Biodegradable multilayer film comprising outer layer and core layer for wall of disposable bag

Abstract

The present disclosure relates to a biodegradable multilayer film comprising stacked on each other in the following order: a) a first outer layer comprising a first polymer selected from the group consisting of poly (butylene adipate-co-terephthalate) (PBAT), poly (butylene succinate) (PBS), poly (butylene succinate-co-adipate) (PBSA), and mixtures thereof; b) a core layer comprising a blend of thermoplastic starch (TPS) and polybutylene adipate terephthalate (PBAT); c) a second outer layer comprising a second polymer selected from the group consisting of poly (butylene adipate-co-terephthalate) (PBAT), poly (butylene succinate) (PBS), poly (butylene succinate-co-adipate) (PBSA), and mixtures thereof. The present disclosure also relates to a disposable bag, a bioreactor and a container suitable for mixing, packaging, storing or transporting biopharmaceutical substances, wherein the disposable bag, bioreactor and container comprise the biodegradable multilayer film.

Description

Technical Field

[0001] This disclosure relates to a biodegradable multilayer membrane that can be used to manufacture single-use bags for use as bioreactors or in bioprocesses, or for manufacturing containers for mixing, packaging, storing or transporting, particularly for preparing, storing or transporting biopharmaceutical fluids, and a method for manufacturing such a multilayer membrane.

[0002] This disclosure also relates to a disposable bag made from the biodegradable multilayer film and a method for manufacturing the same. Background Technology

[0003] Biopharmaceuticals are drugs produced using molecular biology methods in biotechnology processes. Unlike synthetic drugs, which are products of chemical processes, biopharmaceuticals are produced in living cells. Biopharmaceuticals, such as biological products, include, for example, vaccines, therapeutic proteins, blood and blood components, tissues, etc. Compared to small-molecule chemically synthesized drugs with well-defined structures, biological products are derived from living materials (human, animal, microorganism, or plant) and are structurally larger and more complex. Currently, most commercially available biopharmaceuticals contain recombinant proteins as their active pharmaceutical ingredient. These proteins are typically produced in prokaryotic systems, primarily *E. coli*, or fungal-based eukaryotic systems (*Saccharomyces cerevisiae* and *Pichia pastoris*), mammalian cells, or insect cell lines.

[0004] Single-use or disposable technologies, especially single-use bags, are widely used in the manufacture of biopharmaceuticals because they avoid cleaning reusable containers or other equipment that comes into contact with the product, thus preventing cross-contamination in multi-product manufacturing plants.

[0005] Single-use bags can be in the form of 2D or 3D flexible bags (D stands for demensions). 2D bags are made by sealing two pieces of film together, while 3D bags are typically made by sealing at least three pieces of film together. Single-use bags can have volumes up to 3000 liters or more. 2D and 3D single-use bags are, for example, trademarked by Sartorius. and Sales. 2D and 3D disposable bags are available for use in a variety of bioreactors:

[0006] - A stirred tank bioreactor in which a 3D bag is mounted within a cylindrical frame to secure the bag, and a top-driven impeller is placed inside the bag to mix and agitate the biopharmaceutical fluid contained within the bag.

[0007] - A shaking or wave-mixing bioreactor in which 2D bags are mounted on a shaking platform to provide agitation and gas transfer.

[0008] - An orbital oscillating bioreactor in which a 3D bag is placed on a platform capable of circular horizontal movement.

[0009] This causes the fluid inside the bag to undergo orbital motion.

[0010] The wall of a single-use bag can be composed of multiple layers of film, including a core layer sandwiched between two outer layers. The outer layers can be in direct contact with the core layer. Alternatively, an adhesive layer can be disposed between the core layer and the outer layers.

[0011] If the bag is to contain biopharmaceutical products, the outer layer that comes into contact with the biopharmaceutical products should be made of a material that will not cause degradation of the biopharmaceutical products upon contact. Furthermore, it must be self-sealing. For this purpose, the material is typically selected from polyolefins, such as polyethylene.

[0012] The core layer provides a barrier to the passage of gases such as oxygen and carbon dioxide. The core layer is typically made of ethylene-vinyl alcohol copolymer (EVOH).

[0013] The outer layer, which comes into contact with the external environment, contributes to the mechanical strength of the bag wall. For this, it must be flexible enough to withstand high mechanical stresses. However, it should not be overly stretchable to prevent the bag from deforming when filled with fluid, especially large volumes of fluid.

[0014] In the field of bioreactors, as we have previously mentioned, there are various types of bioreactors. Their main difference lies in their mixing and agitation methods, which may involve, for example, oscillating motion (shaking motion), orbital motion, or axial motion. Therefore, the mechanical stress between different systems can vary considerably. For the transport of large volumes of liquids, the mechanical stress can also be very high. In both of these applications, membranes unable to withstand these mechanical stresses may rupture and leak product out of the bag.

[0015] Therefore, the films used to manufacture 2D or 3D bags must be able to withstand a variety of mechanical stresses to suit a wide range of applications, such as bioreactors suitable for any stirring mode, or for storing or transporting fluids, or for mixing fluids or fluids with powders. These bags must also be suitable for small or large volumes.

[0016] Furthermore, in the biopharmaceutical field, bags must be sterilized before use. Sterilization is typically performed using gamma or X-ray irradiation. Therefore, the materials used in the film that makes up the bag must be able to withstand gamma or X-ray radiation without degrading the film's physical properties.

[0017] Traditional bags are made of polymers, such as polyolefins (e.g., polyethylene, polypropylene) or copolymers containing ethylene units and, for example, acrylic acid or vinyl alcohol. These polymers or copolymers are derived from fossil fuels, which are non-renewable and unsustainable raw materials. Furthermore, bags made from these raw materials are neither compostable nor biodegradable. Moreover, because these bags are used to process, store, or transport biopharmaceutical fluids that are highly sensitive to contamination, the likelihood of reusing them is quite low. Therefore, most bags are used only once; they are single-use bags. Consequently, the vast majority of these bags end up in landfills or incinerators. In addition, the multilayer membranes of traditional bags may contain compounds that raise concerns due to the risk of migration into biopharmaceutical fluids.

[0018] Therefore, a multilayer membrane suitable for manufacturing 2D or 3D bags is needed. This membrane is biodegradable and capable of withstanding the mechanical stresses experienced by 2D and 3D bags during use as bioreactors, mixing containers, packaging containers, storage containers, or transport containers. The multilayer membrane is also expected to have little or no effect on cell growth of cultures after sterilization by gamma or X-ray irradiation. The membrane should not release significant amounts of degradation compounds, such as those that interfere with cell growth after gamma or X-ray irradiation. Simultaneously, the biodegradable multilayer membrane should have a bio-based carbon content of at least 20%. Summary of the Invention

[0019] Traditional bio-based and / or biodegradable polymer films have limited mechanical and barrier properties, making them unsuitable for bioprocessing, mixing, packaging, storage, or transport applications in 2D or 3D bags.

[0020] The multilayer film disclosed herein overcomes these limitations and is therefore suitable for biopharmaceutical packaging and bioprocessing applications.

[0021] Through extensive testing, the applicant of this application has found that multilayer films based on thermoplastic starch and certain bio-based polyesters are biodegradable and meet the requirements for single-use bioreactors or bioprocess bags (robustness, flexibility, sealing strength, no leaching compounds), as well as the requirements for containers for mixing, packaging, storage or transport.

[0022] In one aspect, this disclosure provides a biodegradable multilayer membrane comprising layers stacked on top of each other in the following order:

[0023] a) A first outer layer comprising a first polymer selected from the group consisting of polybutylene terephthalate (PBAT), polybutylene succinate (PBS), polybutylene succinate-adipate (PBSA), and mixtures thereof.

[0024] b) Core layer, comprising a blend of thermoplastic starch (TPS) and polybutylene terephthalate (PBAT);

[0025] c) A second outer layer comprising a second polymer selected from the group consisting of polybutylene terephthalate (PBAT), polybutylene succinate (PBS), polybutylene succinate-adipate (PBSA), and mixtures thereof.

[0026] In one implementation, the first outer layer is in direct contact with the core layer, and the second outer layer is in direct contact with the core layer.

[0027] In one embodiment, the multilayer film further includes one or more adhesive layers between the first outer layer and the core layer, and between the second outer layer and the core layer.

[0028] The one or more adhesive layers may comprise a blend of thermoplastic starch (TPS) and a polymer selected from the group consisting of polybutylene terephthalate (PBAT), polybutylene succinate (PBS), polybutylene succinate-adipate (PBSA), and mixtures thereof.

[0029] In one aspect, this disclosure provides a method for manufacturing a biodegradable multilayer film according to any one of claims 1 to 16, comprising the following steps:

[0030] - Introduce and melt a blend of thermoplastic starch (TPS) and polybutylene terephthalate (PBAT) into the first extruder to form a core laminar flow;

[0031] - Introducing and melting the first polymer into the second extruder to form a first outer laminar flow;

[0032] - Introduce and melt the second polymer into the third extruder to form a second outer laminar flow;

[0033] Alternatively, if the first polymer and the second polymer are the same, the second extruder is connected to the feed block to form a first outer laminar flow and a second outer laminar flow;

[0034] - Introduce the core laminar flow, the first outer laminar flow, and the second outer laminar flow into the feed block to form a three-layer melt flow;

[0035] - Introduce the three-layer melt flow into the die head to form a multilayer film.

[0036] In another aspect, this disclosure provides a disposable bag whose wall comprises a multilayer film of this disclosure.

[0037] In another aspect, this disclosure provides a bioreactor comprising the disposable bag of this disclosure.

[0038] In another aspect, this disclosure provides a container suitable for mixing, packaging, storing or transporting biopharmaceutical substances, which includes the single-use bag of this disclosure.

[0039] The advantages of the multilayer membrane disclosed herein lie in its ability to meet a wide range of mechanical performance requirements. This multilayer membrane is biodegradable and bio-based. Furthermore, it has little or no effect on cell growth after sterilization by gamma irradiation. Therefore, it is particularly suitable for 2D or 3D bags used for the preparation, storage, or transportation of biopharmaceutical fluids (e.g., cell culture media). The multilayer membrane disclosed herein is particularly suitable for the production of bags used as bioreactors or as containers for mixing, packaging, storage, or transportation.

[0040] The following projects are disclosed in this article.

[0041] (1) A biodegradable multilayer membrane comprising layers stacked on top of each other in the following order:

[0042] a) A first outer layer comprising a first polymer selected from the group consisting of polybutylene terephthalate (PBAT), polybutylene succinate (PBS), polybutylene succinate-adipate (PBSA), and mixtures thereof.

[0043] b) Core layer, comprising a blend of thermoplastic starch (TPS) and polybutylene terephthalate (PBAT);

[0044] c) A second outer layer comprising a second polymer selected from the group consisting of polybutylene terephthalate (PBAT), polybutylene succinate (PBS), polybutylene succinate-adipate (PBSA), and mixtures thereof.

[0045] (2) The biodegradable multilayer membrane according to (1), wherein the first outer layer is in direct contact with the core layer and the second outer layer is in direct contact with the core layer.

[0046] (3) The biodegradable multilayer membrane according to (1), wherein the multilayer membrane further comprises one or more adhesive layers between the first outer layer and the core layer and between the second outer layer and the core layer.

[0047] (3a). The biodegradable multilayer film according to (3), wherein one or more adhesive layers comprise a blend of thermoplastic starch (TPS) and a polymer selected from the group consisting of polybutylene terephthalate (PBAT), polybutylene succinate (PBS), polybutylene succinate-adipate (PBSA), and mixtures thereof.

[0048] (4). A biodegradable multilayer film according to any one of (1) to (3a), wherein the blend of thermoplastic starch (TPS) and polybutylene terephthalate (PBAT) in the core layer comprises TPS and PBAT in a weight ratio of 60:40 to 40:60, preferably from 55:45 to 45:55, more preferably 50:50.

[0049] (5). The biodegradable multilayer film according to any one of (3a) to (4), wherein the thermoplastic starch (TPS) of the one or more adhesive layers and the blend of a polymer selected from the group consisting of polybutylene terephthalate (PBAT), polybutylene succinate (PBS), polybutylene succinate-adipate (PBSA) and mixtures thereof comprise TPS and the polymer in a weight ratio of from 60:40 to 20:80, more preferably from 45:55 to 40:60.

[0050] (6). The biodegradable multilayer membrane according to (5), wherein the polymer of one or more adhesive layers is PBAT.

[0051] (7) A biodegradable multilayer film according to any one of (1) to (6), wherein the thermoplastic starch (TPS) comprises 15 to 45% by weight, preferably 22 to 35%, more preferably 25 to 35% by weight of a plasticizer based on the weight of the thermoplastic starch.

[0052] (8). The biodegradable multilayer film according to (7), wherein the plasticizer is selected from the group consisting of polyols or amides and mixtures thereof, and water.

[0053] (9) The biodegradable multilayer film according to (7) or (8), wherein the plasticizer is selected from the group consisting of glycerol, sorbitol, ethylene glycol, urea, formamide and mixtures thereof, and water; preferably glycerol and water.

[0054] (10). A biodegradable multilayer film according to any one of (1) to (9), wherein the thermoplastic starch (TPS) comprises starch selected from the group consisting of corn starch, tapioca starch, cassava starch, wheat starch, natural wheat starch, potato starch, rice starch, sorghum starch, seaweed starch and mixtures thereof, preferably natural wheat starch.

[0055] (11). A biodegradable multilayer film according to any one of (1) to (10), wherein the first outer layer and / or the second outer layer comprises polybutylene terephthalate (PBAT).

[0056] (12). The biodegradable multilayer film according to (6) or (11), wherein the polybutylene adipate terephthalate (PBAT) comprises 55 to 45 mol%, preferably 53 to 47 mol%, of butylene adipate units and 45 to 55 mol%, preferably 47 to 53 mol%, of butylene terephthalate units.

[0057] (13). The biodegradable multilayer film according to (6) or (11) or (12), wherein the polybutylene terephthalate (PBAT) has a melting point of 105 to 125 degrees Celsius, preferably 110 to 120 degrees Celsius, as measured by DSC (ISO) according to ASTM E793-06.

[0058] (14). A biodegradable multilayer film according to any one of (6) or (11) to (13), wherein the melt volume flow rate of the polybutylene terephthalate (PBAT) is 2.0 to 5.0 ml / 10 min, preferably 2.5 to 4.5 ml / 10 min, as measured according to ISO 1133, 190 degrees Celsius and 2.16 kg.

[0059] (15.) A biodegradable multilayer film according to any one of (6) or (11) to (14), wherein the melt flow index of the polybutylene terephthalate (PBAT) is 2 to 6 g / 10 min, preferably 2.7 to 4.9 g / 10 min, as measured according to ISO 1133, 190 degrees Celsius and 2.16 kg.

[0060] (16.) A biodegradable multilayer film according to any one of (6) or (11) to (15), wherein the polybutylene adipate terephthalate (PBAT) has a number-average molecular weight of 10 to 70, preferably 20 to 60, more preferably 30 to 50 kg / mol as measured by size exclusion chromatography.

[0061] (17). A biodegradable multilayer film according to any one of (6) or (11) to (16), wherein the polybutylene terephthalate adipate (PBAT) has a dispersion of 1.5 to 2.5, preferably 1.6 to 2.3, more preferably 1.8 to 2.2, as measured by size exclusion chromatography.

[0062] (18). A biodegradable multilayer film according to any one of (1) to (17), wherein the ultimate tensile strength of the polybutylene terephthalate (PBAT) extruded into a single layer is 14 to 20 MPa in the machine direction (MD) and 12 to 15 MPa in the transverse direction (CD).

[0063] (19). A biodegradable multilayer film according to any one of (1) to (18), wherein the elongation at break of the polybutylene terephthalate (PBAT) extruded into a single layer is 350 to 800% in the machine direction (MD) and 500 to 700% in the transverse direction (CD).

[0064] (20). A biodegradable multilayer membrane according to any one of (1) to (19), wherein the core layer further comprises a compatibilizer.

[0065] (21). The biodegradable multilayer film according to (20), wherein the compatibilizer is selected from the group consisting of maleic anhydride grafted polyester, polyvinyl acetate, and copolymers comprising styrene monomer and epoxy-functionalized (meth)acrylate or (meth)acrylate monomer.

[0066] (22). The biodegradable multilayer film according to (21), wherein the compatibilizer is a copolymer comprising styrene monomer and epoxy-functionalized (meth)acrylate or (meth)acrylate monomer.

[0067] (23). A biodegradable multilayer film according to any one of (20) to (22), wherein the compatibilizer content is 0.05 to 0.7 phr, 0.1 to 0.6 phr or 0.2 to 0.5 phr, preferably 0.1 to 0.3 phr, wherein each hundred parts by weight is based on the blend of TPS and PBAT.

[0068] (24). A biodegradable multilayer film according to any one of (1) to (23), wherein the biodegradable multilayer film contains no additives other than plasticizers and compatibilizers.

[0069] (25). A biodegradable multilayer film according to any one of (1) to (24), wherein the thickness of the biodegradable multilayer film is from 300 micrometers to 500 micrometers, preferably from 350 to 450 micrometers, and more preferably from 380 to 420 micrometers.

[0070] (26). A biodegradable multilayer membrane according to any one of (1) to (25), wherein the thickness of the core layer is from 200 micrometers to 300 micrometers, preferably from 215 to 285 micrometers, more preferably from 230 to 270 micrometers.

[0071] (27). A biodegradable multilayer film according to any one of (1) to (26), wherein the thickness of the first outer layer and / or the second outer layer is from 50 micrometers to 100 micrometers, preferably from 60 to 90 micrometers, more preferably from 70 to 80 micrometers.

[0072] (28). A biodegradable multilayer film according to any one of (1) to (19) and (24) to (27), wherein when hot-pressed into a single layer and without compatibilizer, the core layer has an elongation at break of 25 to 45%, preferably 30 to 40%, and an ultimate tensile strength of 3 to 5 MPa, preferably 3.5 to 4.5 MPa.

[0073] (28a). A biodegradable multilayer film according to any one of (1) to (19) and (24) to (27), wherein when extruded into a single layer film without compatibilizer, the core layer has an elongation at break of 2 to 20% in the transverse (CD) direction and 2 to 15% in the machine direction (MD) direction, and an ultimate tensile strength of 3 to 6 MPa in the transverse (CD) direction and 5 to 10 MPa in the machine direction (MD) direction.

[0074] (29). A biodegradable multilayer film according to any one of (20) to (23), wherein when hot-pressed into a single layer and containing a compatibilizer, the core layer has an elongation at break of 50 to 110%, preferably 70 to 90%, and an ultimate tensile strength of 4 to 6 MPa, preferably 4.5 to 5.5 MPa.

[0075] (29a). A biodegradable multilayer film according to any one of (20) to (23), wherein when extruded into a single-layer film and containing a compatibilizer, the core layer has an elongation at break of 2 to 30% on CD and 4 to 35% on MD, and an ultimate tensile strength of 3 to 7 MPa on CD and 6 to 11 MPa on MD.

[0076] (30). A biodegradable multilayer film according to any one of (1) to (29), wherein the elongation at break of the multilayer film is up to 200% in the machine direction (MD) and up to 100% in the transverse direction (CD).

[0077] (31). A biodegradable multilayer film according to any one of (1) to (19) and (24) to (28), wherein when extruded and without compatibilizer, the biodegradable multilayer film has an elongation at break of up to 200% in the machine direction (MD), preferably 10 to 150%, and up to 100% in the transverse direction (CD), preferably 10 to 80%.

[0078] (32). A biodegradable multilayer film according to any one of (20) to (27) and (29), wherein when extruded and containing a compatibilizer, the biodegradable multilayer film has an elongation at break of up to 20% in the machine direction (MD), preferably 5 to 20%, and up to 70% in the transverse direction (CD), preferably 20 to 70%.

[0079] (33). A biodegradable multilayer film according to any one of (1) to (19) and (24) to (28) and (31), wherein when extruded and without compatibilizer, the ultimate tensile strength of the biodegradable multilayer film is 7 to 15 MPa on MD and 4 to 8 MPa on CD.

[0080] (34). A biodegradable multilayer film according to any one of (20) to (27) and (29) and (32), wherein when extruded and containing a compatibilizer, the ultimate tensile strength of the biodegradable multilayer film is 4 to 10 MPa on MD and 4 to 10 MPa on CD.

[0081] (35). A biodegradable multilayer membrane according to any one of (1) to (34), wherein the biodegradable multilayer membrane has a bio-based carbon content of at least 20%.

[0082] (36). A biodegradable multilayer film according to any one of (1) to (35), wherein the biodegradable multilayer film has a flexural strength of less than 10 pinholes as measured on an A4 size sheet according to standard ASTM F392-A (2004).

[0083] (37). A biodegradable multilayer membrane according to any one of (1) to (36), which is suitable for manufacturing bags that can be used as bioreactors.

[0084] (38). A method for manufacturing a biodegradable multilayer film according to any one of (1) to (37), comprising the following steps:

[0085] - Introduce and melt a blend of thermoplastic starch (TPS) and polybutylene terephthalate (PBAT) into the first extruder to form a core laminar flow;

[0086] - Introducing and melting the first polymer into the second extruder to form a first outer laminar flow;

[0087] - Introduce and melt the second polymer into the third extruder to form a second outer laminar flow;

[0088] Alternatively, if the first polymer and the second polymer are the same, the second extruder is connected to the feed block to form a first outer laminar flow and a second outer laminar flow;

[0089] - Introduce the core laminar flow, the first outer laminar flow, and the second outer laminar flow into the feed block to form a three-layer melt flow;

[0090] - Introduce the three-layer melt flow into the die head to form a multilayer film.

[0091] (39). The method according to (38), wherein the multilayer film is introduced into a calender.

[0092] (40). The method according to (38) or (39), wherein the thermoplastic starch (TPS) introduced into the extruder contains 10 to 15 wt.% moisture content.

[0093] (41). A biodegradable multilayer membrane obtained by the method according to (38) to (40).

[0094] (42). A disposable bag, the wall of which comprises a multilayer film according to any one of (1) to (37).

[0095] (43). A bioreactor comprising a single-use bag according to (42).

[0096] (44). A container suitable for mixing, packaging, storing or transporting biopharmaceutical substances, comprising a single-use bag as described in (42). Detailed Implementation

[0097] Further aspects, features, and advantages of the exemplary embodiments will become apparent from the following detailed description.

[0098] The patents, published applications and scientific literature cited in this article establish the knowledge of those skilled in the art and are hereby incorporated in their entirety by reference as if each of them were specifically and individually indicated to be incorporated by reference.

[0099] As used herein, whether in transitional phrases or in the text of the claims, the terms "comprise(s)" and "comprising" should be interpreted as having an open-ended meaning. That is, these terms should be interpreted as synonymous with the phrases "having at least" or "including at least". When used in the context of a method, the term "comprise" means that the method includes at least the stated steps, but may include additional steps.

[0100] The terms "consists essentially of" or "consisting essentially of" have a partially closed meaning, meaning they do not allow the inclusion of steps, features, or components that would substantially alter the essential characteristics of the method or composition; for example, steps, features, or components that would significantly interfere with the desired properties of the compound or composition described herein. That is, the method or composition is limited to the specified steps or materials and those steps or materials that do not substantially affect the essential and novel characteristics of the method or composition. The terms "consists of" and "consists" are closed terms, allowing only the inclusion of the stated steps, features, or components.

[0101] As used herein, the singular form “a, an, the” explicitly includes the plural form of the term it refers to, unless the context explicitly states otherwise.

[0102] The term “about” as used herein means approximately, within a certain range, roughly, or around. When the term “about” is used with a numerical range, it modifies the range by extending the upper and lower boundaries of the stated value.

[0103] The terms "dissolved" or "substantially dissolved" as used in this article refer to the dissolution of a solid in a solution. When the resulting solution is clear or substantially clear, the solid can be considered to have "dissolved" or "substantially dissolved" in that solution.

[0104] As used herein, statements about the numerical range of a variable are intended to convey that the variable can be equal to any value within that range. Therefore, for a variable that is inherently discrete, the variable can be equal to any integer value within that range, including the endpoints of the range. Similarly, for a variable that is inherently continuous, the variable can be equal to any real value within that range, including the endpoints of the range. For example, a variable described as having values ​​between 0 and 2 could be 0, 1, or 2 for an inherently discrete variable, and could be 0.0, 0.1, 0.01, 0.001, or any other real value for an inherently continuous variable.

[0105] In the specification and claims, unless the context clearly specifies otherwise, the singular form includes the plural reference.

[0106] Unless otherwise defined, the technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this description pertains.

[0107] According to this disclosure, the expression "a layer includes X" means that the layer contains X in any amount or is essentially composed of X.

[0108] Core layer

[0109] The biodegradable multilayer membrane disclosed herein comprises a core layer.

[0110] The core layer comprises a blend of thermoplastic starch (TPS) and polybutylene terephthalate (PBAT).

[0111] The blend of thermoplastic starch (TPS) and polybutylene terephthalate (PBAT) in the core layer may contain TPS and PBAT in a weight ratio of 60:40 to 40:60, preferably 55:45 to 45:55, and more preferably 50:50.

[0112] The thermoplastic starch (TPS) in the core layer preferably comprises plant-based starch. This starch can be selected from the group consisting of corn starch, tapioca starch, cassava starch, wheat starch, natural wheat starch, potato starch, rice starch, sorghum starch, seaweed starch, and mixtures thereof. In one example, the thermoplastic starch may comprise natural wheat starch. Plant-based starches contain polysaccharides, which are of particular interest, primarily in short-term applications, due to their biodegradability in standardized environments, low cost, and widespread availability.

[0113] Since the thermal degradation of starch occurs below its theoretical melting temperature, starch is typically plasticized to obtain processable thermoplastic-like materials. Blends of plasticizers and starch are called thermoplastic starch (TPS). Plasticizers can be added to the thermoplastic starch (TPS) in amounts of 15 to 45% by weight, preferably 22 to 35% by weight, more preferably 25 to 35% or 27 to 32% by weight, based on the weight of the thermoplastic starch.

[0114] The plasticizer can be a non-volatile plasticizer, such as a polyol or amide or a mixture thereof. These can be added in an amount of 10 to 25% by weight, preferably 15 to 22% by weight, based on the weight of the thermoplastic starch. The plasticizer can be selected from the group consisting of glycerol, sorbitol, ethylene glycol, urea, formamide, and mixtures thereof. Preferably, glycerol can be added as a plasticizer.

[0115] Another common plasticizer is water, which is volatile, so the water concentration in starch depends to some extent on the ambient atmosphere, i.e., its moisture content and temperature. During the production of thermoplastic starch, 5 to 20% by weight or 7 to 15% by weight of water can be added.

[0116] However, to maintain the required water concentration and preserve thermoplasticity and mechanical properties in thermoplastic starch, moisture content must be strictly controlled during and after processing. However, compared to fossil-based engineered polymers, thermoplastic starch has significant limitations, such as mechanical properties, critical aging issues, high hydrophilicity, and reduced water vapor barrier properties. To overcome these limitations while maintaining biodegradability, several strategies have been developed, such as developing multiphase materials incorporating biodegradable polyesters or developing multilayer films containing biodegradable polyester layers.

[0117] In this disclosure, thermoplastic starch (TPS) is blended with polybutylene terephthalate (PBAT) to form the core layer. PBAT is a biodegradable polyester.

[0118] The core layer may comprise TPS and PBAT in a weight ratio of 60:40 to 40:60, preferably 55:45 to 45:55, and more preferably 50:50. The properties and composition of the PBAT in the core layer may be the same as those of the PBAT used in the first and second outer layers.

[0119] In some cases, blends of thermoplastic starch (TPS) and polybutylene terephthalate (PBAT) in the core layer may also include a compatibilizer. Due to the interfacial tension between the polyester and the polysaccharide, the TPS / PBAT multiphase system may exhibit limited compatibility, resulting in blends with limited mechanical and barrier properties. Therefore, some TPS / PBAT blends may require compatibilization, i.e., the addition of a compatibilizer.

[0120] The compatibilizer may be selected from the group consisting of maleic anhydride-grafted polyesters, polyvinyl acetate, and copolymers comprising styrene monomers and epoxy-functionalized (meth)acrylates or (meth)acrylate monomers. Preferably, the compatibilizer may be a copolymer comprising styrene monomers and epoxy-functionalized (meth)acrylates or (meth)acrylate monomers, a product marketed under a trade name... Reactive compatibilizers for sale. Such compatibilizers are well known in the art and are included in formulations of biodegradable blends.

[0121] The compatibilizer content can be 0.05 to 0.7 phr, 0.1 to 0.6 phr or 0.2 to 0.5 phr, preferably 0.1 to 0.3 phr, wherein parts per hundred (phr) refers to the blend of TPS and PBAT.

[0122] Use such as Compatibilizers can be compatibilized via a two-step process: a) first, the compatibilizer is mixed with and reacted with PBAT, and then b) it is blended with TPS or a TPS / PBAT blend. Alternatively, the compatibilizer can be added to the TPS / PBAT blend. The inventors of this disclosure have found that, in some cases, the two-step process results in favorable mechanical properties for both the core layer and the multilayer film of this disclosure. Adding a compatibilizer to the core layer improves the adhesion between the layers in the multilayer film.

[0123] Regarding the properties of the core layer, a core layer that does not contain a compatibilizer and has been hot-pressed into a monolayer film has an elongation at break of 25% to 45%, preferably 30% to 40%, and an ultimate tensile strength of 3 to 5 MPa, preferably 3.5 to 4.5 MPa. A core layer that does not contain a compatibilizer and has been extruded into a monolayer film has an elongation at break of 2% to 20% in the cross direction (CD) and 2% to 15% in the machine direction (MD), and an ultimate tensile strength of 3% to 6 MPa in the cross direction (CD) and 5% to 10 MPa in the machine direction (MD).

[0124] The core layer, which has been hot-pressed into a single-layer film and contains a compatibilizer, has an elongation at break of 50 to 110%, preferably 70 to 90%, and an ultimate tensile strength of 4 to 6 MPa, preferably 4.5 to 5.5 MPa.

[0125] The core layer, which has been extruded into a single-layer film and contains a compatibilizer, has an elongation at break of 2 to 30% on CD and 4 to 35% on MD, and an ultimate tensile strength of 3 to 7 MPa on CD and 6 to 11 MPa on MD.

[0126] The thickness of the core layer can be from 200 micrometers to 300 micrometers, preferably from 215 to 285 micrometers, and more preferably from 230 to 270 micrometers.

[0127] The core layer can be produced by first mixing starch with a plasticizer (e.g., glycerol) in a turbine mixer. Mixing can then be continued with the addition of water to obtain a powder. This powder can then be extruded, for example, through a twin-screw extruder, to obtain TPS granules. The TPS granules can be equilibrated for a given time in a dryer with the desired humidity. TPS granules and PBAT granules can be manually mixed and then extruded to obtain granules containing a blend of TPS and PBAT. During the production of multilayer films, granules containing a blend of TPS and PBAT are introduced into the extruder and co-extruded with the first and second outer layers via cast extrusion.

[0128] First and second outer layers

[0129] A biodegradable multilayer membrane comprises a first outer layer and a second outer layer.

[0130] The first outer layer comprises a first polymer selected from the group consisting of polybutylene terephthalate (PBAT), polybutylene succinate (PBS), polybutylene succinate-adipate (PBSA), and mixtures thereof.

[0131] The second outer layer comprises a second polymer selected from the group consisting of polybutylene terephthalate (PBAT), polybutylene succinate (PBS), polybutylene succinate-adipate (PBSA), and mixtures thereof.

[0132] The first and second outer layers may contain the same or different polymers. The first and second outer layers may consist essentially of the aforementioned polymers, meaning that, in addition to the aforementioned polymers, the first and second outer layers contain only additives present in commercially available polymer products, wherein these additives account for less than 5%, preferably less than 3%, of the weight of one layer, i.e., the first or second outer layer, and impurities contained during the polymer manufacturing process. In particular, since one outer layer of the multilayer membrane is intended to be in contact with and remain in contact with biopharmaceutical fluids (e.g., cell cultures) for an extended period, this outer layer should not contain slip agents or phthalates as plasticizers, as these additives may contain or generate low-molecular-weight molecules capable of migrating to the layer surface after sterilization by gamma or X-ray irradiation, thereby contaminating the cell culture medium.

[0133] In addition, the outer layer should not contain additives, or should contain only a limited amount (less than 0.10 wt.%, preferably less than 0.07 wt%) of additives that would form degradation compounds under gamma irradiation in the range of 25 kGy to 50 kGy, which would slow down or delay cell growth.

[0134] These degradation compounds, which can slow down or delay cell growth, can be produced by Sartorius Stedim Biotech. In the Cultibag RM system, the Per-C6 cell line was used to compare the test membrane with a control membrane (such as the EVA membrane BF1400 sold by Renolit, which does not delay or slow cell growth) for 7 days to detect the membrane.

[0135] These degradation compounds may be released from the antioxidant. Antioxidants are used to prevent polymer degradation that may be caused by heat, light, impurities (such as catalyst residues), or mechanical stress. Therefore, the first and second outer layers preferably do not contain antioxidants or contain only a limited amount of antioxidants, i.e., less than 0.3 wt.% (preferably less than 0.10 wt.%, more preferably less than 0.07 wt.%).

[0136] In one example, the first outer layer and / or the second outer layer comprises polybutylene terephthalate (PBAT).

[0137] The PBAT contained in the first outer layer and / or the second outer layer, as well as the TPS / PBAT blend in the core layer, may contain 55 to 45 mol%, preferably 53 to 47 mol%, of butylene adipate blocks and 45 to 55 mol%, preferably 47 to 53 mol%, of butylene terephthalate blocks.

[0138] The melting point of the PBAT, as measured by DSC according to ASTM E793-06, can be between 105 and 125 degrees Celsius, preferably between 110 and 120 degrees Celsius.

[0139] The melt volumetric flow rate of the PBAT (measured according to ISO 1133 at 190 degrees Celsius and 2.16 kg) can be from 2.0 to 5.0 ml / 10 min, preferably from 2.5 to 4.5 ml / 10 min.

[0140] The melt flow index of the PBAT (measured according to ISO 1133 at 190 degrees Celsius and 2.16 kg) can be 2 to 6 g / 10 min, preferably 2.7 to 4.9 g / 10 min.

[0141] The number-average molecular weight of the PBAT, as measured by size exclusion chromatography, can be 10 to 70, preferably 20 to 60, and more preferably 30 to 50 kg / mol.

[0142] The dispersion of the PBAT, as measured by size exclusion chromatography, can be 1.5 to 2.5, preferably 1.6 to 2.3, and more preferably 1.8 to 2.2.

[0143] When the PBAT is extruded into a single-layer film, its ultimate tensile strength, measured according to ISO 527-3 or ASTM D638, is 14 to 20 MPa in the machine direction (MD) and 12 to 15 MPa in the cross direction (CD).

[0144] When the PBAT is extruded into a single-layer film, its elongation at break, measured according to ISO 527-3 or ASTM D638, is 350 to 800% in the machine direction (MD) and 500 to 700% in the transverse direction (CD).

[0145] The thickness of the first outer layer and / or the second outer layer can be from 40 micrometers to 100 micrometers, preferably from 60 to 90 micrometers, and more preferably from 70 to 80 micrometers.

[0146] The first and second outer layers contribute to the mechanical strength of the bag wall. For this, they must be flexible enough to withstand high mechanical stresses, but not overly stretchable. Simultaneously, the outer layers must be sufficiently rigid to prevent the bag or container from deforming when filled with biopharmaceutical fluids. In addition to the required balance of flexibility and rigidity, the outer layers must possess a certain degree of adhesiveness so that they can self-seal to form 2D or 3D bags. Furthermore, as mentioned above, the outer layers must be made of a material capable of maintaining and being in contact with the biopharmaceutical for extended periods, such as during cell culture (e.g., several days) or during storage, without causing degradation of the film or the biopharmaceutical product.

[0147] The outer layer can be manufactured by extrusion. Preferably, the outer layer is manufactured by casting extrusion with other multilayer film layers.

[0148] Adhesive layer

[0149] The biodegradable multilayer membrane may also include one or more adhesive layers between the first outer layer and the core layer, and between the second outer layer and the core layer.

[0150] The adhesive layer must be able to seal the outer layer to the core layer and prevent these layers from separating during bag use.

[0151] The one or more adhesive layers may comprise a blend of thermoplastic starch (TPS) and a polymer selected from the group consisting of polybutylene terephthalate (PBAT), polybutylene succinate (PBS), polybutylene-succinate adipate (PBSA), and mixtures thereof. Preferably, the polymer of the one or more adhesive layers is PBAT. The properties and composition of TPS and PBAT are as described above.

[0152] In the one or more adhesive layers, TPS and the polymer may be contained in a weight ratio of 60:40 to 20:80, more preferably from 45:55 to 40:60.

[0153] In one example, when there are adhesive layers between the first and second outer layers and the core layer, these two or more adhesive layers may have the same composition.

[0154] The adhesive layer is added to improve the interlayer adhesion between the outer layer and the core layer, so as to prevent layer separation when using the bag.

[0155] The thickness of the adhesive layer can be from 5 μm to 50 μm, preferably from 10 μm to 30 μm, and more preferably from 15 μm to 25 μm.

[0156] The adhesive layers can be manufactured by extrusion. Preferably, they are manufactured by casting extrusion with other multilayer film layers.

[0157] Multilayer film

[0158] The biodegradable multilayer membrane disclosed herein comprises layers stacked on top of each other in the following order:

[0159] a) A first outer layer comprising a first polymer selected from the group consisting of polybutylene terephthalate (PBAT), polybutylene succinate (PBS), polybutylene succinate-adipate (PBSA), and mixtures thereof.

[0160] b) Core layer, comprising a blend of thermoplastic starch (TPS) and polybutylene terephthalate (PBAT);

[0161] c) A second outer layer comprising a second polymer selected from the group consisting of polybutylene terephthalate (PBAT), polybutylene succinate (PBS), polybutylene succinate-adipate (PBSA), and mixtures thereof.

[0162] In one implementation, the first outer layer is in direct contact with the core layer, and the second outer layer is in direct contact with the core layer.

[0163] In one example, the biodegradable multilayer membrane of this disclosure comprises layers stacked on top of each other in the following order:

[0164] a) A first outer layer comprising PBAT as a first polymer.

[0165] b) Core layer, comprising a blend of thermoplastic starch (TPS) and PBAT.

[0166] c) A second outer layer comprising PBAT as a second polymer.

[0167] In one example, the biodegradable multilayer film described above does not contain a compatibilizer.

[0168] In another example, the aforementioned film contains a compatibilizer, such as a copolymer comprising styrene monomers and epoxy-functionalized (meth)acrylate or (meth)acrylate monomers. In one example, the aforementioned biodegradable multilayer film, measured by ISO 527 or ASTM D882, exhibits an elongation at break of up to 200% in the machine direction (MD) and up to 100% in the transverse direction (CD).

[0169] In another embodiment, the biodegradable multilayer film further includes one or more adhesive layers between the first outer layer and the core layer, and between the second outer layer and the core layer, wherein the one or more adhesive layers comprise a blend of thermoplastic starch (TPS) and a polymer selected from the group consisting of polybutylene terephthalate (PBAT), polybutylene succinate (PBS), polybutylene succinate-adipate (PBSA), and mixtures thereof.

[0170] In one example, the biodegradable multilayer membrane of this disclosure comprises layers stacked on top of each other in the following order:

[0171] a) A first outer layer comprising a first polymer selected from the group consisting of polybutylene terephthalate (PBAT), polybutylene succinate (PBS), polybutylene succinate-adipate (PBSA), and mixtures thereof.

[0172] b) A first intermediate layer comprising a blend of thermoplastic starch (TPS) and a polymer selected from the group consisting of polybutylene terephthalate (PBAT), polybutylene succinate (PBS), polybutylene succinate-adipate (PBSA), and mixtures thereof.

[0173] c) Core layer, comprising a blend of thermoplastic starch (TPS) and polybutylene terephthalate (PBAT);

[0174] d) A second intermediate layer comprising a blend of thermoplastic starch (TPS) and a polymer selected from the group consisting of polybutylene terephthalate (PBAT), polybutylene succinate (PBS), polybutylene succinate-adipate (PBSA), and mixtures thereof;

[0175] e) A second outer layer comprising a second polymer selected from the group consisting of polybutylene terephthalate (PBAT), polybutylene succinate (PBS), polybutylene succinate-adipate (PBSA), and mixtures thereof.

[0176] In another example, the biodegradable multilayer membrane of this disclosure comprises layers stacked on top of each other in the following order:

[0177] a) A first outer layer comprising polybutylene terephthalate (PBAT) as a first polymer;

[0178] b) The first intermediate layer, which contains PBAT;

[0179] c) Core layer, comprising a blend of thermoplastic starch (TPS) and PBAT;

[0180] d) The second intermediate layer, containing PBAT;

[0181] e) A second outer layer comprising PBAT as a second polymer. In one example, the above-described biodegradable multilayer film does not contain a compatibilizer.

[0182] In another example, the aforementioned film contains a compatibilizer, such as a copolymer comprising styrene monomer and epoxy-functionalized (meth)acrylate or (meth)acrylate monomer.

[0183] In one example, the aforementioned biodegradable multilayer film, measured by ISO 527 or ASTM D882, exhibits an elongation at break of up to 200% in the machine direction (MD) and up to 100% in the transverse direction (CD).

[0184] The thickness of the biodegradable multilayer film can be from 300 micrometers to 500 micrometers, preferably from 350 to 450 micrometers, and more preferably from 380 to 420 micrometers.

[0185] Performance of multilayer films

[0186] The multilayer membrane disclosed herein is biodegradable. Biodegradability is measured according to standard NF EN 13432.

[0187] The biodegradable multilayer membrane may have a bio-based carbon content of at least 20%, wherein the at least 20% is the result of selecting the individual components constituting the various layers and components of the multilayer membrane. The bio-based content can be measured according to standard ASTM D6866.

[0188] The multilayer films disclosed herein typically exhibit elongation at break up to 200% in the machine direction (MD) and up to 100% in the transverse direction (CD).

[0189] In one example, the extruded multilayer film, which does not contain a compatibilizer, has an elongation at break of up to 200%, preferably 10 to 150%, in the machine direction (MD) and up to 100%, preferably 10 to 80%, in the transverse direction (CD). The ultimate tensile strength of the multilayer film is 7 to 15 MPa in the MD and 4 to 8 MPa in the CD.

[0190] In one example, the extruded and compatibilized biodegradable multilayer film has an elongation at break of up to 20%, preferably 5 to 20%, in the machine direction (MD) and up to 70%, preferably 20 to 70%, in the transverse direction (CD).

[0191] The ultimate tensile strength of the multilayer film can be 4 to 10 MPa on MD and 4 to 10 MPa on CD.

[0192] The biodegradable multilayer film exhibits a flexural strength of less than 10 pinholes when measured on an A4-sized sheet according to standard ASTM F392-A (2004).

[0193] The outer layer of the biodegradable multilayer membrane does not produce, or produces only small amounts of, degradation compounds that form when the membrane is exposed to gamma irradiation in the range of 25 kGy to 50 kGy. These degradation compounds can slow down or delay cell growth, thereby interfering with the reactions occurring in the bioreactor.

[0194] Multilayer film production

[0195] Each layer can be processed into a multilayer film using standard extrusion techniques well known to those skilled in the art, including extrusion or co-extrusion, such as casting or blow extrusion, extrusion coating, extrusion coating and lamination or a combination thereof, for example by co-extruding at least two layers and then coating another layer, or by co-extruding at least two layers, extruding another layer and then coating and laminating the co-extruded layer and the extruded layer together.

[0196] Preferably, the multilayer film is manufactured using a cast co-extrusion process.

[0197] Therefore, this disclosure provides a method for manufacturing the biodegradable multilayer film as described above, comprising the following steps:

[0198] - Introduce and melt a blend of thermoplastic starch (TPS) and polybutylene terephthalate (PBAT) into the first extruder to form a core laminar flow;

[0199] - Introducing and melting the first polymer into the second extruder to form a first outer laminar flow;

[0200] - Introduce and melt the second polymer into the third extruder to form a second outer laminar flow;

[0201] - Introduce the core laminar flow, the first outer laminar flow, and the second outer laminar flow into the feed block to form a three-layer melt flow;

[0202] - Introduce the three-layer melt flow into the die head to form a multilayer film.

[0203] Alternatively, a multilayer film having the same first and second outer layers can be produced by a method comprising the steps of connecting the aforementioned second extruder to a feed block to form a first and second outer layer flow, rather than introducing and melting the second polymer into a third extruder to form a second outer layer flow. The remaining steps of the method are as described in the preceding paragraph.

[0204] In the above method, the rotational frequency of the extruder can be selected as 30 to 50 rpm, preferably 35 to 45 rpm, and more preferably 37 to 40 rpm.

[0205] In the above method, a temperature gradient of 80 to 130 degrees Celsius, preferably 100 to 130 degrees Celsius, can be used in the first extruder to form a core laminar flow. Temperature gradients of 80 to 120 degrees Celsius, preferably 100 to 120 degrees Celsius, can be used in the second and third extruders to form the first and second outer laminar flows, respectively.

[0206] In the above method, the feed block can be maintained at 120 to 140 degrees Celsius, preferably 125 to 135 degrees Celsius, and more preferably 130 degrees Celsius when forming a three-layer flow. The feed block can be configured as a 25 / 50 / 25 plate.

[0207] In the above method, the die head can be maintained at 120 to 140 degrees Celsius, preferably 125 to 135 degrees Celsius, and more preferably 130 degrees Celsius to obtain a multilayer film.

[0208] The method may include a step in which the multilayer film is introduced into a calender after exiting the die. The calender may be set to 30 degrees Celsius.

[0209] The method may include introducing thermoplastic starch (TPS) with a moisture content of 10 to 15 wt.%, preferably 11 to 13 wt.%, or about 12 wt.% into an extruder.

[0210] This method should be carried out in the absence of slip agents and other low molecular weight additives that may increase the extractable to unacceptable levels. Generally, the use of additives should be avoided in production and processing, except for plasticizers used to form thermoplastic starch (TPS) and compatibilizers that may be added to TPS and PBAT blends. Additives contained in commercially available raw material polymers may not be unavoidable, but are acceptable as long as their concentrations do not interfere with the suitability and technical application of this disclosure.

[0211] This disclosure also provides a biodegradable multilayer membrane obtained by the above method.

[0212] Bags produced by this method

[0213] Due to the aforementioned characteristics, the biodegradable multilayer film disclosed herein is particularly suitable for manufacturing single-use bags, including 2D bags or 3D bags.

[0214] Therefore, this disclosure provides a disposable bag whose wall comprises a biodegradable multilayer film as described above.

[0215] The biodegradable multilayer membranes disclosed herein can withstand a variety of mechanical stresses, making them suitable for a wide range of applications, such as bioreactors in any stirred mode or for storing or transporting fluids. These bags must also be suitable for small or large volumes.

[0216] Such bags can be manufactured according to standard techniques known to those skilled in the art.

[0217] The biodegradable multilayer film disclosed herein is particularly suitable for manufacturing single-use bags intended to contain cell cultures because it has little or no effect on cell growth after sterilization by gamma or X-ray irradiation. Specifically, the contact layer of the multilayer film of the present invention releases little or no amount of degradation compounds due to gamma irradiation, which could interfere with cell growth.

[0218] The biodegradable multilayer film disclosed herein is also suitable for manufacturing containers for mixing, packaging, storage, or transportation.

[0219] Brief description of the attached figures

[0220] Figure 1 : Screw configuration used for extruding TPS and TPS / polyester blends.

[0221] Figure 2 Oxygen barrier properties of single-layer and multi-layer membranes: permeability coefficient (top), diffusion coefficient (middle), and solubility coefficient (bottom).

[0222] Figure 3 Water vapor adsorption isotherm of extruded thin film

[0223] Figure 4 Cell culture growth evaluation results of extruded monolayer films

[0224] The present invention will be further described in the following embodiments. These embodiments are for illustrative purposes and should not be construed as limiting the invention.

[0225] Example

[0226] Material

[0227] Natural wheat starch, 25wt% amylose, initial moisture content <12.5%, protein content <0.5%, purchased from Roquette (France).

[0228] Glycerin (purity >98%) – used as a non-volatile plasticizer in starch, purchased from Fisher Scientific (France).

[0229] Poly(butylene adipate-co-terephthalate) (PBAT) - extrusion grade, number average molecular weight Mn = 38 kg / mol, dispersity D = 2.0 as determined by size exclusion chromatography (SEC) – BASF's commercial product Ecoflex

[0230] compatibilizer ADR4368-CS (purity >99%, Mw 6800 g / mol, from BASF)

[0231] Polybutylene succinate (PBS), extrusion grade, Mn = 71 kg / mol, dispersity D of 2.4 as determined by SEC, sourced from NaturePlast (France).

[0232] Poly(butylene succinate-co-butylene adipate) (PBSA), extrusion grade, Mn = 74 kg / mol, dispersity D of 2.4 as determined by SEC, from NaturePlast (France).

[0233] Natural wheat starch, glycerin, and polyester are dried in a ventilated oven at 70 degrees Celsius for about 15 hours before use.

[0234] Measurement methods

[0235] Unless otherwise stated, the polymer densities mentioned in this application are measured in accordance with the standard test method ASTM D792–08.

[0236] Biodegradability was measured according to standard NF EN 13432.

[0237] The biodegradable multilayer membrane may have a bio-based carbon content of at least 20%. This at least 20% is a result of selecting the individual components constituting the various layers and components of the multilayer membrane, and can be calculated accordingly. Alternatively, the bio-based content can be measured according to standard ASTM D6866.

[0238] Average values ​​of ultimate tensile strength (UTS) and elongation at break (εmax):

[0239] Uniaxial tensile tests were performed on an Instron 5567H universal testing machine (USA) using a 10kN load cell and a 20mm minimum cross-section. -1 A constant crosshead speed was maintained. The room temperature was kept constant at 23°C. For each formulation, at least four dumbbell-shaped samples, approximately 45×5×1mm in size, were tested from the film. 3 The average values ​​of ultimate tensile strength (UTS) and elongation at break (εmax) were finally obtained from the curves. Statistical analysis of the tensile test data was performed using the Welch t-test with unequal variances, with a p-value <0.01 considered statistically significant. The test conditions were highly similar to ASTM D638 or ISO 527-3.

[0240] Number-average molecular weight (Mn) and dispersion

[0241] Size exclusion chromatography (SEC) measurements were performed in chloroform (HPLC grade) using a PLGEL Mixed-C and PLGEL... A Shimadzu liquid chromatograph with a Shimadzu RID-20A refractive index detector. Number-average molecular weight (Mn) and dispersity. The determination was made using a calibration curve based on polystyrene standards. 8 ± 1 mg of sample was dissolved in chloroform and filtered through a 0.2 μm PTFE membrane before injection. The injection volume was set to 50 μL, and the flow rate was 0.8 mL / min. -1 All analyses were performed at 25°C.

[0242] Melting point:

[0243] Differential scanning calorimetry (DSC) analysis was performed using a TA Instruments (USA) Q2000 DSC instrument. Samples ranging from 1.3 to 2.6 mg were placed in a standard aluminum dish and incubated in 50 mL for 1 minute. -1 Analysis was performed under a nitrogen flow. The sample was rapidly heated to 150°C, then cooled to -80°C, and then reheated to 150°C, with each heating / cooling rate being 10°C / min. -1 .

[0244] Flexural strength was measured on A4 size sheet according to standard ASTM F392-A (2004).

[0245] The melt volumetric flow rate was measured according to ISO 1133 at 190 degrees Celsius and 2.16 kg.

[0246] Melt flow index was measured according to ISO 1133 at 190 degrees Celsius and 2.16 kg.

[0247] Oxygen permeation:

[0248] The permeation of oxygen (99.9% purity, Air Liquide) was evaluated at 25°C using a self-made laboratory apparatus based on time-delayed pressure measurement (Métayer, M., Labbé, M., Marais, S., Langevin, D., Chappey, C., Dreux, F., Brainville, M., Belliard, P., 1999. Diffusion of water through various polymer films: a new high-performance method of characterization. Polym. Test. 18, 533–549). First, the measurement cell containing the sample was degassed under high vacuum. The amount of gas transported through the membrane was monitored while gas pressure was applied until a steady state of permeation was reached. This was determined by detecting a constant increase in gas pressure using a differential pressure sensor on the permeation side. The permeation coefficient P was directly determined by Equation 2.2.

[0249] P = Jst * L / Δp (2.2)

[0250] Where Jst is the steady-state flux, Δp is the pressure difference across the membrane, L is the membrane thickness, and P is expressed in bar (1 bar = 10⁻⁶ bar). -10 cm 3 (STP)cm cm -2 s -1 cmHg -1 Considering that gas molecules do not cause plasticization during the measurement, the diffusion coefficient D can be assumed to be constant and determined by formula 2.3:

[0251] D = L 2 / (6*t1) (2.3)

[0252] Where t1 is the time delay, determined by the intercept of the asymptotic line of the steady-state flux with the time axis. Since the permeability coefficient is the product of the diffusion coefficient and the solubility coefficient, the solubility coefficient S can be derived from Equation 2.4:

[0253] S = P / D (2.4)

[0254] Three different samples were taken for each test film and measured three times to ensure repeatability.

[0255] Water vapor adsorption kinetics measurement:

[0256] Water vapor adsorption kinetics measurements were performed using a Cahn D200 electronic microbalance in a gravimetric dynamic vapor adsorption analyzer (Surface Measurement Systems, Ltd., London, UK), as described by Follian et al., "Water Transport Properties of Bio-nanocomposites Reinforced by Luffa Cylindrica Cellulose Crystals". Journal of Membrane Science, 2013, vol. 427, pp. 218–229. The measurement temperature was set at 25.0 (±0.1) °C. Water activity was regulated by mixing a dry and water-saturated nitrogen gas stream (N2 gas, 99.999% purity, Air Liquide) using an electronic mass flow controller. Approximately 8 mg of polymer film (8 mm in diameter) was first dried until no change in dry weight could be measured, and then hydrated by exposure to a selected water vapor pressure. The changes in film mass with time and water activity were recorded, and adsorption kinetics were gradually tracked until the equilibrium weight was reached. The water vapor adsorption isotherm was determined based on the dry weight at each water activity and the mass at equilibrium. The water mass gain Ceq was determined by equation (2.5):

[0257] Ceq(%)=(meq-m0) / m0×100 (2.5)

[0258] Where m0 and meq are the mass of the dried sample and the mass of the sample under equilibrium conditions, respectively.

[0259] Cell culture growth assessment:

[0260] Cell culture growth assessment was performed according to ASTM E3231-19. The membrane was first transferred to a borosilicate glass vial and irradiated with gamma at 48 kGy. Ten days later, extractable extraction was performed using ActiCHO-SM (a protein-free, hydrolysate-free, and animal-derived cell culture medium (Cellca, Germany)) at 36.8 °C and constant shaking at 120 rpm for 3 days. In this setup, the applied surface area to liquid volume ratio was 0.3 cm². 2 ml -1Empty borosilicate glass vials were used as a reference, and 2% DMSO solution was used as a negative control. The extracts were then transferred to six-well plates (ThinCert™, Greiner Bio-one, Germany) for further growth studies. The Chinese hamster ovary cell line CHO-DG44 (Cellca, Germany) was selected for cell culture. The cell density was 0.2 × 10⁻⁶ cells / well. 6 cells ml -1 Each membrane was tested three times in a CO2 incubator (CERTOMAT CTplus, Sartorius, Germany) for four consecutive days. Live cell counts and viability were measured using a NucleoCounter (Chemometec, Denmark).

[0261] Preparation Examples

[0262] 1. Preparation of plasticized starch

[0263] The TPS formulation is based on 70 wt% natural starch, 18 wt% glycerol, and 12 wt% distilled water. Plasticized starch (TPS) granules are prepared according to the following procedure: First, natural wheat starch is dried overnight at 70°C in a ventilated oven. Then, the starch powder is introduced into a Papenmeier turbine mixer, and glycerol is slowly added while stirring (approximately 700 rpm). After complete addition of glycerol, the mixture is mixed at high speed (approximately 1400 rpm) to obtain a uniform dispersion. The mixture is then placed in a ventilated oven at 145°C for 60 minutes, with occasional stirring to allow moisture to evaporate and glycerol to diffuse into the starch granules. After cooling, the dry mixture is reintroduced into the Papenmeier turbine mixer, and a suitable amount of distilled water is slowly added while stirring. The mixture is then dispersed at high speed again. Subsequently, the powder is extruded on a co-rotating twin-screw extruder (Thermo Fisher Scientific, Haake Thermo Fisher Scientific, France), equipped with a mixing element, a screw diameter of 16 mm, and a length-to-diameter ratio of 40:1. Figure 1 As shown in the schematic diagram, the two screws have identical configurations designed to ensure proper melting and mixing of the extruded material. The screw speed is set to 80 rpm, and the temperature profile from the feed hopper to the circular die is 80 / 105 / 110 / 115 / 120 / 120 / 115 / 110 / 105 / 100°C. The feed strip is cooled and granulated by air jet cooling. The TPS granules are stored at room temperature in sealed polyethylene bags until the blend is prepared.

[0264] 2. Preparation of incompatible TPS / polyester blends

[0265] Beforehand, TPS granules were equilibrated for 8 days in a desiccator at 23°C and 58% relative humidity (RH), and polyester granules were dried overnight in a ventilated oven at 70°C. The TPS and polyester granules were manually mixed and then melt-blended in a twin-screw extruder equipped with a mixing element. The screw speed was set to 20 rpm, and the temperature profile from the feed hopper to the round die was 80 / 105 / 130 / 135 / 145 / 145 / 135 / 130 / 125 / 120°C. The granules were cooled and granulated by air jet cooling. The TPS / polyester granules were stored at room temperature in sealed polyethylene bags.

[0266] 3. Preparation of compatible TPS / polyester blends

[0267] The first step is to Dry polyester granules were manually mixed at a ratio of 100:1 and then melt-blended in a twin-screw extruder equipped with a mixing element. The screw speed was set to 15 rpm, and the temperature profile from the feed hopper to the round die was 80 / 110 / 140 / 170 / 200 / 200 / 200 / 180 / 170 / 150°C. The granules were cooled and granulated by air jet cooling. The resulting polyester / The granules are stored in sealed polyethylene bags. Then, they are manually mixed with TPS, polyester, and polyester / Particles were melt-blended using the same extruder in ratios of 50:40:10, 50:30:20, or 50:0:50 to prepare products containing 0.1, 0.2, or 0.5 phr, respectively. Compatible TPS / polyester blends were prepared. The screw speed was set to 20 rpm, and the temperature profile from the feed hopper to the circular die was 80 / 105 / 130 / 135 / 145 / 145 / 135 / 130 / 125 / 120°C. The feed strip was cooled and granulated by air jet cooling. The compatible blend granules were equilibrated for 8 days in a desiccator at 23°C and 58% relative humidity (RH) and finally stored in sealed polyethylene bags.

[0268] 4. Production of single-layer and multi-layer films by (co)extrusion.

[0269] Use the following settings to co-extrude containing TPS / PBAT 50 / 50 or TPS / PBAT / A three-layer thin film consisting of a core layer of 50 / 50 / 0.1 and an outer layer of PBAT:

[0270] TPS / PBAT 50 / 50 is extruded on a single-screw extruder with a screw diameter of 25 mm, an L / D ratio of 20:1, and a screw speed set to 40 rpm. The extruder is divided into 5 heating zones, set at 100 / 130 / 130 / 130 / 130°C from the feed hopper to the feed block. The extruder used for PBAT extrusion consists of a single screw with a diameter of 20 mm, an L / D ratio of 20:1, and a screw speed set to 37 rpm. The extruder is divided into 4 heating zones, set at 100 / 120 / 120 / 120°C from the feed hopper to the feed block. Each extruder is connected to a feed block at its output end. Both feed blocks are connected to a central feed block consisting of 4 plates, allowing for the distribution of the PBAT flow and layer distribution. At the output end of the central feed block, the three-layer melt flow is conveyed to a 150 mm wide coat hanger-type flat die. The die lip thickness is set to 0.6 mm. The final part of the co-extrusion setup is a three-roll calendering system with the roll temperature set at 30°C. The target film thickness is 400 μm, adjusted by modifying the calender's traction speed.

[0271] 4.1 Single-layer films obtained by hot pressing:

[0272] In preliminary tests aimed at validating and optimizing the blends before producing three-layer films, the core and outer layers were produced separately to obtain monolayer films for each individual layer. Finally, all films containing TPS were equilibrated for 4 weeks in a desiccator at 23°C and 58% RH prior to characterization for post-processing and material stabilization.

[0273] Incompatible TPS / PBAT blends were prepared by twin-screw extrusion using the aforementioned setup to produce feedstock, which was then granulated to obtain pellets, followed by compression molding to obtain hot-pressed films. The effects of polyester structure and content on the mechanical properties of the blends were investigated by testing three TPS blends of each selected polyester (polyester contents of 10%, 30%, and 50%). Ultimate tensile strength and elongation at break were then measured.

[0274]

[0275]

[0276] Subsequently, a TPS / PBAT ratio of 50:50 was applied, with BASF's TPS / PBAT ratio of 0.1, 0.2, or 0.5 phr added. Compatibilizers (per 100 parts by weight based on a 50:50 TPS / PBAT blend) were used to prepare compatible TPS / PBAT blends. Ultimate tensile strength and elongation at break were then measured.

[0277] Compatibilizer content (wt.%) Ultimate tensile strength (MPa) Elongation at break (%) 0.1 4.8 to 5.0 55% to 110% 0.2 4.9 to 5.4 57% to 115% 0.5 3.8 to 4.3 30 to 50%

[0278] In preliminary tests aimed at validating and optimizing the blends before producing three-layer films, the core and outer layers were produced separately to obtain monolayer films for each individual layer. Finally, all films containing TPS were equilibrated for 4 weeks in a desiccator at 23°C and 58% RH prior to characterization for post-processing and material stabilization.

[0279] 4.2. Multilayer thin films:

[0280] Two blends were selected: one incompatible and one compatible, namely TPS / PBAT 50 / 50 and TPS / PBAT / 50 / 50 / 0.1, as the core layer of a multilayer thin film with pure PBAT as the outer layer, are respectively called Core (incompatible) and Core-J (compatible).

[0281] First, each layer was extruded individually to obtain a reference monolayer film. Initial testing showed that films extruded at low screw speeds (e.g., 15 rpm) were rough and irregular. This is because TPS particles require sufficient shear force, specific mechanical energy (SME), temperature, and overall thermomechanical input to achieve the appropriate viscosity at the extruder exit. At higher screw speeds, such as approximately 40 rpm, the resulting films were all smooth and more transparent than those extruded at lower SME. The speed of each extruder was then configured to achieve approximately equal flow rates at their respective outputs.

[0282] The co-extrusion of multilayer films is performed using the settings described in item 4 above, as follows:

[0283] A blend of thermoplastic starch (TPS) and polybutylene terephthalate (PBAT) is introduced into a first extruder to form a core laminar flow. The first extruder has a temperature gradient of 100 to 130 degrees Celsius. The rotation frequency is set to 40 rpm, which results in an outlet pressure P exit Approximately 78 bar. Extruder specifications: 25mm / 20D.

[0284] PBAT is introduced into the second and third co-extruders to form the outer layer of material flow. These extruders have a temperature gradient of 100 to 120 degrees Celsius and a rotation frequency of 37 rpm. The extruder specifications are 20 mm / 20D.

[0285] A core laminar flow and two outer laminar flows are introduced into the feed block to form a three-layer melt flow. The feed block is maintained at 130 degrees Celsius and is set to a 25 / 50 / 25 plate. The three-layer melt flow is then introduced into the die to form a multilayer film. The die is set at 130 degrees Celsius, with a size of 150 mm and a die lip size (ε) of approximately 0.6 mm. The film is then calendered at 30 degrees Celsius. The target film thickness is 400 μm, adjusted by modifying the traction speed of the calender.

[0286] Co-extruded multilayer films were obtained, with the multilayer film labeled M containing wt% TPS / PBAT 50 / 50 core layers. The multilayer thin film with a 50 / 50 / 0.1 core layer is labeled MJ.

[0287] At the edges of the multilayer film, a highly transparent PBAT cap was observed to encapsulate the core layer, due to its lower viscosity compared to the core blend layer. This phenomenon has been previously reported and analyzed (L. Avérous, "Multilayer Coextrusion of Starch / Biopolyester," in *Biodegradable Polymer Blends and Composites from Renewable Sources*, John Wiley & Sons Ltd., 2008, pp. 435-464). Therefore, the edges of the multilayer film were removed, retaining only the central portion for subsequent analysis and characterization.

[0288] Then, the thickness, elongation at break (EB%), machine direction (MD), and ultimate tensile strength (UTS (MPa)) in the transverse direction (TD, also known as cross direction (CD)) were measured for single-layer and three-layer films.

[0289] The results are shown in Table 1 below.

[0290]

[0291] 5. Oxygen barrier properties

[0292] To evaluate the suitability of co-extruded multilayer films for packaging applications, their oxygen permeability was assessed. For each test film, the O2 permeability coefficient and its two main related parameters, namely the diffusion coefficient and the solubility coefficient, were calculated, such as... Figure 2 As shown. As expected, the TPS / PBAT blend significantly reduced oxygen permeability compared to pure PBAT. Unsurprisingly, the oxygen permeability of the multilayer film was slightly higher than that of the monolayer core layer due to the presence of the more oxygen-permeable PBAT cap layer. Nevertheless, its permeability coefficient was still lower than that of pure PBAT, thus preserving the advantages of TPS incorporation and confirming the relevance of such multiphase systems for packaging applications. However, surprisingly, the diffusion coefficient of the MJ film was more than twice that of the other films, even higher than that of the pure PBAT film. Nevertheless, its permeability coefficient was still lower than that of PBAT and relatively close to that of the M film, meaning that the increase in diffusivity was largely offset by a significant reduction in solubility. Overall, the O2 barrier performance of all multiphase systems was satisfactory, with all permeability coefficients below 0.5 bar.

[0293] 6. Water vapor adsorption

[0294] The water vapor adsorption behavior of the extruded films was also investigated. For each film, the equilibrium water mass gain was measured at given water activities (0.0, 0.1, 0.3, 0.5, and 0.9), and the resulting water vapor adsorption isotherms were plotted. Figure 3 .

[0295] First, the hydrophobic behavior of PBAT is confirmed by its low water mass gain, which remains below 1% even at the highest water activity (0.9) due to its aromatic rings and low ester density. As for the monolayer core layer and co-extruded multilayer films, the water mass gain is low and similar in all multiphase systems up to the intermediate water activity (0.5), increasing sharply at the highest water activity. Therefore, the shape of the resulting water vapor adsorption isotherm corresponds to a Flory-Huggins type curve, also known as Type III in the Brunauer-Emmett-Teller classification. This result is entirely consistent with the known water vapor adsorption characteristics of carbohydrate-based systems. However, at all tested water activities, the water mass gain of the monolayer core layer is significantly lower than that of other TPS / PBAT blends reported in the literature. This excellent result indicates that the blend formulation and processing parameters are appropriately selected to minimize its water vapor affinity. Overall, these films exhibit reasonable water vapor barrier properties at intermediate water activities, which appropriately simulates the ambient humidity of potential packaging applications.

[0296] 7. Cell culture growth assessment:

[0297] The cell compatibility of extruded films was evaluated to assess their potential for biomedical applications. Three replicate tests were performed on extruded monolayer films from PBAT, Core, and Core-J. Results are summarized in... Figure 4 The reference was an empty borosilicate glass vial, and the negative control was a 2% DMSO solution. All three films allowed for adequate cell growth, as indicated by a relative cell growth percentage of approximately 100% in all cases. No significant differences were obtained between the three tested films, and based on cell growth results, all can be considered biocompatible. These definitive results confirm... It is a compatibilizer suitable for applications requiring biocompatibility. However, incubation of films containing TPS (Core and Core-J) resulted in a slightly brownish turbidity in the solution due to the TPS phase. Pure PBAT films, on the other hand, produced a completely clear and colorless solution. This difference in tolerance to extraction media further supports the argument for designing multilayer films with external pure PBAT cap layers in applications involving liquid contact.

Claims

1. A biodegradable multilayer membrane comprising layers stacked on top of each other in the following order: a) A first outer layer comprising a first polymer selected from the group consisting of polybutylene terephthalate (PBAT), polybutylene succinate (PBS), polybutylene succinate-adipate (PBSA), and mixtures thereof. b) Core layer, comprising a blend of thermoplastic starch (TPS) and polybutylene terephthalate (PBAT); c) A second outer layer comprising a second polymer selected from the group consisting of polybutylene terephthalate (PBAT), polybutylene succinate (PBS), polybutylene succinate-adipate (PBSA), and mixtures thereof.

2. The biodegradable multilayer membrane according to claim 1, wherein the first outer layer is in direct contact with the core layer, and the second outer layer is in direct contact with the core layer.

3. The biodegradable multilayer membrane according to claim 1, wherein the multilayer membrane further comprises one or more adhesive layers between the first outer layer and the core layer, and between the second outer layer and the core layer.

4. The biodegradable multilayer film according to any one of claims 1 to 3, wherein the blend of thermoplastic starch (TPS) and polybutylene terephthalate (PBAT) in the core layer comprises TPS and PBAT in a weight ratio of 60:40 to 40:60, preferably 55:45 to 45:55, more preferably 50:

50.

5. The biodegradable multilayer film according to any one of claims 1 to 4, wherein the thermoplastic starch (TPS) comprises starch selected from the group consisting of corn starch, tapioca starch, cassava starch, wheat starch, natural wheat starch, potato starch, rice starch, sorghum starch, seaweed starch, and mixtures thereof, preferably natural wheat starch.

6. The biodegradable multilayer film according to any one of claims 1 to 5, wherein the core layer further comprises a compatibilizer, such as maleic anhydride-grafted polyester, polyvinyl acetate, or a copolymer comprising styrene monomer and epoxy-functionalized (meth)acrylate or (meth)acrylate monomer.

7. The biodegradable multilayer film according to any one of claims 1 to 6, wherein the thickness of the biodegradable multilayer film is 300 micrometers to 500 micrometers, preferably 350 to 450 micrometers, more preferably 380 to 420 micrometers.

8. The biodegradable multilayer membrane according to any one of claims 1 to 7, wherein the elongation at break of the multilayer membrane is up to 200% in the machine direction (MD) and up to 100% in the cross direction (CD).

9. The biodegradable multilayer membrane according to any one of claims 1 to 8, wherein the biodegradable multilayer membrane has a bio-based carbon content of at least 20%.

10. The biodegradable multilayer film according to any one of claims 1 to 9, wherein the biodegradable multilayer film has a flexural strength of less than 10 pinholes as measured on an A4 size sheet according to standard ASTM F392-A (2004).

11. The biodegradable multilayer membrane according to any one of claims 1 to 10, which is suitable for manufacturing bags that can be used as bioreactors.

12. A method for manufacturing a biodegradable multilayer film according to any one of claims 1 to 11, comprising the following steps: - Introduce and melt a blend of thermoplastic starch (TPS) and polybutylene terephthalate (PBAT) into the first extruder to form a core laminar flow; - Introducing and melting the first polymer into the second extruder to form a first outer laminar flow; - Introduce and melt the second polymer into the third extruder to form a second outer laminar flow; or, if the first and second polymers are the same, connect the second extruder to the feed block to form a first and a second outer laminar flow; - Introduce the core laminar flow, the first outer laminar flow, and the second outer laminar flow into the feed block to form a three-layer melt flow; - Introduce the three-layer melt flow into the die head to form a multilayer film.

13. A biodegradable multilayer membrane obtained by the method according to claim 12.

14. A disposable bag, the wall of which comprises a multilayer film according to any one of claims 1 to 11.

15. A bioreactor comprising a disposable bag according to claim 14.

16. A container suitable for mixing, packaging, storing or transporting biopharmaceutical substances, comprising a single-use bag as claimed in claim 14.

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