WO2022257252A1 - 一种一次性生物工艺袋用膜材及其制备方法 - Google Patents

一种一次性生物工艺袋用膜材及其制备方法 Download PDF

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WO2022257252A1
WO2022257252A1 PCT/CN2021/108644 CN2021108644W WO2022257252A1 WO 2022257252 A1 WO2022257252 A1 WO 2022257252A1 CN 2021108644 W CN2021108644 W CN 2021108644W WO 2022257252 A1 WO2022257252 A1 WO 2022257252A1
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layer
disposable
thickness
gas barrier
liquid contact
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PCT/CN2021/108644
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English (en)
French (fr)
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杨宇明
徐江宁
宋金沛
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浙江金仪盛世生物工程有限公司
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Priority claimed from CN202110815733.0A external-priority patent/CN113580703A/zh
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C48/00—Extrusion moulding, i.e. expressing the moulding material through a die or nozzle which imparts the desired form; Apparatus therefor
    • B29C48/16—Articles comprising two or more components, e.g. co-extruded layers
    • B29C48/18—Articles comprising two or more components, e.g. co-extruded layers the components being layers
    • B29C48/21—Articles comprising two or more components, e.g. co-extruded layers the components being layers the layers being joined at their surfaces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B32—LAYERED PRODUCTS
    • B32B—LAYERED PRODUCTS, i.e. PRODUCTS BUILT-UP OF STRATA OF FLAT OR NON-FLAT, e.g. CELLULAR OR HONEYCOMB, FORM
    • B32B27/00—Layered products comprising a layer of synthetic resin
    • B32B27/06—Layered products comprising a layer of synthetic resin as the main or only constituent of a layer, which is next to another layer of the same or of a different material
    • B32B27/08—Layered products comprising a layer of synthetic resin as the main or only constituent of a layer, which is next to another layer of the same or of a different material of synthetic resin
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B32—LAYERED PRODUCTS
    • B32B—LAYERED PRODUCTS, i.e. PRODUCTS BUILT-UP OF STRATA OF FLAT OR NON-FLAT, e.g. CELLULAR OR HONEYCOMB, FORM
    • B32B27/00—Layered products comprising a layer of synthetic resin
    • B32B27/30—Layered products comprising a layer of synthetic resin comprising vinyl (co)polymers; comprising acrylic (co)polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B32—LAYERED PRODUCTS
    • B32B—LAYERED PRODUCTS, i.e. PRODUCTS BUILT-UP OF STRATA OF FLAT OR NON-FLAT, e.g. CELLULAR OR HONEYCOMB, FORM
    • B32B27/00—Layered products comprising a layer of synthetic resin
    • B32B27/32—Layered products comprising a layer of synthetic resin comprising polyolefins
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B32—LAYERED PRODUCTS
    • B32B—LAYERED PRODUCTS, i.e. PRODUCTS BUILT-UP OF STRATA OF FLAT OR NON-FLAT, e.g. CELLULAR OR HONEYCOMB, FORM
    • B32B27/00—Layered products comprising a layer of synthetic resin
    • B32B27/34—Layered products comprising a layer of synthetic resin comprising polyamides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B32—LAYERED PRODUCTS
    • B32B—LAYERED PRODUCTS, i.e. PRODUCTS BUILT-UP OF STRATA OF FLAT OR NON-FLAT, e.g. CELLULAR OR HONEYCOMB, FORM
    • B32B7/00—Layered products characterised by the relation between layers; Layered products characterised by the relative orientation of features between layers, or by the relative values of a measurable parameter between layers, i.e. products comprising layers having different physical, chemical or physicochemical properties; Layered products characterised by the interconnection of layers
    • B32B7/04—Interconnection of layers
    • B32B7/12—Interconnection of layers using interposed adhesives or interposed materials with bonding properties
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus

Definitions

  • the invention belongs to the field of membrane materials for bioreactors, and in particular relates to a membrane material for disposable biotechnology bags and a preparation method thereof.
  • the disposable bioreactor adopts an efficient oxygen transfer mechanism for oxygen transfer, which has high oxygen transfer efficiency and is easy to achieve high-density and high-activity cell culture.
  • the reactor does not require additional engineering pipeline support, does not require in-place cleaning, sterilization and disinfection equipment, and occupies a small area. It can be used for various types of cell suspension culture, research and development and production of recombinant proteins and other bioengineering products, and is suitable for production enterprises. , scientific research institutes, etc. for large-scale cell culture and sample preparation.
  • Disposable biotechnology bags can be made into liquid storage bags, mixing bags, bioreactor bags, weighing bags, and feeding bags, etc., which are useful in the development and production of various types of cell suspension cultures, recombinant proteins, and other bioengineering products.
  • Wide range of applications, no cleaning, disposable, high barrier and biocompatibility not only greatly improve work efficiency, but also avoid cross-contamination between some processes.
  • the disposable bioprocess bags used in conjunction with the disposable bioreactors are disposable and do not require cleaning, disinfection, and verification, which greatly improves work efficiency and avoids uncertain factors such as cross-infection between processes.
  • the film material for disposable bioprocess bags is a polymer multilayer composite film, and its basic structure is "liquid contact layer/adhesive layer/gas barrier layer/adhesive layer/protective layer.
  • the liquid contact layer needs to meet the needs of cells. Normal growth and metabolism, creating a suitable environment for cell growth, low extractables/leachables, no animal-derived materials, good biocompatibility, suitable for cell culture.
  • the liquid contact layer also needs to have a certain heat sealability, which can provide heat sealing and welding functions for the processing of the process bag, and provide sufficient heat seal strength to avoid liquid leakage during a cycle of cell culture. In addition, it must also have a certain Puncture resistance and bending resistance ensure reliability during use.
  • the membrane material should have wide adaptability, or meet the needs of cell growth, and at the same time, there should be no precipitation and adsorption of small molecules during use, which will affect the normal growth and metabolism of cells;
  • the gas barrier layer provides oxygen, water vapor, carbon dioxide and other gases to isolate Function; it can be used as the protective layer of the welding layer and the liquid contact layer. It needs to provide the heat sealing function of the splicing methods such as lap joint and butt joint of the film itself, to meet the high frequency welding and direct heat welding, and to provide sufficient heat sealing strength to avoid cell damage. Liquid leaks during one cycle of culture; the adhesive layer provides good interlayer bonding between the above functional layers.
  • the volume of a disposable bioreactor determines the production efficiency, and a large reactor needs to be equipped with a large-volume process bag. This requires that the membrane material needs to have sufficient strength, the welded structure needs to be sufficiently reliable, and it needs to withstand the high pressure of use and various bends that may occur during the production, folding packaging, transportation, unfolding, installation and normal use of the craft bag. breakage and possible damage.
  • the gas barrier layer provides oxygen, carbon dioxide and other gas isolation functions, and is an important part of the process bag membrane material. The oxygen permeability must be less than 1.0mL/m 2 ⁇ d ⁇ atm.
  • the gas barrier layer in the process bag can be a single layer, such as ethylene-vinyl alcohol copolymer (EVOH).
  • EVOH has good gas barrier properties, but EVOH has poor water resistance and bending resistance.
  • most of the barrier layers in domestic and foreign disposable biotechnology bags use single-layer or two-layer ethylene-vinyl alcohol copolymer (EVOH), and the barrier performance of water vapor is relatively poor, especially when applied to liquid storage bags.
  • EVOH ethylene-vinyl alcohol copolymer
  • long-term and cross-regional transportation and storage are very likely to cause antibody inactivation and failure due to lack of barrier properties.
  • Table 1 EVOH is used as the barrier layer in the composition and structure of some foreign craft bag membrane materials.
  • Table 2 There are also domestic patents involving this type of multilayer composite membrane material, as shown in Table 2.
  • the membrane materials of PALL, GE and CN201821441762.5 use nylon (PA) as its wear-resistant layer (outer layer).
  • PA nylon
  • PA is not suitable for the lap joint heat sealing welding process, which increases the processing cost of the biotechnology bag. The production efficiency is reduced, and PA is not suitable for use as the outermost protective layer.
  • there are some patents related to cell culture composite membrane materials but none of these membrane materials have a high gas barrier material layer. Therefore, it is still difficult to obtain good gas barrier properties in the existing bioprocess bags, and the water resistance, bending resistance, and puncture resistance are difficult to meet the needs, and at the same time, the cell culture effect and the culture solution storage effect are also affected.
  • Table 1 The composition and structure of some foreign craft bag film materials
  • design a comprehensive multi-layer structure membrane material to make its gas barrier layer have better water resistance, bending resistance, puncture resistance, and good biocompatibility, so that its liquid contact layer has better Excellent puncture resistance and heat sealing performance, as well as good biocompatibility, so it is suitable for obtaining disposable biobags with high strength, puncture resistance, water resistance, bending resistance, high barrier, and better cell culture effect Membranes are of great importance.
  • the present invention provides a membrane material for a disposable biotechnology bag, which uses a three-layer composite structure of nylon/ethylene-vinyl alcohol copolymer/nylon to prepare a gas barrier layer, with ULDPE or EVA as the liquid contact layer, And selected the best protective layer, adhesive layer, and appropriate thickness, the prepared disposable bioprocess bag has good gas barrier performance, water resistance, strength, bending resistance, puncture resistance, and excellent Excellent heat sealing performance, and has good biocompatibility, high cell culture density, better cell culture effect, suitable for the culture of most types of cells, simple preparation process, easy operation, high efficiency, can be used to prepare one-time Bioreactor bags, liquid storage bags, mixing bags, weighing bags and feeding bags, etc.
  • the present invention provides a use of a gas barrier layer for preparing a membrane material for a disposable biotechnology bag
  • the gas barrier layer is composed of a PA/EVOH/PA three-layer composite film.
  • the disposable biological process bag provided by the invention is specially used for cell culture.
  • the cell culture process has very strict requirements on membrane materials, which are extremely sensitive to the permeation of oxygen, water vapor, carbon dioxide and other gases, and the membrane materials must have good biocompatibility, without the adsorption and precipitation of small molecular substances, in order to obtain good results. cultivation effect.
  • Ethylene-vinyl alcohol copolymer is the polymer material with the best gas barrier properties, but EVOH has poor water resistance and bending resistance.
  • Nylon (PA) is also a polymer material with good gas barrier properties. Its barrier properties to oxygen and carbon dioxide are an order of magnitude lower than EVOH, but PA has the characteristics of puncture resistance, good flexibility, and high strength.
  • the present invention creatively adopts PA/EVOH/PA three-layer composite film as the gas barrier layer of the disposable biotechnology bag film material, which greatly improves the comprehensive performance of the film material, wherein PA and EVOH have good compatibility, and PA No adhesive layer is required between EVOH layers.
  • the PA/EVOH/PA three-layer composite barrier film can obtain excellent comprehensive performance in terms of tensile strength, gas barrier properties, puncture resistance, flexibility, and bending resistance.
  • the nylon is preferably aromatic PA.
  • PA partially modified aromatic nylon
  • aromatic PA is more suitable for the preparation of disposable bioprocess bag membrane materials, so that the prepared disposable bioprocess bags have higher strength and barrier properties, and have better water resistance, bending resistance, and puncture resistance. Excellent for culturing specific cells.
  • the present invention provides a kind of film material for disposable biological process bag, comprises protective layer/adhesive layer/gas barrier layer/adhesive layer/liquid contact layer, and described gas barrier layer is made of PA/EVOH/ Composed of PA three-layer composite film.
  • PA is aromatic PA.
  • EVOH is preferably the relevant brand of Japan Kuraray, Synthetic Chemistry and Taiwan Changchun Company, and PA recommends the use of related brand resins of DuPont of the United States, BASF of Germany, Mitsubishi of Japan, Shanghai Yinggu and Shandong Xianglong.
  • the thickness of the PA/EVOH/PA is 30-80 ⁇ m
  • the thickness of the PA is 10-30 ⁇ m
  • the thickness of the EVOH is 10-20 ⁇ m.
  • the barrier layer is too thin to meet the requirements; if it is too thick, it does not have much significance for the barrier performance, but will affect the overall processing performance. Therefore, it is preferably 30 ⁇ 80 ⁇ m. And further experiments to determine the respective thickness of PA and EVOH.
  • liquid contacted by the liquid contact layer includes cells used for culture.
  • the liquid contact layer is a layer in direct contact with the cell culture medium, which must have good biocompatibility, and at the same time, no small molecules can be precipitated during use, which will affect the normal growth and metabolism of cells.
  • the liquid contact layer is ULDPE, and the thickness of the ULDPE is 30-200 ⁇ m.
  • Ultra-low density polyethylene (ULDPE) is the most suitable liquid contact layer material found so far, and it has good compatibility for the culture of many cells.
  • the ultra-low density polyethylene (ULDPE) of the liquid contact layer is preferably Dow Engage 8480K Health+, Dow Health+ series and Sabic PCG series from Saudi Arabia.
  • the liquid contact layer is EVA, and the thickness of the EVA is 50-250 ⁇ m.
  • the primary function of the liquid contact layer is to protect and promote cell growth, and at the same time, no small molecules can be precipitated during use, which will affect the normal growth and metabolism of cells. It has been found in the actual production process that ethylene-vinyl acetate copolymer (EVA) Some cell growth is more suitable than ultra-low-density polyethylene (ULDPE). For example, for human lymphocyte PBMC, the cultivation effect of ULDPE is relatively poor; when EVA is used as the liquid contact layer, the cell concentration growth effect is very good.
  • EVA ultra-low-density polyethylene
  • the grades of EVA that can be selected include resins such as Celanese company and related grades of Yangzi Petrochemical.
  • the liquid contact layer is a blend of linear low density polyethylene and ultra low density polyethylene.
  • linear low-density polyethylene accounts for 10-100%; the thickness of the liquid contact layer is 50-250 ⁇ m.
  • the liquid contact layer should not have small molecules precipitated during use, which will affect the normal growth and metabolism of cells. Practice has proved that both LLDPE and ULDPE have good cell compatibility.
  • LLDPE has higher strength, and ULDPE has better puncture resistance and bending resistance.
  • Using the mixture of LLDPE and ULDPE as the liquid contact layer can better balance welding strength, puncture resistance and bending resistance.
  • the amount of ULDPE can be increased appropriately, so preferably, the content of LLDPE is 10-100%.
  • the thickness of the liquid contact layer is 50-250 ⁇ m. If the liquid contact layer is too thin, the welding strength may be insufficient during butt heat sealing, resulting in liquid leakage of the bio-bag, making it unusable; if the liquid contact layer is too thick, it will affect the flexibility of the bio-bag.
  • the protective layer is any one of EVA, TPU, LDPE, LLDPE, or a mixture of LDPE and LLDPE.
  • the protective layer is EVA, polyurethane elastomer (TPU), low-density polyethylene (LDPE) or linear low-density polyethylene (LLDPE) with a thickness between 50 ⁇ m and 100 ⁇ m, or low-density polyethylene (LDPE) and linear low density polyethylene.
  • TPU polyurethane elastomer
  • LDPE low-density polyethylene
  • LLDPE linear low-density polyethylene
  • the preferred LDPE and LLDPE are the relevant brands of Dow, Sabic, Borealis, Shanghai Petrochemical, Maoming Petrochemical, Lanzhou Petrochemical and other companies.
  • the preferred EVA includes resins of related brands from Celanese Company and Yangzi Petrochemical.
  • preferred TPUs include resins of related brands from companies such as Lubrizol, Bayer, Huntsman, and Basf.
  • the protective layer is a mixture of LDPE and LLDPE, and in the mixture of LDPE and LLDPE, the LDPE component is 20% to 30% in parts by mass.
  • LDPE should account for 20-30% of the mixture, which can achieve a more beautiful welding effect and excellent welding strength.
  • the mixture of LDPE and LLDPE can also improve the transparency of the membrane, which is convenient for users to observe cell growth during use.
  • LDPE and LLDPE materials for the protective layer can be selected from but not limited to the following brands of resins, such as Dow, Sabic, Borealis Bormed, Shanghai Petrochemical, Maoming Petrochemical, Lanzhou Petrochemical and other companies.
  • the protective layer has a thickness of 50-150 ⁇ m.
  • the adhesive layer is one or a mixture of EAA, EVA or EMA, and the thickness of the adhesive layer is 5-20 ⁇ m.
  • the primary function of the adhesive layer is adhesion, and one or more of ethylene-acrylic acid copolymer (EAA) or ethylene-methacrylate copolymer (EMA) can be selected.
  • EAA ethylene-acrylic acid copolymer
  • EMA ethylene-methacrylate copolymer
  • DuPont Bynel series Dow Chemical Primacor series
  • Mitsui Petrochemical ADMER series Mitsubishi Petrochemical MODIC series .
  • EVA can also be used as an adhesive layer in small and medium-sized disposable bioprocess bags; preferred EVA There are related brands of resins from Celanese and Yangzi Petrochemical.
  • the total thickness of the disposable bioprocess bag film and the thickness of each layer are mainly considered for the stability of physical properties and the strength of welding to ensure that the integrity test items of the bag are compliant.
  • the liquid contact layer in contact with the cells and the outermost protective layer are relatively thick to ensure the thickness of the butt or lap welded layer to ensure the heat seal strength and the integrity of the bag; the thickness of the barrier layer reaches the corresponding The gas transmission rate is sufficient, and the thickness is moderate.
  • the adhesive layer only needs to provide good adhesion between the layers, so the layer is relatively thin.
  • the present invention provides a membrane material for a disposable bioprocess bag with a simplified structure, including a protective layer/gas barrier layer/liquid contact layer, and the gas barrier layer is composed of a three-layer composite film of PA/EVOH/PA .
  • PA is aromatic PA.
  • the thickness of the PA/EVOH/PA is 30-80 ⁇ m
  • the thickness of the PA is 10-30 ⁇ m
  • the thickness of the EVOH is 10-20 ⁇ m.
  • liquid contact layer and the protective layer are EVA.
  • the thickness of the liquid contact layer is 50-250 ⁇ m
  • the thickness of the protective layer is 50-100 ⁇ m.
  • the composition structure of the simplified structure membrane is EVA/PA/EVOH/PA/EVA.
  • This membrane can be prepared by blow molding or casting.
  • the required equipment has 3 screws to prepare the membrane with this symmetrical structure. , easy to prepare and high in production efficiency.
  • the present invention provides a membrane material for a disposable bioprocessing bag with a simplified structure, comprising a protective layer/adhesive layer/gas barrier layer/adhesive layer/liquid contact layer, and the gas barrier layer is made of nylon Layer/ethylene-vinyl alcohol copolymer layer is composed of two-layer composite film; the liquid contact layer is a mixture of linear low-density polyethylene and ultra-low-density polyethylene.
  • linear low-density polyethylene accounts for 10-100%; the thickness of the gas barrier layer is 30-80 ⁇ m; wherein the thickness of the nylon layer is 20-70 ⁇ m, and the thickness of the ethylene-vinyl alcohol copolymer layer is 10-60 ⁇ m.
  • the present invention can also adopt PA/EVOH two-layer composite film as the gas barrier layer of disposable biotechnology bag film material, this is because the liquid contact layer provided by the present invention is the mixture of LLDPE and ULDPE, has certain The anti-puncture ability can make up for part of the effect of the gas barrier layer, and can be used to prepare some smaller bioprocess bags.
  • the present invention provides a method for preparing the above-mentioned film material, which is characterized in that it is prepared by co-extrusion casting method, and the specific steps are: 1) each layer of raw materials is respectively added to the corresponding extruder Medium; 2) It is produced by melting, co-extrusion blow molding or casting under the condition of head temperature of 120-220 °C.
  • the preparation method provided by the present invention mainly adopts the co-extrusion casting method, and the specific steps are: 1) adding the raw materials of each layer into the corresponding extruder; , co-extrusion casting and other processes, by adjusting the screw speed to obtain a specific thickness of the film material for disposable biotechnology bags.
  • Multi-layer co-extrusion blow molding is a molding method of multi-layer polymer composite film materials. It can melt two or more polymers using more than two extruders, and then pass through separate flow channels. Enter the multi-layer die to co-extrude the "film tube" with multi-layer structure, and form a polymer composite film material with multi-layer structure.
  • the polymer film prepared by the casting method is first to plasticize and melt the raw material through an extruder, and then extrude through a T-shaped structure forming die. The sheet is cooled and shaped on the cooling roller, and then rolled after traction and edge trimming.
  • the thickness of the film when the thickness of the film is less than 300 ⁇ m, it is produced by co-extrusion blow molding or casting; when the thickness of the film is greater than 300 ⁇ m, it is produced by co-extrusion casting.
  • the multi-layer co-extrusion blow molding process is simple and suitable for films with an overall thickness of less than 300 ⁇ m.
  • the blow molding method becomes somewhat difficult, and the transparency of the film material will decrease.
  • the casting method is more suitable for obtaining a film material with uniform thickness and good transparency.
  • co-extruded cast film Compared with co-extruded blown film, co-extruded cast film has better film transparency and better thickness uniformity, and is more suitable for the production of thicker disposable bioprocess bags.
  • the thickness of the film material used for disposable bioreactor process bags is much larger than that of ordinary packaging films, generally exceeding 300 ⁇ m. It is very difficult to use co-extrusion blow molding, and it needs to be produced by co-extrusion casting.
  • the film material for disposable biotechnology bags provided by the present invention is preferably prepared by co-extrusion casting method.
  • the co-extrusion casting unit should have a suitable number of screws and distributor inserts.
  • the present invention provides the use of nylon for preparing a gas barrier layer of a membrane material for a disposable bioprocessing bag.
  • the gas barrier layer is a three-layer composite structure of nylon/ethylene-vinyl alcohol copolymer/nylon copolymer; the nylon is aromatic nylon; the membrane material is used to prepare a primary membrane for culturing cells in a bioreactor Sexual biotechnology bags.
  • the tensile strength of the membrane material for the disposable biotechnology bag provided by the invention can reach 20MPa, the oxygen permeability is less than 0.1mL/m 2 ⁇ d ⁇ atm, the water vapor transmission rate is less than 0.5mL/m 2 ⁇ d ⁇ atm,
  • the overall membrane material has high transparency, good flexibility, good puncture resistance, strong and durable, and is suitable for direct heating or high frequency welding to realize lap or butt welding of membrane materials.
  • manufacturers of this kind of polymer multilayer composite membrane can provide membrane materials with different composition and structure designs. Due to the special functional requirements of biomedical membrane materials and the high level of membrane manufacturing environment requirements, there are strict restrictions on the functional layers of the membrane materials.
  • this kind of membrane material is used for cell culture, especially for ultra-large-capacity cell culture, and the requirements for its membrane material are particularly strict.
  • strict Control the gas exchange between the membrane material and the outside world. If there is an irregular gas exchange, it will seriously affect the quality and output of the cell culture in the membrane material.
  • the process bag needs to be sterilized by ⁇ -ray irradiation, electron beam irradiation or ethylene oxide before use.
  • the physical and chemical properties of the film cannot change. Chemical changes, or the leakage of chemical substances, will pollute the culture medium and affect the quality of cell culture; within a cycle of cell culture (such as 5 days, 7 days, 10 days, or within 30 days, etc.) etc.), the membrane material must not have any precipitation or dissolution of low-molecular substances that affect the effect of cell culture.
  • the cell culture functionality, tensile strength, gas barrier performance, and heat sealing performance of the membrane material need to be considered as a whole to make the membrane material more practical.
  • the preparation process is simple, the operation is convenient, and the efficiency is high, and it can be used for preparing disposable bioreactor bags, liquid storage bags, mixing bags, weighing bags, and feeding bags, etc.
  • Fig. 1 the schematic diagram of the structure of each layer of the membrane material used in the disposable bioprocessing bag in Examples 1, 3, and 4
  • Fig. 3 the schematic diagram of the structure of each layer of the multi-layer co-extruded disposable biotechnology bag film material in Example 15
  • FIG. 4 the schematic diagram of the structure of each layer of the multi-layer co-extruded disposable biotechnology bag film material in Example 16
  • Embodiment 1 Containing the membrane material of EVA disposable bioprocess bag
  • the co-extrusion casting method is used to prepare a film material for a disposable biotechnology bag containing EVA with a thickness of 325 ⁇ m.
  • the composition and structure of the film material include as shown in Figure 1, including a protective layer 1, an adhesive layer 2, Gas barrier layer 3/4/5, adhesive layer 6 and liquid contact layer 7.
  • the protective layer 1 adopts the Dow series LDPE and LLDPE mixture of Dow, LDPE accounts for 30%, and LLDPE accounts for 70%.
  • the thickness of this layer is 50 ⁇ m.
  • the adhesive layers 2 and 6 are made of Primacor resin from Dow Chemical, with a thickness of 10 ⁇ m.
  • the gas barrier layers 3 and 5 are aromatic PA, which are made by American DuPont, and the thickness of the layers is 20 ⁇ m.
  • the gas barrier layer 4 is EVOH using Kuraray EVAL resin, and the thickness of this layer is 15 ⁇ m.
  • the liquid contact layer 7 is made of Celanese EVA, and the thickness of this layer is 200 ⁇ m.
  • the preparation method of the disposable biotechnology bag membrane material containing EVA is as follows:
  • the thickness of the membrane material is precisely controlled by an online thickness measuring device, and the thickness of each layer is controlled by adjusting the screw speed.
  • the oxygen, water vapor permeability, tensile strength, transparency and cell culture effect (biocompatibility) of the prepared EVA-containing disposable bioprocess bag membrane were investigated.
  • the oxygen and water vapor permeability of the disposable bioprocess bag containing EVA are tested by the pressure difference method, and the samples are cut into discs with a diameter of not less than 100mm, and the test is carried out at room temperature according to the GB/T-1038 standard. Test, test the oxygen and water vapor permeability of the membrane. There are 3 samples in each group, and the average value is calculated.
  • the tensile strength and welding strength of the film material for disposable biotechnology bags containing EVA are tested by Instron universal testing machine. For testing, at least 5 splines are tested for each sample, and the average value is calculated.
  • the transparency of the film material for disposable bioprocess bags containing EVA is tested by the haze meter method, according to GB/T-2410-2008, and 3 samples are tested for each group, and the average value is calculated.
  • the biocompatibility of the membrane material for disposable biotechnology bags containing EVA is characterized by statistical cell density.
  • the prepared membrane material is welded by direct heating lap joint, it is prepared into a 50L disposable biological culture bag. , and sterilized by gamma-ray irradiation.
  • Human lymphocyte PBMC was used as the cultured cells, and the initial concentration was 1 ⁇ 10 5 /ml.
  • the disposable biological culture bag is packed in the bioreactor, and the human lymphocyte PBMC is cultured in the disposable bioprocess bag for 5 days.
  • Embodiment 2 The disposable bioprocess bag film material containing EVA of simplified structure
  • the composition and structure of the EVA-containing disposable bioprocessing bag membrane material provided in this example is shown in Figure 2, including a protective layer 8, a gas barrier layer 9/10/11, and a liquid contact layer 12.
  • a film material for a disposable biotechnology bag with a thickness of 220 ⁇ m was prepared by co-extrusion casting method
  • the protective layer 8 and the liquid contact layer 12 adopt Celanese EVA, both of which have a thickness of 90 ⁇ m;
  • the middle gas barrier layer 10 is EVOH of Kuraray EVAL series, with a thickness of 10 ⁇ m;
  • the 9 and 11 of the gas barrier layers on both sides are aromatic PA, using the relevant brand of DuPont in the United States, and the thickness of this layer is 15 ⁇ m.
  • the thickness of the membrane material is precisely controlled by an online thickness measuring device, and the thickness of each layer is controlled by adjusting the screw speed.
  • the thickness of the film is precisely controlled by the online thickness measuring device, and the thickness of each layer is controlled by the screw speed adjustment.
  • the oxygen, water vapor permeability, tensile strength, transparency and cell culture effect (biocompatibility) of the prepared multilayer co-extruded disposable bioprocess bag membrane were investigated. Among them, the detection methods of oxygen, water vapor permeability, tensile strength, transparency and cell culture effect are shown in Example 1, and the test results of cultured cells are shown in Table 4.
  • a disposable biotechnology bag film with a thickness of 250 ⁇ m is prepared by co-extrusion blow molding method.
  • the composition and structure of the film are as shown in Fig. Layers 3/4/5, adhesive layer 6 and liquid contact layer 7.
  • the heat-sealing layer LDPE adopts the Dow series of Dow, and the thickness of this layer is 50 ⁇ m.
  • the adhesive layers 2 and 6 are made of Dow Chemical Primacor resin with a thickness of 5 ⁇ m.
  • the gas barrier layers 3 and 5 are aromatic PA, which are made by American DuPont, and the thickness of the layers is 15 ⁇ m.
  • the gas barrier layer 4 is EVOH using Kuraray EVAL resin, and the thickness of this layer is 10 ⁇ m.
  • the ULDPE of the liquid contact layer 7 is Engage 8480K from American Dow Company, and the thickness of this layer is 150 ⁇ m.
  • the preparation method of the multi-layer co-extruded disposable biotechnology bag film material containing ULDPE is as follows:
  • the oxygen, water vapor permeability, tensile strength, transparency and cell culture effect (biocompatibility) of the prepared multilayer co-extruded disposable bioprocess bag membrane were investigated.
  • the oxygen and water vapor permeability of a multi-layer co-extruded disposable biotechnology bag film was tested by the pressure difference method, and the sample was cut into a disc with a diameter of not less than 100mm, according to the GB/T-1038 standard.
  • the test is carried out at room temperature to test the oxygen and water vapor permeability of the membrane. There are 3 samples in each group, and the average value is calculated.
  • the tensile strength of a multi-layer co-extruded disposable biotechnology bag film is tested by an Instron universal testing machine.
  • the sample is cut into a dumbbell-shaped sample and tested at a rate of 200mm/min.
  • Each sample is tested for at least 5 splines and calculate the average.
  • the transparency of a film material for multi-layer co-extruded disposable biotechnology bags is tested by the haze meter method according to GB/T-2410-2008, and 3 samples are tested for each group, and the average value is calculated.
  • the biocompatibility of a multi-layer co-extruded disposable biotechnology bag membrane is characterized by statistical cell density, and the prepared membrane is welded by direct heating lap joints to prepare a 5L disposable Biological culture bags, and sterilized by gamma ray irradiation.
  • 293 human embryonic kidney cells were used as cultured cells, and the initial concentration was 100,000 cells/ml.
  • Disposable biological culture bags were packed in a bioreactor, and 293 human embryonic kidney cells were cultured in the disposable bioprocess bags for 5 days under the conditions of temperature 37°C and stirring speed of 200rpm, and the cell density was detected after the culture.
  • the composition and structure of the ULDPE-containing disposable bioprocess bag film material provided in this example is shown in Figure 1, including a heat-sealing layer 1, an adhesive layer 2, a gas barrier layer 3/4/5, an adhesive layer 6 and Liquid contact layer 7 .
  • the co-extrusion casting method is used to prepare a disposable biotechnology bag film material with a thickness of 400 ⁇ m;
  • Heat-sealing layer 1 adopts the EVA of Celanese company, and the thickness of this layer is 100 ⁇ m;
  • Adhesive layers 2 and 6 use Yangzi Petrochemical EVA with a thickness of 10 ⁇ m;
  • the gas barrier layers 3 and 5 on both sides are EVOH of the Kuraray EVAL series, with a thickness of 30 ⁇ m;
  • the intermediate gas barrier layer 4 is made of aromatic PA of the relevant brand of DuPont in the United States, and the thickness of this layer is 20 ⁇ m;
  • the liquid contact layer 7 is Dow Chemical Engage 8480K series ULDPE with a thickness of 200 ⁇ m.
  • the thickness of the film is precisely controlled by the online thickness measuring device, and the thickness of each layer is controlled by the screw speed adjustment.
  • the oxygen, water vapor permeability, tensile strength, transparency and cell culture effect (biocompatibility) of the prepared multilayer co-extruded disposable bioprocess bag membrane were investigated.
  • the detection methods of oxygen, water vapor permeability, tensile strength, transparency and cell culture effect are as shown in Example 1, and 293 human embryonic kidney cells are used as the cultured cells, and the detection results are shown in Table 6.
  • Embodiment 5 The influence of adopting different gas barrier layers on the performance of disposable bioprocess bags containing EVA
  • Example 1 the method provided in Example 1 is used to prepare EVA as a liquid contact layer with a total thickness of 325 ⁇ m for disposable biotechnology bag membranes, and different gas barrier layers as shown in Table 5 are used respectively, wherein the thickness is The thickness of the gas barrier layer was used to investigate the oxygen, water vapor permeability, tensile strength, transparency and cell culture effect (biocompatibility) of the prepared multilayer co-extruded disposable bioprocess bag membrane.
  • EVOH adopts Kuraray EVAL series
  • PA adopts aromatic PA, aliphatic PA, and aliphatic-aromatic PA of DuPont related brands in the United States
  • PE uses related brands of Dow Company in the United States.
  • Example 1 oxygen, water vapor permeability, tensile strength, welding strength, transparency and cell culture effect detection methods are as shown in Example 1, the cultured cells are human lymphocytes PBMC, and the inspection results are shown in Table 7.
  • PE/EVOH/PE which is also in the form of a gas barrier layer of a three-layer composite film, has far less strength, gas barrier performance and cell culture function than PA/EVOH/PA Combination; most preferably a three-layer composite film gas barrier layer of PA/EVOH/PA, which can achieve the best tensile strength, gas barrier performance and cell culture function.
  • the disposable biotechnology bag membrane material prepared by the gas barrier layer with the serial number of 6 in Example 5 is used, that is, PA/EVOH/PA is used as the gas barrier layer, and the disposable biotechnology bag is prepared according to the method provided in Example 1.
  • EVOH adopts Kuraray EVAL series
  • PA adopts aromatic PA of DuPont related brand in the United States.
  • Example 1 oxygen, water vapor permeability, tensile strength, transparency and cell culture effect detection methods are as shown in Example 1, and the cultured cells are human lymphocytes PBMC, and the investigation results are shown in Table 8.
  • the different thicknesses of the gas barrier layer have a great influence on the performance of the prepared disposable bioprocess bag membrane, and choosing an appropriate thickness can further improve the strength and gas barrier performance of the disposable bioprocess bag membrane And cell culture function, studies have proved that the thickness of the gas barrier layer is 30-80 ⁇ m, the thickness of the gas barrier layer in the middle is 10-20 ⁇ m, and the thickness of the gas barrier layer on both sides is 10-30 ⁇ m.
  • the strength, gas barrier performance and cell culture function of the bag film material are significantly improved, and the optimal thickness of the gas barrier layer PA/EVOH/PA is 30/20/30 ⁇ m.
  • Embodiment 7 The impact of adopting different protective layers on the performance of disposable bioprocess bags containing EVA
  • the disposable biotechnology bag membrane material prepared by the gas barrier layer with the serial number of 6 in Example 5 is used, that is, PA/EVOH/PA is used as the gas barrier layer, and the disposable biotechnology bag is prepared according to the method provided in Example 1.
  • LDPE and LLDPE use American Dow DOW; EVA uses EVA from Celanese; TPU uses TPU from Lubrizol; PA uses aromatic PA of DuPont related brands in the United States.
  • oxygen, water vapor permeability, tensile strength, transparency and cell culture effect detection method are as shown in embodiment 1, heat-sealing welding strength is obtained by preparing it into a standard spline, and performing a peel strength test, and the cultured cells are Human lymphocyte PBMC, the investigation results are shown in Table 9.
  • the different thicknesses of the protective layer will obviously affect the heat-sealing and welding performance of the prepared disposable bioprocessing bag membrane, which will also affect the cell culture effect , the preferred thickness of the protective layer is 30-150 ⁇ m; when the protective layer is prepared with a mixture of LDPE:LLDPE of 2:8, the cell culture effect is better when the thickness of the protective layer is 50 ⁇ m.
  • the disposable biotechnology bag film material prepared by the serial number 6 of Example 7 is used, that is, PA/EVOH/PA is used as the gas barrier layer, and the LDPE:LLDPE is a mixture of 2:8 to prepare the protective layer, according to Example 1
  • the method provided prepares the film material for disposable biotechnology bags, and adopts different body contact layers as shown in Table 10, wherein the thickness is the thickness of the liquid contact layer, and investigates the obtained multilayer co-extruded disposable biotechnology bags Dissolution of small molecular substances, oxygen, water vapor permeability, tensile strength, transparency and cell culture effect (biocompatibility) of membrane materials.
  • LDPE adopts LDPE from Dow DOW in the United States; EVA adopts EVA from Celanese Company; ULDPE adopts ULDPE from Dow Chemical Engage8480K series.
  • Example 1 oxygen, water vapor permeability, tensile strength, transparency and cell culture effect detection methods are as shown in Example 1, and the cultured cells are two kinds of human lymphocyte PBMC and human embryonic kidney cell 293; Chromatography/liquid chromatography-mass spectrometry method detection; investigation results are shown in Table 10.
  • the different thicknesses of the liquid contact layer will obviously affect the cell culture effect of the prepared disposable bioprocess bag membrane material, and the preferred thickness of the liquid contact layer is 30 ⁇ 200 ⁇ m; when using ULDPE and EVA as the liquid contact layer, the preferred thickness of the liquid contact layer is 200 ⁇ m.
  • Example 9 The effect of using different adhesive layers on the performance of disposable bioprocess bags containing EVA
  • the disposable bioprocess bag film material prepared by No. 3 of Example 8 is used, that is, PA/EVOH/PA is used as the gas barrier layer, LDPE:LLDPE is a mixture of 2:8 to prepare the protective layer, and EVA is used to prepare the liquid contact layer.
  • PA/EVOH/PA is used as the gas barrier layer
  • LDPE:LLDPE is a mixture of 2:8 to prepare the protective layer
  • EVA is used to prepare the liquid contact layer.
  • EVA adopts the EVA of Celanese Company
  • EAA and EMA adopt the relevant brand products of DuPont Company of the United States and Dow Chemical respectively.
  • Example 1 oxygen, water vapor permeability, tensile strength, transparency and cell culture effect detection methods are as shown in Example 1; the investigation results are as shown in Table 11, because the adhesive layer includes two layers of 2 and 6, so the adhesive layer is used.
  • Layer 2/barrier layer/adhesive layer 6, the thickness is the thickness of the two layers respectively.
  • the different thicknesses of the adhesive layer will obviously affect the cell culture effect of the prepared disposable bioprocessing bag membrane material, and too thick or too thin will cause cell culture.
  • the density is reduced, and the preferred thickness of the adhesive layer is 5-20 ⁇ m; when EVA is used to prepare the adhesive layer, the preferred thickness of the liquid contact layer is 10 ⁇ m.
  • Example 4 the method provided in Example 4 is used to prepare a disposable biotechnology bag film material with a total thickness of 400 ⁇ m, and different gas barrier layers as shown in Table 12 are used respectively, where the thickness is the thickness of the gas barrier layer,
  • the oxygen, water vapor permeability, tensile strength, transparency and cell culture effect (biocompatibility) of the prepared multilayer co-extruded disposable bioprocess bag membrane were investigated.
  • EVOH adopts Kuraray EVAL series
  • PA adopts aromatic PA, aliphatic PA, and aliphatic-aromatic PA of DuPont related brands in the United States
  • PE uses related brands of Dow Company in the United States.
  • Example 4 oxygen, water vapor permeability, tensile strength, welding strength, transparency and cell culture effect detection methods are shown in Example 4, and 293 human embryonic kidney cells were used as cultured cells, and the investigation results are shown in Table 12.
  • EVOH has poor water resistance, so it is used as the outer barrier In the case of water, the barrier performance will decrease, resulting in a decrease in the tensile strength, oxygen permeability, water vapor transmission rate, transparency and cell culture density of the disposable bioprocess bag film; the same is a three-layer composite film PE/EVOH/PE in the form of a gas barrier layer is far inferior to PA/EVOH/PA or the combination of EVOH/PA/EVOH in terms of strength, gas barrier performance and cell culture function; the most preferred is a three-layer composite of PA/EVOH/PA Membrane gas barrier for optimum tensile strength, gas barrier properties and cell culture functionality.
  • the disposable biotechnology bag film material prepared by the gas barrier layer with the serial number of 6 in Example 10 is used, that is, PA/EVOH/PA is used as the gas barrier layer, and the disposable biotechnology bag is prepared according to the method provided in Example 4
  • Use film material and adopt the gas barrier layer of different thicknesses as shown in Table 13 respectively, wherein the thickness is the thickness of gas barrier layer, investigate the oxygen, water content of the multilayer co-extruded disposable biotechnology bag film material that makes. Vapor permeability, tensile strength, transparency and cell culture effect (biocompatibility).
  • EVOH adopts Kuraray EVAL series
  • PA adopts aromatic PA of DuPont related brand in the United States.
  • Example 4 oxygen, water vapor permeability, tensile strength, transparency and cell culture effect detection methods are shown in Example 4, and 293 human embryonic kidney cells were used as cultured cells, and the investigation results are shown in Table 13.
  • the different thicknesses of the gas barrier layer have a great influence on the performance of the prepared disposable bioprocess bag membrane, and choosing an appropriate thickness can further improve the strength and gas barrier performance of the disposable bioprocess bag membrane And cell culture function, studies have proved that the thickness of the gas barrier layer is 30-80 ⁇ m, the thickness of the gas barrier layer in the middle is 10-20 ⁇ m, and the thickness of the gas barrier layer on both sides is 10-30 ⁇ m.
  • the strength, gas barrier performance and cell culture function of the bag film material are significantly improved; comprehensively, the optimal thickness of the gas barrier layer PA/EVOH/PA is 30/20/30 ⁇ m.
  • Example 12 Effects of different heat-sealing layers on the performance of ULDPE-containing disposable bioprocess bags
  • the disposable biotechnology bag film material prepared by the gas barrier layer with the serial number of 6 in Example 10 is used, that is, PA/EVOH/PA is used as the gas barrier layer, and the disposable biotechnology bag is prepared according to the method provided in Example 4 Using film materials, and adopting different heat-sealing layers as shown in Table 14 respectively, wherein the thickness is the thickness of the heat-sealing layer, investigate the oxygen and water vapor permeation of the prepared multi-layer co-extruded disposable biotechnology bag film materials rate, tensile strength, transparency, heat seal welding performance and cell culture effect (biocompatibility).
  • LDPE and LLDPE use American Dow DOW; EVA uses EVA from Celanese; TPU uses TPU from Lubrizol; PA uses aromatic PA of DuPont related brands in the United States.
  • Example 4 oxygen, water vapor permeability, tensile strength, transparency and cell culture effect detection methods are as shown in Example 4, cultured cells use 293 human embryonic kidney cells, and the heat-sealing welding strength is prepared into standard splines, carried out The peel strength test was obtained, and the investigation results are shown in Table 14.
  • the different thicknesses of the heat-sealing layer will obviously affect the heat-sealing and welding performance of the prepared disposable bioprocess bag membrane, which will also affect the cell culture effect. Influence, the preferred thickness of the heat-seal layer is 30-150 ⁇ m; when the heat-seal layer is prepared with a mixture of LDPE:LLDPE of 2:8, the preferred thickness of the heat-seal layer is 100 ⁇ m.
  • Example 13 The effect of using different liquid contact layers on the performance of disposable bioprocess bags containing ULDPE
  • the disposable bioprocess bag film material prepared by serial number 6 in Example 12 is used, that is, PA/EVOH/PA is used as the gas barrier layer, and the heat-sealing layer is prepared with a mixture of LDPE:LLDPE of 2:8, according to the example
  • the method provided in 4 prepares membrane materials for disposable biotechnology bags, and adopts different body contact layers as shown in Table 8, the thickness of which is the thickness of the liquid contact layer, and investigates the prepared multilayer co-extruded disposable biotechnology bags. Dissolution of small molecular substances, oxygen, water vapor permeability, tensile strength, transparency and cell culture effect (biocompatibility) of film materials for bags.
  • LDPE adopts LDPE from Dow DOW in the United States; EVA adopts EVA from Celanese Company; ULDPE adopts ULDPE from Dow Chemical Engage8480K series.
  • Example 4 oxygen, water vapor permeability, tensile strength, transparency and cell culture effect detection methods are as shown in Example 4; cultured cells use 293 human embryonic kidney cells, and are detected by the method of gas chromatography/liquid chromatography-mass spectrometry ; The investigation results are shown in Table 15.
  • the disposable bioprocess bag film material prepared by No. 1 of Example 13 is used, that is, PA/EVOH/PA is used as the gas barrier layer, the heat-sealing layer is prepared by a mixture of LDPE:LLDPE at 2:8, and the liquid contact layer is prepared by ULDPE.
  • layer according to the method provided in Example 4 to prepare a disposable bioprocess bag film material, and adopt different adhesive layers as shown in Table 15 respectively, wherein the thickness is the thickness of the adhesive layer, and investigate the obtained multi-layer total Oxygen, water vapor permeability, tensile strength, transparency and cell culture effect (biocompatibility) of membrane materials for extruded disposable bioprocess bags.
  • EVA adopts the EVA of Celanese Company
  • EAA and EMA adopt the relevant brand products of DuPont Company of the United States and Dow Chemical respectively.
  • the detection method of oxygen, water vapor permeability, tensile strength, transparency and cell culture effect is as shown in Example 4; the cultured cells use 293 human embryonic kidney cells, and the investigation results are as shown in Table 16, because the adhesive layer contains 2 and 6 are two layers, so it is represented by adhesive layer 2/barrier layer/adhesive layer 6, and the thickness is the thickness of the two layers respectively.
  • Example 15 Multi-layer co-extruded film material for disposable bioprocess bags containing LLDPE and ULDPE
  • the co-extrusion casting method is used to prepare a disposable biotechnology bag membrane material with a thickness of 325 ⁇ m.
  • the composition and structure of the membrane material are as shown in Figure 3, including a protective layer 1, an adhesive layer 2, and a gas barrier layer. 3/4/5, adhesive layer 6 and liquid contact layer 7.
  • the protective layer is a mixture of LDPE and LLDPE, LDPE accounts for 30%, and LLDPE accounts for 70%.
  • LDPE adopts Dow's DOW series
  • LLDPE adopts American Dow's DOW
  • the thickness of this layer is 50 ⁇ m.
  • the adhesive layers 2 and 6 are made of EVA, and the EVA is made of Celanese EVA, and the thickness of this layer is 10 ⁇ m.
  • the gas barrier layers 3 and 5 are aromatic PA, which are made by American DuPont, and the thickness of the layers is 20 ⁇ m.
  • the gas barrier layer 4 is EVOH using Kuraray EVAL resin, and the thickness of this layer is 15 ⁇ m.
  • the liquid contact layer 7 is a mixture of LLDPE and ULDPE, LLDPE accounts for 10%, ULDPE accounts for 90%, LLDPE adopts Dow DOW of the United States, ULDPE adopts Engage 8480K of Dow Company of the United States, and the thickness of this layer is 200 ⁇ m.
  • the preparation method of the film material for multi-layer co-extrusion disposable biotechnology bag is as follows:
  • the thickness of the film is precisely controlled by the online thickness measuring device, and the thickness of each layer is controlled by the screw speed adjustment.
  • the oxygen, water vapor permeability, tensile strength, welding strength, puncture resistance, transparency and cell culture effect (biocompatibility) of the prepared multi-layer co-extruded disposable bioprocess bag film were investigated.
  • the oxygen and water vapor permeability of a multi-layer co-extruded disposable biotechnology bag film is tested by the pressure difference method, and the sample is cut into a disc with a diameter of not less than 100mm, according to the GB/T-1038 standard
  • the test was carried out at room temperature to test the oxygen and water vapor permeability of the membrane. There are 3 samples in each group, and the average value is calculated.
  • the tensile strength and welding strength of a multi-layer co-extruded disposable biotechnology bag film are tested by an Instron universal testing machine.
  • the samples are respectively cut into dumbbell-shaped specimens and prepared into corresponding welding specimens.
  • /min rate for testing at least 5 splines were tested for each sample, and the average value was calculated.
  • the transparency of a multi-layer co-extruded disposable biotechnology bag film is tested by the haze meter method, according to GB/T-2410-2008, and 3 samples are tested for each group, and the average value is calculated.
  • the puncture resistance performance test of the prepared membrane material for disposable bioprocess bags was carried out at a speed of 200mm/min ⁇ 20mm/min. Record the maximum force applied to puncture the sample, test 10 samples each time, and calculate the average value.
  • the biocompatibility of the film material for multi-layer co-extrusion disposable biotechnology bags is characterized by statistical cell density.
  • the culture bag was sterilized by gamma radiation.
  • Human embryonic kidney cells 293 (hereinafter referred to as 293 cells) and SF9 insect cells were used as cultured cells, and the initial concentration was 1 ⁇ 10 5 /ml.
  • the disposable biological culture bag was packed in the bioreactor, and the 293 cells and SF9 insect cells were cultured in the disposable bioprocess bag for 5 days, the culture condition was 37°C, the stirring speed was 200rpm, and the cell density was detected after the culture.
  • Table 17 shows the physical properties and cell density results of the prepared multilayer co-extruded disposable bioprocess bag film.
  • Example 16 Multi-layer co-extruded film material for disposable bioprocess bags containing LLDPE and ULDPE
  • a film material for a disposable biotechnology bag with a thickness of 305 ⁇ m is prepared by co-extrusion casting method.
  • the composition and structure of the film material include as shown in Figure 4, including a protective layer 13, an adhesive layer 14, and a gas barrier layer. 15/16, adhesive layer 17 and liquid contact layer 18.
  • the protective layer 13 is a mixture of LDPE and LLDPE, LDPE accounts for 30%, and LLDPE accounts for 70%.
  • LDPE adopts Dow series of Dow
  • LLDPE adopts American Dow DOW
  • the thickness of this layer is 50 ⁇ m.
  • the adhesive layers 14 and 17 are made of EVA
  • the EVA is made of Celanese EVA
  • the thickness of this layer is 10 ⁇ m.
  • the gas barrier layer 15 is aromatic PA, which adopts the relevant brand of American DuPont, and the thickness of this layer is 20 ⁇ m.
  • the gas barrier layer 16 is EVOH using Kuraray EVAL resin, and the thickness of this layer is 15 ⁇ m.
  • the liquid contact layer 18 adopts a mixture of LLDPE and ULDPE, LLDPE accounts for 10%, ULDPE accounts for 90%, LLDPE adopts American Dow DOW, ULDPE adopts American Dow Engage 8480K, and the thickness of this layer is 200 ⁇ m.
  • the preparation method of the film material for multi-layer co-extrusion disposable biotechnology bag is as follows:
  • the thickness of the film is precisely controlled by the online thickness measuring device, and the thickness of each layer is controlled by the screw speed adjustment.
  • the oxygen, water vapor permeability, tensile strength, welding strength, puncture resistance, transparency and cell culture effect (biocompatibility) of the prepared multi-layer co-extruded disposable bioprocess bag film were investigated.
  • the detection method is as shown in Example 1. 293 cells and SF9 insect cells were used as cultured cells, and the initial concentration was 1 ⁇ 10 5 /ml.
  • the disposable biological culture bag was packed in the bioreactor, and the 293 cells and SF9 insect cells were cultured in the disposable bioprocess bag for 5 days, the culture condition was 37°C, the stirring speed was 200rpm, and the cell density was detected after the culture.
  • Table 18 shows the physical properties and cell density results of the prepared multilayer co-extruded disposable bioprocess bag film.
  • the disposable biotechnology bag film material is prepared according to the method of Example 15, that is, PA/EVOH/PA is used as the gas barrier layer, and the mixture of LDPE:LLDPE is 3:7 to prepare the protective layer, according to the method provided in Example 15
  • the detection method of oxygen, water vapor permeability, tensile strength, transparency and cell culture effect is as shown in embodiment 15, uses SF9 insect cell and human embryonic kidney cell 293 (hereinafter referred to as 293 cell) respectively for culture cell, initial concentration is 1 ⁇ 10 5 /ml; the dissolution of small molecular substances was detected by gas chromatography/liquid chromatography-mass spectrometry; the investigation results are shown in Table 19.
  • Example 18 Film materials for disposable bioprocess bags containing LLDPE and ULDPE with different barrier layer structures
  • Example 15 the method provided in Example 15 is used to prepare a disposable bioprocess bag film material with a total thickness of 325 ⁇ m, and the structure is protective layer/adhesive layer/barrier layer 1/barrier layer 2/barrier layer 3/adhesive layer/ The liquid contact layer, and adopt different gas barrier layers as shown in Table 20, wherein PA refers to aromatic PA, and the thickness is the thickness of the gas barrier layer.
  • EVOH adopts Kuraray EVAL series
  • PA adopts aromatic PA of DuPont related brand in the United States.
  • the oxygen, water vapor permeability, tensile strength, welding strength, transparency and cell culture effect detection methods of the prepared membrane are as shown in Example 15, and SF9 insect cells are used for culture cells, and the inspection results are shown in Table 20.
  • the barrier performance will drop sharply, resulting in the tensile strength and oxygen permeability of the disposable bioprocess bag membrane. Rate, water vapor transmission rate, transparency and cell culture density are all decreased; the most preferred is the gas barrier layer of the three-layer composite film of PA/EVOH/PA, which can achieve the best tensile strength, gas barrier performance and cell culture function .
  • Example 19 Effect of Different Thicknesses of the Gas Barrier Layer on the Performance of Disposable Bioprocess Bags Containing LLDPE and ULDPE
  • the disposable biotechnology bag film material prepared by the gas barrier layer with the serial number of 8 in Example 18 is used, that is, PA/EVOH/PA is used as the gas barrier layer, and the disposable biotechnology bag is prepared according to the method provided in Example 15.
  • LDPE and LLDPE use American Dow DOW; EVA uses EVA from Celanese; PA uses aromatic PA of DuPont related brands in the United States.
  • Example 15 the detection methods of oxygen, water vapor permeability, tensile strength, puncture resistance, transparency and cell culture effect are shown in Example 15, and SF9 insect cells were used for cultured cells, and the inspection results are shown in Table 21.
  • the different thicknesses of the gas barrier layer have a great influence on the performance of the prepared disposable bioprocess bag membrane, and choosing an appropriate thickness can further improve the strength and gas barrier performance of the disposable bioprocess bag membrane And cell culture function, studies have proved that the thickness of the gas barrier layer is 30-80 ⁇ m, the thickness of the gas barrier layer in the middle is 10-20 ⁇ m, and the thickness of the gas barrier layer on both sides is 10-30 ⁇ m.
  • the strength, gas barrier performance and cell culture function of the bag film material are significantly improved, and the optimal thickness of the gas barrier layer PA/EVOH/PA is 30/20/30 ⁇ m.
  • the disposable bioprocess bag film material is prepared according to the method provided in Example 15, and different protective layers as shown in Table 22 are used respectively, and the thickness is the thickness of the protective layer, and the obtained multi-layer co-extruded Oxygen, water vapor permeability, tensile strength, puncture resistance, transparency, heat sealing welding performance and cell culture effect (biocompatibility) of membrane materials for disposable bioprocess bags.
  • LDPE and LLDPE adopt American Dow DOW.
  • Example 15 oxygen, water vapor permeability, tensile strength, puncture resistance, welding strength, transparency, and cell culture effect detection methods are as shown in Example 15, and the cultured cells use SF9 insect cells and human embryonic kidney cells 293 (hereinafter referred to as 293 cells), the initial concentration was 1 ⁇ 10 5 /ml; the dissolution of small molecular substances was detected by gas chromatography/liquid chromatography-mass spectrometry; the investigation results are shown in Table 22.
  • the preferred thickness of the protective layer is 50-150 ⁇ m.
  • this example prepares a membrane material for a disposable bioprocess bag, and adopts different adhesive layers as shown in Table 23, wherein the thickness is the thickness of the adhesive layer, and the obtained multi-layer Oxygen, water vapor permeability, tensile strength, welding strength, puncture resistance, transparency and cell culture effect (biocompatibility) of membrane materials for co-extruded disposable bioprocess bags.
  • EVA adopts the EVA of Celanese Company
  • EAA and EMA adopt the relevant brand products of DuPont Company of the United States and Dow Chemical respectively.
  • the adhesive layer includes two layers 2 and 6, so it is represented by adhesive layer 2/barrier layer/adhesive layer 6, and the thickness is the respective thickness of the two layers.

Abstract

一种一次性生物工艺袋用膜材及其制备方法，采用尼龙／乙烯-乙烯醇共聚物／尼龙的三层复合结构制备气体阻隔层（3，4，5），以ULDPE或EVA为液体接触层（7），并选用了最佳的防护层（1）、粘合层（2，6），以及合适的厚度，制得的一次性生物工艺袋同时具备良好的气体阻隔性能、耐水性、强度、抗弯折性能、抗穿刺性能，以及优异的热封性能和透明度，并且具备良好的生物相容性，细胞培养密度高，具有更好的细胞培养效果，制备工艺简单、操作简便、效率高，对特定细胞培养功能极佳。

Description

一种一次性生物工艺袋用膜材及其制备方法
本申请主张中国在先申请,申请号:202110644600.1，申请日2021年6月9日的优先权；主张中国在先申请,申请号:202110644601.6，申请日2021年6月9日的优先权；主张中国在先申请,申请号:2021108157330，申请日2021年7月19日的优先权；其所有的内容作为本发明的一部分其所有的内容作为本发明的一部分。
技术领域
本发明属于生物反应器用膜材领域，具体而言，涉及一种一次性生物工艺袋用膜材及其制备方法。
背景技术
一次性生物反应器采用高效传氧机制进行传氧，传氧效率高，易于实现高密度高活性细胞培养。该反应器无需附加工程化管道支持，无需在位清洗、灭菌消毒设备，占地面积小，可用于多种类型细胞悬浮培养，重组蛋白及其它生物工程制品的研发和生产，适用于生产企业、科研院所等进行细胞大规模培养和样品制备。
一次性生物工艺袋可以制成储液袋、搅拌袋、生物反应器袋、称量袋以及投料袋等，在多种类型的细胞悬浮培养、重组蛋白及其它生物工程制品的研发和生产中具有广泛的应用，其无需清洗、即用即弃、高阻隔性以及生物相容性不仅极大地提高了工作效率，同时也避免了一些工艺流程之间的交叉污染。
与一次性生物反应器配套使用的一次性生物工艺袋即用即弃，无需清洗、消毒、验证，极大地提高了工作效率，也避免了工艺流程之间交叉感染等不确定因素的发生。
一次性生物工艺袋用膜材是一种聚合物多层复合膜，基本组成结构为“液体接触层/粘合层/气体阻隔层/粘合层/防护层。其中，液体接触层需要满足细胞的正常生长和代谢，为细胞生长营造适宜的环境，较低的可提取物/浸出物，无动物源性材料，有较好的生物相容性，适于细胞培养。同时，和防护层一样，液体接触层也需要具有一定的热封性，能为工艺袋的加工提供热合焊接功能，并提供足够的热封强度，避免在细胞培养一个周期内液体渗漏。此外，还要具有一定的耐穿刺、耐弯折能力，确保在使用过程中的可靠性。
膜材应具有广泛适应性，或者满足细胞生长的使用需求，同时在使用过程中不能有小分子析出和吸附，影响细胞的正常生长和代谢；气体阻隔层提供氧气、水蒸气、二氧化碳等气体隔绝功能；可作为焊接层的防护层及液体接触层需要提供膜材自身搭接、对接等拼接方式的热合功能，满足高频焊接和直热式焊接，并提供足够的热封强度，避免在细胞培养一个周期内液体渗漏；粘合层提供上述功能层间良好的层间粘结。
一次性生物反应器的容积决定了生产效率，大型反应器需要配备大容积工艺袋。这要求膜材需要具备足够的强度，焊接结构需要足够的可靠性，需要经受住较大的使用压力以及工艺袋在制作、折叠包装、运输、展开安装和正常使用过程中可能产生的各种弯折和可能的损伤。气体阻隔层提供氧气、二氧化碳等气体隔绝功能，是工艺袋膜材的重要组成部分，氧气渗透率必须小于1.0mL/m 2·d·atm。工艺袋中气体阻隔层可以是单一的一层，如采用乙烯-乙烯醇共聚物(EVOH)。EVOH具有较好的气体阻隔性能， 但EVOH的耐水性和耐弯折性能较差。当前，国内外的一次性生物工艺袋中的阻隔层大多选用单层或两层结构的乙烯-乙烯醇共聚物(EVOH)，水蒸气的阻隔性能相对较差，尤其是当应用于储液袋时，长时间及跨区域的运输和储存极有可能会因为阻隔性的欠缺导致抗体失活失效等。如表1所示，国外的一些工艺袋膜材的组成结构都采用EVOH为阻隔层。国内也有专利涉及这类多层复合膜材，如表2所示。其中，PALL、GE和CN201821441762.5的膜材使用尼龙(PA)为其耐磨层(外层)，然而PA并不适合搭接热合焊接工艺，从而使得生物工艺袋的加工成本有所提高，生产效率下降，PA不适合用来作为最外层的防护层。此外，还有一些专利涉及细胞培养复合膜材料，但这些膜材中都没有高气体阻隔性材料层。因此，现有的生物工艺袋还难以获得良好的气体阻隔性能，而且耐水性、抗弯折性能、抗穿刺性能都难以满足需要，同时还影响了细胞培养效果和培养液储存效果。
表1 国外的一些工艺袋膜材的组成结构
Figure PCTCN2021108644-appb-000001
表2 国内的一些复合膜材的组成结构
专利 组成结构
CN201811633602.5 ULDPE/粘合层/EVOH/粘合层/LDPE
申请号：201910303806.0 聚乳酸/粘合□/EVOH/粘合□/聚□□□
CN201821441762.5 PA/PE/EVOH/PE/EVA
因此，设计综合性的多层结构膜材，使其气体阻隔层具备更好的耐水性、抗弯折性能、抗穿刺性能，并且具备良好的生物相容性，使其液体接触层具有更好的抗穿刺性能和热封性能，同时具备良好的生物相容性，从而对于获得高强度、抗穿刺、耐水、抗弯折、高阻隔，并具有更好的细胞培养效果的一次性生物袋用膜材具有重要意义。
发明内容
为解决上述问题，本发明提供了一种一次性生物工艺袋用膜材，采用尼龙/乙烯-乙烯醇共聚物/尼龙的三层复合结构制备气体阻隔层，以ULDPE或EVA为液体接触层，并选用了最佳的防护层、粘合层，以及合适的厚度，制得的一次性生物工艺袋同时具备良好的气体阻隔性能、耐水性、强度、抗弯折性能、抗穿刺性能，以及优异的热封性能，并且具备良好的生物相容性，细胞培养密度高，具有更好的细胞培养效果，适于大部分种类细胞的培养，制备工艺简单、操作简便、效率高，可用于制备一次性生物反应 器袋、储液袋、搅拌袋、称量袋以及投料袋等。
一方面，本发明提供了一种气体阻隔层用于制备一次性生物工艺袋用膜材的用途，所述气体阻隔层由PA/EVOH/PA三层复合膜组成。
本发明提供的一次性生物工艺袋专用于细胞培养。细胞培养过程对膜材要求非常严格，对氧气、水蒸气、二氧化碳等气体的渗透都极为敏感，且膜材必须有很好的生物相容性，无小分子物质的吸附与析出，才能获得好的培养效果。
乙烯-乙烯醇共聚物(EVOH)是气体阻隔性能最好的聚合物材料，但EVOH的耐水性和耐弯折性能较差。
尼龙(PA)也是气体阻隔性较好的聚合物材料，其对氧气、二氧化碳的阻隔性能比EVOH低一个数量级，但PA具有抗穿刺、柔韧性好、强度高等特性。
本发明创造性地采用PA/EVOH/PA三层复合膜作为一次性生物工艺袋膜材的气体阻隔层，大幅提高了膜材的综合性能，其中的PA与EVOH间具有良好的相容性，PA与EVOH层间不需要粘合层。
PA/EVOH/PA三层复合阻隔膜能够在拉伸强度、气体阻隔性、抗穿刺、柔韧性、抗弯折等特性方面获得优异的综合性能。
进一步地，所述尼龙为优选芳香族PA。研究证明，相比于脂肪族PA和脂肪-芳香族PA，对于部分改性后的芳香族尼龙(PA)而言，几乎不受水汽的影响，因此兼具有优异的水蒸气、氧气、二氧化碳阻隔性。因此芳香族PA更适合用于制备一次性生物工艺袋膜材，使制得的一次性生物工艺袋具备更高强度和阻隔性，具备更好的耐水性、抗弯折性能、抗穿刺性能，对特定细胞的培养功能极佳。
另一方面，本发明提供了一种一次性生物工艺袋用膜材，包括防护层/粘合层/气体阻隔层/粘合层/液体接触层，所述的气体阻隔层由PA/EVOH/PA三层复合膜组成。
进一步地，所述PA为芳香族PA。
在一些方式中，EVOH优选为日本可乐丽、合成化学和台湾长春公司的相关牌号，PA推荐使用美国杜邦、德国BASF、日本三菱、上海盈固和山东祥龙的相关牌号树脂。
进一步地，所述PA/EVOH/PA厚度为30～80μm，所述PA厚度为10～30μm，所述EVOH厚度为10～20μm。
由于生物工艺袋需要低氧气透过量和低水蒸气透过量，阻隔层太薄则不能满足；太厚之后对于阻隔性能的意义并不大，反而会影响整体的加工性能，因此，优选为30～80μm。并进一步经试验确定PA和EVOH分别的厚度。
进一步地，所述液体接触层接触的液体中包括用于培养的细胞。
液体接触层是与细胞培养液直接接触的一层，需具有良好的生物相容性，同时在使用过程中不能有小分子析出，影响细胞的正常生长和代谢。
进一步地，所述液体接触层为ULDPE，所述ULDPE厚度为30～200μm。
超低密度聚乙烯(ULDPE)是目前发现的最适宜的液体接触层材料，对很多细胞的培养都有较好的相容性。
在一些方式中，所述液体接触层的超低密度聚乙烯(ULDPE)优选为陶氏Engage 8480K Health+、Dow Health+系列和沙特基础Sabic PCG系列。
进一步地，所述液体接触层为EVA，所述EVA厚度为50～250μm。
液体接触层的首要功能是保护和促进细胞生长，同时在使用过程中不能有小分子析出，影响细胞的正常生长和代谢，在实际生产过程中已经发现，乙烯-醋酸乙烯共聚物(EVA)对一些细胞生长比超低密度聚乙烯(ULDPE)更适合，比如对于人体淋巴细胞PBMC，ULDPE的培养效果比较差；而选用EVA作为液体接触层时，细胞浓度增长效果非常好。
EVA可供选择的牌号有Celanese公司以及杨子石化的相关牌号等树脂。
在一些方式中，所述液体接触层为线性低密度聚乙烯和超低密度聚乙烯的混合物。
进一步地，所述线性低密度聚乙烯和超低密度聚乙烯的混合物中，线性低密度聚乙烯占比为10～100％；所述液体接触层的厚度为50～250μm。
液体接触层在使用过程中不能有小分子析出，影响细胞的正常生长和代谢，实践证明LLDPE和ULDPE均具有良好的细胞相容性。
LLDPE具有更高的强度，ULDPE具有更佳的抗穿刺、耐弯折性能，将LLDPE与ULDPE混合物作为液体接触层，能更好地兼顾焊接强度、抗穿刺与抗弯折性能。
在一些方式中，对于强度要求不严格的应用场景，ULDPE的量可以适量增多，因此优选的，LLDPE的含量为10～100％。
在一些方式中，液体接触层的厚度为50～250μm。若液体接触层太薄，在对接热合时，可能会出现焊接强度不够而导致生物袋发生漏液现象，从而无法使用；若液体接触层太厚，则会影响生物袋的柔韧性。
进一步地，所述防护层为EVA、TPU、LDPE、LLDPE、或LDPE与LLDPE的混合物中的任意一种。
一些方式中，所述的防护层为厚度在50μm～100μm之间的EVA、聚氨酯弹性体(TPU)、低密度聚乙烯(LDPE)或线性低密度聚乙烯(LLDPE)，或低密度聚乙烯(LDPE)与线性低密度聚乙烯两者的混合物。
在一些方式中，优选的LDPE及LLDPE有美国陶氏Dow、沙特基础Sabic、北欧化工Bormed、上海石化、茂名石化、兰州石化等公司的相关牌号。
在一些方式中，优选的EVA有Celanese公司、杨子石化的相关牌号树脂。
在一些方式中，优选的TPU有Lubrizol、Bayer、Huntsman、Basf等公司的相关牌号树脂。
进一步地，所述防护层为LDPE与LLDPE的混合物，所述LDPE与LLDPE的混合物中，按质量份数计，LDPE成分为20％～30％。
若选用LDPE与LLDPE的混合物，LDPE应占混合物的20～30％，可以实现更美观的焊接效果及优异的焊接强度。采用LDPE与LLDPE的混合物还可以提高膜材的透明度，便于用户在使用过程中观察 细胞生长。防护层材料LDPE与LLDPE可以选用但不限于如下牌号的树脂，美国陶氏Dow、沙特基础Sabic、北欧化工Bormed、上海石化、茂名石化、兰州石化等公司的相关牌号树脂。
进一步地，所述防护层的厚度为50～150μm。
进一步地，所述粘合层为EAA、EVA或EMA中的一种或几种组成的混合物，所述粘合层厚度为5～20μm。
粘合层的首要功能是粘合作用，可选用乙烯-丙烯酸共聚物(EAA)或乙烯-甲基丙烯酸酯共聚物(EMA)中的一种或几种。在一些方式中，乙烯-丙烯酸共聚物(EAA)或乙烯-甲基丙烯酸酯共聚物(EMA)可选用的树脂有杜邦Bynel系列、陶氏化学Primacor系列、三井石化ADMER系列、三菱油化MODIC系列。
在一些方式中，EVA与聚乙烯之间具有良好的相容性，EVA与PA间也有部分相容性，因此，EVA也可以作为粘合层应用于中小型一次性生物工艺袋；优选的EVA有Celanese公司、杨子石化的相关牌号树脂。
一次性生物工艺袋膜的总厚度、每一层的厚度的考量主要是物性的稳定性、焊接的强度，以确保袋子的完整性测试项合规。其中，作为与细胞接触的液体接触层和最外层的防护层相对较厚，以保证在对接或搭接的焊接层的厚度从而保证热合强度，确保袋子的完整性；阻隔层厚度达到相应的气体透过率要求即可，厚度适中，粘合层只需要提供各层之间的良好粘合，因此该层较薄。
另一方面，本发明提供了一种简化结构的一次性生物工艺袋用膜材，包括防护层/气体阻隔层/液体接触层，所述气体阻隔层由PA/EVOH/PA三层复合膜组成。
在一些小型的一次性生物工艺袋用膜材中，由于强度要求不高，EVA与PA间有较好的粘合，可以用EVA代替粘合层，从而得到简化结构的膜材，不需要再添加粘合层。
进一步地，所述PA为芳香族PA。
进一步地，所述PA/EVOH/PA厚度为30～80μm，所述PA厚度为10～30μm，所述EVOH厚度为10～20μm。
进一步地，所述液体接触层和防护层都为EVA。
进一步地，其中液体接触层厚度为50～250μm，防护层的厚度为50～100μm。
简化结构膜材的组成结构为EVA/PA/EVOH/PA/EVA，这种膜材可以采用吹塑或流延方式制备，所需设备有3个螺杆即可制备这种对称结构形式的膜材，制备简便、生产效率高。
再一方面，本发明提供了一种简化结构的一次性生物工艺袋用膜材，包括防护层/粘合层/气体阻隔层/粘合层/液体接触层，所述的气体阻隔层由尼龙层/乙烯-乙烯醇共聚物层两层复合膜组成；所述液体接触层为线性低密度聚乙烯和超低密度聚乙烯的混合物。
进一步地，所述线性低密度聚乙烯和超低密度聚乙烯的混合物中，线性低密度聚乙烯占比为10～100％；所述气体阻隔层厚度为30～80μm；其中尼龙层的厚度为20～70μm，乙烯-乙烯醇共聚物层的厚度为10～60μm。
在一些方式中，本发明也可采用PA/EVOH两层复合膜作为一次性生物工艺袋膜材的气体阻隔层， 这是因为本发明提供的液体接触层为LLDPE与ULDPE的混合物，具备一定的抗穿刺能力，可以弥补一部分气体阻隔层的功效，可用于制备一些体积较小的生物工艺袋。
再一方面，本发明提供了一种如上所述的膜材的制备方法，其特征在于，采用共挤出流延方法制备，具体步骤为：1)各层原料分别加入至对应的挤出机中；2)在机头温度120～220℃条件下，经熔融、共挤出吹塑或流延制得。
本发明提供的制备方法，主要采用共挤出流延方法，具体步骤为：1)各层原料分别加入至对应的挤出机中；2)在机头温度160～220℃条件下，经熔融、共挤出流延等过程，通过调整螺杆转速得到特定厚度的一次性生物工艺袋用膜材料。
在聚合物功能膜材料的工业化生产中，吹塑、流延等薄膜挤出工艺是最常用的工艺技术。多层共挤出吹塑是多层聚合物复合膜材料的一种成型方法，它可以将两种以上的聚合物，使用两台以上的挤出机，分别熔融后经过各自独立的流道，进入多层口模共同挤出具有多层结构的“膜管”，并成型为多层结构的聚合物复合膜材料。通过流延方法制备的聚合物薄膜，是先经过挤出机把原料塑化熔融，通过T型结构成型模具挤出，聚合物熔体呈片状流延至平稳旋转的冷却辊筒表面上，膜片在冷却辊筒上经冷却降温定型，再经牵引、切边后收卷。
进一步地，当膜材厚度小于300μm时，采用共挤出吹塑或流延制得；当膜材厚度大于300μm时，采用共挤出流延制得。
多层共挤出吹塑工艺简便，适合整体厚度小于300μm的膜材。当厚度超过300μm时，吹塑方式变得有些困难，同时膜材的透明度会有所下降，此时，流延法更适合获得厚度均匀、透明度好的膜材。
共挤出流延薄膜与共挤出吹塑薄膜相比，薄膜透明度好，厚度均匀性好，更适合生产厚度较大的一次性生物工艺袋用膜材。
一次性生物反应器工艺袋用膜材的厚度远大于普通包装膜，一般超过300μm，采用共挤出吹塑方式很困难，需要采用共挤出流延方式生产。
本发明提供的一次性生物工艺袋用膜材优选采用共挤出流延方法制备，共挤出流延机组应具有合适数量的螺杆和分配器插块，通过将各层原料分别加入至对应的挤出机中，在机头温度120～220℃条件下，分别熔融挤出液体接触层、粘合层树脂、阻隔层PA//EVOH/PA、粘合层树脂、防护层LDPE与LLDPE共混物，通过T型结构成型模具挤出成型，得到结构为防护层/粘合层/PA/EVOH/PA/粘合层/液体接触层的膜材。
再一方面，本发明提供了尼龙用于制备一次性生物工艺袋用膜材的气体阻隔层的用途。
进一步地，所述气体阻隔层为尼龙/乙烯-乙烯醇共聚物/尼龙共聚物的三层复合结构；所述尼龙为芳香族尼龙；所述膜材用于制备生物反应器中培养细胞的一次性生物工艺袋。
本发明提供的一次性生物工艺袋用膜材的拉伸强度可达到20MPa，氧气渗透率小于0.1mL/m 2·d·atm，水蒸气透过率小于0.5mL/m 2·d·atm，膜材整体透明度高，柔韧性好，抗穿刺能力好，结实耐用，适用于直热式或高频焊接实现膜材的搭接或对接焊接。
基于自身的技术条件和优势，这种高分子多层复合膜制造者可以提供不同组成结构设计的膜材。由 于生物医用膜材特殊功能性要求和膜材制造环境要求等级高等因素，对膜材的各组成功能层有较严格的限制。特别的，该种膜材用于细胞培养，特别是超大容量的细胞培养，对其膜材的要求特别严格，对于某些细胞的培养，当膜材经过长时间培养和运输过程中，需要严格控制膜材内与外界的气体交换，如果存在不符规定的气体交换，则会严重影响膜材内的细胞培养的质量和产量。另外，当细胞培养中，也要严格控制这些多层膜材中的有机物质被培养细胞的培养基所溶解，这些有机物大多数对细胞具有毒性的物质。再者，工艺袋在使用前需要经历γ射线辐照、电子束辐照或环氧乙烷等方式进行灭菌处理，在这一过程中，膜材不能出现物理和化学的性能变化，如果发生化学变化，或者有化学物质的渗出，则会对培养中的培养基造成污染，从而影响细胞培养的质量；在细胞培养一个周期内(比如5天、7天、10天，或者30天内等等)，膜材不能有任何影响细胞培养效果的低分子物析出或溶出。此外，对膜材的细胞培养功能性、拉伸强度、气体阻隔性能、热封性能需要进行整体考虑，使膜材更具有实用性。
本发明提供的多层共挤一次性生物工艺袋用膜材具有以下有益效果：
(1)具备良好的气体阻隔性能、耐水性、强度、抗弯折性能、抗穿刺性能，拉伸强度可达到20MPa，氧气渗透率小于0.1mL/m 2·d·atm，水蒸气透过率小于0.5mL/m 2·d·atm；
(2)优异的热封性能，通过焊接强度测试发现，热封强度可达40N/15mm以上；
(3)透明度高，达到96％以上；抗穿刺能力强，穿刺力值可达4.5N以上；
(4)具备良好的生物相容性，细胞培养密度高，达到约233*10 5/ml，具有更好的细胞培养效果，适于多种细胞培养；
(5)灭菌处理及细胞培养过程中，析出物或溶出物含量极低；
(6)制备工艺简单、操作简便、效率高，可用于制备一次性生物反应器袋、储液袋、搅拌袋、称量袋以及投料袋等。
附图说明
图1、实施例1、3、4中的一次性生物工艺袋用膜材各层结构示意图
图2、实施例2中的简化结构的一次性生物工艺袋用膜材各层结构示意图
图3、实施例15中的多层共挤一次性生物工艺袋用膜材各层结构示意图
[根据细则91更正 11.08.2021]　
图4、实施例16中的多层共挤一次性生物工艺袋用膜材各层结构示意图
具体实施方式
下面结合附图和实施例对本发明作进一步详细描述，需要指出的是，以下所述实施例旨在便于对本发明的理解，而对其不起任何限定作用。
实施例1 含EVA的一次性生物工艺袋用膜材
本实施例采用共挤出流延法制备厚度为325μm的含EVA的一次性生物工艺袋用膜材，该膜材的组成结构包括如图1所示，包括防护层1、粘合层2、气体阻隔层3/4/5、粘合层6和液体接触层7。
其中防护层1采用陶氏的Dow系列的LDPE和LLDPE混合物，LDPE占比为30％，LLDPE占比 为70％。该层的厚度为50μm。
其中粘合层2和6采用陶氏化学的Primacor树脂，厚度为10μm。
气体阻隔层3和5为芳香族PA，采用美国杜邦相关牌号，该层的厚度为20μm。
气体阻隔层4为EVOH采用可乐丽EVAL树脂，该层的厚度为15μm。
液体接触层7采用Celanese公司的EVA，该层的厚度为200μm。
含EVA的一次性生物工艺袋用膜材的制备方法如下：
1)将所述各层原料分别加入至对应的挤出机中，防护层对应的螺杆温度设置在180～210℃；粘合层对应的螺杆温度设置在160～200℃；阻隔层尼龙对应的螺杆温度设置在200～240℃；阻隔层EVOH对应的螺杆温度设置在190～220℃；EVA对应的螺杆温度设置在160～200℃；机头温度设置为220℃。
2)膜材的厚度由在线测厚装置精确控制，各层厚度由螺杆转速调节控制。
考察制得的含EVA的一次性生物工艺袋用膜材的氧气、水蒸气渗透率、拉伸强度、透明度和细胞培养效果(生物相容性)。
其中，含EVA的一次性生物工艺袋用膜材的氧气和水蒸气渗透率采用压差法进行测试，将样品裁至直径不小于100mm的圆片，依据GB/T-1038标准于室温下进行测试，测试膜材的氧气和水蒸气渗透率。每组试样为3个，求取平均值。
含EVA的一次性生物工艺袋用膜材的拉伸强度及焊接强度采用Instron万能试验机进行测试，将样品分别裁成哑铃状样条和制备成相应的焊接样条，以200mm/min的速率进行测试，每个样品至少测试5个样条，并求取平均值。
含EVA的一次性生物工艺袋用膜材的透明度采用雾度计法，依据GB/T-2410-2008进行测试，每组测试3个样品，并求取平均值。
含EVA的一次性生物工艺袋用膜材的生物相容性通过统计细胞密度进行表征，将制备好的膜材通过直热式搭接形式进行焊接后，制备成50L规格的一次性生物培养袋，并经过γ射线辐照灭菌。培养细胞使用人体淋巴细胞PBMC，初始浓度为1×10 5/ml。一次性生物培养袋装在生物反应器内，人体淋巴细胞PBMC在一次性生物工艺袋中培养5天，培养条件为37℃，搅拌速度为200rpm，培养结束后检测细胞密度。
制得的含EVA的一次性生物工艺袋用膜材的物理性能及细胞密度结果如表3所示。
表3、含EVA的一次性生物工艺袋用膜材的物理性能及人体淋巴细胞PBMC密度结果
Figure PCTCN2021108644-appb-000002
实施例2 简化结构的含EVA的一次性生物工艺袋用膜材
本实施例提供的简化结构的含EVA的一次性生物工艺袋用膜材的组成结构如图2所示，包括防护层8、气体阻隔层9/10/11、液体接触层12。
本实施例采用共挤出流延法制备厚度为220μm的一次性生物工艺袋用膜材；
防护层8和液体接触层12采用Celanese公司的EVA，厚度都为90μm；
中间气体阻隔层10为可乐丽EVAL系列的EVOH，厚度为10μm；
两侧气体阻隔层9个11为芳香族PA，采用美国杜邦相关牌号，该层的厚度为15μm。
其中制备方法如下：
1)将所述各层原料分别加入至对应的挤出机中，气体阻隔层PA对应的螺杆温度设置在200～240℃；气体阻隔层EVOH对应的螺杆温度设置在190～220℃；EVA对应的螺杆温度设置在160～200℃；机头温度设置为220℃。
2)膜材的厚度由在线测厚装置精确控制，各层厚度由螺杆转速调节控制。
膜材的厚度由在线测厚装置精确控制，各层厚度由螺杆转速调节控制。
考察制得的多层共挤一次性生物工艺袋用膜材的氧气、水蒸气渗透率、拉伸强度、透明度和细胞培养效果(生物相容性)。其中，氧气、水蒸气渗透率、拉伸强度、透明度和细胞培养效果检测方法如实施例1所示，培养细胞使用检测结果如表4所示。
表4、简化结构的含EVA的一次性生物工艺袋用膜材的物理性能及人体淋巴细胞PBMC密度结果
Figure PCTCN2021108644-appb-000003
实施例3 采用共挤出吹塑法制备的含ULDPE的一次性生物工艺袋用膜材
本实施例采用共挤出吹塑法制备厚度为250μm的一次性生物工艺袋用膜材，该膜材的组成结构包括如图1所示，包括热封层1、粘合层2、气体阻隔层3/4/5、粘合层6和液体接触层7。
其中热封层LDPE采用陶氏的Dow系列，该层的厚度为50μm。
其中粘合层2和6采用陶氏化学的Primacor树脂，厚度为5μm。
气体阻隔层3和5为芳香族PA，采用美国杜邦相关牌号，该层的厚度为15μm。
气体阻隔层4为EVOH采用可乐丽EVAL树脂，该层的厚度为10μm。
液体接触层7的ULDPE采用美国陶氏公司的Engage 8480K，该层的厚度为150μm。
含ULDPE的多层共挤出一次性生物工艺袋用膜材的制备方法如下：
1)将所述各层原料分别加入至对应的挤出机中，热封层对应的螺杆温度设置在180～210℃；粘合层对应的螺杆温度设置在160～200℃；气体阻隔层PA对应的螺杆温度设置在200～240℃；气体阻隔层EVOH对应的螺杆温度设置在190～220℃；ULDPE对应的螺杆温度设置在160～200℃；模头温度设置为220℃。
2)待各层原料熔融后，在氮气氛围下进行吹膜成型，冷却后收卷。
考察制得的多层共挤一次性生物工艺袋用膜材的氧气、水蒸气渗透率、拉伸强度、透明度和细胞培养效果(生物相容性)。
其中，一种多层共挤一次性生物工艺袋用膜材的氧气和水蒸气渗透率采用压差法进行测试，将样品裁至直径不小于100mm的圆片，依据GB/T-1038标准于室温下进行测试，测试膜材的氧气和水蒸气渗透率。每组试样为3个，求取平均值。
一种多层共挤一次性生物工艺袋用膜材的拉伸强度采用Instron万能试验机进行测试，将样品裁成哑铃状样条，以200mm/min的速率进行测试，每个样品至少测试5个样条，并求取平均值。
一种多层共挤一次性生物工艺袋用膜材的透明度采用雾度计法，依据GB/T-2410-2008进行测试，每组测试3个样品，并求取平均值。
一种多层共挤一次性生物工艺袋用膜材的生物相容性通过统计细胞密度进行表征，将制备好的膜材通过直热式搭接形式进行焊接后，制备成5L规格的一次性生物培养袋，并经过γ射线辐照灭菌。培养细胞使用293人胚胎肾细胞，初始浓度为10万细胞/ml。一次性生物培养袋装在生物反应器内，293人胚胎肾细胞在一次性生物工艺袋中培养5天，培养条件为温度37℃，搅拌速度为200rpm，培养结束后检测细胞密度。
制得的多层共挤出一次性生物工艺袋用膜材的物理性能及细胞密度结果如表5所示。
表5、共挤出吹塑法制备的膜材的物理性能及293人胚胎肾细胞密度结果
Figure PCTCN2021108644-appb-000004
实施例4 采用共挤出流延法制备的含ULDPE的一次性生物工艺袋用膜材
本实施例提供的含ULDPE的一次性生物工艺袋用膜材的组成结构如图1所示，包括热封层1、粘合层2、气体阻隔层3/4/5、粘合层6和液体接触层7。
本实施例采用共挤出流延法制备厚度为400μm的一次性生物工艺袋用膜材；
热封层1采用Celanese公司的EVA，该层的厚度为100μm；
粘合层2和6选用扬子石化的EVA，厚度为10μm；
两侧气体阻隔层3和5为可乐丽EVAL系列的EVOH，厚度为30μm；
中间气体阻隔层4采用美国杜邦相关牌号的芳香族PA，该层的厚度为20μm；
液体接触层7为陶氏化学的Engage 8480K系列的ULDPE，厚度为200μm。
其中制备方法如下：
1)将所述各层原料分别加入至对应的挤出机中，气体阻隔层芳香族PA对应的螺杆温度设置在200～240℃；气体阻隔层EVOH对应的螺杆温度设置在190～220℃；EVA和ULDPE对应的螺杆温度设置在160～200℃；机头温度设置为220℃。
2)熔融后的各层原料，通过T型结构成型模具挤出，熔体呈片状流延至平稳旋转的冷却辊筒表面上，膜片在冷却辊筒上经冷却降温定型，再经牵引、切边后收卷，一次性生物工艺袋用膜材。
膜材的厚度由在线测厚装置精确控制，各层厚度由螺杆转速调节控制。
考察制得的多层共挤一次性生物工艺袋用膜材的氧气、水蒸气渗透率、拉伸强度、透明度和细胞培 养效果(生物相容性)。其中，氧气、水蒸气渗透率、拉伸强度、透明度和细胞培养效果检测方法如实施例1所示，其中的培养细胞使用293人胚胎肾细胞，检测结果如表6所示。
表6、共挤出流延法制备得到的膜材的物理性能及293人胚胎肾细胞细胞密度数据
Figure PCTCN2021108644-appb-000005
实施例5 采用不同气体阻隔层对含EVA的一次性生物工艺袋性能的影响
本实施例采用实施例1提供的方法制备EVA为液体接触层的总厚度为325μm的一次性生物工艺袋用膜材，并分别采用如表5所示的不同的气体阻隔层，其中的厚度为气体阻隔层的厚度，考察制得的多层共挤一次性生物工艺袋用膜材的氧气、水蒸气渗透率、拉伸强度、透明度和细胞培养效果(生物相容性)。
EVOH采用可乐丽EVAL系列，PA采用美国杜邦相关牌号的芳香族PA、脂肪族PA、脂肪-芳香族PA，PE采用美国陶氏公司相关牌号的产品。
其中，氧气、水蒸气渗透率、拉伸强度、焊接强度、透明度和细胞培养效果检测方法如实施例1所示，培养的细胞为人体淋巴细胞PBMC，考察结果如表7所示。
表7、不同气体阻隔层对含EVA的一次性生物工艺袋性能的影响
Figure PCTCN2021108644-appb-000006
由表7可见，当防护层、粘合层、液体接触层及厚度都一样时，采用不同的气体阻隔层对一次性生物工艺袋膜材的拉伸强度、氧气渗透率、水蒸气透过量、透明度和细胞培养密度都存在较大的区别。相比不同种类的PA，芳香族PA的强度和气体阻隔性能明显更优，生物相容性也较好，因此优先选用芳香族PA制备一次性生物工艺袋；相比单独采用EVOH或单独采用芳香族PA作为气体阻隔层时，PA和EVOH组合可以明显提升强度、气体阻隔性能和细胞培养功能；而采用PA/EVOH/PA，则可进一步提升一次性生物工艺袋膜材的性能；采用EVOH/PA/EVOH时，由于EVOH的耐水性比较差，因此将 其作为外层阻隔层时，如遇水，阻隔性能会发生急剧下降，导致一次性生物工艺袋膜材的拉伸强度、氧气渗透率、水蒸气透过量、透明度和细胞培养密度都出现下降；同样为三层复合膜气体阻隔层形式的PE/EVOH/PE，其强度、气体阻隔性能和细胞培养功能则远不如PA/EVOH/PA的组合；最优选为PA/EVOH/PA的三层复合膜气体阻隔层，可使拉伸强度、气体阻隔性能和细胞培养功能都达到最佳。
实施例6 气体阻隔层的不同厚度对含EVA的一次性生物工艺袋性能的影响
本实施例采用实施例5的序号为6的气体阻隔层制备的一次性生物工艺袋膜材，即以PA/EVOH/PA作为气体阻隔层，按照实施例1提供的方法制备一次性生物工艺袋用膜材，并分别采用如表8所示的不同厚度的气体阻隔层，其中的厚度为气体阻隔层的厚度，考察制得的多层共挤一次性生物工艺袋用膜材的氧气、水蒸气渗透率、拉伸强度、透明度和细胞培养效果(生物相容性)。
EVOH采用可乐丽EVAL系列，PA采用美国杜邦相关牌号的芳香族PA。
其中，氧气、水蒸气渗透率、拉伸强度、透明度和细胞培养效果检测方法如实施例1所示，培养的细胞为人体淋巴细胞PBMC，考察结果如表8所示。
表8、不同气体阻隔层厚度对含EVA的一次性生物工艺袋性能的影响
Figure PCTCN2021108644-appb-000007
由表8可见，气体阻隔层的不同厚度，对制备的一次性生物工艺袋膜材的性能存在较大影响，选用合适的厚度，可进一步提升一次性生物工艺袋膜材的强度、气体阻隔性能和细胞培养功能，研究证明，采用气体阻隔层的厚度为30～80μm，其中中间的气体阻隔层厚度为10～20μm，两侧的气体阻隔层厚度为10～30μm时，制备的一次性生物工艺袋膜材的强度、气体阻隔性能和细胞培养功能明显提高，气体阻隔层PA/EVOH/PA的最佳厚度选择为30/20/30μm。
实施例7 采用不同防护层对含EVA的一次性生物工艺袋性能的影响
本实施例采用实施例5的序号为6的气体阻隔层制备的一次性生物工艺袋膜材，即以PA/EVOH/PA作为气体阻隔层，按照实施例1提供的方法制备一次性生物工艺袋用膜材，并分别采用如表9所示的不同防护层，其中的厚度为防护层的厚度，考察制得的多层共挤一次性生物工艺袋用膜材的氧气、水蒸气渗透率、拉伸强度、透明度、热封焊接性能和细胞培养效果(生物相容性)。
LDPE和LLDPE采用美国陶氏DOW；EVA采用Celanese公司的EVA；TPU采用Lubrizol的TPU；PA采用美国杜邦相关牌号的芳香族PA。
其中，氧气、水蒸气渗透率、拉伸强度、透明度和细胞培养效果检测方法如实施例1所示，热封焊 接强度通过将其制备成标准样条，进行剥离强度测试得到，培养的细胞为人体淋巴细胞PBMC，考察结果如表9所示。
表9、不同防护层对含EVA的一次性生物工艺袋性能的影响
Figure PCTCN2021108644-appb-000008
由表9可见，采用不同的防护层及不同厚度的防护层，对一次性生物工艺袋用膜材的氧气、水蒸气渗透率、拉伸强度、透明度、热封焊接性能和细胞培养效果都存在一定的影响，尤其是对透明度和热封焊接性能，并且由于焊接性能不佳也影响了细胞培养效果。
对比表9中的序号1-5可以看出，采用LDPE、LLDPE、EVA、TPU作为防护层时，制备的一次性生物工艺袋用膜材的氧气、水蒸气渗透率、拉伸强度、透明度、热封焊接性能和细胞培养效果都较合适，但采用PA作为防护层时，其熔点较高，焊接困难，使得加工成本明显提高，生产效率下降，同时还影响了细胞培养效果。而采用EVA时，针对人体淋巴细胞PBMC的培养效果更好，细胞培养密度更高。
对比表9中的序号1、2、6可以看出，采用LDPE、LLDPE、或是LDPE和LLDPE的混合物作为防护层时，LDPE和LLDPE的混合物可明显提高一次性生物工艺袋膜材的透明度，同时热封焊接性能也非常好，强度、氧气、水蒸气渗透率和细胞培养效果都能符合一次性生物工艺袋膜材的制备需求。
对比表9中的序号6-9可见，LDPE和LLDPE的混合物的不同比例关系会明显影响制备的一次性生物工艺袋膜材的透明度，优选LDPE和LLDPE的混合物的比例关系为2～3:7～8。
对比表9中的序号6、10、11、12、13可见，防护层的不同厚度，会明显影响制备的一次性生物工艺袋膜材的热封焊接性能，从而也会对细胞培养效果产生影响，防护层的优选厚度为30～150μm；采用LDPE：LLDPE为2:8的混合物制备防护层时，防护层的厚度为50μm时，细胞培养效果更好。
实施例8 采用不同液体接触层对含EVA的一次性生物工艺袋性能的影响
本实施例采用实施例7的序号为6制备的一次性生物工艺袋膜材，即以PA/EVOH/PA作为气体阻隔层，LDPE：LLDPE为2:8的混合物制备防护层，按照实施例1提供的方法制备一次性生物工艺袋用膜材，并分别采用如表10所示的不同体接触层，其中的厚度为液体接触层的厚度，考察制得的多层共挤一次性生物工艺袋用膜材的小分子物质溶出、氧气、水蒸气渗透率、拉伸强度、透明度和细胞培养效果(生物相容性)。
LDPE采用美国陶氏DOW的LDPE；EVA采用Celanese公司的EVA；ULDPE采用陶氏化学的Engage8480K系列的ULDPE。
其中，氧气、水蒸气渗透率、拉伸强度、透明度和细胞培养效果检测方法如实施例1所示，培养的细胞为人体淋巴细胞PBMC和人胚肾细胞293两种；小分子物质溶出通过气相色谱/液相色谱-质谱联用的方法检测；考察结果如表10所示。
表10、不同液体接触层对含EVA的一次性生物工艺袋性能的影响
Figure PCTCN2021108644-appb-000009
Figure PCTCN2021108644-appb-000010
由表10可见，采用不同的液体接触层及不同厚度的液体接触层，对一次性生物工艺袋用膜材的氧气、水蒸气渗透率、拉伸强度、透明度、热封焊接性能和细胞培养效果都存在一定的影响，尤其是对细胞培养效果影响明显。
对比表10中的序号1-3可以看出，采用LDPE、ULDPE、EVA作为液体接触层时，制备的一次性生物工艺袋用膜材的氧气、水蒸气渗透率、拉伸强度、透明度都较合适，但对于人胚肾细胞293，采用ULDPE作为液体接触层时，细胞培养密度较高，EVA的培养效果较差；而对于人体淋巴细胞PBMC，ULDPE的培养效果比较差，EVA作为液体接触层，培养效果更好。
对比表11中的序号1、4、5、6、7可见，液体接触层的不同厚度，会明显影响制备的一次性生物工艺袋膜材的细胞培养效果，液体接触层的优选厚度为30～200μm；采用ULDPE和EVA作为液体接触层时，液体接触层的优选厚度为200μm。
实施例9 采用不同粘合层对含EVA的一次性生物工艺袋性能的影响
本实施例采用实施例8的序号3制备的一次性生物工艺袋膜材，即以PA/EVOH/PA作为气体阻隔层，LDPE：LLDPE为2:8的混合物制备防护层，EVA制备液体接触层，按照实施例1提供的方法制备一次性生物工艺袋用膜材，并分别采用如表11所示的不同粘合层，其中的厚度为粘合层的厚度，考察制得的多层共挤一次性生物工艺袋用膜材的氧气、水蒸气渗透率、拉伸强度、透明度和细胞培养效果(生物相容性)。
EVA采用Celanese公司的EVA；EAA和EMA分别采用美国杜邦公司和陶氏化学的相关牌号产品。
其中，氧气、水蒸气渗透率、拉伸强度、透明度和细胞培养效果检测方法如实施例1所示；考察结果如表11所示，因粘合层包含2和6两层，因此采用粘合层2/阻隔层/粘合层6来表示，厚度为两层分别的厚度。
表11、不同粘合层对含EVA的一次性生物工艺袋性能的影响
Figure PCTCN2021108644-appb-000011
由表11可见，采用不同的粘合层及不同厚度的粘合层，对一次性生物工艺袋用膜材的氧气、水蒸 气渗透率、拉伸强度、透明度、热封焊接性能和细胞培养效果都存在一定的影响。
对比表11中的序号1-5可以看出，采用EVA、EMA、EAA作为粘合层时，制备的一次性生物工艺袋用膜材的氧气、水蒸气渗透率、拉伸强度、透明度、细胞培养效果都较合适。
对比表11中的序号1、6、7、8、9可见，粘合层的不同厚度，会明显影响制备的一次性生物工艺袋膜材的细胞培养效果，过厚或过薄都会使细胞培养密度降低，粘合层的优选厚度为5～20μm；采用EVA制备粘合层时，液体接触层的优选厚度为10μm。
实施例10 采用不同气体阻隔层对含ULDPE的一次性生物工艺袋性能的影响
本实施例采用实施例4提供的方法制备总厚度为400μm的一次性生物工艺袋用膜材，并分别采用如表12所示的不同的气体阻隔层，其中的厚度为气体阻隔层的厚度，考察制得的多层共挤一次性生物工艺袋用膜材的氧气、水蒸气渗透率、拉伸强度、透明度和细胞培养效果(生物相容性)。
EVOH采用可乐丽EVAL系列，PA采用美国杜邦相关牌号的芳香族PA、脂肪族PA、脂肪-芳香族PA，PE采用美国陶氏公司相关牌号的产品。
其中，氧气、水蒸气渗透率、拉伸强度、焊接强度、透明度和细胞培养效果检测方法如实施例4所示，培养细胞使用293人胚胎肾细胞，考察结果如表12所示。
表12、不同气体阻隔层对含ULDPE的一次性生物工艺袋性能的影响
Figure PCTCN2021108644-appb-000012
由表12可见，当热封层、粘合层、液体接触层及厚度都一样时，采用不同的气体阻隔层对一次性生物工艺袋膜材的拉伸强度、氧气渗透率、水蒸气透过量、透明度和细胞培养密度都存在较大的区别。相比不同种类的PA，芳香族PA的强度和气体阻隔性能明显更优，生物相容性也较好，因此优先选用芳香族PA制备一次性生物工艺袋；相比单独采用EVOH或单独采用芳香族PA作为气体阻隔层时，PA和EVOH组合可以明显提升强度、气体阻隔性能和细胞培养功能；而采用PA/EVOH/PA或EVOH/PA/EVOH的三层复合膜气体阻隔层形式，则可进一步提升一次性生物工艺袋膜材的性能；而PA/EVOH/PA与EVOH/PA/EVOH相比，采用EVOH/PA/EVOH时，由于EVOH的耐水性比较差，因此将其作为外层阻隔层时，如遇水，阻隔性能会发生下降，导致一次性生物工艺袋膜材的拉伸强度、氧气渗透率、水蒸气透过量、透明度和细胞培养密度都出现下降；同样为三层复合膜气体阻隔层形式的PE/EVOH/PE，其强度、气体阻隔性能和细胞培养功能则远不如PA/EVOH/PA或EVOH/PA/EVOH的组 合；最优选为PA/EVOH/PA的三层复合膜气体阻隔层，可使拉伸强度、气体阻隔性能和细胞培养功能都达到最佳。
实施例11 气体阻隔层的不同厚度对含ULDPE一次性生物工艺袋性能的影响
本实施例采用实施例10的序号为6的气体阻隔层制备的一次性生物工艺袋膜材，即以PA/EVOH/PA作为气体阻隔层，按照实施例4提供的方法制备一次性生物工艺袋用膜材，并分别采用如表13所示的不同厚度的气体阻隔层，其中的厚度为气体阻隔层的厚度，考察制得的多层共挤一次性生物工艺袋用膜材的氧气、水蒸气渗透率、拉伸强度、透明度和细胞培养效果(生物相容性)。
EVOH采用可乐丽EVAL系列，PA采用美国杜邦相关牌号的芳香族PA。
其中，氧气、水蒸气渗透率、拉伸强度、透明度和细胞培养效果检测方法如实施例4所示，培养细胞使用293人胚胎肾细胞，考察结果如表13所示。
表13、不同气体阻隔层厚度对含ULDPE一次性生物工艺袋性能的影响
Figure PCTCN2021108644-appb-000013
由表13可见，气体阻隔层的不同厚度，对制备的一次性生物工艺袋膜材的性能存在较大影响，选用合适的厚度，可进一步提升一次性生物工艺袋膜材的强度、气体阻隔性能和细胞培养功能，研究证明，采用气体阻隔层的厚度为30～80μm，其中中间的气体阻隔层厚度为10～20μm，两侧的气体阻隔层厚度为10～30μm时，制备的一次性生物工艺袋膜材的强度、气体阻隔性能和细胞培养功能明显提高；综合考虑，气体阻隔层PA/EVOH/PA的最佳厚度选择为30/20/30μm。
实施例12 采用不同热封层对含ULDPE一次性生物工艺袋性能的影响
本实施例采用实施例10的序号为6的气体阻隔层制备的一次性生物工艺袋膜材，即以PA/EVOH/PA作为气体阻隔层，按照实施例4提供的方法制备一次性生物工艺袋用膜材，并分别采用如表14所示的不同热封层，其中的厚度为热封层的厚度，考察制得的多层共挤一次性生物工艺袋用膜材的氧气、水蒸气渗透率、拉伸强度、透明度、热封焊接性能和细胞培养效果(生物相容性)。
LDPE和LLDPE采用美国陶氏DOW；EVA采用Celanese公司的EVA；TPU采用Lubrizol的TPU；PA采用美国杜邦相关牌号的芳香族PA。
其中，氧气、水蒸气渗透率、拉伸强度、透明度和细胞培养效果检测方法如实施例4所示，培养细胞使用293人胚胎肾细胞，热封焊接强度通过将其制备成标准样条，进行剥离强度测试得到，考察结果如表14所示。
表14、不同热封层对含ULDPE一次性生物工艺袋性能的影响
Figure PCTCN2021108644-appb-000014
Figure PCTCN2021108644-appb-000015
由表14可见，采用不同的热封层及不同厚度的热封层，对一次性生物工艺袋用膜材的氧气、水蒸气渗透率、拉伸强度、透明度、热封焊接性能和细胞培养效果都存在一定的影响，尤其是对透明度和热封焊接性能，并且由于焊接性能不佳也影响了细胞培养效果。
对比表14中的序号1-5可以看出，采用LDPE、LLDPE、EVA、TPU作为热封层时，制备的一次性生物工艺袋用膜材的氧气、水蒸气渗透率、拉伸强度、透明度、热封焊接性能和细胞培养效果都较合适，但采用PA作为热封层时，因其熔点较高，焊接困难，而且使加工成本明显提高，生产效率下降，同时还影响了细胞培养效果。
对比表14中的序号1、2、6可以看出，采用LDPE、LLDPE、或是LDPE和LLDPE的混合物作为热封层时，LDPE和LLDPE的混合物可明显提高一次性生物工艺袋膜材的透明度，同时热封焊接性能也非常好，强度、氧气、水蒸气渗透率和细胞培养效果都能符合一次性生物工艺袋膜材的制备需求。
对比表14中的序号6-9可见，LDPE和LLDPE的混合物的不同比例关系会明显影响制备的一次性生物工艺袋膜材的透明度，优选LDPE和LLDPE的混合物的比例关系为2～3:7～8。
对比表14中的序号6、10、11、12、13可见，热封层的不同厚度，会明显影响制备的一次性生物工艺袋膜材的热封焊接性能，从而也会对细胞培养效果产生影响，热封层的优选厚度为30～150μm；采用LDPE：LLDPE为2:8的混合物制备热封层时，热封层的优选厚度为100μm。
实施例13 采用不同液体接触层对含ULDPE一次性生物工艺袋性能的影响
本实施例采用实施例12的序号为6制备的一次性生物工艺袋膜材，即以PA/EVOH/PA作为气体阻隔层，LDPE：LLDPE为2:8的混合物制备热封层，按照实施例4提供的方法制备一次性生物工艺袋用膜材，并分别采用如表8所示的不同体接触层，其中的厚度为液体接触层的厚度，考察制得的多层共挤一次性生物工艺袋用膜材的小分子物质溶出、氧气、水蒸气渗透率、拉伸强度、透明度和细胞培养效果(生物相容性)。
LDPE采用美国陶氏DOW的LDPE；EVA采用Celanese公司的EVA；ULDPE采用陶氏化学的Engage8480K系列的ULDPE。
其中，氧气、水蒸气渗透率、拉伸强度、透明度和细胞培养效果检测方法如实施例4所示；培养细胞使用293人胚胎肾细胞，通过气相色谱/液相色谱-质谱联用的方法检测；考察结果如表15所示。
表15、不同液体接触层对含ULDPE一次性生物工艺袋性能的影响
Figure PCTCN2021108644-appb-000016
由表15可见，采用不同的液体接触层及不同厚度的液体接触层，对一次性生物工艺袋用膜材的氧气、水蒸气渗透率、拉伸强度、透明度、热封焊接性能和细胞培养效果都存在一定的影响，尤其是对细胞培养效果影响明显。
对比表15中的序号1-3可以看出，采用LDPE、ULDPE、EVA作为液体接触层时，制备的一次性生物工艺袋用膜材的氧气、水蒸气渗透率、拉伸强度、透明度都较合适，但采用ULDPE作为液体接触层时，细胞培养密度出现大幅提升，达到了233万/ml，其原因可能是ULDPE具有更好的生物相容性，且无小分子溶出，因此优选采用ULDPE作为液体接触层。
对比表15中的序号1、4、5、6、7可见，液体接触层的不同厚度，会明显影响制备的一次性生物工艺袋膜材的细胞培养效果，液体接触层的优选厚度为30～200μm；采用ULDPE制备液体接触层时，液体接触层的优选厚度为200μm。
实施例14 采用不同粘合层对含ULDPE一次性生物工艺袋性能的影响
本实施例采用实施例13的序号1制备的一次性生物工艺袋膜材，即以PA/EVOH/PA作为气体阻隔层，LDPE：LLDPE为2:8的混合物制备热封层，ULDPE制备液体接触层，按照实施例4提供的方法制备一次性生物工艺袋用膜材，并分别采用如表15所示的不同粘合层，其中的厚度为粘合层的厚度，考察制得的多层共挤一次性生物工艺袋用膜材的氧气、水蒸气渗透率、拉伸强度、透明度和细胞培养效果(生物相容性)。
EVA采用Celanese公司的EVA；EAA和EMA分别采用美国杜邦公司和陶氏化学的相关牌号产品。
其中，氧气、水蒸气渗透率、拉伸强度、透明度和细胞培养效果检测方法如实施例4所示；培养细胞使用293人胚胎肾细胞，考察结果如表16所示，因粘合层包含2和6两层，因此采用粘合层2/阻隔层/粘合层6来表示，厚度为两层分别的厚度。
表16、不同粘合层对含ULDPE一次性生物工艺袋性能的影响
Figure PCTCN2021108644-appb-000017
由表16可见，采用不同的粘合层及不同厚度的粘合层，对一次性生物工艺袋用膜材的氧气、水蒸气渗透率、拉伸强度、透明度、热封焊接性能和细胞培养效果都存在一定的影响，尤其是对拉伸强度和细胞培养效果影响明显。
对比表16中的序号1-5可以看出，采用EVA、EMA、EAA作为粘合层时，制备的一次性生物工艺袋用膜材的氧气、水蒸气渗透率、拉伸强度、透明度、细胞培养效果都较合适，但采用EVA作为液体接触层时，细胞培养密度更高，其原因可能是EVA和液体接触层ULDPE之间具有更好的相容性，因此优选采用EVA作为粘合层。
对比表16中的序号1、6、7、8、9可见，粘合层的不同厚度，会明显影响制备的一次性生物工艺袋膜材的细胞培养效果，过厚或过薄都会使细胞培养密度降低，粘合层的优选厚度为5～20μm；采用EVA制备粘合层时，液体接触层的优选厚度为10μm。
实施例15 含LLDPE与ULDPE的多层共挤出一次性生物工艺袋用膜材
本实施例采用共挤出流延法制备厚度为325μm的一次性生物工艺袋用膜材，该膜材的组成结构包括如图3所示，包括防护层1、粘合层2、气体阻隔层3/4/5、粘合层6和液体接触层7。
其中防护层采用LDPE和LLDPE混合物，LDPE占比为30％，LLDPE占比为70％。LDPE采用陶 氏的DOW系列，LLDPE采用美国陶氏DOW，该层的厚度为50μm。
其中粘合层2和6采用EVA，EVA采用Celanese公司的EVA，该层的厚度为10μm。
气体阻隔层3和5为芳香族PA，采用美国杜邦相关牌号，该层的厚度为20μm。
气体阻隔层4为EVOH采用可乐丽EVAL树脂，该层的厚度为15μm。
液体接触层7采用LLDPE与ULDPE的混合物，LLDPE占比10％，ULDPE占比90％，LLDPE采用美国陶氏DOW，ULDPE采用美国陶氏公司的Engage 8480K，该层的厚度为200μm。
多层共挤出一次性生物工艺袋用膜材的制备方法如下：
1)将所述各层原料分别加入至对应的挤出机中，防护层对应的螺杆温度设置在180～210℃；粘合层对应的螺杆温度设置在160～200℃；气体阻隔层尼龙对应的螺杆温度设置在200～240℃；气体阻隔层EVOH对应的螺杆温度设置在190～220℃；液体接触层对应的螺杆温度设置在180～200℃；模头温度设置为220℃。
2)熔融后的各层原料，通过T型结构成型模具挤出，熔体呈片状流延至平稳旋转的冷却辊筒表面上，膜片在冷却辊筒上经冷却降温定型，再经牵引、切边后收卷，得到一次性生物工艺袋用膜材。
膜材的厚度由在线测厚装置精确控制，各层厚度由螺杆转速调节控制。
考察制得的多层共挤出一次性生物工艺袋用膜材的氧气、水蒸气渗透率、拉伸强度、焊接强度、耐穿刺、透明度和细胞培养效果(生物相容性)。
其中，一种多层共挤出一次性生物工艺袋用膜材的氧气和水蒸气渗透率采用压差法进行测试，将样品裁至直径不小于100mm的圆片，依据GB/T-1038标准于室温下进行测试，测试膜材的氧气和水蒸气渗透率。每组试样为3个，求取平均值。
一种多层共挤出一次性生物工艺袋用膜材的拉伸强度及焊接强度采用Instron万能试验机进行测试，将样品分别裁成哑铃状样条和制备成相应的焊接样条，以200mm/min的速率进行测试，每个样品至少测试5个样条，并求取平均值。
一种多层共挤出一次性生物工艺袋用膜材的透明度采用雾度计法，依据GB/T-2410-2008进行测试，每组测试3个样品，并求取平均值。
依据YBB 00322004-2015对制得的一次性生物工艺袋用膜材进行耐穿刺性能测试，在200mm/min±20mm/min的速度进行穿刺。记录穿刺样品所施加的最大力值，每次测试10个样品，并求取平均值。
多层共挤出一次性生物工艺袋用膜材的生物相容性通过统计细胞密度进行表征，将制备好的膜材通过直热式搭接形式进行焊接后，制备成一定规格的一次性生物培养袋，并经过γ射线辐照灭菌。培养细胞分别使用人胚肾细胞293(以下简称293细胞)和SF9昆虫细胞，初始浓度为1×10 5/ml。一次性生物培养袋装在生物反应器内，293细胞和SF9昆虫细胞在一次性生物工艺袋中培养5天，培养条件为37℃，搅拌速度为200rpm，培养结束后检测细胞密度。
制得的多层共挤出一次性生物工艺袋用膜材的物理性能及细胞密度结果如表17所示。
表17、共挤出流延法制备的膜材的物理性能及细胞培养密度结果
Figure PCTCN2021108644-appb-000018
实施例16 含LLDPE与ULDPE的多层共挤出一次性生物工艺袋用膜材
本实施例采用共挤出流延法制备厚度为305μm的一次性生物工艺袋用膜材，该膜材的组成结构包括如图4所示，包括防护层13、粘合层14、气体阻隔层15/16、粘合层17和液体接触层18。
其中防护层13采用LDPE和LLDPE混合物，LDPE占比为30％，LLDPE占比为70％。LDPE采用陶氏的Dow系列，LLDPE采用美国陶氏DOW，该层的厚度为50μm。
其中粘合层14和17采用EVA，EVA采用Celanese公司的EVA，该层的厚度为10μm。
气体阻隔层15为芳香族PA，采用美国杜邦相关牌号，该层的厚度为20μm。
气体阻隔层16为EVOH采用可乐丽EVAL树脂，该层的厚度为15μm。
液体接触层18采用LLDPE与ULDPE的混合物，LLDPE占比10％，ULDPE占比90％，LLDPE采用美国陶氏DOW，ULDPE采用美国陶氏公司的Engage 8480K，该层的厚度为200μm。
多层共挤出一次性生物工艺袋用膜材的制备方法如下：
1)将所述各层原料分别加入至对应的挤出机中，防护层对应的螺杆温度设置在180～210℃；粘合层对应的螺杆温度设置在160～200℃；气体阻隔层尼龙对应的螺杆温度设置在200～240℃；气体阻隔层EVOH对应的螺杆温度设置在190～220℃；液体接触层对应的螺杆温度设置在180～200℃；模头温度设置为220℃。
2)熔融后的各层原料，通过T型结构成型模具挤出，熔体呈片状流延至平稳旋转的冷却辊筒表面上，膜片在冷却辊筒上经冷却降温定型，再经牵引、切边后收卷，一次性生物工艺袋用膜材。
膜材的厚度由在线测厚装置精确控制，各层厚度由螺杆转速调节控制。
考察制得的多层共挤出一次性生物工艺袋用膜材的氧气、水蒸气渗透率、拉伸强度、焊接强度、耐穿刺、透明度和细胞培养效果(生物相容性)。检测方法如实施例1所示。培养细胞分别使用293细胞和SF9昆虫细胞，初始浓度为1×10 5/ml。一次性生物培养袋装在生物反应器内，293细胞和SF9昆虫细胞在一次性生物工艺袋中培养5天，培养条件为37℃，搅拌速度为200rpm，培养结束后检测细胞密度。
制得的多层共挤出一次性生物工艺袋用膜材的物理性能及细胞密度结果如表18所示。
表18、共挤出流延法制备的膜材的物理性能及细胞培养密度结果
Figure PCTCN2021108644-appb-000019
实施例17 不同液体接触层对一次性生物工艺袋性能的影响
本实施例按照实施例15的方法制备一次性生物工艺袋膜材，即以PA/EVOH/PA作为气体阻隔层，LDPE：LLDPE为3:7的混合物制备防护层，按照实施例15提供的方法制备一次性生物工艺袋用膜材，并 分别采用如表19所示的液体接触层，其中的厚度为液体接触层的厚度，考察制得的多层共挤一次性生物工艺袋用膜材的小分子物质溶出、氧气、水蒸气渗透率、拉伸强度、抗穿刺性能、透明度和细胞培养效果(生物相容性)。
LDPE和LLDPE均采用美国陶氏DOW；ULDPE采用陶氏化学的Engage 8480K系列的ULDPE。
其中，氧气、水蒸气渗透率、拉伸强度、透明度和细胞培养效果检测方法如实施例15所示，培养细胞分别使用SF9昆虫细胞和人胚肾细胞293(以下简称293细胞)，初始浓度为1×10 5/ml；小分子物质溶出通过气相色谱/液相色谱-质谱联用的方法检测；考察结果如表19所示。
表19、不同液体接触层对一次性生物工艺袋性能的影响
Figure PCTCN2021108644-appb-000020
Figure PCTCN2021108644-appb-000021
由表19可见，采用不同类型的聚乙烯作为液体接触层及不同厚度的液体接触层，对一次性生物工艺袋用膜材的氧气、水蒸气渗透率、拉伸强度、透明度、热封焊接性能和细胞培养效果都存在一定的影响。
对比表19中的序号1-4可以看出，相比于单独采用LDPE、或LLDPE作为液体接触层时，采用ULDPE与LLDPE的混合物作为液体接触层制备的一次性生物工艺袋用膜材的氧气、水蒸气渗透率、拉伸强度、透明度都较合适，而且对SF9昆虫细胞的培养效果明显更好，证明采用ULDPE与LLDPE的混合物作为液体接触层能明显提升细胞相容性；细胞培养效果与单独使用ULDPE作为液体接触层时的效果差别虽不明显，但抗穿刺能力更优于ULDPE。
对比表19中的序号4-8可见，采用ULDPE和LLDPE混合物作为液体接触层时，ULDPE和LLDPE的不同配比，对抗穿刺能力和细胞培养效果都有一定的影响。优选的ULDPE与LLDPE的配比为2:8，此时抗穿刺能力和细胞培养效果都较好。
对比表19中的序号8-13可见，采用ULDPE和LLDPE混合物作为液体接触层时，不同的液体接触层厚度对焊接强度会有明显的影响，当液体接触层厚度小于50μm时，焊接强度明显下降，也会影响细胞培养效果，厚度大于250μm时，培养效果明显下降，因此液体接触层优选的厚度为50～250μm。
实施例18 不同阻隔层结构的含LLDPE与ULDPE的一次性生物工艺袋用膜材
本实施例采用实施例15提供的方法制备总厚度为325μm的一次性生物工艺袋用膜材，结构为防护层/粘合层/阻隔层1/阻隔层2/阻隔层3/粘合层/液体接触层，并分别采用如表20所示的不同的气体阻隔层，其中PA指芳香族PA，厚度为气体阻隔层的厚度。
EVOH采用可乐丽EVAL系列，PA采用美国杜邦相关牌号的芳香族PA。
制得的膜材的氧气、水蒸气渗透率、拉伸强度、焊接强度、透明度和细胞培养效果检测方法如实施例15所示，培养细胞使用SF9昆虫细胞，考察结果如表20所示。
表20、不同阻隔层结构对一次性生物工艺袋性能的影响
Figure PCTCN2021108644-appb-000022
Figure PCTCN2021108644-appb-000023
由表20可见，当防护层、粘合层、液体接触层及厚度都一样时，采用不同的气体阻隔结构对一次性生物工艺袋膜材的拉伸强度、氧气渗透率、水蒸气透过量、透明度和细胞培养密度都存在较大的区别。相比不同种类的PA，芳香族PA的强度和气体阻隔性能明显更优，生物相容性也较好，因此优先选用芳香族PA制备一次性生物工艺袋；相比单独采用EVOH或单独采用芳香族PA作为气体阻隔层时，PA和EVOH组合可以明显提升强度、气体阻隔性能和细胞培养功能，PA/EVOH或EVOH/PA结构获得的生物袋膜的各性能差别不大；采用PA/EVOH/PA，则可进一步提升一次性生物工艺袋膜材的性能；同样为三层复合膜气体阻隔层形式的EVOH/PA/EVOH，其强度、气体阻隔性能和细胞培养功能则远不如PA/EVOH/PA的组合，这主要是因为EVOH的耐水性差导致，因此将其作为外层阻隔层时，如遇水，阻隔性能会发生急剧下降，导致一次性生物工艺袋膜材的拉伸强度、氧气渗透率、水蒸气透过量、透明度和细胞培养密度都出现下降；最优选为PA/EVOH/PA的三层复合膜气体阻隔层，可使拉伸强度、气体阻隔性能和细胞培养功能都达到最佳。
实施例19 气体阻隔层的不同厚度对含LLDPE与ULDPE的一次性生物工艺袋性能的影响
本实施例采用实施例18的序号为8的气体阻隔层制备的一次性生物工艺袋用膜材，即以PA/EVOH/PA作为气体阻隔层，按照实施例15提供的方法制备一次性生物工艺袋用膜材，并分别采用如表21所示的不同厚度的气体阻隔层，其中的厚度为气体阻隔层的厚度，考察制得的多层共挤一次性生物工艺袋用膜材的氧气、水蒸气渗透率、拉伸强度、抗穿刺性能、透明度和细胞培养效果(生物相容性)。
LDPE和LLDPE采用美国陶氏DOW；EVA采用Celanese公司的EVA；PA采用美国杜邦相关牌号的芳香族PA。
其中，氧气、水蒸气渗透率、拉伸强度、抗穿刺性能、透明度和细胞培养效果检测方法如实施例15所示，培养细胞使用SF9昆虫细胞，考察结果如表21所示。
表21、不同气体阻隔层厚度对一次性生物工艺袋性能的影响
Figure PCTCN2021108644-appb-000024
由表21可见，气体阻隔层的不同厚度，对制备的一次性生物工艺袋膜材的性能存在较大影响，选用合适的厚度，可进一步提升一次性生物工艺袋膜材的强度、气体阻隔性能和细胞培养功能，研究证明，采用气体阻隔层的厚度为30～80μm，其中中间的气体阻隔层厚度为10～20μm，两侧的气体阻隔层厚度为10～30μm时，制备的一次性生物工艺袋膜材的强度、气体阻隔性能和细胞培养功能明显提高，气体阻隔层PA/EVOH/PA的最佳厚度选择为30/20/30μm。
实施例20 不同防护层对含LLDPE与ULDPE的一次性生物工艺袋性能的影响
本实施例按照实施例15提供的方法制备一次性生物工艺袋用膜材，并分别采用如表22所示的不同防护层，其中的厚度为防护层的厚度，考察制得的多层共挤一次性生物工艺袋用膜材的氧气、水蒸气渗透率、拉伸强度、抗穿刺性能、透明度、热封焊接性能和细胞培养效果(生物相容性)。
LDPE和LLDPE采用美国陶氏DOW。
其中，氧气、水蒸气渗透率、拉伸强度、抗穿刺性能、焊接强度、透明度和细胞培养效果检测方法如实施例15所示，培养细胞分别使用SF9昆虫细胞和人胚肾细胞293(以下简称293细胞)，初始浓度为1×10 5/ml；小分子物质溶出通过气相色谱/液相色谱-质谱联用的方法检测；考察结果如表22所示。
表22、不同防护层对一次性生物工艺袋性能的影响
Figure PCTCN2021108644-appb-000025
由表22可见，采用不同的防护层及不同厚度的防护层，对一次性生物工艺袋用膜材的拉伸强度、透明度、热封焊接性能和细胞培养效果都存在一定的影响，尤其是对透明度和热封焊接性能，并且由于焊接性能不佳也影响了细胞培养效果。
对比表22中的序号1和2可以看出，采用LDPE作为防护层时，制备的一次性生物工艺袋用膜材的透明度最高，但是拉伸强度和热封焊接性能比较差；采用LLDPE作为防护层时，虽然透明度不及LDPE作为防护层的生物工艺袋，但是拉伸强度、焊接强度和细胞培养效果均远高于以LDPE为防护层的生物工艺袋膜。由此可见，LLDPE比LDPE更适合作为生物工艺袋膜材的外层防护层。
对比表22中的序号3-7可以看出，采用LDPE、LLDPE、或是LDPE和LLDPE的混合物作为防护层时，LDPE和LLDPE的混合物可明显提高一次性生物工艺袋膜材的透明度，同时热封焊接性能也非常好，强度、氧气、水蒸气渗透率和细胞培养效果都能符合一次性生物工艺袋膜材的制备需求。其中，LDPE和LLDPE的混合物的不同比例关系会明显影响制备的一次性生物工艺袋膜材的透明度，优选LDPE和LLDPE的混合物的比例关系为1：9～3:7。
对比表22中的序号4和10以及3、8、9可见，防护层的不同厚度，会明显影响制备的一次性生物工艺袋膜材的热封焊接性能，从而也会对细胞培养效果产生影响，防护层的优选厚度为50～150μm。
实施例21 采用不同粘合层对一次性生物工艺袋性能的影响
本实施例按照实施例15提供的方法制备一次性生物工艺袋用膜材，并分别采用如表23所示的不同粘合层，其中的厚度为粘合层的厚度，考察制得的多层共挤一次性生物工艺袋用膜材的氧气、水蒸气渗透率、拉伸强度、焊接强度、抗穿刺性能、透明度和细胞培养效果(生物相容性)。
EVA采用Celanese公司的EVA；EAA和EMA分别采用美国杜邦公司和陶氏化学的相关牌号产品。
其中，氧气、水蒸气渗透率、拉伸强度、焊接强度、抗穿刺性能、透明度和细胞培养效果检测方法如实施例15所示；培养细胞使用SF9昆虫细胞，考察结果如表23所示，因粘合层包含2和6两层，因此采用粘合层2/阻隔层/粘合层6来表示，厚度为两层分别的厚度。
表23、不同粘合层对一次性生物工艺袋性能的影响
Figure PCTCN2021108644-appb-000026
Figure PCTCN2021108644-appb-000027
由表23可见，采用不同的粘合层及不同厚度的粘合层，对一次性生物工艺袋用膜材的氧气、水蒸气渗透率、拉伸强度、透明度、热封焊接性能和细胞培养效果都存在一定的影响。
对比表23中的序号1-5可以看出，采用EVA、EMA、EAA作为粘合层时，制备的一次性生物工艺袋用膜材的氧气、水蒸气渗透率、拉伸强度、透明度、穿刺最大力，细胞培养效果都较合适。
对比表23中的序号1、6、7、8、9可见，粘合层的不同厚度，会明显影响制备的一次性生物工艺袋用膜材的细胞培养效果，过厚或过薄都会使细胞培养密度降低，粘合层的优选厚度为5～20μm；采用EVA制备粘合层时，粘合层的优选厚度为10μm。
虽然本发明披露如上，但本发明并非限定于此。任何本领域技术人员，在不脱离本发明的精神和范围内，均可作各种更动与修改，因此本发明的保护范围应当以权利要求所限定的范围为准。

Claims (24)

  1. 一种气体阻隔层用于制备一次性生物工艺袋用膜材的用途，其特征在于，所述气体阻隔层由PA/EVOH/PA三层复合膜组成。
  2. 一种一次性生物工艺袋用膜材，其特征在于，包括防护层/粘合层/气体阻隔层/粘合层/液体接触层，所述的气体阻隔层由PA/EVOH/PA三层复合膜组成。
  3. 如权利要求2所述的一次性生物工艺袋用膜材，其特征在于，所述PA为芳香族PA。
  4. 如权利要求3所述的一次性生物工艺袋用膜材，其特征在于，所述PA/EVOH/PA厚度为30～80μm，所述PA厚度为10～30μm，所述EVOH厚度为10～20μm。
  5. 如权利要求4所述的一次性生物工艺袋用膜材，其特征在于，所述液体接触层接触的液体中包括用于培养的细胞。
  6. 如权利要求5所述的一次性生物工艺袋用膜材，其特征在于，所述液体接触层为ULDPE，所述ULDPE厚度为30～200μm。
  7. 如权利要求5所述的一次性生物工艺袋用膜材，其特征在于，所述液体接触层为EVA，所述EVA厚度为50～250μm。
  8. 如权利要求5所述的一次性生物工艺袋用膜材，其特征在于，所述液体接触层为线性低密度聚乙烯和超低密度聚乙烯的混合物。
  9. 如权利要求8所述的一次性生物工艺袋用膜材，其特征在于，所述线性低密度聚乙烯和超低密度聚乙烯的混合物中，线性低密度聚乙烯占比为10～100％；所述液体接触层的厚度为50～250μm。
  10. 如权利要求1-9任一项所述的一次性生物工艺袋用膜材，其特征在于，所述防护层为EVA、TPU、LDPE、LLDPE、或LDPE与LLDPE的混合物中的任意一种。
  11. 如权利要求10所述的一次性生物工艺袋用膜材，其特征在于，所述防护层为LDPE与LLDPE的混合物，所述LDPE与LLDPE的混合物中，按质量份数计，LDPE成分为20％～30％。
  12. 如权利要求11所述的一次性生物工艺袋用膜材，其特征在于，所述防护层的厚度为50～150μm。
  13. 如权利要求12所述的一次性生物工艺袋用膜材，其特征在于，所述粘合层为EAA、EVA或EMA中的一种或几种组成的混合物，所述粘合层厚度为5～20μm。
  14. 一种简化结构的一次性生物工艺袋用膜材，其特征在于，包括防护层/气体阻隔层/液体接触层，所述气体阻隔层由PA/EVOH/PA三层复合膜组成。
  15. 如权利要求14所述的简化结构的一次性生物工艺袋用膜材，其特征在于，所述PA为芳香族PA。
  16. 如权利要求15所述的简化结构的一次性生物工艺袋用膜材，其特征在于，所述PA/EVOH/PA厚度为30～80μm，所述PA厚度为10～30μm，所述EVOH厚度为10～20μm。
  17. 如权利要求16所述的简化结构的一次性生物工艺袋用膜材，其特征在于，所述液体接触层和防护层都为EVA。
  18. 如权利要求17所述的简化结构的一次性生物工艺袋用膜材，其特征在于，其中液体接触层厚度为50～250μm，防护层的厚度为50～100μm。
  19. 一种简化结构的一次性生物工艺袋用膜材，其特征在于，包括防护层/粘合层/气体阻隔层/粘合层/ 液体接触层，所述的气体阻隔层由尼龙层/乙烯-乙烯醇共聚物层两层复合膜组成；所述液体接触层为线性低密度聚乙烯和超低密度聚乙烯的混合物。
  20. 如权利要求19所述的简化结构的一次性生物工艺袋用膜材，其特征在于，所述线性低密度聚乙烯和超低密度聚乙烯的混合物中，线性低密度聚乙烯占比为10～100％；所述气体阻隔层厚度为30～80μm；其中尼龙层的厚度为20～70μm，乙烯-乙烯醇共聚物层的厚度为10～60μm。
  21. 一种如权利要求2～20任一项所述的膜材的制备方法，其特征在于，采用共挤出流延方法制备，具体步骤为：1)各层原料分别加入至对应的挤出机中；2)在机头温度120～220℃条件下，经熔融、共挤出吹塑或流延制得。
  22. 如权利要求21所述的方法，其特征在于，当膜材厚度小于300μm时，采用共挤出吹塑或流延制得；当膜材厚度大于300μm时，采用共挤出流延制得。
  23. 尼龙用于制备一次性生物工艺袋用膜材的气体阻隔层的用途。
  24. 如权利要求23所述的用途，其特征在于，所述气体阻隔层为尼龙/乙烯-乙烯醇共聚物/尼龙共聚物的三层复合结构；所述尼龙为芳香族尼龙；所述膜材用于制备生物反应器中培养细胞的一次性生物工艺袋。
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