude other elements from the claim as a whole. The phrase "consisting essentially of" as used herein limits the scope of the claim to those specified elements or method steps, plus those that do not materially affect the basic and novel characteristics of the claimed subject matter.

[0064] With respect to the terms "comprising," "consisting of," and "consisting essentially of," when one of these three terms is used herein, the subject matter disclosed and claimed herein may include the use of either of the other two terms, except as used by the Markush Group. Thus, in some embodiments not expressly stated otherwise, any instance of "comprising" may be replaced by "consisting of" or alternatively with "consisting essentially of."

[0065] The present invention is premised on the use of UV light, or other forms of non-ionizing radiation, to effectively sterilize bioreactors. The disclosed technology is a fundamental advance in the field of sparged bioreactors for many types of commercial fermentation. The bioreactors can incorporate materials that are not explicitly compatible with steam sterilization, allowing for the use of lower cost materials, among many other benefits that accrue from UV sterilization.

[0066] Traditionally, steam sterilization is used to create sterile conditions in bioreactor systems. Such bioreactor systems are evacuated and then pressurized with steam, typically at 121° C. The steam environment is maintained until adventitious microorganisms are inactivated, at which point the steam is removed from the system and bioreactor operation can begin. This steam sterilization process creates significant costs for system operators. First, it requires a large amount of energy to generate the steam, which is expensive and very carbon intensive unless renewable energy is used to generate the steam, which is even more costly to use. Also, designing bioreactors for water steam compatibility creates significant additional costs and complexity. The present inventors have recognized the problem and need for a better way to create sterile operating conditions within a bioreactor environment.

[0067] It is known that ultraviolet light can kill or inactivate living organisms. Ultraviolet light, mainly UV-C, is one of the powerful factors that can change the normal state of life by inducing various mutagenic and cytotoxic DNA damages, such as cyclobutane pyrimidine dimers, 6-4 photoproducts, DNA strand breaks by disrupting genome integrity, etc. See Rastogi et al., "Molecular Mechanisms of Ultraviolet Radiation-Induced DNA Damage and Repair", Journal of Nucleic Acids, Volume 2010, Article ID 592980, Pages 1-32 (2010), which is incorporated herein by reference.

[0068] In some variations, the present invention provides a method for producing a composition comprising: (a) a chamber configured to carry out a reaction; (b) a component configured to introduce a gas into the chamber; (c) one or more UV light sources configured to irradiate UV light onto surfaces within the chamber; The present invention provides a UV sterilizable bioreactor system comprising:

[0069] As used herein, "UV sterilizable" means that a system or component can be sterilized, at least to some extent, by UV light. As used herein, "sterilized," "sterilize," "sterilization," and the like, refer to various degrees of killing, inactivating, or removing microorganisms. Sterilization does not mean that absolutely all microorganisms have been killed, inactivated, or removed (this may be referred to as "sterility"). In some embodiments, a sterile condition can be provided to the system or component being sterilized.

[0070] The chamber is preferably a bioreactor chamber configured to carry out a biological reaction such as fermentation or enzymatic conversion. As used herein, a "bioreactor" is a fermenter that utilizes at least one gas, typically air or oxygen, in a fermentation reaction. In other embodiments, the chamber is a reactor chamber configured to carry out an anaerobic biological reaction, or an abiotic reaction that does not utilize microorganisms or enzymes.

[0071] In a preferred embodiment, the chamber is configured to maintain a sterile boundary with the environment. A sterile boundary means that no adventitious microorganisms can enter the bioreactor chamber from the environment. An "adventitious microorganism" is any microorganism (e.g., yeast, bacteria, fungi, mold) that is not the desired microorganism (biocatalyst) to catalyze the intended reaction in the chamber. Adventitious microorganisms are sometimes referred to as contaminating microorganisms. Adventitious microorganisms may compete with the biocatalyst for resources, introduce undesirable properties, or generate by-products within the bioreactor environment.

[0072] The gas introduced into the chamber may be a reactant, such as in aerobic or microaerobic fermentation, or may be a catalyst, promoter, reaction rate modifier, or other reactant. The gas introduced into the chamber is air, oxygen, syngas, hydrogen, carbon monoxide, methane, natural gas, or combinations thereof. In typical embodiments, the gas is oxygen, air, oxygen-enriched air, or oxygen-depleted air.

[0073] External oxygen concentrations can be used to increase the oxygen content in the air beyond the normal 21 vol% O2 concentration. In various embodiments employing O2 in fermentation, the O2 concentration in the gas stream supplied to the chamber (e.g., through a sparger) is about, at least about, or at most about 1 vol%, 2 vol%, 5 vol%, 10 vol%, 15 vol%, 20 vol%, 21 vol%, 22 vol%, 25 vol%, 30 vol%, 40 vol%, 50 vol%, 60 vol%, 70 vol%, 80 vol%, 90 vol%, 95 vol%, or 100 vol%, including all intermediate ranges.

[0074] In some embodiments, the component for introducing a gas is a gas sparger. A sparger can be defined as a component for introducing a gas into the liquid in the bioreactor. The three basic types of spargers are porous spargers, orifice spargers, and nozzle spargers. Spargers are adapted to introduce the desired gas in a controlled manner to provide mass transfer between the liquid and gas phases while simultaneously introducing mechanical energy into the system.

[0075] In some embodiments, the component for introducing gas is a membrane. These components serve to introduce the gas substrate through a semipermeable membrane, allowing selective mass transfer of the desired component between the gas phase and the liquid phase. In some cases, this mass transfer occurs via diffusion within the polymer matrix, while in other cases, this mass transfer occurs via diffusion within the pores in the membrane itself. In some embodiments, the gas component transported across the membrane is oxygen.

[0076] In some embodiments, the component for introducing gas is a pipe or tube that is placed within the bioreactor volume and injects the gas directly into the liquid phase. The pipe or tube can have a single outlet, such as near the bottom of the chamber, near the top of the chamber, or elsewhere within the chamber. The pipe or tube can have multiple outlets, such as a perforated pipe with multiple holes, through which the gas enters the liquid phase.

[0077] In some embodiments, the component for introducing gas is a plate sparger, which is typically located at the bottom of the chamber and has multiple holes through which the gas enters the liquid phase.

[0078] In a preferred embodiment, the components for introducing gas are UV sterilizable. The components for introducing gas (e.g., spargers) may be UV sterilizable by exposure to a UV light source that is external to the component (and internal or external to the chamber). Alternatively or additionally, the components may be UV sterilizable by incorporating a UV light source into the component itself.

[0079] In some embodiments, the component for introducing gas comprises a UV-transparent material. As used herein, a "UV-transparent material" is not necessarily completely UV-transparent, and some absorption of UV radiation may occur. In particular, "UV-transparent" means that a sheet of material 1 millimeter thick absorbs less than 50%, preferably about 25% or less, more preferably about 10% or less, and most preferably about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of incident (normal) UV light at the wavelength of interest. The actual material or component need not be 1 millimeter, i.e., its thickness is specified only when measuring UV transmittance for the purposes of this paragraph. When a wavelength range is used, the UV transmittance is the average value of the range. UV transmittance can be determined experimentally, for example, using a Perkin Elmer UV-Vis-IR spectrometer. It is noted that UV transmittance may generally include normal UV transmittance and diffuse UV transmittance, both of which may contribute to the total UV transmittance.

[0080] The UV-transparent material (in the component for introducing gas) may include a UV-transparent polymer, a UV-transparent ceramic, a UV-transparent glass, or a combination thereof. The UV-transparent polymer may be selected from the group consisting of polyacrylates, silicones, fluoropolymers, and combinations thereof. The fluoropolymer may be selected from the group consisting of polyvinylidene fluoride, polytetrafluoroethylene, perfluoroalkoxyalkanes, and combinations thereof. The UV-transparent ceramic may be selected from the group consisting of quartz, fused silica, borosilicates, and combinations thereof. The silica or borosilicates may be doped to modify their UV transparency.

[0081] The number of UV light sources may vary widely. In some embodiments, there is a single UV light source configured to irradiate surfaces within the chamber with ultraviolet light. In other embodiments, there are multiple UV light sources, each configured to irradiate surfaces within the chamber with ultraviolet light. The number of individual UV light sources can vary widely, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 100, or more. In some embodiments employing one or more microarrays of UV LED light sources, the number of individual UV light sources can be hundreds or thousands.

[0082] In some embodiments, the one or more UV light sources are located within the chamber. The one or more UV light sources can be permanently located within the chamber. Alternatively or additionally, the one or more UV light sources can be reversibly located within the chamber. The UV light sources can be removed from the chamber in such a way that sterile conditions are maintained within the chamber.

[0083] In some embodiments, the one or more UV light sources are located in a UV-transparent well located in the chamber. The UV-transparent well can be made of a UV-transparent material including a UV-transparent polymer, a UV-transparent ceramic, a UV-transparent glass, or a combination thereof. The UV-transparent polymer can be selected from the group consisting of polyacrylates, silicones, fluoropolymers, and combinations thereof. The fluoropolymer can be selected from the group consisting of polyvinylidene fluoride, polytetrafluoroethylene, perfluoroalkoxyalkanes, and combinations thereof. The UV-transparent ceramic can be quartz. The UV-transparent glass can be fused silica, borosilicate, or a combination thereof. The silica or borosilicate can be doped to modify their UV transparency.

[0084] In some embodiments, at least some (including at least one) of the one or more UV light sources are external to the chamber, hi some embodiments, all of the one or more UV light sources are external to the chamber.

[0085] Typically, the chamber walls facing the interior of the chamber need to be sterilized, but the outer chamber walls exposed to the environment do not need to be sterilized. However, in some embodiments, it may be beneficial to have at least one external UV light source that irradiates the outer wall or selected portions thereof with UV light to kill or inactivate foreign microorganisms before they can enter the chamber through small openings. For example, in some embodiments, UV LEDs can be utilized continuously during bioreactor operation to prevent foreign microorganisms from entering the bioreactor environment.

[0086] In some embodiments, the one or more UV light sources are configured to provide a UV radiant flux to a corner of the chamber, such as a corner defined by a portion of the chamber wall and an internal component (e.g., the chamber lid or the agitator shaft). In some embodiments, separate UV LED fixtures are utilized for certain components of the bioreactor that are not accessible to a primary UV light source, such as a UV mercury lamp.

[0087] In some embodiments, the UV sterilizable bioreactor system further comprises one or more conduits configured to supply input materials into the chamber and / or withdraw output materials from the chamber. Preferably, at least one of the one or more conduits is UV sterilizable. In some embodiments, there are two, three or more conduits, and each of the conduits is UV sterilizable.

[0088] In some embodiments, the chamber has walls made of a metal, a metal alloy, a polymer, a ceramic, a composite, glass, concrete, or a combination thereof. In some embodiments, the metal is aluminum. In some embodiments, the metal alloy is carbon steel or stainless steel. In some embodiments, the polymer is selected from the group consisting of polyolefins, polyacrylates, polycarbonates, fluoropolymers, silicones, and combinations thereof. In some embodiments, the composite is a polymer reinforced with glass fibers. In some embodiments, the polymer is selected from high density polyethylene, polypropylene, polycarbonate, or combinations thereof. The polymer can be selected from the group consisting of poly(methyl methacrylate), polyvinylidene fluoride, hexafluoropropylene-tetrafluoroethylene copolymer, perfluoroether-tetrafluoroethylene copolymer, and combinations thereof.

[0089] In some embodiments, the chamber has a UV-transparent chamber wall comprising a UV-transparent material. The UV-transparent material can include a UV-transparent polymer, a UV-transparent ceramic (e.g., quartz), a UV-transparent glass (e.g., fused silica and / or borosilicate), or a combination thereof. The UV-transparent polymer can be selected from the group consisting of a polyacrylate (e.g., poly(methyl methacrylate)), a silicone, a fluoropolymer, and a combination thereof. The fluoropolymer can be selected from the group consisting of polyvinylidene fluoride, hexafluoropropylene-tetrafluoroethylene copolymer, perfluoroether-tetrafluoroethylene copolymer, poly(ethylene-co-tetrafluoroethylene), or a combination thereof.

[0090] In some embodiments, the chamber has UV-reflective chamber walls that include or are internally coated with a UV-reflective material, which may be selected from, for example, aluminum, stainless steel, polytetrafluoroethylene, or combinations thereof.

[0091] In some embodiments, the chamber is configured with a chamber top that is not sealed from the environment, but the chamber top is UV sterilizable and forms a sterile, and potentially aseptic, barrier with the environment.

[0092] The chamber volume can vary widely for a given bioreactor system, including laboratory, pilot, demonstration and commercial scales, in various embodiments, the chamber volume is about, at least about, or at most about 10 mL, 100 mL, 250 mL, 1 L, 2 L, 5 L, 10 L, 25 L, 50 L, 100 L, 500 L, 1,000 L, 5,000 L, 10,000 L, 50,000 L, 100,000 L, 500,000 L, 1,000,000 L, 2,000,000 L, 5,000,000 L or more.

[0093] The shape of the chamber can also vary. A typical chamber is cylindrical with rounded (circular with respect to the vertical axis) walls. The chamber can have rounded walls, flat walls, or a combination thereof. The shape of the chamber can be generally cylindrical, tubular, conical, spherical, or rectangular, etc. The aspect ratio of the chamber can vary, such as tall (longer than lateral) or short (longer than lateral). The orientation of the chamber can be vertical, horizontal, or inclined. The chamber can be designed to have a continuously stirred flow pattern, plug flow, or a flow distribution between these extremes.

[0094] The one or more UV light sources can be configured to irradiate at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the total surface area of ​​the chamber, including its internal components. The chamber surface area can include, for example, spargers, impellers, impeller shafts, clean-in-place arms, internal portions of valves, and / or internal portions of sensors. The total surface area of ​​all internal parts can be less than, about the same as, or greater than the surface area of ​​the chamber walls facing the interior of the chamber.

[0095] The one or more UV light sources can be configured to irradiate at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the total volume of the chamber with UV light, where the total volume of the chamber is calculated from a nominal volume based on the inner diameter and height, not the working volume.

[0096] In some embodiments, the one or more UV light sources each have a UV wavelength selected from about 100 nm to about 400 nm. In some embodiments, the UV wavelength is selected from about 220 nm to about 300 nm for at least one of the one or more UV light sources, such as for all of the UV light sources. In various embodiments, the UV wavelength is about, at least about, or at most about 100, 110, 120, 125, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, or 400 nm, including all intermediate ranges. In some embodiments, the one or more UV light sources each have a UV wavelength selected from 125 nm to 400 nm.

[0097] In some embodiments, the one or more UV light sources each have a UV wavelength selected from UV-C wavelengths, which for purposes of this specification are between 200 and 290 nm. In some embodiments, the one or more UV light sources each have a UV wavelength selected from UV-B wavelengths, which for purposes of this specification are between 291 and 320 nm. In some embodiments, the one or more UV light sources each have a UV wavelength selected from UV-A wavelengths, which for purposes of this specification are between 321 and 400 nm.

[0098] Each of the one or more UV light sources can be selected from the group consisting of, for example, a UV light emitting diode, a UV mercury lamp, a UV xenon lamp, and a UV krypton lamp. In some embodiments, all of the UV light sources are UV light emitting diodes (UV LEDs). The UV light sources can be configured to generate pulsed UV light. For example, the pulsed UV light source can be pulsed several times per second, with each pulse lasting for about 100 nanoseconds to 10 milliseconds.

[0099] In some embodiments, the bioreactor system includes an impeller located within the chamber, the impeller preferably being UV sterilizable. In other embodiments, the chamber does not include an impeller. When the chamber does not include an impeller, the bioreactor is known as a bubble column. In a bubble column, there is no impeller and internal mixing is achieved via momentum transfer from the gas.

[0100] In some embodiments, the UV sterilizable bioreactor system further comprises a filtration unit configured to filter an input material (e.g., gas being supplied to the sparger) before it is supplied into the chamber. In some embodiments, the filtration unit is UV sterilizable.

[0101] In some embodiments, the UV sterilizable bioreactor system includes a bioreactor sensor located within the chamber. The bioreactor sensor can be configured to detect or measure a bioreactor parameter selected from the group consisting of, for example, pH, temperature, oxygen, carbon dioxide, foaming, mixing, cell density, feed substrate concentration, reaction intermediate concentration, and product concentration. There may be multiple bioreactor sensors located within the chamber. In some embodiments, the bioreactor sensor is configured to transmit a wireless signal.

[0102] In some embodiments, the bioreactor sensor is UV sterilizable. The bioreactor sensor can be configured with a UV light guide for sterilizing the bioreactor sensor. The UV light guide can be, for example, a UV optical fiber.

[0103] In some embodiments, the bioreactor sensor is disposed in or through a probe port, and the probe port is UV sterilizable. In some embodiments, the bioreactor sensor is contained within a UV sterilizable housing located within the chamber. The UV sterilizable housing can include a UV transparent material, such as a UV transparent polymer, a UV transparent ceramic, a UV transparent glass, or a combination thereof. The UV transparent polymer can be selected from the group consisting of polyacrylates, silicones, fluoropolymers, and combinations thereof. An exemplary polyacrylate is poly(methyl methacrylate). The fluoropolymer can be selected from the group consisting of polyvinylidene fluoride, hexafluoropropylene-tetrafluoroethylene copolymer, perfluoroether-tetrafluoroethylene copolymer, poly(ethylene-co-tetrafluoroethylene), and combinations thereof. An exemplary UV transparent ceramic is quartz. An exemplary UV radiation transparent glass includes fused silica, borosilicate, or a combination thereof. The silica or borosilicate may be doped to modify their UV transparency.

[0104] In some embodiments, the bioreactor system includes a clean-in-place arm located within the chamber. A retractable clean-in-place arm can be utilized to clean the bioreactor prior to UV sterilization. In some embodiments, the clean-in-place arm is configured to facilitate preparation of the bioreactor system for fermentation.

[0105] The cleaning-in-place arm may be UV sterilizable. In these embodiments, the cleaning-in-place arm may be exposed to a UV light source that also irradiates the chamber walls or other components with UV light. Alternatively or additionally, the cleaning-in-place arm itself may be configured with a UV sterilizer located within or directly on the cleaning-in-place arm. In some embodiments, an array of UV LED light sources is mounted internally within the cleaning-in-place arm.

[0106] Additional components may be desirable for the proper operation of a bioreactor system, including (but not limited to) components for the introduction of reagents (e.g., conduits), components for aeration (e.g., spargers), components for agitation (e.g., impellers), components for thermal management, components for sensing, and components for control.

[0107] In some embodiments, the port is a component in the bioreactor system that is adapted to be used during operation without compromising the sterile operating environment. The port can be isolated from the external environment, for example, via a pinch valve, diaphragm valve, or ball valve. In some embodiments, the valve can be designed to sterilize reagents at the point of use during operation of the bioreactor. In some embodiments, a UV light source is located in or on the valve sterilizer. In some embodiments, the UV light source is mounted internally within the valve sterilizer. The input that enters the valve typically comes through a tube from a source container. In some embodiments, the tube or source container is subjected to UV sterilization.

[0108] In some embodiments, aeration is accomplished using a gas input component configured to introduce gas into the chamber. The gas input component may be a sparger, a membrane, or another means to effectively distribute the gas into the chamber. It is possible to feed the gas into the chamber through a port, but this typically results in a highly non-uniform gas concentration within the chamber. In some embodiments, the gas input is sterilized via UV sterilization, filtration, or a combination thereof. In some embodiments, the gas input component may have a UV light source mounted within the component such that a sterile condition is created inside the gas input component.

[0109] A stirring component promotes mixing of the internal components of the bioreactor chamber. In some embodiments, the stirring component is an impeller that rotates within the chamber and achieves mixing by transferring kinetic energy to the chamber contents (which can be laminar, but usually in turbulent flow). Power to the impeller is usually provided by an electric motor, i.e., electrically driven, although the impeller could in principle also be driven by magnetic induction or even compressed air. If an electric motor is used, the motor is preferably mounted outside the chamber and can be sealed using a mechanical seal, a lip seal or a magnetic seal, all of which can be UV sterilized.

[0110] Thermal management components can introduce or remove thermal energy (heat) from the bioreactor's operating environment. In some embodiments, heat transfer can occur through the reactor walls using a heat transfer fluid, a steam jacket, a heating coil, a cooling coil, another thermal management component, or a combination thereof. In some embodiments, the chamber contents are heated or cooled using internal baffles and / or internal coils. An external flow loop may be used to heat or cool the chamber contents.

[0111] Various components for sensing can be used within the bioreactor system, such as bioreactor sensors. In various embodiments, the bioreactor sensors detect or measure a bioreactor parameter selected from the group consisting of, for example, pH, temperature, dissolved oxygen, dissolved air, dissolved hydrogen, dissolved carbon monoxide, dissolved carbon dioxide, dissolved methane, foaming, mixing, cell density, feed substrate concentration, reaction intermediate concentration, product concentration, density, or weight. The bioreactor sensors can measure various spectrophotometric properties. In some embodiments, the bioreactor sensors are configured to transmit wireless signals to a computer for monitoring and control of the system.

[0112] Sensors can also be used to measure properties within the product stream or a property of a sample. For example, a product or sample sensor can detect or measure a parameter selected from the group consisting of pH, temperature, dissolved oxygen, dissolved air, dissolved hydrogen, dissolved carbon monoxide, dissolved carbon dioxide, dissolved methane, cell density, product concentration, density, or weight.

[0113] Measurements taken by the sensors can be used to make dynamic or future process adjustments that can utilize well-known principles of process control, such as proportional feedback control, proportional-integral-derivative (PID) feedback control, feedforward control, etc. Computers can be employed to automatically make process and system adjustments based on one or more measurements from the sensors.

[0114] Several embodiments will now be described with reference to the accompanying drawings of FIGS. 1-5, which are intended to illustrate various examples of the present invention and should not be construed as limiting the present invention.

[0115] FIG. 1 is a schematic (side view) of a UV sterilizable bioreactor system in some embodiments of the present invention. In FIG. 1, there is a chamber configured to perform a fermentation reaction, a sparger configured to introduce gas into the chamber volume, and a tank sterilizer (UV light source) configured to irradiate surfaces within the chamber volume with UV light. Although FIG. 1 shows two tank sterilizers, it is understood that the number of tank sterilizers shown is merely exemplary. The sparger is equipped with a sparger sterilizer configured to irradiate the sparger with UV light. There is a sugar valve for introducing sugar substrates and medium components. There is an acid / base valve for introducing acid, base and / or buffer into the chamber. The acid / base valve is equipped with a dedicated valve sterilizer configured to irradiate the valve with UV light. A sampling valve allows samples to be extracted before, during or after fermentation. A discharge valve allows fermentation broth to be discharged during or after fermentation. The discharge valve is equipped with a discharge valve sterilizer configured to irradiate the discharge valve with UV light. An exhaust valve allows gas to be discharged during or after fermentation. At the top of the chamber, near the agitator motor, is a lid / seal sterilizer configured to apply UV light to the lid and seal. At or near the top of the chamber is a clean-in-place arm. The clean-in-place arm is equipped with a clean-in-place arm sterilizer configured to apply UV light to the clean-in-place arm. While the diagram in FIG. 1 shows four impellers, it is understood that the number of impellers shown is merely exemplary. Near the top of the chamber, a defoamer is configured. It should be noted that while FIG. 1 explicitly shows a tank sterilizer, a sparger sterilizer, an acid / base valve sterilizer, a UV clean-in-place arm sterilizer, a lid / seal sterilizer, and a drain valve sterilizer, other embodiments of the invention employ fewer UV sterilizers (e.g., only a tank sterilizer) or additional UV sterilizers (e.g., a sugar valve sterilizer is also used).

[0116] FIG. 2 is a schematic (isometric) diagram of a UV sterilizable bioreactor system according to some embodiments of the present invention. In FIG. 2, there is a chamber configured to carry out a fermentation reaction, a sparger configured to introduce gas into the chamber volume, and a UV light source configured to irradiate surfaces within the chamber volume with UV light. FIG. 2 shows two UV light sources, although it is understood that the number of UV light sources shown is merely exemplary. The diagram in FIG. 1 shows six impellers, however it is understood that the number of impellers shown is merely exemplary.

[0117] FIG. 3 is a schematic (side view) of a UV sterilizable bioreactor top with a stirrer seal in some embodiments of the invention. The UV sterilizable bioreactor top is designed to maintain a sterile boundary with the environment. In FIG. 3, the fermenter top plate is configured to allow the agitator stirrer shaft to pass through the plate while maintaining a sterile barrier. The lower bearing for the stirrer shaft is mounted in the center of the fermenter top plate. The upper bearing for the stirrer shaft is mounted in a yoke just above the fermenter top plate. The bearing is pressed into a housing within the yoke. A skirt provides a shield to prevent airborne contaminants from settling on the lower bearing and ultimately entering the chamber. There are multiple UV sterilizers (UV light sources) configured to shine UV light through the assembly.

[0118] FIG. 4 is a schematic (top view) of a UV sterilizable sparger in some embodiments of the present invention. The sparger can be UV sterilized by exposure to a UV light source external to the sparger. The sparger may also be UV sterilizable by incorporating a UV light source into the sparger itself. The sparger can be made from a UV transparent material such as a UV transparent polymer, a UV transparent ceramic, a UV transparent glass, or a combination thereof. In FIG. 4, a gas such as air or oxygen enters through a top port shown in the center of the figure from a pipe or tube not shown. The gas circulates around the curved tube shown as having an inner and outer circle. The gas is dispersed into the chamber through small holes (not shown) in the tube.

[0119] FIG. 5 is a schematic (isometric view) of another UV sterilizable sparger in some embodiments of the present invention. The sparger can be UV sterilized by exposure to a UV light source external to the sparger. The sparger may be UV sterilizable by incorporating a UV light source into the sparger itself. The sparger can be made from a UV transparent material such as a UV transparent polymer, a UV transparent ceramic, a UV transparent glass, or a combination thereof. In FIG. 5, a gas such as air or oxygen enters through the top tube (hub) and is dispersed into the chamber through outlets contained in each of the outwardly directed spokes. The gas can also be dispersed into the chamber through small holes in the tube, for example. The drawing shows six spokes (additional spokes are implied but may be hidden when viewed from the hub).

[0120] Some variations provide a method for cleaning and sterilizing a bioreactor, the method comprising: (i) providing a UV sterilizable bioreactor system comprising: (a) a chamber configured to carry out a reaction; (b) a component configured to introduce a gas into the chamber; and (c) one or more UV light sources configured to irradiate surfaces within the chamber with ultraviolet light; (ii) cleaning the chamber; and (iii) exposing the chamber to UV radiation to sterilize the chamber; Includes.

[0121] In some methods, the UV sterilizable bioreactor system further comprises one or more conduits configured to supply input materials into the chamber and / or withdraw output materials from the chamber. The conduits may be valves, inlets or outlets. In some methods, at least one of the conduits is UV sterilizable. The conduits may be UV sterilizable when exposed to a UV light source external to the conduit. Alternatively or additionally, the conduits may have an integrated UV light source (e.g., UV LED) integrated on or within the conduit so that they can be UV sterilized when desired.

[0122] In some methods, one or more UV light sources, such as all of the UV light sources, are located within the chamber. In some methods, one or more UV light sources, such as all of the UV light sources, are permanently located within the chamber. In some methods, one or more UV light sources, such as all of the UV light sources, are reversibly located within the chamber.

[0123] In some methods, one or more UV light sources, for example all of the UV light sources, are located within a UV-transparent well that is disposed within the chamber.

[0124] In some methods, at least some of the one or more UV light sources are external to the chamber. In some methods, all of the one or more UV light sources are external to the chamber.

[0125] In some methods, the chamber has walls made from a metal, a metal alloy, a polymer, a ceramic, a composite material, glass, concrete, or a combination thereof.

[0126] In some methods, the chamber has UV-transparent chamber walls that include a UV-transparent material, as previously discussed herein.

[0127] In some methods, the chamber has UV-reflective chamber walls that include or are internally coated with a UV-reflective material.

[0128] In some methods, the chamber is configured with a chamber top that is not sealed from the environment, and the chamber top is UV sterilizable to form a sterile barrier with the environment.

[0129] In some methods, an impeller is located within the chamber, and the impeller is UV sterilizable.

[0130] In some methods, the UV sterilizable bioreactor system further comprises a filtration unit configured to filter the input material before it is provided into the chamber. In some methods, the filtration unit is UV sterilizable.

[0131] In some methods, a bioreactor sensor is located in the chamber. The bioreactor sensor can be configured to detect or measure a bioreactor parameter selected from the group consisting of pH, temperature, oxygen, carbon dioxide, foaming, mixing, cell density, feed substrate concentration, reaction intermediate concentration, and product concentration. The bioreactor sensor can be configured to have an optical microscope, camera, or IR scope that images mass and / or thermal distribution at several locations in the bioreactor, such as near the sparger. The bioreactor sensor can be configured to transmit a wireless signal during or after the sensor measurement. During operation, the bioreactor sensor can actually detect or measure the bioreactor parameter. A control strategy can be used to adjust the bioreactor parameter, for example, if dissolved oxygen is measured to be too low, air or O2 with an increased sparging rate can be fed to the bioreactor chamber.

[0132] In some methods, the bioreactor sensor is UV sterilizable. The bioreactor sensor can be configured with a UV light guide for sterilizing the bioreactor sensor. Alternatively or additionally, the bioreactor sensor is contained within a UV sterilizable housing located within the chamber. The UV sterilizable housing can include a UV transparent material. Alternatively or additionally, the bioreactor sensor can be disposed within or through a probe port, and the probe port is UV sterilizable.

[0133] In some methods, the bioreactor includes a wash-in-place arm located within the chamber, and the wash-in-place arm is UV sterilizable. The wash-in-place arm may be UV sterilized during step (iii) or in a separate step.

[0134] In some methods, step (ii) utilizes a cleaning agent selected from the group consisting of hot water, alkaline detergent, sodium hydroxide, sodium percarbonate, acidic detergent, phosphoric acid, peracetic acid, isopropanol, ethanol, sodium hypochlorite, hydrogen peroxide, ethylene oxide, chlorine dioxide, ozone, formaldehyde, peracetic acid, glutaraldehyde, and combinations thereof. Other cleaning agents can be used in place of or in addition to these chemicals. The cleaning agent can be a liquid, a vapor, a gas, or a combination thereof (e.g., a liquid / vapor mixture, or a liquid followed by a gas, etc.).

[0135] In some methods, step (iii) is performed at 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, or 5000 mJ / cm, including all intermediate ranges. 2 , or more, of sterilization energy per unit total area within the chamber. Sterilization energy per unit total area is also known as light intensity. The light intensity may be constant or may vary over time.

[0136] In some methods, step (iii) is about 0.5 mW / cm 2 ~About 1000mW / cm 2 The total UV power output may be about, at least about, or at most about 0.5, 1, 2, 3, 4, 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mW / cm, including all intermediate ranges. 2 The total UV power output may be constant or may vary over time.

[0137] In some methods, step (iii) is about 0.1 W / m 3 ~about 5000W / m 3 The total UV power capacity is calculated as the input UV power divided by the volume of the chamber. The total UV power capacity may be about, or at most about 5000, 4000, 3000, 2000, 1000, 500, 400, 300, 200, 100, 50, 40, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.4, 0.3, 0.2 or 0.1 W / m, including all intermediate ranges. 3 It could be.

[0138] In some methods, step (iii) utilizes a UV sterilization time of about 1 minute to about 24 hours. The UV sterilization time is the time that the UV light source is irradiated. In various embodiments, the UV sterilization time is about, at least about, or at most about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 20 hours, or 24 hours, including all intermediate ranges.

[0139] In some methods, step (iii) is performed at a sterilization temperature of about 10° C. to about 95° C. In various embodiments, the sterilization temperature is about, at least about, or at most about 10° C., 15° C., 20° C., 25° C., 30° C., 35° C., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., 80° C., 85° C., 90° C., or 95° C., including all intermediate ranges. In some embodiments, the sterilization temperature is not controlled and is at or near ambient temperature, such as about 25° C. In other embodiments, it is desirable to increase the temperature to aid in sterilization.

[0140] In a typical process, step (iii) is carried out in an atmosphere that consists essentially of air, which may be dry or moist air. In a typical process, step (iii) is carried out in an atmosphere that is free of steam, although water vapour may be present, for example due to humidity in the air.

[0141] In some methods, step (iii) is performed in an inert gas atmosphere, such as CO, N, Ar, or a combination thereof. When step (iii) is performed with air present in the chamber, the air includes N, which is inert, and O, which may or may not be inert.

[0142] In some methods, step (iii) is carried out in an atmosphere containing a vapor or gas that promotes sterilization, such as ethylene oxide, chlorine dioxide, hydrogen peroxide, ozone, formaldehyde, peracetic acid, or glutaraldehyde. Ozone can be generated in situ (e.g., from air) from the photolysis of oxygen to produce O3 from O2, such as when one or more UV wavelengths in the range of 160-240 nm are used for UV sterilization.

[0143] In some methods, step (iii) exposes at least 80% of the total surface area of ​​the chamber, including its internal components, to UV radiation. Step (iii) may expose at least 85%, at least 90%, at least 95%, or at least 99% (including 100%) of the total surface area of ​​the chamber, including its internal components, to UV radiation. An "internal component" of a chamber is a component or portion thereof that is inside the boundary with the environment.

[0144] In some methods, step (iii) exposes at least 80% of the total volume of the chamber to UV radiation. Step (iii) may expose at least 85%, at least 90%, at least 95% or at least 99% (including 100%) of the total volume of the chamber to UV radiation.

[0145] In some methods, step (iii) utilizes a UV wavelength selected from about 100 nm to about 400 nm, such as a UV wavelength selected from about 220 nm to about 300 nm. In various methods, step (iii) utilizes a UV wavelength selected from about, at least about, or at most about 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290 , 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, or 400 nanometers (including intervening ranges (e.g., 200-300 nm), including each 1 nanometer increment within each subrange (e.g., including 227-304 nm, 188-242 nm, etc.)). In some embodiments, such as (but not limited to) those that utilize pulsed light, the light source may provide multiple wavelengths of radiation, some of which are in the range of 100-400 nm, some of which are higher than 400 nm, such as in the visible or infrared ranges of the electromagnetic spectrum.

[0146] With respect to wavelength, 100-400 nm is a preferred radiation wavelength range, although it will be recognized that in some embodiments, higher wavelengths may also be effective, depending on the specific exogenous microorganism and radiation parameters other than wavelength (e.g., time, light intensity, total power, or absorbed energy). Wavelengths in the visible light band (about 400-700 nm) and / or infrared band (about 700 nm-1 mm) may be able to inactivate exogenous microorganisms, such as by inducing DNA damage.

[0147] Some variations are generally: (a) a chamber configured to carry out a reaction; (b) a component configured to introduce a gas into the chamber; (c) one or more light sources configured to irradiate a surface within the chamber with radiation, the radiation having a wavelength greater than 1 nm and less than 1 mm; The present invention provides a radiation sterilizable bioreactor system comprising:

[0148] Generally, some variations provide a method for cleaning and sterilizing a bioreactor, the method comprising: (i) providing a radiation sterilizable bioreactor system comprising: (a) a chamber configured to carry out a reaction; (b) a component configured to introduce a gas into the chamber; and (c) one or more light sources configured to irradiate a surface within the chamber; (ii) cleaning the chamber; and (iii) sterilizing the chamber by exposing the chamber to radiation, the radiation having a wavelength longer than 1 nm and less than 1 mm; Includes.

[0149] In a preferred embodiment of the radiation sterilizable bioreactor system and the method for cleaning and sterilizing the bioreactor, the one or more light sources are configured to irradiate the surfaces in the chamber with non-ionizing UV radiation, visible light, IR radiation, or a combination thereof. X-rays and gamma rays are examples of ionizing radiation, and therefore, X-rays or gamma rays are not included in the scope of non-ionizing UV radiation. In a preferred method, the light source is a UV light source, which may be selected from the group consisting of UV light emitting diodes, UV mercury lamps, UV xenon lamps, and UV krypton lamps.

[0150] In various methods, step (iii) is effective to achieve a 4-log reduction in adventitious microorganisms present prior to step (iii). As used herein, "log" refers to logarithms to the base 10. Thus, a 4-log reduction is a reduction in the proportion of adventitious microorganisms remaining that is 10 -4 and log10 -4=-4, resulting in a 99.99% reduction in the population of adventitious microorganisms. In some methods, step (iii) is effective to achieve a 6-log reduction in live adventitious microorganisms present prior to step (iii). In some methods, step (iii) is effective to achieve an 8-log reduction in adventitious microorganisms present prior to step (iii). In particular methods, step (iii) is effective to achieve a 10-log reduction in adventitious microorganisms present prior to step (iii). In various embodiments, step (iii) is effective to achieve a reduction in adventitious microorganisms initially present prior to step (iii) that is about or at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 logs. In some embodiments, step (iii) is effective to achieve a 100% reduction, which may be referred to as the complete elimination of adventitious microorganisms initially present prior to step (iii), i.e., the achievement of a sterility state. In general, different microorganisms have different susceptibility to UV light. When multiple species of adventitious microorganisms are present, the kill rates of different species may differ.

[0151] In a bioreactor system, the exogenous microorganisms may include, for example, spore-forming gram-positive or gram-negative bacilli, non-spore-forming bacteria, fungal contamination, or cocci. The microorganisms for the desired fermentation may or may not be more UV-resistant than the exogenous microorganisms. If the exogenous microorganisms are particularly UV-resistant and can withstand UV sterilization, there will be no problem with the final fermentation in the bioreactor.

[0152] The method may further include introducing a gas into the chamber after step (iii). The gas may be, for example, air, oxygen, syngas, hydrogen, carbon monoxide, methane, natural gas, or a combination thereof. The gas may be a mixture of O2 and N2, with various concentrations of O2 above or below 21 vol% O2. The gas may be a reactant, such as in aerobic or microaerobic fermentation, or may be a catalyst, promoter, reaction rate modifier, or other reactant. The gas may be first sterilized by filtration, exposure to UV light, or a combination thereof (e.g., by filtering through a UV sterilizing filter) before being fed into the chamber.

[0153] To carry out a desired fermentation, several inputs are generally required, including but not limited to culture medium, a feedstock that acts as a carbon source, an acid input, a base input, an antifoam agent, a buffer, vitamins, a gaseous input, or any bolus of a substance intended to affect a biochemical process.

[0154] Fermentation can be carried out at any suitable fermentation temperature, such as from about 10° C. to about 60° C. In various embodiments, the fermentation temperature is about, at least about, or at most about 10° C., 15° C., 20° C., 25° C., 30° C., 35° C., 40° C., 45° C., 50° C., 55° C., or 60° C., including any intermediate ranges. Multiple fermentation temperatures can be used, such as an initial temperature for the cell growth phase and a second temperature during the production phase.

[0155] The fermentation temperature can be monitored and controlled by thermal management. In some embodiments, thermal management is achieved by heat exchange through the chamber walls. In some embodiments, thermal management is achieved by heat exchange through an internal cooling baffle. In some embodiments, thermal management is achieved by heat exchange through an external cooling loop.

[0156] The fermentation can be carried out at any suitable fermentation pH, such as from about 2 to about 12. In various embodiments, the fermentation pH is about, at least about, or at most about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, including any intermediate ranges. Multiple fermentation pH values ​​can be used, such as an initial pH during a first product stage and a second pH during a second product stage.

[0157] The fermentation can be carried out at any suitable fermentation pressure, such as from about 0.1 bar (vacuum) to about 20 bar. In various embodiments, the fermentation pressure is about, at least about, or at most about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 1.0, 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bar, including any intermediate ranges. Higher pressures can enhance microbial growth rates and product formation, such as when fermenting syngas, because higher pressures increase the driving force for mass transfer of H2 and CO to the liquid phase containing the microbial cells.

[0158] Fermentation can be carried out at any suitable gas (e.g., O2) concentration, such as from about 0.1 mg / L to about 100 mg / L (milligrams of gas per liter of liquid present). In various embodiments, the gas concentration is about, at least about, or at most about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 mg / L, including any intermediate ranges. Multiple fermentation gas concentration values ​​can be used, such as an initial gas concentration in the cell growth phase and a second gas concentration in the production phase.

[0159] The fermentation can be carried out at any suitable cell concentration, such as from about 0.1 g / L to about 200 g / L (grams of cells of the desired biocatalyst per liter of liquid present). In various embodiments, the cell concentration is about, at least about, or at most about 0, 0.1, 0.5, 1, 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 150, or 200 g / L, including any intermediate ranges. In some embodiments, the cell concentration remains approximately constant during the fermentation. In other embodiments, there is a cell growth phase in which active cells of the biocatalyst are produced, followed by a production phase in which the cells make a product (e.g., organic acid, enzyme, etc.) that is released from the cells. In still other embodiments, the cells are the desired product and the cell concentration can increase throughout the fermentation.

[0160] The fermentation can be carried out for any suitable fermentation time, such as about, at least about, or at most about 8, 12, 16, 24, 36, 48, 72, or 96 hours, such as about 8 hours to about 350 hours, including any intermediate ranges. For batch or fed-batch fermentation, the fermentation time begins when all fermentation components are present at the fermentation temperature, pressure, pH, and gas concentrations, and the fermentation time ends when one or more parameters are adjusted to stop the fermentation and / or the bioreactor is emptied to harvest product. For continuous fermentation, the fermentation time is the residence time, which is the inverse of the dilution rate.

[0161] The bioreactor can be operated batch, semi-batch, fed-batch, semi-continuous, continuous, or combinations or hybrids thereof.

[0162] When the bioreactor operates in batch mode, typically step (ii) and step (iii) are each performed after each batch. In other embodiments, step (ii) may be performed after some batches but not others. In these or other embodiments, step (iii) may be performed after some batches but not others. Various protocols can be used and measurements (e.g., compositional or biological analyses) can be performed to determine whether to perform step (ii) and / or step (iii) after a given batch.

[0163] When the bioreactor operates continuously, typically step (ii) is performed to clean the chamber, then step (iii) is performed to sterilize the chamber, followed by a period of continuous operation to produce the product of the bioreactor. During such continuous operation, the UV light source can be used to sterilize a variety of inputs or products, such as (but not limited to) substrates (e.g., sugars), gases (e.g., air), vitamins, minerals, acids, bases, buffers, antifoam agents, samples or products. Such inputs or products can be sterilized intermittently or, more preferably, continuously.

[0164] In general, the principles of the present invention can be applied to a wide variety of commercial processes and products, including (but not limited to) industrial chemicals, biochemicals, biofuels, pharmaceuticals, dietary supplements, vitamins, food ingredients, protein products, enzymes and cells.

[0165] Optionally, sucrose and oxygen are combined to synthesize riboflavin. Optionally, glucose and oxygen are combined to synthesize hyaluronic acid. Optionally, molasses and oxygen are combined to synthesize citric acid. Optionally, complex media and oxygen are combined to produce bovine cells. Optionally, gaseous carbon monoxide or carbon dioxide and gaseous hydrogen are combined to produce ethanol. Optionally, formic acid and oxygen are combined to produce lactic acid. Optionally, glycerol and oxygen are combined to produce glycolic acid. In some embodiments, methanol and oxygen are combined to produce microbial biomass. In some embodiments, methane and oxygen are combined to produce organic acids. Optionally, feedstock and oxygen are combined to produce multicellular organisms. In some embodiments, carbon feedstock and oxygen are combined to produce filamentous fungi. In some embodiments, waste carbon (e.g., waste oil) and oxygen are combined to produce polyhydroxyalkanoates. In some embodiments, glucose and oxygen are combined to produce insulin, hi some embodiments, sucrose and oxygen are combined to make farnesene.

[0166] In a preferred method, the chamber maintains a sterile boundary with the environment. The sterile boundary can be maintained using an airtight seal. Alternatively or additionally, the open areas of the chamber (e.g., the open chamber top) can be UV sterilized to maintain the sterile boundary by forming a sort of virtual, rather than physical, sterile barrier with the environment.

[0167] In some embodiments, after step (iii) and during the operation to produce the fermentation product, one or more UV light sources are still used. The UV light sources used during the operation of the bioreactor may be less than all the UV light sources used for UV sterilization in step (ii). For example, UV LEDs can be used continuously or intermittently during the operation of the bioreactor to prevent the ingress of adventitious microorganisms. If the active (desired) biocatalyst is more UV resistant than the adventitious microorganism, or if some UV exposure of the biocatalyst is desired, for example during a mutagenesis campaign, it is conceptually possible to use all the UV light sources used in step (ii) during the operation of the bioreactor.

[0168] After production of the fermentation product, the product is usually present in a dilute solution or broth. Product recovery can be performed using known techniques, such as (but not limited to) evaporation, distillation, centrifugation, liquid-liquid extraction, which produce a concentrated form of the desired product. In some embodiments, concentration of the product is not required, as the fermentation broth as is is a product that can be stored, shipped, or used elsewhere at the plant site.

[0169] In some embodiments, one or more materials recovered from the dilute solution or broth are recovered and recycled, for example, for reuse at the plant site or an adjacent site. For example, water can be recovered and reused to improve water balance. Vitamins and minerals can be recovered and reused in another fermentation. Recovered materials can be UV sterilized prior to reuse or UV sterilized at the point of reintroduction into the process, such as through a UV sterilized filter. In some embodiments, biological materials are inactivated as they are removed from the bioreactor system by UV sterilization.

[0170] In this detailed description, reference has been made to several embodiments which illustrate, by way of example, specific exemplary embodiments of the invention. These embodiments have been described in sufficient detail to enable those skilled in the art to practice the invention, and it is to be understood that modifications to the various disclosed embodiments may be made by those skilled in the art.

[0171] While the methods and steps described above show some events occurring in a particular order, those skilled in the art will recognize that the order of some steps may be changed and that such changes are in accordance with variations of the invention. Furthermore, some steps may be performed simultaneously in a parallel process, rather than just sequentially, where possible.

[0172] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein. This specification is hereby incorporated by reference into Stanbury et al., Principles of Fermentation Technology, 3rd Edition, Elsevier, 2017.

[0173] The above-described embodiments and variations are illustrative of the utility and versatility of the present invention. Other embodiments that do not provide all of the features and advantages set forth herein may be utilized without departing from the spirit and scope of the present invention. Such modifications and variations are considered to be within the scope of the present invention as defined by the claims. EXAMPLES

[0174] Working Example A 4 L glass vessel with a custom-made head plate is manufactured. The system is cleaned with Alcojet® detergent and water and then dried. The head plate and vessel are joined using a silicone gasket to achieve an air-tight seal.

[0175] UV sterilization is performed in glass containers as follows: Three 11 W UV-C bulbs are inserted into UV-transmitting wells that are fixedly attached to the acrylic head plate. The bulbs are powered for 60 minutes while maintaining an air-tight chamber seal from the outside environment.

[0176] Next, 2 L of sterile trypticase soy broth (TSB) medium is introduced into the bioreactor volume using the sterile interface located on the head plate. 1 VVM (vessel volume per minute) of air is introduced into the TSB medium through a 0.22 micron sterile filter within the bioreactor volume for 5 days. During this period, samples are removed from the bioreactor volume under sterile conditions.

[0177] Optical density at 600 nm (OD600) is used to characterize the microbial growth within the bioreactor volume. OD600=0 readings are obtained on days 0, 1, 2, 3, 4, and 5. Additionally, samples were plated on both TSB and liquid thioglycolate plates and incubated at temperatures of 22° C. and 32° C., respectively, for 2 weeks. No observable growth is detected.

[0178] Comparative Example A 4 L glass vessel with a custom-made head plate is manufactured. The system is cleaned with Alcojet® detergent and water and then dried. The head plate and vessel are joined using a silicone gasket to achieve an airtight seal. No UV sterilization is performed on or in the glass vessel.

[0179] Next, introduce 2 L of sterile Trypticase Soy Broth (TSB) medium into the bioreactor volume using the sterile interface located on the head plate. Introduce 1 VVM of air into the TSB medium through a 0.22 micron sterile filter within the bioreactor volume for 5 days. During this period, remove samples from the bioreactor volume in sterile conditions.

[0180] Optical density at 600 nm (OD600) is used to characterize the microbial growth within the bioreactor volume. The OD600 reading is significantly greater than 0 from day 1 onwards, after which point the experiment can be terminated. Additionally, samples are plated on both TSB and liquid thioglycolate plates and incubated at temperatures of 22°C and 32°C respectively for 2 weeks. Significant observable growth is detected.

Claims

1. (a) A chamber configured to carry out a reaction, (b) A component configured to introduce gas into the chamber, (c) One or more UV light sources configured to irradiate the surface inside the chamber with UV light, A UV-sterilizable bioreactor system equipped with [specific feature / technology].

2. The UV-sterilizable bioreactor system according to claim 1, wherein the one or more UV light sources are reversibly positioned within the chamber.

3. The UV-sterilizable bioreactor system according to claim 1, wherein the chamber is configured to have a chamber top that is not sealed from the environment, and the chamber top is UV-sterilizable to form a sterilization barrier with respect to the environment.

4. The UV-sterilizable bioreactor system according to claim 1, wherein the one or more UV light sources are configured to irradiate ultraviolet light to at least 80% of the total surface area of ​​the chamber, including its internal components.

5. The UV-sterilizable bioreactor system according to claim 1, wherein the bioreactor system includes an impeller located within the chamber, and the impeller is UV-sterilizable.

6. The UV-sterilizable bioreactor system according to claim 1, further comprising a filtration unit configured to filter the input substance before it is supplied into the chamber.

7. The UV-sterilizable bioreactor system according to claim 1, wherein the UV-sterilizable bioreactor system includes a bioreactor sensor located in the chamber, and the bioreactor sensor is UV-sterilizable.

8. The UV-sterilizable bioreactor system according to claim 7, wherein the bioreactor sensor is configured to transmit a wireless signal.

9. The UV-sterilizable bioreactor system according to claim 1, wherein the bioreactor system includes a stationary cleaning arm located within the chamber.

10. A method for cleaning and sterilizing a bioreactor, (i) Prepare a UV-sterilizable bioreactor system comprising (a) a chamber configured to carry out a reaction, (b) a component configured to introduce a gas into the chamber, and (c) one or more UV light sources configured to irradiate the surface inside the chamber with ultraviolet light. (ii) Cleaning the chamber, (iii) Sterilizing the chamber by exposing it to UV radiation, A method that includes this.

11. Step (iii) is approximately 50 mJ / cm 2 The method according to claim 10, wherein the sterilization energy per unit total area in the chamber described above is utilized.

12. The method according to claim 10, wherein step (iii) irradiates at least 80% of the total surface area of ​​the chamber, including its internal components, with UV radiation.

13. The method according to claim 10, further comprising introducing the gas into the chamber after step (iii).

14. The method according to claim 13, wherein the gas is oxygen.

15. The method according to claim 13, wherein the gas is synthesis gas, hydrogen, carbon monoxide, methane, natural gas, or a combination thereof.

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