Biocompatible epoxy coatings for bioreactors and their application in cellular manufacturing

Abstract

Disclosed herein is a 2D or 3D bioreactor, comprising a vessel forming an interior volume, wherein at least one interior surface of the vessel comprises an epoxy coating in contact with the contents of the bioreactor, wherein said epoxy coating is biocompatible by way of suitable adhesion, physical stability, absence of leaching into the contents of the bioreactor, and allows for the maintenance of cell viability. Also described are methods of manufacturing such bioreactors.

Description

BIOCOMPATIBLE EPOXY COATINGS FOR BIOREACTORS AND THEIR APPLICATION IN CELLULAR MANUFACTURINGRELATED APPLICATIONS INFORMATION

[0001] This application claims priority from US Provisional Patent Application No. 63 / 573,612, filed April 3, 2024, and entitled EPOXY-COATED BIOREACTORS FOR CELLULAR MANUFACTURING, which is expressly incorporated herein by reference.FIELD OF INVENTION

[0002] The present invention relates to biocompatible epoxy coatings applied to the interior surfaces of two-dimensional or three-dimensional vessels, enabling their use as multi-use bioreactors. These biocompatible epoxy-coated bioreactors are suitable for cellular manufacturing environments.BACKGROUND OF THE INVENTION

[0003] The bioreactor vessel plays a pivotal role in cellular manufacturing by offering a controlled environment for cell development and growth. This environment can be either two-dimensional (2D), where cell growth is typically limited to a single plane, or three-dimensional (3D), where the cells are typically grown within a growth liquid or media. The choice of 2D or 3D bioreactor is tailored to support the specific needs of cellular cultivation.

[0004] The selection of materials for constructing bioreactor vessels is crucial, with biocompatibility and sterility being the primary considerations. This has led to the identification of optimal materials suited for different bioreactor sizes.

[0005] For benchtop applications, where volumes range from 100 milliliters to 25 liters, glass is the preferred material due to its cost-effectiveness and ability to withstand repeated sterilization through high-temperature steam without degradation. However, its fragility limits its use to vessels smaller than 100 liters. Thermoplastics offer a versatile volume range like glass, with the additional advantage of lining larger rigid vessels. Standalone thermoplastic vessels range from 10 milliliters to 25 liters, standalone plastic bags (wave bioreactors) range from 100 milliliters to 500 liters, and plastic-lined rigid vessels extend this range up to 1000 liters. Despite their versatility,thermoplastic vessels are predominantly single-use, as they cannot endure multiple cleaning or sterilization cycles, making them cost-prohibitive over time.

[0006] For larger production-scale operations (500 liters and above), 316L stainless steel, a high-quality alloy known for its resistance to carbide precipitation during welding and high temperatures, has become the standard in the industry. It combines durability with the ability to undergo repeated sterilization, supporting large-volume manufacturing. Nonetheless, the high cost associated with 316L stainless steel presents significant challenges for widespread adoption in large-scale applications.

[0007] While smaller bioreactors have access to economical and biocompatible materials, scaling beyond 25 liters introduces financial and technical challenges. Achieving a balance between structural integrity, biocompatibility, reusability, and cost becomes increasingly difficult with scale. Thus, there is a need in the art for novel, cost-effective, and scalable bioreactor vessels that can be sterilized and reused without compromising sterility or cell viability.SUMMARY OF THE INVENTION

[0008] The present invention introduces epoxy coatings as a novel solution for bioreactors by enabling non-traditional structural materials such as aluminum, carbon steel, reinforced polymer composites, and other cost-effective substrates to be used in cellular manufacturing. These materials, while structurally viable, are not inherently suitable for bioreactor applications due to sterility, chemical leaching, and / or cytotoxicity concerns. The application of an epoxy coating overcomes these limitations, creating a functional bioreactor surface that is durable, reusable, and resistant to degradation.

[0009] However, not all epoxies are suitable for use in bioreactors. While epoxies are widely used in industrial applications for their durability, chemical resistance, and adhesion properties, their application in cellular manufacturing has not been explored due to concerns about sterility, chemical leaching, and their effects on cell viability. Some epoxies degrade under sterilization, release toxic compounds into the bioreactor environment, or fail to maintain a stable biological interface. In addition to introducing epoxy-coated bioreactors as a novel concept, this invention defines thekey parameters that enable an epoxy to be biocompatible for use in cell-based manufacturing.

[0010] This invention addresses two critical challenges in the field. First, it provides a novel application of epoxy coatings that allows non-traditional materials to serve as scalable, cost-effective bioreactor vessels. Second, it establishes parameters that determine whether an epoxy is biocompatible, ensuring its suitability for cellular manufacturing. By defining these parameters, this invention enables the selection and application of epoxies that meet the demands of bioreactor environments. The result is an alternative to traditional materials that maintain biocompatibility and reusability while allowing for greater flexibility in bioreactor construction and scalability.

[0011] The present invention relates to multi-use, biocompatible epoxy-coated bioreactors. According to the present invention there is provided a bioreactor comprising (a) a vessel formed of a substrate, and (b) a biocompatible epoxy-coating. The vessel forms a three-dimensional shape, having one or more interior surfaces, and the epoxy coating is disposed on the substrate of at least one interior surface of said vessel.

[0012] In a further embodiment, there is provided a biocompatible epoxy-coated bioreactor, wherein the epoxy coating withstands sterilization in accordance with ISO 11135:2014 standard and / or the ISO 17665:2024 standard.

[0013] In a further embodiment, there is provided a biocompatible epoxy-coated bioreactor, wherein the epoxy coating meets the NSF600 standard.

[0014] In a further embodiment, the epoxy surface can be further functionalized through surface modifications. One such example of surface modifications is to improve the smoothness of the interior surface of the epoxy coating to achieve lower surface roughness.

[0015] In a further embodiment, there is a method of preparing a biocompatible epoxy-coated bioreactor, comprising forming a vessel from a substrate of aluminum, aluminum alloys, carbon steel, carbon steel alloys, stainless steel, or coated carbon steels (including but not limited to zinc-coated, zinc-nickel, and other corrosionresistant steel coatings). The vessel may also be made of thermoplastics or polymercomposites, including but not limited to high-density polyethylene (HDPE), polypropylene (PP), fiberglass-reinforced plastic, fiberglass, reinforced polymer composites, and polyurethane, as well as concrete, ceramics, or mixtures thereof. A biocompatible epoxy coating is adhered to at least part of one interior surface of the substrate of said vessel, preferably at least one interior surface of the substrate of said vessel, and more preferably all interior surfaces of the substrate of said vessel, to obtain a biocompatible epoxy-coated bioreactor.

[0016] In a further embodiment, there is provided a biocompatible epoxy-coated bioreactor wherein the epoxy coating exhibits good adhesion to the substrate, as assessed by ASTM D4541 or D3359. The coating must remain intact, without any detachments, blisters, cracks, or wear, even after prolonged exposure to standard operating conditions and multiple cleaning cycles by way of pasteurization or sterilization.

[0017] In a further embodiment, there is provided a biocompatible epoxy-coated bioreactor wherein the bioreactor’s weight varies by no more than 0-5% (higher or lower) after pasteurization or sterilization. Preferably the bioreactor’s weight varies by no more than 0-3% (higher or lower).

[0018] In a further embodiment, there is provided a biocompatible epoxy-coated bioreactor wherein said bioreactor is capable of withstanding at least 10 pasteurization or sterilization cycles while maintaining structural and chemical integrity.

[0019] In a further embodiment, the cleaning cycle is preferably a sterilization cycle, and the biocompatible epoxy-coated bioreactor is capable of withstanding at least 10 sterilization cycles.

[0020] In a further embodiment, there is provided a biocompatible epoxy-coated bioreactor wherein the epoxy is disposed on all interior surfaces of the vessel.

[0021] In a further embodiment, there is provided a use of a biocompatible epoxy for coating at least one surface of a vessel, said epoxy-coated vessel configured for use as a bioreactor.

[0022] In a further embodiment, the biocompatible epoxy coating must meet or exceed the standards set by ASTM G31 for corrosion resistance, ASTM D543 for chemical resistance, and / or ASTM D1308 for coating durability against chemicals. The biocompatible epoxy coating must withstand exposure to cleaning agents, sterilizing solutions, substances used for operation, such as media and nutrients, and byproducts from bioreactor use without any degradation, such as cracking, blistering, peeling, or loss of adhesion to the substrate. It should retain its integrity after repeated cycles of exposure to chemicals such as alkalis, acids, solvents, and detergents.

[0023] In a further embodiment, the biocompatible epoxy coating must have a cleanable surface with a surface roughness of 63 pin Ra (3.2 pm Ra) or less to prevent harboring bacteria, fungi, or other contaminants.

[0024] In one embodiment, there is a bioreactor comprising: a vessel, and a biocompatible epoxy coating, wherein the vessel forms a three-dimensional shape, an interior volume and one or more interior surfaces, wherein the biocompatible epoxy coating is disposed on at least one interior surface of said vessel, and wherein the biocompatible epoxy coating: a. meets or exceeds the chemical resistance standard as set forth by ASTM D1308 and / or ASTM D543; b. achieves a rating of 10 for adhesion to the substrate per ASTM D6677, achieves a rating of 5 for both methods A or B according to ASTM D3359, achieves at least 1000 psi per testing ASTM D4541 (Protocol 1), and / or achieves a pass according to ASTM D4541 (Protocol 2); c. exhibits few or less blisters, all smaller than size 8, as defined by ASTM D714, after any process directly or indirectly involved in cell manufacturing; d. exhibits a degree of rusting of 10 as defined by ASTM D610 after any process directly or indirectly involved in cell manufacturing;e. exhibits a hardness variation of lower than 1.0% in accordance with ASTM D2240 after any process directly or indirectly involved in cell manufacturing; f. exhibits an absorption rate of 5.0% or less according to ASTM C413 using mold method A and water as a covering, exhibits no alterations form when tested according to ASTM D2247, and / or exhibits no alterations form when tested according to ASTM D870; g. withstands sterilization in accordance with ISO 11135:2014, ISO 14937:2009 and / or ISO 17665:2024; h. ensures that the any products with direct contact with the coating meets or exceeds NSF600; and i. maintains cell viability for cultured cells for at least 3 days.

[0025] In a further embodiment, the vessel is formed of substrate of aluminum, aluminum alloys, carbon steel, carbon steel alloys, stainless steel or coated carbon steels zinc-coated steel, zinc-nickel coated steel, high-density polyethylene (HDPE), polypropylene (PP), fiberglass-reinforced plastic, fiberglass, reinforced polymer composites, polyurethane, concrete, ceramics, or mixtures thereof.

[0026] In yet a further embodiment the bioreactor is a 3D bioreactor.

[0027] In yet a further embodiment the biocompatible epoxy-coating is disposed on at least some portion of each interior surface of the vessel that are in contact with the bioreactor contents.

[0028] In yet a further embodiment the biocompatible epoxy-coating is disposed on the entire portion of each interior surface of the vessel that is in contact with bioreactor contents.

[0029] In yet a further embodiment the epoxy coating is modified to reduce surface roughness.

[0030] In yet a further embodiment the biocompatible epoxy coating has a cleanable and sterilizable surface with a surface roughness of 63 pin Ra (3.2 pm Ra) or less.

[0031] In a different embodiment of the invention, there is provided a method for manufacturing a 2D or 3D bioreactor, comprising: forming a vessel from a substrate, and applying a biocompatible epoxy to at least one inner surface of the vessel that would be in contact with the contents of the bioreactor, wherein the biocompatible epoxy: a. meets or exceeds the chemical resistance standard as set forth by ASTM D1308 and / or ASTM D543; b. achieves a rating of 10 for adhesion to the substrate per ASTM D6677, achieves a rating of 5 for both methods A or B according to ASTM D3359, achieves at least 1000 psi per testing ASTM D4541 (Protocol 1), and / or achieves a pass according to ASTM D4541 (Protocol 2); c. exhibits few or less blisters, all smaller than size 8, as defined by ASTM D714, after any process directly or indirectly involved in cell manufacturing; d. exhibits a degree of rusting of 10 as defined by ASTM D610 after any process directly or indirectly involved in cell manufacturing; e. exhibits a hardness variation of lower than 1.0% in accordance with ASTM D2240 after any process directly or indirectly involved in cell manufacturing; f. exhibits an absorption rate of 5.0% or less according to ASTM C413 using mold method A and water as a covering, exhibits no alterations form when tested according to ASTM D2247, and / or exhibits no alterations form when tested according to ASTM D870; g. withstands sterilization in accordance with ISO 11135:2014, ISO 14937:2009 and / or ISO 17665:2024; h. ensures that the any products with direct contact with the coating meets or exceeds NSF600; and i. maintains cell viability for cultured cells for at least 3 days.

[0032] This summary of the invention does not necessarily describe all features of the invention.BRIEF DESCRIPTION OF THE DRAWINGS

[0033] These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings, wherein:

[0034] FIGURE 1 illustrates a schematic representation of two potential applications for the biocompatible epoxy coating. In one embodiment, the biocompatible epoxy coating is applied in a 2D cell culture system (1). In another embodiment, the biocompatible epoxy coating is applied in a 3D cell culture method (2). Both applications utilize a substrate (3), which is coated with biocompatible epoxy (4), creating a stable and safe surface that contacts the cell culture (5).

[0035] FIGURE 2 shows a schematic representation of a biocompatible epoxy coating (4) applied to a 3D bioreactor vessel (2), in which a substrate (3), has a biocompatible epoxy coating (4) attached to it. Other surfaces, such as an impeller shaft (6) and the impeller (7), may or may not be coated.

[0036] FIGURE 3 shows a schematic representation of a biocompatible epoxy coating applied to a 2D bioreactor (1) in which a substrate (3) made of a thermoplastic material, has a biocompatible epoxy coating (4) attached to all surfaces having direct contact with live cells.

[0037] FIGURE 4 illustrates a schematic representation of a biocompatible epoxy coating applied to a 3D bioreactor (2). In this design, only a portion of the interior surface of the vessel is coated with a biocompatible epoxy coating, as only the coated surface is exposed to cell culture (5). Neither the top of the 3D bioreactor (8), nor the upper region of the interior wall substrate (3) contain a biocompatible epoxy coating. In contrast the lower region of the interior wall of the substrate (3), which is in direct contact with the cells, are coated with a biocompatible epoxy coating (4). Other components, such as the impeller shaft (6) and impeller (7), may or may not be coated.

[0038] FIGURE 5 illustrates the measured weight variations observed over 50 autoclave cycles in biocompatible epoxy-coated aluminum cups. Measurements were taken of epoxy-coated vessels using the following epoxies: Series 21 and 22 Epoxoline™ from TNEMEC, Series 1432 ProPolymer™ from TNEMEC, Pro Industrial™ High-Performance Epoxy from Sherwin-Williams™ and Axis 921 from ResinDesigns™, as described in the within Examples.

[0039] FIGURE 6 presents the measured changes in metabolite concentrations of (a) glucose, (b) ammonia, (c) calcium, (d) potassium, and (e) magnesium, over a four-day period of cell culture, comparing the biocompatible epoxy-coated aluminum cups with a glass vessel, as described in the within Examples.

[0040] FIGURE 7 shows the observed cell viability over a 4-day period of cell culture, comparing the biocompatible epoxy-coated aluminum cups with a glass vessel, as described in the within Examples.

[0041] FIGURE 8 depicts measured cell growth, as cell count per milliliter, over a four-day period of cell culture, again comparing the biocompatible epoxy-coated aluminum cups (using the epoxies Series 21 and 22 Epoxoline™ from TNEMEC, Series 1432 ProPolymer™ from TNEMEC, Pro Industrial™ High-Performance Epoxy from Sherwin-Williams™, and Axis 921 from ResinDesigns™) with a glass vessel, as described in the within Examples.DETAILED DESCRIPTION

[0042] The following description represents a preferred embodiment(s) of the invention and should not be understood as limiting the invention.Definitions

[0043] The following definitions apply to the terms used in this application.

[0044] The term “appropriate diffusion” means that the biocompatible epoxy may, in accordance with ASTM DI 653, present a water vapor transmission rate (WVT) < 25 mg / m2per day when following test method B with a 100 pm thickness layer under high humidity conditions.

[0045] The term “biologically active environment” refers to an environment within a bioreactor that is capable of promoting cell growth / differentiation or the activity of biological processes. The biocompatible epoxy-coated vessels are constructed to minimize chemical leaching and prevent active inhibition of cell growth / differentiation or cellular processes.

[0046] The term “bioreactor” refers to a device or system that supports a biologically active environment. Bioreactors are generally vessels or tanks in which whole cells or cell-free enzymes convert raw materials into biochemical products.

[0047] The term "cell viability" refers to the proportion of live cells in a population that exhibit sustained physiological function, including, but not limited to, metabolic activity, membrane integrity, and the ability to replicate or perform specific biological functions.

[0048] The term “chemical resistance” means that the biocompatible epoxy coating after being tested according to ASTM D543 for distilled water, sodium chloride solution (10%), ethyl alcohol (50%), acetic acid (5%), and ammonium hydroxide (10%), exhibits few or less blisters, all of number 8 or smaller (following ASTM D714), no loss of adhesion (at least 1000 psi in accordance to ASTM D4541), hardness variations smaller than 1.0% in accordance with ASTM D2240, and no weight or dimension variation of 0.5% or larger.

[0049] The term “chemical sterilants” means chemicals that are used to eliminate microbes and biological material. This could include peracetic acid (PAA) (0.2%), glutaraldehyde (>2.4%), ortho-phthalaldehyde (OP A) (0.55%), and hydrogen peroxide (7.5%), without being limited.

[0050] The term “high adhesion to the substrate” means that the biocompatible epoxy coating may achieve a rating of 10 for adhesion to the substrate per ASTM D6677, and / or it may achieve a rating of 5 for both methods A and B according to ASTM D3359. In addition, it may achieve at least 1000 psi per testing ASTM D4541 (Protocol 1) and pass according to ASTM D4541 (Protocol 2). One or more of these test methods can be utilized.

[0051] The term “high water resistance” means that when the biocompatible epoxy coating is subjected to testing according to ASTM D870, the epoxy must present few or less blisters, all of number 8 or smaller (following ASTM D714), no loss of adhesion (at least 1000 psi in accordance to ASTM D4541), and hardness variations of lower than 1.0% in accordance with ASTM D2240.

[0052] The term “low absorption” means the epoxy exhibits an absorption rate of 5.0% or less according to ASTM C413 using mold method A and water as a covering. Higher water absorption rates could result in the biocompatible epoxy absorbing media and other fluids, potentially compromising sterilization efficiency and raising corrosion concerns if the absorbed liquid interacts with the metal substrate.

[0053] The term “negatively affecting the biocompatible epoxy coating” refers to the degradation, cracking, leaching, and / or peeling of the epoxy coating, without being limiting.

[0054] The term “radiation” in the context of sterilization refers to energy that is used to eliminate microbes or biological material. The radiation could be in the form of UV light, gamma rays, or other forms of radiation known in the art to lead to sterilization.

[0055] The term “resistance (or resistant) to humidity” means that the biocompatible epoxy coating, as tested following the ASTM D2247, exhibits few or less blisters, all of number 8 or smaller (following ASTM D714), show no loss of adhesion (at least 1000 psi in accordance to ASTM D4541), a degree of rusting of 10 following ASTM D610 (applicable for metallic substrates), and no occurrence of other special phenomena.ASTM Standards

[0056] The ASTM is an organization that develops and publishes technical standards, covering the procedures for testing and classification of a wide range of materials. ASTM standards are used worldwide. The ASTM standards identified below are specific standards used for determining, for the present invention, the epoxies that meet the desired properties for use in a bioreactor.

[0057] Reference to a specific standard within this application is intended as a reference to the below indicated standard. However, it is understood that these standards are updated over time. While it is unexpected that updates would materially change the nature of the standard, the below reference is intended to be the standard at the time of filing. It is expected that compliance with these standards would also result in compliance with updated versions of these standards.

[0058] All of the below standards are hereby incorporated by reference in their entirety.

[0059] ASTM International. ASTM C413: Standard Test Method for Absorption of Chemical-Resistant Mortars, Grouts, Monolithic Surfacings, and Polymer Concretes. Digital Object Identifier (“DOI”): 10.1520 / C0413-18.

[0060] ASTM International. ASTM D2247: Standard Practice for Testing Water Resistance of Coatings in 100 % Relative Humidity. D01:10.1520 / D2247-15R20.

[0061] ASTM International. ASTM D714: Standard Test Method for Evaluating Degree of Blistering of Paints. D01:10.1520 / D0714-02R17.

[0062] ASTM International. ASTM D4541: Standard Test Method for Pull-Off Strength of Coatings Using Portable Adhesion Testers. DOI:10.1520 / D4541-22.

[0063] ASTM International. ASTM D610: Standard Practice for Evaluating Degree of Rusting on Painted Steel Surfaces. D01:10.1520 / D0610-08R19.

[0064] ASTM International. ASTM D870: Standard Practice for Testing Water Resistance of Coatings Using Water Immersion. DOE10.1520 / D0870-15R20.

[0065] ASTM International. ASTM D2240: Standard Test Method for Rubber Property-Durometer Hardness. DOI: 10.1520 / D2240-15.

[0066] ASTM International. ASTM D543: Standard Practices for Evaluating the Resistance of Plastics to Chemical Reagents. DOE10.1520 / D0543-21.

[0067] ASTM International. ASTM D6677: Standard Test Method for Evaluating Adhesion by Knife. DOE 10.1520 / D6677-18R22.

[0068] ASTM International. ASTM D3359: Standard Test Methods for Measuring Adhesion by Tape Test. D01:10.1520 / D3359-09E02.

[0069] ASTM International. ASTM D1653: Standard Test Methods for Water Vapor Transmission of Organic Coating Films. DOI:10.1520 / D1653-13R21.ISO Standards

[0070] ISO, the International Organization for Standardization, is an organization that develops and publishes a wide range of standards. The ISO standards indicated below are specific standards used for sterilization. Reference to a specific standard within this application is intended as a reference to the below indicated standard. However, it is understood that these standards are updated over time. While it is unexpected that updates would materially change the nature of the standard, the below reference is intended to be the standard at the time of filing. It is expected that compliance with these standards would also result in compliance with updated versions of these standards.

[0071] All of the below standards are hereby incorporated by reference in their entirety.

[0072] ISO 11135:2014: Sterilization of health-care products - Ethylene oxide - Requirements for the development, validation and routine control of a sterilization process for medical devices. Publication date: 2014-07, Edition 2.

[0073] ISO 17665:2024: Sterilization of Health Care Products-Moist Heat- Requirements for the Development, Validation and Routine Control of a Sterilization Process for Medical Devices. (2024). Publication date: 2024-03, Edition 1.

[0074] ISO 14937:2009 - Sterilization of health care products — General requirements for characterization of a sterilizing agent and the development, validation and routine control of a sterilization process for medical devices. (2009). Publication date: 2209- 10, Edition 2.NSF600 Standard

[0075] Bioreactors used to produce products for human use requires stringent safety considerations. These measures are expected to extend to the epoxy coating, ensuring that it does not introduce harmful contaminants into the process environment. NSF600 standards therefore provide a crucial benchmark for selecting coatings that ensure biocompatibility and safety. Epoxy coatings that satisfy NSF600 standards, ensures that all materials in contact with process water do not leach harmful substances, thereby safeguarding the quality and purity of bio-products. As such, the epoxy coating containing epoxy components, hardeners, enhancers, pigments, and any other additives / possible contaminants, shall conform to NSF600 standards.

[0076] This NSF600 standard is entitled “Health Effects Evaluation And Criteria For Chemicals In Drinking Water”. The Standard defines the toxicological review and evaluation procedures for the evaluation of substances imparted to drinking water through contact with drinking water system components (and drinking water additives). It is intended to establish the human health risk, if any, of the substances imparted to drinking water under the anticipated use conditions of the product. With regard to the present invention, this standard was used to ensure that all epoxy components, additives, and / or contaminants remain within the prescribed limits established by the standard. The standard is cited below: a. NSF International. NSF / ANSI / CAN 600 - 2023 - Health Effects Evaluating and Criteria for Chemicals in Drinking Water.

[0077] The NSF600 Standard is hereby incorporated by reference, in its entirety.Description of preferred embodiments

[0078] Embodiments of the present invention relate to biocompatible epoxy-coated bioreactors, which enable the use of non-traditional vessel materials (or substrates) for bioreactor applications. Unlike conventional industrial epoxies, which may degrade under sterilization, leach harmful chemicals, or fail to maintain structural stability, the epoxies described in this invention are characterized by low absorption, high structural and chemical stability, high adhesion to the substrate, chemical resistance, and multi-use durability. The following embodiments illustrate the criteria that definea biocompatible epoxy suitable for repeated sterilization and long-term cell culture applications.

[0079] The present invention also relates to the identification of biocompatible epoxies, suitable for use in coating bioreactors, and their method of application to reactor substrates to form biocompatible epoxy-coated bioreactors.

[0080] In an embodiment of the present invention, there is provided a bioreactor comprising a vessel formed from a substrate (3), coated with a biocompatible epoxy on an interior surface (4), wherein the epoxy coating is capable of enduring multiple sterilizations without degradation.

[0081] A vessel, whether formed as a 2D or 3D bioreactor, is configured to contain an interior volume for housing the contents of the bioreactor. These bioreactor contents will make contact with at least one interior surface of the vessel.

[0082] Epoxy coatings are thermoset polymers known for their mechanical strength, chemical resistance, and adhesion, achieved through the mixing of epoxy with a curing agent (hardener). Central to their functionality are epoxide groups within the resin, which, upon reacting with the hardener, initiate a curing process that transforms the mixture into a solid, durable material. The versatility of epoxy coatings can be further enhanced by the incorporation of various additives, enabling customization for specific applications.

[0083] In reference to Figure 1, an embodiment of the present invention may follow the design of a traditional bioreactor, both as a 2D system (1) or 3D system (2). Examples include a stirred tank, air-lift, vertical wheel, packed bed, bubble column, roller bottle, fluidized bed, and / or hollow fiber reactors, or may be of a unique design, without being limiting. The epoxy may coat any metallic or non-metallic surface of the substrate (3), which would convert that surface or vessel into one that can maintain a biologically active environment, be sterilized, and be durable for multi-use applications. This bioreactor design is advantageous over what is currently known in the art due to the versatility in the material that may be used for the vessel, the cost reduction associated with multi-use bioreactors, and the positive environmental impact of reducing single-use bioreactors.

[0084] As a result of the invention, the vessel can be formed from a variety of potential substrates (3). Unlike conventional bioreactors constructed from materials such as glass, polymers, or 316L stainless steel alloys, the described bioreactors can be manufactured from a broader range of metallic alloys or non-metallic materials, including those that are typically considered unsuitable for traditional bioreactor applications. These metallic alloys or non-metallic materials are selected for their ability to provide the necessary structural support, forming the external shell (substrate) of the bioreactor. These materials could include aluminum, aluminum alloys, carbon steel, carbon steel alloys, stainless steel, or coated carbon steel, including but not limited to zinc-coated, zinc-nickel, and other corrosion-resistant steel coatings. Also food-grade thermoplastics or polymer composites, including but not limited to high-density polyethylene (HDPE), polypropylene (PP), fiberglass- reinforced plastic, fiberglass, reinforced polymer composites, and polyurethane, as well as concrete, ceramics, or mixtures thereof, without being limiting. The use of non-metallic materials offers advantages such as corrosion resistance, light weight, and ease of customization. This versatility in material selection opens possibilities for tailored bioreactor designs to meet specific application requirements while maintaining cost-effectiveness and performance.

[0085] The invention further permits the use of materials as substrate of the vessel of the bioreactor which, without use of the invention, would not be suitable for use in a bioreactor. For example, the vessel of the bioreactor can be manufactured using a substrate (3) comprising: concrete, stainless and carbon steel and their alloys, aluminum and zinc alloys, non-laboratory -grade glass, thermoplastics and their reinforced versions, or combinations thereof (without being limiting). These materials would not be suitable for use as substrates (3) in forming a bioreactor on their own, however through the application of a biocompatible epoxy coating (4) these materials become suitable for use in a bioreactor.

[0086] Figure 2 is a representation of one embodiment of the invention, in the form of a 3D bioreactor system (2). In Figure 2, the substrate (3) is configured to form a vessel, acting as the primary structural support of a 3D bioreactor system (2). The substrate (3) provides the bioreactor with mechanical strength and durability to withstand the internal pressure, weight, and stresses exerted during operation. Thebiocompatible epoxy coating (4) is found on all interior surfaces of the vessel, attached to said substrate (3), and ensures that there is no direct contact between the substrate (3) and the cell culture (5) within the bioreactor. As a result of this epoxy coating, the containment and isolation of the cell culture (5) environment is maintained, thereby minimizing the risk of contamination and chemical reactions caused by or with the substrate (3). A similar embodiment to the above can be carried out in a 2D bioreactor system (1), as shown in Figure 3, wherein the biocompatible epoxy coating (4) is located on only one interior surface of the vessel.

[0087] In a further embodiment of the present invention, the epoxy coating is found on at least one, but not all, of the interior surfaces of the vessel. In a further embodiment, the biocompatible epoxy coating (4) may cover only a portion of a given interior surface. One such embodiment is shown in Figure 4. Whereby the biocompatible epoxy coating (4) is found on some portion of the interior surface, but both the top of the 3D bioreactor (8), and the upper region of the interior surface contain uncoated substrate (3). These surfaces of uncoated substrate (3) are not in contact with the cell culture (5), and therefore coating is unnecessary. Such an embodiment minimizes the required coverage of the epoxy coating, minimizing costs of the epoxy-coated reactor, provided that the remaining uncoated substrate does not prevent sterilization and biocompatibility of the bioreactor.

[0088] Alternative embodiments would include the use of a biocompatible epoxy coating (4) in 2D bioreactor vessels such as flasks, petti dishes, or culture plates.

[0089] The selection of epoxy for coating the vessel must consider both physical and chemical properties to ensure compatibility with the chosen substrate material and biocompatibility with the cultivation process. This compatibility is essential for maintaining the integrity of the cell culture environment within the bioreactor, and ensuring no leaching occurs from the epoxy coating into the content of the vessel.

[0090] In a preferred embodiment of the invention, an epoxy is considered biocompatible if it can withstand repeated use in a bioreactor involving a cleaning process after each use. The cleaning process can be a pasteurization step or sterilization step, and in a preferred embodiment a sterilization step is used.

[0091] One method of determining if an epoxy is biocompatible is determining that there is no material change in weight of the coated vessel after several sterilization cycles. A material increase of weight is considered undesirable as it is believed to represent chemical modification of the epoxy and / or uptake of water. A material decrease of weight is considered undesirable as it is believed to represent a loss of the epoxy coating, or a leaching of epoxy materials.

[0092] Some changes in weight (higher or lower) are expected without negatively impacting the cell culture, such as a result of measurement variability. A change in weight is generally not considered to be material if does not exceed 5% and preferably does not exceed 3%. As such, a biocompatible epoxy coating should exhibit a change in weight (higher or lower) of no more than 0-5%, preferably no more than 0-3%, after a cleaning cycle has been performed and preferably after 10 cleaning cycles have been performed.

[0093] In an embodiment of the invention, an epoxy is considered biocompatible for use in coating a substrate in accordance with the present invention if the epoxy satisfies certain ASTM and ISO standards.

[0094] In an embodiment, an epoxy is considered suitable if it satisfies minimal required results when tested by: ASTM C413, ASTM D2247, ASTM D4541, ASTM D870, ASTM D543, and ASTM D1653. For the proper results interpretation ASTM D714, ASTM D6677, ASTM D3359, and ASTM D610 should be used.

[0095] In an embodiment, an epoxy is considered biocompatible if it satisfies the following standards: a. Withstands at least one of the sterilization methods described by ISO 11135:2014 or ISO 17665:2024, and b. NSF600.

[0096] An epoxy is considered biocompatible for use in the within invention if the epoxy coating exhibits high adhesion to the substrate. This could include achieving a rating of 10 for adhesion to the substrate per ASTM D6677 and / or achieves a rating of 5 for both methods A and B according to ASTM D3359. In addition, the epoxypreferably achieves at least 1000 psi per testing ASTM D4541 (Protocol 1) and pass according to ASTM D4541 (Protocol 2). One or more of these test methods can be utilized to determine epoxy applicability for use in a bioreactor. Adequate adhesion is important in epoxy selection, as any issues with adhesion may result in the peeling of the epoxy from the walls of the bioreactor into the cell culture media.

[0097] An epoxy is considered biocompatible for use in the invention if the epoxy coating can withstand repeated cleaning cycles, such as by way of sterilization. The most common sterilization process is steam sterilization, whereby steam is applied under pressure for a specified time. The typical cycle for plastic materials lasts at least 15 minutes, while the cycle for glass usually extends for 30 minutes. In a preferred embodiment the epoxy coating can withstand steam application for at least 15 minutes, and more preferably at least 30 minutes. In a further preferred embodiment, the temperature of the steam during the cleaning cycle is greater than 70°C, more preferably the temperature is greater than 100°C, and even more preferably the temperature is 121 °C.

[0098] The effective sterilization of equipment using steam has been defined under ISO 17665:2024. The epoxy coating should possess the capability of being sterilized under this standard and exhibit few or less blisters smaller than size 8 under ASTM D714 and maintain adhesion of at least 1000 psi under ASTM D4541.

[0099] Preferably, a biocompatible epoxy also withstands repeated sterilization by other means, including ethylene oxide gas, chemical sterilization, radiation, dry heat, and other sterilization means known in the art. Further preferred biocompatible epoxy coatings are able to withstand repeated sterilization by a combination of sterilization techniques known in the art. The ability to withstand repeated sterilization can be determined through the absence of blisters bigger than size 8 (ASTM D714), and adhesion of at least 1000 psi (ASTM D4541) after one or more sterilization cycles, preferably after 10 or more sterilization cycles, and more preferably after 20 or more sterilization cycles.

[0100] An epoxy is considered biocompatible for use in this invention if the epoxy coating can withstand extended periods in cell culture solution and / or high humidity without any adverse effects on the integrity of the application to thesubstrate. In these cases, the epoxy coating chosen for the bioreactor may exhibit few or less blisters smaller than size 8 (ASTM D714) and maintain adhesion of at least 1000 psi (ASTM D4541) under three conditions: for humidity resistance, as tested by ASTM D2247, with no special phenomena and a rusting degree of 10 for metallic substrates (ASTM D610); for water immersion, tested according to ASTM D870, requiring a hardness variation below 1.0% (ASTM D2240); and for chemical resistance against distilled water, 10% sodium chloride, 50% ethyl alcohol, 5% acetic acid, and 10% ammonium hydroxide, as specified by ASTM D543, showing no significant weight or dimension changes over 2%, alongside the consistent standards for blisters and adhesion across these specific ASTM tests.

[0101] An epoxy is considered biocompatible for use in the within invention if the epoxy coating of a bioreactor vessel meets all or some of the NSF600 standards and conditions. Further, the epoxy may possess some or all of the following properties to be a suitable epoxy coating for a bioreactor: ability to be sterilized, have low absorption, resistance to humidity, high water resistance, chemical resistance, high adhesion to the substrate, resistance to delaminaton, and appropriate diffusion properties, without being limiting.

[0102] The proper application of the biocompatible epoxy coating onto the interior walls of the substrate ensures the containment and protection of biological materials within the system. The uniformity of the epoxy covering and the strength of adherence to the substrate are two factors in obtaining an adequate coating. Prior to application of the epoxy, the surface of the bioreactor’s interior walls undergoes thorough preparation, which may include sanding, and cleaning of any contaminants or debris that could impair adhesion. Subsequently, the epoxy coating mixture is prepared, considering variables such as curing time, humidity and temperature as appropriate for the selected epoxy. The preferred epoxy coating thickness can be readily determined based upon the specific properties of the epoxy, such as viscosity and the volume of solids, to ensure that full coverage of the substrate can be obtained with a uniform epoxy surface.

[0103] The epoxy is applied to the prepared surface using a suitable application method. Examples of application methods include but are not limited to air spraying, airless spraying, rolling, or brushing. The epoxy is applied to achieve thedesired thickness and coverage. Application of the epoxy preferably results in uniform application and complete coverage of the interior walls to prevent any areas of weakness or vulnerability. Once applied, the epoxy coating is allowed to cure and bond to the substrate, forming a durable and protective barrier against moisture, chemicals, and biological agents. The bioreactor vessel is then cleaned and sterilized before liquid culture is introduced to the system.

[0104] In a further embodiment, the epoxy surface can be functionalized through surface modifications to reduce the surface roughness of the interior surface of the epoxy coating. Applying a glass coating, such as enamel, can lower surface roughness, making it suitable for applications that require extremely low surface roughness, such as the pharmaceutical industry which demands an Ra of around 0.25 pm. In comparison, the dairy industry typically requires an Ra of around 0.8 pm. Such application methods are known in the art.

[0105] In a preferred embodiment of the present invention, a 2D or 3D bioreactor vessel contains at least one interior surface covered by an epoxy coating, whereby said epoxy coating is considered biocompatible as it satisfies the following standards: a. the epoxy coating meets or exceeds the chemical resistance standard as set forth by ASTM D1308 and / or ASTM D543; b. the epoxy coating achieve a rating of 10 for adhesion to the substrate per ASTM D6677, or a rating of 5 for both methods A or B according to ASTM D3359, or achieve at least 1000 psi per testing ASTM D4541 (Protocol 1) or pass according to ASTM D4541 (Protocol 2); c. the epoxy coating, after any process directly or indirectly involved in cell manufacturing, exhibits few or less blisters, all smaller than size 8, as defined by ASTM D714; d. the epoxy coating, after any process directly or indirectly involved in cell manufacturing, exhibits a degree of rusting of 10 as defined by ASTM D610 (applicable for metallic substrates);e. the epoxy coating, after any process directly or indirectly involved in cell manufacturing, exhibits a hardness variation of lower than 1.0% in accordance with ASTM D2240; f. the epoxy coating exhibits an absorption rate of 5.0% or less according to ASTM C413 using mold method A and water as a covering, and / or no alterations form when tested according to ASTM D2247 or ASTM D870; g. the epoxy coating withstands sterilization in accordance with the following standards regarding a cleaning process: ISO 11135:2014, ISO 14937:2009 and / or ISO 17665:2024; h. the epoxy coating meets or exceeds the leaching standard set forth by NSF600; and i. the epoxy coating maintains cell viability for cultured cells for at least 3 days (as determined by standard methods known in the art).

[0106] In a further preferred embodiment, the substrate used to form the 2D or 3D bioreactor, and to which a biocompatible epoxy is applied, is aluminum.

[0107] In a further preferred embodiment, the method is used to create a 2D or 3D bioreactor from an aluminum substrate. In a further preferred embodiment, the bioreactor is a 3D bioreactor, and all surfaces in contact with the cell media are coated with epoxy.

[0108] In a preferred embodiment a suitable biocompatible epoxy coating is formed from the application of Series 21 Epoxoline™ from TNEMEC (NSF600 extraction requirement compliant) to at least one interior surface of a vessel.

[0109] In another preferred embodiment a suitable biocompatible epoxy coating is formed from the application of Series 22 Epoxoline™ from TNEMEC (NSF600 extraction requirement compliant) to at least one interior surface of a vessel.

[0110] In a further preferred embodiment, the substrate of the vessel is aluminum, and in a further preferred embodiment all interior surfaces of the vessel are epoxy coated.

[0111] In an embodiment of the present invention, the biocompatible epoxy-coated bioreactor is capable of providing an environment that allows for the growth, differentiation, and / or cultivation of prokaryotic and / or eukaryotic cells, such as, but not limited to, bacterial, fungal, plant, and mammalian cells. The epoxy-coated bioreactor may also provide an adequate environment for virus replication.

[0112] In certain embodiments, the biocompatible epoxy-coated bioreactor provides an environment for cell-free biochemical reactions. In further embodiments, these biocompatible epoxy-coated bioreactors may be utilized in the production of cellular products and / or cell-as-products. For example, a bioreactor system can support repeated cultivation of cells grown in suspension or in adherent cultures for cell-based meat, bacterial fermentation, or CHO cell for protein production.

[0113] In particular embodiments of the present invention, the epoxy coating is compatible with air, liquid, or air-liquid interfaces for cell cultivation.

[0114] Epoxy-coated vessels in accordance with this invention permit the preparation of bioreactor vessels ranging from 100 milliliters to 10,000,000 liters in size, preferably ranging from 100 liters to 100,000 liters. In preferred embodiments, the use of an epoxy-coating in accordance with this invention permits the preparation of bioreactor vessels of 25 liters or more, more preferably 100 liters or more, more preferably 500 liters or more, and more preferably 1000 liters or more.

[0115] The present invention will be further illustrated through the following examples.Example 1 - Identification of biocompatible epoxy-coatingsMaterials and MethodsEpoxy Selection

[0116] Six different types of epoxies were evaluated to assess their potential as biocompatible coatings for cellular manufacturing vessels. The tested epoxies were: a. Axis 921 from ResinDesigns™ b. Epoxibond™ EB-153 from Epoxy Set Inc. c. Pro Industrial™ High-Performance Epoxy from Sherwin-Williams™ d. Series 1432 ProPolymer™ from TNEMEC e. Series 21 Epoxoline™ from TNEMEC (NSF600 extraction requirement compliant) f. Series 22 Epoxoline™ from TNEMEC (NSF600 extraction requirement compliant)Sample Preparation

[0117] All the tested materials were 2-part epoxies prepared according to the specifications provided by the manufacturers, including mixing rates and times, curing times and temperature conditions. The epoxies were subsequently applied to the interior surface of anodized aluminum cups, having an interior volume of approximate 15 oz. The application was performed by hand-coating the surfaces with a brush to ensure the deposition of a uniform, thin layer. Each epoxy formulation was applied to two separate cups to allow for reproducibility and consistency in the evaluation process.

[0118] Additionally, nine flat coupons were coated with Series 22 Epoxoline™ from TNEMEC on 3 mm thick 6061 aluminum sheets (3" x 3" in size). The epoxy was applied using a brush, in the same manner as for the cups, to ensure a uniform and thin layer. The coated coupons were then subjected to coating adhesion, surface roughness, and chemical resistance / immersion tests.Steam Sterilization Testing

[0119] One set of the epoxy-coated aluminum cups underwent a total of 50 autoclave cycles, performed in increments of 10 cycles using a Tuttnauer autoclave, model 3870EL. There was a one-hour waiting period between cycles, while they were kept inside the autoclave. After every 10 cycles, the cups were cooled to ambient temperature, weighed, and visually inspected for any signs of coating damage, including cracks, blisters, peeling, color changes or other undesired effects. The autoclave cycles followed a plastic cycle protocol - 121°C (250 °F) with a pressure of 17 psig for 15 minutes, followed by gradual depressurization, in accordance with the recommendations of ISO 17665:2024. This protocol minimized thermal shock, making it suitable for evaluating the performance of coatings under prolonged sterilization conditions.Suspension Cell Culture

[0120] A separate set of cups was used to evaluate the biocompatibility of the epoxy coatings in regard to their impact on the cell culture. After undergoing a single plastic autoclave cycle, where the sterilization temperature was maintained at 121 °C for 15 minutes at approximately 17 psig, the cups were tested for their impact on cell behavior.

[0121] CultiSpher™ S gelatin microcarriers were selected for cell seeding. These microcarriers were hydrated using phosphate-buffered saline (PBS) and sterilized following the same sterilization cycle as the epoxy-coated vessel. A total of 200 mg of CultiSpher™ S gelatin microcarriers, loaded with approximately 1,600,000 primary porcine skeletal muscle stem cells (SMSCs), were seeded into the epoxy-coated aluminum cups and two controls, a standard 150 ml laboratory borosilicate glass spinner flask and a 304-stainless steel non-coated cup with an interior volume of approximate 15 oz (the same as the epoxy coated cups). Cell seeding was performed using an intermittent agitation method, with a cycle of 3 minutes on and 20 minutes off at 50 rpm. The seeding process was carried out overnight to ensure cell attachment.

[0122] SMSCs were cultivated using microcarriers in both the epoxy-coated anodized aluminum cups and standard laboratory glass spinner flask (control) toassess any potential effects of the coatings on cell behavior. The seeded cells were cultured in SMSC growth media containing 15% FBS (Fisher Chemical™), 1% Glutamax supplement (Thermo Fisher™), 0.02% animal-free fibroblast growth factor (FGF) (Peprotech™), and 0.1% Gentamicin sulphate (Sigma-Aldrich™). The culture conditions were maintained at standard cell culture parameters (38 °C with 5% CO2). The cultivation period lasted four days within the spinner flasks, allowing for the observation of any differences in cell growth, viability, or morphology induced by the epoxy coatings, compared to the standard glass environment.

[0123] The media was changed every other day to provide fresh nutrients and remove waste products. Samples were collected daily for cell viability assessment and metabolite measurements. A Cedex™ Bio HT system (Roche™) was used to measure baseline metabolites in the media. Metabolite measurements included glucose, ammonia, calcium, potassium, and magnesium. Data obtained from the experiments were analyzed statistically to determine any significant differences between the epoxy-coated vessels and the glass spinner flask in supporting SMSCs growth and function. Cell viability was assessed using both a live / dead staining method and the DNA CyQuant™ Cell Proliferation Assay Kit (Thermo Fisher™). A 500 pL sample of microcarriers and cells was harvested, rinsed with PBS, and stained with a solution containing 4.8 pL fluorescein diacetate (FDA) and 30 pL Hoechst solution in 3 mL minimum essential medium (MEM) for 5 minutes at room temperature. After staining, the cells were rinsed with MEM and imaged using an ECHO microscope. Additionally, cell viability was quantified using the DNA CyQuant™ Cell Proliferation Assay Kit. The readings were performed with a CLARIOstar™ Plus plate reader, utilizing an excitation wavelength of 480 nm, emission wavelength of 520 nm, and a 3x3 matrix scan (9 measurements per well) covering the full bottom of the wells with an enhanced dynamic range.Analysis of Leaching from Biocompatible Epoxy Coatings

[0124] To evaluate the potential leaching from biocompatible epoxy coatings into the media, a two-week incubation test using Series 22 Epoxoline™ from TNEMEC epoxy, which showed the best performance in previous analyses, was conducted. Two conditions were tested, a fresh newly coated cup and an epoxy-coated cup that underwent 30 autoclave cycles. 250 ml of MEM were incubated for a 14-day period- T1 - in the biocompatible epoxy-coated cups and in a glass bottle as control, all at 37 °C.The samples were sent to Eurofins Experchem Laboratories Inc. for analysis of Bisphenol-A (BPA), phthalate compounds and TiCh content.Chemical Resistance / Immersion Test

[0125] The chemical resistance / immersion test was conducted according to ASTM D1308 and ASTM D543 standards. Six specimens were immersed in Dulbecco’s Modified Eagle’s Medium (DMEM) at 38.5 °C for a 21-day exposure period. Every week, two specimens were removed from the DMEM bath, rinsed with 70% ethanol, and dried. The specimens were then evaluated for any changes, anomalies, or defects, including visual appearance (color and gloss change), weight change, blistering, and loss of adhesion. The chemical reagent used in this test was DMEM (high glucose with 4500 mg / L glucose, L-glutamine, sodium pyruvate, and sodium bicarbonate). The testing was conducted by Integrity Testing Laboratory Inc.Analysis of Coating Adhesion by Tape Test

[0126] Evaluation of the adhesion of the epoxy coating to the aluminum substrate was conducted per ASTM D3359, Test Method A - X-Cut Tape Test. An X-cut was made through the coating film to the substrate, pressure-sensitive tape (51596 adhesive tape test - 60 oz / in.) was applied over the cut and then removed, and adhesion was assessed qualitatively on a 0 to 5 scale. Samples were evaluated as initial state, after 7, 14 and 21 days of immersion in DMEM. Testing was conducted by Integrity Testing Laboratory Inc.Surface Roughness (Ra) Measurement

[0127] It is known that the epoxy application methods used here are recommended by the manufacturer exclusively for small areas or hard-to-reach surfaces, as the surface roughness is influenced not only by the substrate but also by the application tool, in this case, the brush. Therefore, this test was conducted to assess the maximum acceptable surface roughness, considering that the same method was used for the biocompatibility tests, where no contamination or cell adhesion to the coated layer was detected.

[0128] Surface roughness (Ra) measurements were conducted on the coating surface of the submitted test coupons using a stylus-type (contact) instrument, the Mitutoyo Surftest 301 Surface Roughness Tester. Signal detection was performed using the differential inductance method. The stylus, made of diamond with a tip radius of 5 pm, applied a measuring force of 4 mN (0.4 gf) over an evaluation length of 4 mm. This test was conducted by Integrity Testing Laboratory Inc.Results and DiscussionSample Preparation

[0129] Apart from Epoxibond™ EB-153 from Epoxy Set Inc. all epoxies examined in this study demonstrated the ability to effectively coat the surface of the aluminum cups. These epoxies exhibited strong adhesion to the substrate, with no instances of delamination, blistering, or other adverse effects observed post-curing the Pro Industrial™ High-Performance Epoxy from Sherwin-Williams™ coating had a low viscosity, as it was specially formulated for flat surfaces rather than vertical walls. Although it was able to fully cover the cup walls, the finish was not ideal, exhibiting irregularities in coating thickness and surface roughness. Epoxibond™ EB-153 from Epoxy Set Inc. exhibited a low viscosity that prevented proper adhesion to the lateral surfaces of the cup. This resulted in a dripping behavior during the curing process, leading to a non-uniform coating. As a result, Epoxibond™ EB-153 from Epoxy Set Inc. was excluded from subsequent analysesSteam Sterilization Testing

[0130] Series 21 and 22 Epoxoline™ from TNEMEC, Pro Industrial™ High- Performance Epoxy from Sherwin-Williams™, and Axis 921 from ResinDesigns™ showed no significant weight changes after consecutive autoclave cycles, as shown in Figure 5. The Series 21 Epoxoline™ from TNEMEC coating exhibited a decrease of 1.1%, in weight while the Pro Industrial™ High-Performance Epoxy from Sherwin- Williams™ coating showed a decrease of 0.3%. These reductions may be the result of measurement variability or may be due to the degradation of organic materials, potentially from substances attached to the cup or from the external identification tags added to them. The Series 21 Epoxoline™ from TNEMEC coating exhibited a pronounced yellowing, suggesting that pigmentation degradation may have occurred,which could similarly contribute to the observed weight change. These decreases were not considered detrimental to the biocompatibility of the epoxy for use with the invention.

[0131] On the other hand, the Series 22 Epoxoline™ from TNEMEC coating demonstrated a slight weight increase of 0.1%, and the Axis 921 from ResinDesigns™ coating showed a weight increase of 0.2%. These increases are considered negligible and are likely caused by minor condensation adsorbed on the surface of the coatings. These increases were not considered detrimental to the biocompatibility of the epoxy for use with the within invention.

[0132] The Series 1432 ProPolymer™ coating from TNEMEC exhibited a 2.5% increase in weight. If this increase had been solely due to weight gain, it would not necessarily disqualify the material as a suitable option. However, the weight gain resulted from surface blistering, with blister sizes of 6 and 8, as classified by ASTM D 714 standards, observed after 40 autoclave cycles. The smaller blister appeared to have ruptured during the sterilization process, exposing the underlying substrate, while the larger blister remained intact but was detached from the substrate, though still fully covering it. By failing to meet the criteria for adhesion to the substrate (here ASTM D 714), the Series 1432 ProPolymer™ from TNEMEC coating was not considered to be a biocompatible epoxy for use with the within invention.Suspension Cell Culture

[0133] The metabolite analysis revealed that the glass and stainless-steel controls consumed the most glucose and produced the most ammonia among all the spinner flasks, indicating, as expected, that it supported higher cell production. Results for stainless steel and glass were statistically identical; therefore, the term 'control' will be used to refer to both. The results for the Pro Industrial™ High-Performance Epoxy from Sherwin-Williams™, Series 21 and 22 Epoxoline™ from TNEMEC coatings were the most comparable to the control, as shown in Figure 6. Glucose consumption showed similar levels to the control for the Pro Industrial™ High-Performance Epoxy from Sherwin-Williams™ coating, while the Series 21 and 22 Epoxoline™ from TNEMEC coatings exhibited 5% and 6% variations, respectively. However, ammonia production showed notable discrepancies across the samples. Compared to thecontrol, the Pro Industrial™ High-Performance Epoxy from Sherwin-Williams™, Series 21 and 22 Epoxoline™ from TNEMEC coatings exhibited reductions in ammonia production of 24%, 28%, and 31%, respectively. Calcium, potassium, and magnesium levels showed similar results across all the samples.

[0134] The live-dead assay results indicated that cell viability peaked on day 2, as shown in Figure 7. On day 1, the Pro Industrial™ High-Performance Epoxy from Sherwin-Williams™, Series 21 and 22 Epoxoline™ from TNEMEC coatings demonstrated comparable results to the control. By day 2, the control exhibited higher cell viability, although the results for Series 21 and 22 Epoxoline™ from TNEMEC, and Pro Industrial™ High-Performance Epoxy from Sherwin-Williams™ remained closely aligned. From day 3 onward, cell viability began to decline across all samples. The Axis 921 from ResinDesigns™ coating consistently showed no cell viability from day 1, which correlated with its extremely low glucose consumption and ammonia production. The Series 1432 ProPolymer™ from TNEMEC coating demonstrated some cell viability during the first two days, but this ceased entirely after day 3.

[0135] The DNA assay showed that the control demonstrated consistent cell growth over the four days, indicating favorable conditions for cell proliferation and serving as the baseline for comparison. The Series 21 Epoxoline™ from TNEMEC coating showed slightly higher cell counts than the control, following a similar growth pattern but experiencing a slight decline after day 2, with a final count about 3-9% higher than the control, as shown in Figure 8. Series 22 Epoxoline™ from TNEMEC and Pro Industrial™ High-Performance Epoxy from Sherwin-Williams™ initially outperformed the control by 59% and 51.2% respectively, though this was likely due to a sampling error. Despite fluctuations in growth, Series 22 Epoxoline™ from TNEMEC showed strong overall proliferation by day 4, aligning closely with the control, with just a 0.8% difference. The Pro Industrial™ High-Performance Epoxy from Sherwin-Williams™ results were similar to those of Series 21 Epoxoline™ from TNEMEC, achieving a final cell concentration 11% lower than the control, indicating moderate cell proliferation.

[0136] As an unsuitable example, Series 1432 ProPolymer™ from TNEMEC coating experienced significant decline in cell growth, with reductions of up to 97% incomparison to the control, indicating highly unfavorable conditions for cell proliferation. These results were consistent with the live-dead assay, further confirming the unsuitability of this material as a biocompatible epoxy coating. The same is valid for Axis 921 from ResinDesigns™, which exhibited minimal cell proliferation, with a 96.9% decrease on day 1, confirming its unsuitability for supporting viable cell cultures.Analysis of Leaching from Biocompatible Epoxy Coatings

[0137] The results indicated that biocompatible epoxy coatings, such as Series 22 Epoxoline™ from TNEMEC, do not pose a risk of contaminating the media with harmful substances, whether in their fresh state or after undergoing multiple autoclave cycles. The analysis of BP A, TiCh and phthalate compounds showed no detectable leaching, with all samples presenting levels of <1.0 pg / kg for BP A, <25.1 ppm for TiCh. and <0.05 mg / kg for phthalate compounds, confirming the safety of the Series 22 Epoxoline™ from TNEMEC epoxy coating in bioreactor applications. As a reference, the U.S. Food and Drug Administration (FDA) (2014) sets the safe exposure limit for BPA at <5 mg / kg of body weight per day, while the U.S. Environmental Protection Agency (EP A) establishes a reference dose of 0.02 mg / kg / day for phthalate compounds due to their potential health risks. Additionally, the FDA permits the safe use of TiCE as a color additive in food, provided it meets regulatory specifications, including a maximum concentration of 1% by weight, as outlined in FDA regulations (21 CFR 73.575). Based upon this, it is expected that these epoxy coatings would satisfy the leaching standard set at NSF600 standard.Chemical Resistance / Immersion Test

[0138] Two specimens were removed every 7 days for evaluation during the 21 -day chemical resistance / immersion test. No changes in visual appearance (color and gloss), no weight variation, no blistering, and no loss of adhesion were observed on any of the test specimens following the chemical resistance / immersion test.Analysis of Coating Adhesion by Tape Test

[0139] The results of the coating adhesion test using the X-cut tape method showed no peeling or removal of the coating on any of the Series 22 Epoxoline™ fromTNEMEC test coupons, neither in their as-received condition and after exposures to DMEM immersion. All coupons received a 5 A classification.Surface Roughness (Ra) Measurement

[0140] From the nine prepared test coupons, three were randomly selected for roughness measurements. The roughness average (Ra) parameter was calculated based on the height deviations within the evaluation length. The results showed an average Ra of 3.2 pm on the coating surface of the test coupons. While this value is notably higher than the standard roughness for sanitary surfaces, which is typically around 0.8 pm (32 pin), it is important to note that this value should be considered as an upper limit for smaller coated areas. For larger surfaces, it is essential to apply the epoxy according to the manufacturer’s recommended procedure (for Series 22 Epoxoline™ - using a spray gun with an atomizing pressure of at least 5500 psi).Following this method will significantly reduce the surface roughness, ensuring better sanitization and compliance with sanitary standards

[0141] Ultimately, Series 22 Epoxoline™ from TNEMEC, and Series 21 Epoxoline™ from TNEMEC are both suitable as biocompatible epoxy coatings for cellular manufacturing applications, as they satisfy standards for adhesion to the aluminum substrate; do not react to the cell culture media, do not leach material into the cell culture, maintained integrity after sterilization cycles, and permitted at least 3 days of cell viability.

[0142] Series 22 is considered the most suitable, as it offers less variation in cell growth compared to the control than other tested epoxies. Pro Industrial™ High- Performance Epoxy by Sherwin-Williams™ also demonstrated good biocompatibility. However, its low viscosity limits its application to coating horizontal planar surfaces.

[0143] In contrast the following epoxies were found to not be biocompatible for use in accordance with the invention: a. Axis 921 from ResinDesigns™ due to the lack of 3-days cell viability,b. Epoxibond™ EB-153 from Epoxy Set Inc. due to the inability to the required standards for adherence to the interior surfaces of the bioreactor, and c. Series 1432 ProPolymer™ from TNEMEC due to the lack of 3-days cell viability, and due to the failure to adhere to the substrate.

[0144] All references and standards referred to in this application are hereby incorporated in their entirety.

[0145] The present invention has been described with regard to one or more embodiments. However, it will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims.

Claims

WHAT IS CLAIMED IS:

1. A bioreactor comprising:(a) a vessel, and(b) a biocompatible epoxy coating; wherein the vessel forms a three-dimensional shape, an interior volume and one or more interior surfaces; wherein the biocompatible epoxy coating is disposed on at least one interior surface of said vessel; and wherein the biocompatible epoxy coating: a. meets or exceeds the chemical resistance standard as set forth by ASTM D1308 and / or ASTM D543; b. achieves a rating of 10 for adhesion to the substrate per ASTM D6677, achieves a rating of 5 for both methods A or B according to ASTM D3359, achieves at least 1000 psi per testing ASTM D4541 (Protocol 1), and / or achieves a pass according to ASTM D4541 (Protocol 2); c. exhibits few or less blisters, all smaller than size 8, as defined by ASTM D714, after any process directly or indirectly involved in cell manufacturing; d. exhibits a degree of rusting of 10 as defined by ASTM D610 after any process directly or indirectly involved in cell manufacturing; e. exhibits a hardness variation of tower than 1.0% in accordance with ASTM D2240 after any process directly or indirectly involved in cell manufacturing; f. exhibits an absorption rate of 5.0% or less according to ASTM C413 using mold method A and water as a covering, exhibits no alterations form when tested according to ASTM D2247, and / or exhibits no alterations form when tested according to ASTM D870;g. withstands sterilization in accordance with ISO 11135:2014, ISO 14937:2009 and / or ISO 17665:2024; h. ensures that the any products with direct contact with the coating meets or exceeds NSF600; and i. maintains cell viability for cultured cells for at least 3 days.

2. The bioreactor of claim 1, wherein the vessel is formed of substrate of aluminum, aluminum alloys, carbon steel, carbon steel alloys, stainless steelor coated carbon steels zinc-coated steel, zinc-nickel coated steel, high-density polyethylene (HDPE), polypropylene (PP), fiberglass-reinforced plastic, fiberglass, reinforced polymer composites, polyurethane, concrete, ceramics, or mixtures thereof.

3. The bioreactor of claims 1 or 2, wherein the bioreactor is a 3D bioreactor.

4. The bioreactor of any one of claims 1 to 3, wherein the biocompatible epoxycoating is disposed on at least some portion of each interior surface of the vessel that are in contact with the bioreactor contents.

5. The bioreactor of claim 4, wherein the biocompatible epoxy-coating is disposed on the entire portion of each interior surface of the vessel that is in contact with bioreactor contents.

6. The bioreactor of any one of claims 1 to 5, wherein the epoxy coating is modified to reduce surface roughness.

7. The bioreactor of any one of claims 1 to 6, wherein the biocompatible epoxy coating has a cleanable and sterilizable surface with a surface roughness of 63 pin Ra (3.2 pm Ra) or less.

8. A method for manufacturing a 2D or 3D bioreactor, comprising: a. forming a vessel from a substrate, and b. applying a biocompatible epoxy to at least one inner surface of the vessel that would be in contact with the contents of the bioreactor, wherein the biocompatible epoxy: a. meets or exceeds the chemical resistance standard as set forth by ASTM D1308 and / or ASTM D543; b. achieves a rating of 10 for adhesion to the substrate per ASTM D6677, achieves a rating of 5 for both methods A or B according to ASTM D3359, achieves at least 1000 psi per testing ASTM D4541 (Protocol 1), and / or achieves a pass according to ASTM D4541 (Protocol 2); c. exhibits few or less blisters, all smaller than size 8, as defined by ASTM D714, after any process directly or indirectly involved in cell manufacturing; d. exhibits a degree of rusting of 10 as defined by ASTM D610 after any process directly or indirectly involved in cell manufacturing; e. exhibits a hardness variation of lower than 1.0% in accordance with ASTM D2240 after any process directly or indirectly involved in cell manufacturing; f. exhibits an absorption rate of 5.0% or less according to ASTM C413 using mold method A and water as a covering, exhibits no alterations form when tested according to ASTM D2247, and / or exhibits no alterations form when tested according to ASTM D870; g. withstands sterilization in accordance with ISO 11135:2014, ISO 14937:2009 and / or ISO 17665:2024; h. ensures that the any products with direct contact with the coating meets or exceeds NSF600; and i. maintains cell viability for cultured cells for at least 3 days.

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