WO2018178937A1 - Multilayer bioreactor bag - Google Patents

Abstract

The disclosure relates to a multilayer bioreactor bag having an inside layer comprising a low density polyethylene/linear low density copolymer, a barrier layer comprising polyvinyl alcohol, and an outside layer comprising a low density polyethylene/linear low density copolymer.

Description

MULTILAYER BIOREACTOR BAG

TECHNICAL FIELD

[0001] The disclosure relates to a multilayer bioreactor bag comprising an inside layer, a barrier layer, and an outside layer.

BACKGROUND

[0002] The stabilization of poly olefin composition is known in the art.

[0003] Suitable stabilizers known in the art are for example synthetic (poly)phenolic compounds such as tetrakis[methylene-3-(3',5')-di-t-butyl-4-hydroxyphenyl)propionate] methane; octadecyl 3,5-di-t-butyl-4-hydroxyhydrocinnamate; 1,1,3 -tris(2 -methyl-4-hydroxy- 5-t-butylphenyl)butane; l,3,5-trimethyl-2,4,6-tris(3,5-di-t-butyl-4-hydroxybenzyl)benzene, bis-[3,3-bis-(4' -hydroxy-3 '-t-butylphenyl butanoic acid]-glycol ester; tris(3,5-di-t-butyl-4- hydroxy benzyl)isocyanurate; l,3,5-tris(4-t-butyl-2,6-dimethyl-3- hydroxybenzyl)isocyanurate; 5-di-t-butyl-4-hydroxy hydrocinnamic acid triester with 1,3,5- tris(2- hydroxyethyl)-s-triazine-2,4,6(lH, 3H, 5H)trione; p-cresol/dicyclopentadiene butylated reaction product and 2,6-bis(2' -bis-hydroxy-3'-t-butyl-5 '-methyl -phenyl-4-methyl- phenol).

[0004] Stabilized polyolefin compounds may be processed via for example injection molding, blow molding, extrusion molding, compression molding or thin-walled injection molding techniques. The obtained products may be applied in a huge amount of applications for example in food contact packaging applications, biomedical applications, health care applications or pharmaceutical applications.

[0005] Most of the synthetic (poly)phenolic antioxidants used are strictly regulated by governments because they are suffering from serious limitations. Synthetic antioxidants can thereby diffuse into the surrounding medium. This can in tum lead to contamination of food and/or other human-used products with potentially toxic by-product substances. This problem may arise simply because some antioxidants are toxic above a certain level of concentration.

[0006] Accordingly, the use of such antioxidants is already an issue in for example food and water contact applications, in medical and pharmaceutical devices. With increased consumer concerns about the amount of chemicals in their foods, processors are looking for improved ways to protect their products. SUMMARY

[0007] The disclosure relates to a multilayer bioreactor bag comprising an inside layer having an inside layer about 150 micrometers (μηι) to about 200 μιη, a barrier layer having a thickness between about 10 μιη to about 30 μιη, and an outside layer having a thickness between about 40 μιη to about 60 μιη. Additionally this disclosure relates to a multilayer bioreactor bag having an inside layer comprising a low density polyethylene/linear low density copolymer, a barrier layer comprising polyvinyl alcohol, and an outside layer comprising a low density polyethylene/linear low density copolymer.

[0008] With improvements in biotechnology and the shift from small-molecule to large- molecule biologic drugs that are used by smaller volumes of customers, there is a need for flexible and scalable facilities for biopharmaceutical production processes that can produce more than one product offering (as opposed to traditional large-scale stainless-steel systems or glass systems).

[0009] Single-Use Systems (SUS) allow smaller production runs with improvements in yield, faster cleaning and sterilization, shorter implementation time, and increased speed to market in biologic drug production.

[0010] In certain embodiments, the present disclosure relates to bioreactor bags for a single use system. Some characteristics of single use systems are (1) flexibility and (2) avoidance of cleaning, sterilization, and validation. This results in advantages such as cost reduction, reduced utilities requirements, improved sustainability, shorter development cycles and higher outputs per unit volume.

[0011] Standard fixed in place process installations are designed for a particular type of reaction or synthesis and are best not used for other purposes. If process units need to be added, removed or modified, this will involve tinkering which introduces high cost through labor and productivity loss. Such installations are best used for (continuous) production of one single product. Discontinuous or batch production as is often the case in

biopharmaceutical process requires cleaning and sterilization in between production batches followed by validation of the cleaning step efficacy. Such processes also add cost through labor, productivity loss and usage of water and cleaning chemicals, which, in addition, have a negative environmental impact.

[0012] Single use systems (SUS) made from plastic are available on the market.

Conventional systems often consist of various components that can be combined into a configuration that is suitable for the target reaction. The systems are complemented by placeholders that keep the single use components in the required shape and a variety of other reusable modular equipment to support process operations such as e.g. shakers. The plastic components are made from, for example, polyethylene or polyvinylidene fluoride films and additional non-contact layers may be added for enhancing barrier properties (such as ethylene vinyl alcohol) or providing more strength (such as elastomeric thermoplastic, thermoplastic polyurethane (TPU)).

[0013] Commercial plastics are complex mixtures of different components. There is variation in chain length and composition between the individual polymer chains in the plastic material. Components may have been added for modification of the properties (e.g. fillers, rubbers) or to facilitate processing (e.g. stabilizers, release agents). Finally, impurities from the production process can be present. These may have come in with the raw materials that have been used, or they may have been generated by side reactions, heating, irradiation, hydrolyses, sterilization and other processes. In the application as single use system, such components may leach into the reaction mixture and influence the reactions taking place.

[0014] For one particular impurity [bis(2,4-di-tert-butylphenyl)phosphate], which is the breakdown product of the commonly used plastics heat stabilizer tris(2,4-di-tert- butylphenyl)phosphite (commercially available under the tradename Irgaphos™ 168 from BASF), it has been found that it has a strong effect on cell growth rate in Chinese hamster ovary (CHO) cell cultures, either reducing that rate or bringing production to a stop, depending on the concentration. ("A Cytotoxic Leachable Compound from Single-Use Bioprocess Equipment that Causes Poor Cell Growth Performance", Hammond et al., Biotechnology Progress, Volume 30, Issue 2, 2014, Pages 332-337).

[0015] The storage and reaction bags are key SUS components. Linear low density polyethylene (LLDPE) is a frequently used material for these components due to its low price, suitable mechanical properties and broad solvent compatibility. As an example, LLDPE may have a density of about 0.916 g/cm³. LLDPE is a substantially linear polymer (polyethylene), with significant numbers of short branches, commonly made by

copolymerization of ethylene with longer-chain olefins. In general, LLDPE is produced at lower temperatures and pressures by copolymerization of ethylene and such higher alpha olefins as butene, hexene, or octene. The copolymerization process produces an LLDPE polymer that has a narrower molecular weight distribution than conventional low density polyethylene, and in combination with the linear structure, significantly different rheological properties. Many LLDPE grades, however, have not been designed for the specific application of SUS and may contain tris(2,4-di-tert- butylphenyl)phosphite, its breakdown product bis(2,4-di-tert-butylphenyl)phosphate and/or have a level of extractables that has not been optimized or controlled. This requires end-users to spend more effort in qualifying SUS components.

[0016] In contrast to metal or glass reactor systems, a plastic reactor system may leech components into the media that are in contact with them during reaction, storage or transport. Such leeched components may contaminate the reactor contents or influence the processed that are conducted in the reactor system. It has been found that bis(2,4-di-tert- butylphenyl)phosphate, a breakdown product of the frequently used polymer stabilizer tris(2,4-di-tert-butylphenyl)phosphite, reduces cell growth rates in CHO cell growth cultures. Though the effect may be different with other cell growth cultures it is undesirable for biopharmaceutical companies to evaluate such potential effects before use. Therefore, there is a clear need for single use reactor systems which are free from bis(2,4-di-tert- butylphenyl)phosphate and from stabilizer tris(2,4-di-tert-butylphenyl)phosphite.

[0017] There is a continuous need to provide improved stabilized poly olefin compositions having no dangerous effects when dispersed in the environment and which also fulfill all requirements related to processing or short term heat stabilization.

[0018] The disclosure, in certain embodiments, relates to a bioreactor bag for single use reaction system Such single use systems are increasingly used in the (bio)pharmaceutical industry for synthesis, cell growth and (bio)reactions. There are many advantages attached to the use of such systems, but one risk that comes with the use of plastics is that components from the plastic may leach into the reaction medium This disclosure describes a single use system made from a plastic which is free of bis(2,4-di-tert-butylphenyl)phosphate and its precursor tris(2,4-di-tert-butylphenyl)phosphite and hence does not require users to test for negative effects on productivity of such components. As referenced herein the materials and methods referencing a bioreactor or bioreactor bag may be applied to other articles such as two- and three-dimensional storage bags, mixing bags, shipping bags, freeze/thaw bags, final filling bags, sampling bags, and components such as tubing, filters, liners, valves, and the like. One or more of the system components may be formed from the disclosed compositions. In certain embodiments, each component of a system may include the same composition.

[0019] The disclosure is characterized in that the stabilized poly olefin composition comprises and/or consists of:

A. a copolymer of ethylene and at least one a-olefin, and B. between 25 and 300 ppm by weight of a-tocopherol relative to the component A.

[0020] The use of this composition results in the ability to introduce a bio-inspired antioxidant package with excellent resistance to oxidative degradation. As used herein, bio- inspired connotes synthetic materials whose structure, properties, or function mimic those of natural materials or living matter. Some examples of bio-inspired materials include light- harvesting photonic materials that mimic photosynthesis, structural composites that imitate the structure of nacre, and metal actuators inspired by the movements of jellyfish. The use of articles including the compositions described herein may minimize the reliance on bis(2,4- di-tert-butylphenyl)phosphate and its precursor tris(2,4-di-tert-butylphenyl)phosphate.

Additional advantages of the stabilizer composition according to the disclosure are the presence of less discoloration of the material after recycling. Furthermore the stabilizer composition according to the disclosure results in improved processing stability and/or improved organoleptic properties.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The following is a brief description of the drawings wherein like elements are numbered alike and which are exemplary of the various aspects described herein.

[0022] FIG. 1 depicts a film having two functional layers according to aspects of the present disclosure.

[0023] FIG. 2 depicts a film having three layers including an intermediate functional layer according to aspects of the present disclosure.

[0024] FIG. 3 depicts a film having three layers including a structural layer according to aspects of the present disclosure.

[0025] FIG. 4 depicts a multi-layer film with structural layers there between according to aspects of the present disclosure.

[0026] FIG. 5 is a comparative graph showing the effect of number of extrusion passes on Melt Flow Rate (MFR) for compositions according to the present disclosure.

[0027] FIG. 6 is a comparative graph showing the effect of number of extrusion passes on Oxidation Induction Time (OIT) for compositions according to the present disclosure.

DETAILED DESCRIPTION

[0028] The stabilized polyolefin composition is efficiently stabilized, while limiting and/or avoiding the use of toxic substances. This may especially, for example, allow the improvement of processing stability, particularly by reducing cross-linking during extrusion and/or multiple extrusions. [0029] A further advantage is that the stabilizer composition according to the disclosure results in improved organoleptic properties. Moreover, the risk related to and/or the amount of compound(s) possibly migrating out of the stabilized polyolefin composition may be reduced.

[0030] Component A may be a copolymer of ethylene with at least one a-olefin, preferably a LLDPE. LLDPE may, for example, be obtained by polymerizing ethylene with at least one a-olefin, which may be selected from 1-butene, 1-pentene, 4-methyl-l-pentene, 1-hexene, 1- heptene and/or 1-octene, preferably 1-butene.

[0031] For example, the polymerized a-olefin may for example account for between 0.05 wt. % and 15 wt. %, preferably between 0.1 wt. % and 1 2 wt. %, further preferred between 1 wt. % and 1 0 wt. % , further preferred between 0.1 wt. % and 5 wt. % or between 5 wt. % and 10 wt. % compared to the total weight of the LLDPE. A copolymer of ethylene and at least one a-olefin may for example have a density as determined according to ISO 1183-1

(2012), method A of 910 kilograms per cubic meter (kg/m 3 ) and 940 kg/m 3 , preferably

3 3

between 913 kg/m and 923 kg/m . This may lead to mechanical properties suitable for the use for film applications.

[0032] A copolymer of ethylene and at least one a-olefin may for example have an MFI represented by the corresponding melt mass flow rate (MFR) as measured according to ISO 1131-1 (2011) at 1 90°C and at a load of 2.16 kilogram (kg) of between 0.1 decigrams per minute (dg/min) and 10 dg/min, preferably between 0.5 dg/min and 5 dg/min, further preferred between 0.5 dg/min and 3 dg/min, even further preferred > 0.5 dg/min and < 2 dg/min. This may lead to a processability suitable for the use for film applications.

[0033] LLDPE may preferably be produced using a gas phase or slurry process. The production processes of polyethylenes are summarized in "Handbook of Polyethylene" by Andrew Peacock (2000; Dekker; ISBN 0824795466) at pages 43-66.

[0034] Component B may be a-tocopherol.

[0035] According to the disclosure the a-tocopherol may be preferably synthetic a- tocopherol.

[0036] A suitable example of α-tocopherol and/or synthetic α-tocopherol according to the disclosure may be is Irganox™ E 201 (supplied by BASF) which is a racemic mixture of equal amounts of all eight possible stereoisomers of α-tocopherol (RRR, SSS, RRS, RSR, SSR, SRS, SRR, RSS) and is referred to as dl-a-tocopherol or all-rac-alpha-tocopherol (racemic mixture). [0037] The use of a-tocopherol may for example allow efficient stabilization with relatively low loadings of a-tocopherol.

[0038] The stabilized poly olefin composition may preferably not comprise other tocopherols and/or no tocotrienol and/or no natural vitamin E and/or no other antioxidant and/or no other compound comprising at least one phenolic motif. This may allow an efficient stabilization of the stabilized poly olefin composition, while reducing the risk associated with and/or the amount of compounds that may migrate out of the stabilized poly olefin composition. This may thus reduce possible health hazards.

[0039] The amount of component B ranges between 25 parts per million (ppm) and 300 ppm by weight, preferably between 50 ppm and 200 ppm by weight, further preferred between > 50 ppm and < 1 00 ppm by weight, relative to the component A. This may allow an efficient stabilization of the stabilized poly olefin composition, while reducing the risk associated with and/or the amount of compounds that may migrate out of the stabilized poly olefin composition. This may thus reduce possible health hazards.

[0040] Besides components A and B, the stabilized polyolefin composition according to the disclosure may also comprise component C, which may be at least one organometallic stearate such as for example magnesium stearate, aluminum stearate, sodium stearate and calcium stearate and/or at least one inorganic hydrotalcites, such as for example DHT-4A™ (Mg4.3Ah(OH)i2.6C03 ^■ mH20), preferably calcium stearate. An organometallic stearate and/or an inorganic hydrotalcite may improve processability.

[0041] The amount of optional component C may range between 100 ppm and 1000 ppm by weight, more preferably between 200 ppm and 800 ppm by weight, further preferred between 400 ppm and 600 ppm by weight, relative to the component A.

[0042] The weight ratio B:C may range for example between 0.05: 1 and 0.5: 1, preferably 0.07: 1 to 0.3: 1, further preferred between 0.1 : 1 and 0.2: 1.

[0043] Another advantage of the stabilized poly olefin composition may be an improved color retention after recycling.

[0044] A stabilized poly olefin composition according to the disclosure may preferably for example comprise and/or consist of:

A. a copolymer of ethylene and at least one a-olefin,

B. between 25 and 300 ppm by weight of α-tocopherol relative to the component A and

C. between 100 ppm and 1000 ppm by weight of a stearate, preferably calcium stearate, relative to the component A.

[0045] That the stabilized poly olefin composition consists of the components listed above may thereby preferably mean that no other antioxidant and/or no other stearate or hydrotalcite and/or no other compound/component is present.

[0046] The composition according to the disclosure may be used in the production of specific articles. Examples of preferred articles are films and/or pouches, especially for packaging applications such as food and/or beverage packaging applications, for biomedical applications for example in-vivo applications, health care applications and/or pharmaceutical applications, medical applications, especially films and/or pouches for biomedical and/or health care, and/or pharmaceutical and/or medical applications.

[0047] The present disclosure also concerns the use of a stabilized poly olefin composition according to the disclosure for films and/or pouches, especially for packaging applications such as food and/or beverage packaging applications, for biomedical applications for example in-vivo applications, health care applications and/or pharmaceutical applications, medical applications, especially films and/or pouches for biomedical and/or health care, and/or pharmaceutical and/or medical applications.

[0048] The present disclosure also concerns use of a stabilized poly olefin composition a bioreactor such as a bag, pouch, or chamber configuration. As an example, the bioreactor may include at least one sheet of polymer material formed from a composition comprising: A. a copolymer of ethylene and at least one a-olefin; and B. between 25 and 300 ppm by weight of a-tocopherol relative to the component A.

[0049] In certain embodiments, a bioreactor bag may be an inflatable bag and may comprise a top sheet of polymer material; and a bottom sheet of the polymer material coupled along at least one edge of the top sheet to form a sealed bag, wherein the polymer material is formed from a composition comprising: A. a copolymer of ethylene and at least one a-olefin; and B. between 25 ppm and 300 ppm by weight of a-tocopherol relative to the component A The top sheet and the bottom sheet may be formed from separate sheets or may be formed from a contiguous sheet folded at least partially upon itself. Other configurations of material may be used. The bioreactor may comprise multiple layer sheets or films including 2, 3, 4, 5 or more layers of material or gaseous spacer layers. The bioreactor bag may further comprise: C. a stearate. The copolymer of ethylene and at least one a-olefin is LLDPE.

[0050] The amount of component B may range between > 50 ppm and < 150 ppm (greater than 50 ppm and less than 150 ppm) by weight relative to the component A. The amount of component C may range between 100 ppm and 1000 ppm by weight relative to the component A.

[0051] In certain embodiments, the stabilized polyolefin composition may be used to make a film. This may be a homogeneous film from the stabilized poly olefin composition, but may include a multilayer film in which the various layers have a different function and in which at least one of the outer layers is made from the stabilized poly olefin composition. In certain embodiments, products derived from the film such as bags are then designed and produced in such a way that the layer consisting of the bio-stabilized poly olefin composition is on the inside of the bag such that this is the layer that is in contact with the reaction medium or the storage solution or the (bio)pharmaceutical product. In other embodiments, products derived from the film such as bags are then designed and produced in such a way that the layer consisting of the bio-stabilized poly olefin composition containing a natural based stabilizer, this poly olefin is on the outside of the bag.

[0052] As an illustrative example, when a multilayer film is used the structure may be as depicted in FIG. 1 where the film has two functional layers. Layer A is the 'inside layer' which is made from the stabilized poly olefin composition. This is the layer that is in contact with the reaction medium L. Layer B is the Outside layer' which may be from the same or from a different composition as A and have the same or a different thickness as A The composition and thickness of layer B can be chosen such that it enhances the certain properties of the film such as ultraviolet (UV) resistance, strength, rigidity, puncture resistance, gas permeability, radiation resistance and/or heat resistance.

[0053] In between the inside layer A and the outside layer B, additional layers may be added. FIG. 2 shows a three layered film having one intermediate barrier layer C. One or more of such intermediate layers may be used to further enhance the performance of the film Layer C can for instance be a polyethylene co-vinyl alcohol (EVOH), such as an ethylene vinylalcohol copolymer which reduces gas permeability.

[0054] In addition to functional layers (A, B, C), also structural layers may be added. Structural layers improve the adhesion between the functional layers and prevent film failure through delamination. Such structural layers may be made from an adhesive. FIGS. 3 and 4 show multifilm layers with two and three functional layers having structural layers (SI, S2) in there between. EXAMPLES

[0055] The disclosure will now be elucidated by way of the following examples without however being limited thereto. Without further elaboration, it is believed that one skilled in the art can, using the description herein, utilize the present disclosure. The following examples are included to provide addition guidance to those skilled in the art of practicing the claimed disclosure. The examples provided are merely representative of the work and contribute to the teaching of the present disclosure. Accordingly, these examples are not intended to limit the disclosure in any manner.

[0056] While aspects of the present disclosure can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present disclosure can be described and claimed in any statutory class. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0057] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.

[0058] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the methods, devices, and systems disclosed and claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in degrees Celsius (°C) or is at ambient temperature, and pressure is at or near atmospheric.

[0059] The following compounds were used:

- LLDPE: SABIC™ LLDPE 11SNE (without additives (no stabilizers, no acid scavengers)

- a-tocopherol purchased from BASF (Irganox™ E 201)

- calcium stearate PLC (PLC referring to non-dusting grade vegetable based) supplied by Paci S.p.A.

Multiple Extrusion Experiments:

[0060] A Leistritz Micro 27/GL24 Schnecke 10 was used under air. Multiple-pass extrusion involves repeatedly passing the polymer through an extruder and then collecting the samples after each pass. After the compounding extrusion (first extrusion step), the pellets were re- extruded five more times with samples being taken after each pass through the extruder. Melt Flow Index Measurements:

[0061] Melt stability was characterized after each extrusion step using melt flow index measurements (MFI) by measuring the MFR after each extruder pass. MFI was measured by applying a standard load of 2.16 kg at a melt temperature of 1 90 °C, in accordance with ASTM D 1238-13. Results are shown below in FIG. 5.

Oxidation Induction Time Measurements:

[0062] Oxidation Induction Time (OIT) has been measured after each extrusion step according to ISO 11357-6: Plastics ~ Differential scanning calorimetry (DSC) ~ Part 6: Determination of oxidation induction time (isothermal OIT) in duplicate.

Temperature: 200 °C

Heating rate: 20 °C/min

Results are shown below in FIG. 6.

Examples I to V and comparative example CI and C2

[0063] Table 1 shows a comparison between stabilized poly olefin compositions according to the disclosure (I, II, III, IV, and V) and comparative examples CI and C2. Table 1

[0064] Irganox™ 1076 is octadecyl 3,5-di-t-butyl-4-hydroxyhydrocinnamate and Irgafos™ 168 is tris (2,4-di-t-butylphenyl) phosphite.

[0065] FIGS. 5 and 6 illustrate comparative graphs. Comparison of compositions I to III with comparative examples CI and C2 shows that processing stability for the compositions according to the disclosure is better than for CI and/or comparable or better than for C2, in that the MFR does not significantly decrease after two consecutive extrusions, preferably even after 3 consecutive extrusions and for composition II and III even after 6 consecutive extrusions. This means that cross-linking occurring during extrusion may be reduced.

[0066] Moreover, OIT can be increased for the compositions I to III compared to CI and/or for compositions II and III even compared to C2. Furthermore, an increased OIT may also be maintained over several consecutive extrusions.

[0067] Each of the component materials disclosed herein are either commercially available and/or the methods for the production thereof are known to those of ordinary skill in the art. It is understood that the compositions disclosed herein have certain functions. Disclosed herein are certain structural requirements for performing the disclosed functions and it is understood that there are a variety of structures that can perform the same function that are related to the disclosed structures, and that these structures will typically achieve the same result.

[0068] The following observations were made with respect to the optical quality of materials described in this disclosure. To measure of the optical film quality of the material, a gel count evaluation was performed. Gel count is used in order to determine the gel level in polyethylene film Gels are optical irregularities in the film which are more predominant if heat stabilization is poor. When measuring gel count, a cast film of 25 μπι is produced from the material. This film is inspected via an optical system in order to determine defects within the film. These defects are measured, classified in size (>300, >450, >600 μπι) and quantified in number of gel class per square meter (m²) film The equipment used for the data included herein was a Gottfert extruder V-type (030 mm, 20 length/diameter, L/D).

[0069] The extrusion settings applied during the gel count analysis provided in Tables 3 and 4 are included in Tables 2A and 2B, respectively, below.

Table 2A

Table 2B

[0070] Table 3 shows the result of gel count measurements of compositions C2 compared with compositions IV and V. Table 3

[0071] Table 4 shows the result of gel count measurements of compositions V compared with composition C2. Gels have been counted on 25 μιη film cast at 230°C and are expressed as gels per square meter (gels/m²) after having been counted on 40 m². If processing takes place at higher temperatures such as those used in the production of these films, a higher level of stabilizer such as composition V is desired.

Table 4

[0072] The films can be used to manufacture SUS components. The films are particularly well suited to make disposable, sterile bioreactor bags. Such bags may be used for storage and/or for executing cell cultures and (bio)chemical reactions. The bioreactor bags can be made in a variety of sizes to suit different process steps and scale-up stages. Bag volumes may range from about 0.1 L to about 5.0 L for laboratory settings and up to about 10,000 L for pilot, pre-production, and production scales.

Table 5

(b) mLLDPE 8112 SABIC Linear low density 0.912 1.1 ethylene- octene

copolymer ethylene- octene copolymer with a

density of 0.912 g/cm³ and

a melt flow rate of 1.1

g/lOmin

(c) LLDPE V055 SABIC Linear low density 0.918 1.0 ethylene- butene

copolymer with a

density of 0.918 g/cm³

and a melt flow rate of

l .O g/lOmin

(d) EVOH Poly(ethylene- co-vinyl

alcohol)

[0073] LDPE 2404N0 is a low density polyethylene tubular grade without additives suitable for a wide variety of cast film applications. mLLDPE8 1 12 is a metallocene linear low density polyethylene grade. More specifically, it is an ethylene-octene copolymer suitable for processing by blown film extrusion. LLDPE V055 is a butene linear low density polyethylene resin that contains a natural based stabilizer and free of phosphor and sulfur additives. V055 is free of bis-(2,4-di-tert.-butylphenol)pentaerythritol diphosphite (Irgafos™ 168) and is stabilized by vitamin E (tocopherol). V055 is suitable for lamination and coextruded films, and contributes to increased tensile properties.

[0074] The density of each polyethylene material was evaluated as grams per cubic centimeter (g/cm³) in accordance with ISO 1183/A. The melt mass-flow rate (MFR) was evaluated at 190° C/2.16 kg in accordance with ISO 133/ASTM D1238.

[0075] The polyethylene co-vinyl alcohol (EVOH) copolymer referred to herein can be obtained from any commercial source. For example, an extrusion grade EVOH is available under the name EVAL™ from Kuraray Co. Ltd. of Japan. The ethylene vinyl alcohol copolymer employed herein can have a vinyl alcohol content ranging from about 40 to about 85 mole percent (mol%), and preferably, from about 50 to about 75 mol%.

[0076] Bioreactor bags were made by extruding film layers as described in Table 6. The individual film layers were prepared using the component materials identified in Table 5. Table 6

[0077] It was observed that combining different polyethylene materials among the various film layers of the bioreactor bag further enhanced the overall properties of the bags, improved processabilty, and increased ability for scaled-up production. In particular, LDPE was selected in part because of its cleanliness and processability. And LLDPE (c) was selected in part because of its preferred tensile properties, namely enhanced flexibility, puncture resistance, and tear resistance, which decrease the potential chance of breach of the bioreactor bag due to mechanical stress. It was observed that by combining these component materials in the ways described in Table 6, the resultant bioreactor bags exhibited an excellent and robust sealability and while maintaining an appropriate balance between toughness and stiffness.

[0078] More particularly, in Table 6, Film Layer A represents the inside layer of the bioreactor bag. Film Layer A was made asymmetrically thicker than the other film layers in an effort to protect Layer C-the barrier layer-from water and humidity. In some aspects, the inside layer comprises LDPE /LLDPE. In some aspects, the inside layer comprises LDPE / linear low density ethylene -octene copolymer. In other aspects, the inside layer comprises LDPE/ linear low density ethylene-butene copolymer. In some aspects, the LDPE comprises about 10 wt% to about 90 wt%, or about 15 wt% to about 85 wt% of the layer. It is understood that various intervening endpoints may be used, including, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, 50 wt%, 55 wt%, 60 wt%, 65 wt%, 70 wt%, 75 wt%, 80 wt%, and 85 wt%. Preferably, the LDPE comprises about 85 wt% of the inside film layer. In other aspects, the LLDPE comprises about 10 wt% to about 90 wt%, or about 15 wt% to about 85 wt% of the inside film layer. In some aspects, the LLDPE is a linear low density ethylene -octene copolymer and comprise about 15 wt% of the inside film layer. In other aspects, the LLDPE is a linear low density ethylene-butene copolymer and comprise about 15 wt% of the inside film layer. [0079] Film Layer A (the inside layer) may be directly adhered to the barrier layer, and may be sufficiently thicker as compared to the barrier layer and the outside layer. Useful inside layer thicknesses include those that range from about 150 μιη to about 200 μιη, about 160 μιη to about 180 μιη. It is understood that various intervening endpoints may be used, including, 155 μιη, 160 μιη, 165 μιη, 170 μιη, 175 μιη, 180 μιη, 185 μιη, 190 μιη, and 195 μιη. Preferably, the inside layer is about 180 μιη.

[0080] Film Layer C (the barrier layer) may be directly adhered the internal surface of the inside layer, or directly adhered to the internal surface of the outside layer, or both, and may be sufficiently thinner than the inside layer and the outside layer. The barrier layer prevents the transmission of moisture and gases between the inside layer and the outside layer. The barrier layer may include polyvinyl alcohol, acrylonitrile-butadiene copolymer,

polyvinylidene chloride, polyalkylene carbonate, polyacrylonitrile, polypropylene, and polyethylene, or a combination thereof. In some aspects, the barrier layer comprises EVOH.

[0081] The specific EVOH in Table 6 was selected because the increased level of vinyl alcohol provided better barrier properties; however, the increased vinyl alcohol content also contributed to decreased moisture resistance, mechanical properties, and processability of this film.

[0082] Useful barrier layer thicknesses include those that range from about 10 μπι to about 30 μπι, about 15 μπι to about 20 μπι. It is understood that various intervening endpoints may be used, including, 11 μπι, 12 μπι, 13 μπι, 14 μπι, 15 μπι, 16 μπι, 17 μπι, 18 μπι, 19 μπι, 20 μπι, 21 μπι, 22 μπι, 23 μπι, 24 μπι, 25 μπι, 26 μπι, 27 μπι, 28 μπι, and 29 μπι. Preferably, the inside layer is about 20 μπι.

[0083] Film Layer B represents the outside layer of the bioreactor bag, and contributes to increased mechanical properties and puncture resistance of the bioreactor bag, and provides additional protections against moisture for Film Layer C (the barrier layer). The outside layer can comprise LDPE, linear low density ethylene-octene copolymer, linear low density ethylene-butene copolymer, or a combination thereof. In some aspects, the outside layer comprises LDPE. In other aspects, the outside layer comprises LDPE/ linear low density ethylene-octene copolymer or LDPE/ linear low density ethylene-butene copolymer. In some aspects, the LDPE comprises about 70 wt% to about 100 wt%, or about 80 wt% to about 90 wt% of the layer. It is understood that various intervening endpoints may be used, including, 75 wt%, 80 wt%, 85 wt%, 90 wt%, and 95 wt%. Preferably, the LDPE comprises about 85 wt% of the outside film layer. In other aspects, the linear low density ethylene-octene copolymer comprises about 15 wt% of the outside film layer. In other aspects, the linear low density ethylene-butene copolymer comprises about 15 wt% of the outside film layer.

[0084] Film Layer B (the outside layer) may be directly adhered to the barrier layer. Useful outside layer film thicknesses include those that range from about 40 μιη to about 60 μιη, about 45 μιη to about 55 μιη. It is understood that various intervening endpoints may be used, including, 41 μιη, 42 μιη, 43 μιη, 44 μιη, 45 μιη, 46 μιη, 47 μιη, 48 μιη, 49 μιη, 50 μιη, 51 μιη, 52 μιη, 53 μιη, 54 μιη, 55 μιη, 56 μm, 57 μιη, 58 μm, and 59 μιη. Preferably, the inside layer is about 55 μιη.

[0085] Additionally, the bioreactor bags may further include a structural layer. The structural layer contributes to the overall structural integrity of the bag, improves adherence among the film layers, and prevents delamination. Useful materials for the structural layer include anhydride-modified polyethylene, ethylene/unsaturated acid copolymer,

ethylene/unsaturated ester copolymer, anhydride -modified polyolefin, polyurethane, and mixtures thereof. Preferably, the structural layer includes anhydride-modified polyethylene. One example of a commercially available anhydride-modified polyethylene is Bynel™ 4157 from the E.I. du Pont de Nemours and Company of Wilmington, Delaware (DuPont).

[0086] The structural layer may be directly adhered to one or both sides of the barrier layer, or directly adhered to the internal surface of the outside layer. The structural layer may be sufficiently thick to provide proper adherence. Useful structural layer thicknesses include those that range from about 1 μπι to about 5 μπι, about 2 μπι to about 4 μπι. It is understood that various intervening endpoints may be used, including, 1.1 μπι, 1.2 μπι, 1.3 μπι, 1.4 μτη, Ι .5 μπι, 1.6 μπι, 1.7 μπι, 1.8 μπι 1.9 μπι, 2.0 μπι, 2.1 μπι, 2.2 μπι, 2.3 μπι, 2.4 μπι, 2.5 μπι, 2.6 μπι, 2.7 μπι, 2.8 μπι, 2.9 μπι, 3.0 μπι, 3.1 μπι, 3.2 μπι, 3.3 μπι, 3.4 μπι, 3.5 μπι, 3.6 μπι, 3.7 μπι, 3.8 μπι, 3.9 μπι, 4.0 μπι, 4.1 μπι, 4.2 μπι, 4.3 μπι, 4.4 μπι, 4.5 μπι, 4.6 μπι, 4.7 μπι, 4.8 μm 4.9 μm, and 5.0 μπι. Preferably, the structural layer is about 3 μπι.

[0087] The bioreactor bag of this disclosure may also include one or more additives useful in films, such as, vitamin E, antiblocking agents, slip agents, antifog agents, colorants, pigments, dyes, flavorants, antimicrobial agents, meat preservatives, antioxidants, fillers, radiation stabilizers, and antistatic agents. Such additives, and their effective amounts, are known in the art. The additives in the individual film layers should be limited to an amount that does not interfere with the desired mechanical properties of the bioreactor bag. Aspects

[0088] In various aspects, the present disclosure pertains to and includes at least the following aspects.

[0089] Aspect 1. A multilayer bioreactor bag comprising: an inside layer having a thickness between about 150 μιη to about 200 μιη; a barrier layer having a thickness between about 10 μιη to about 30 μιη; and an outside layer having a thickness between about 40 μιη to about 60 μιη.

[0090] Aspect 2. A multilayer bioreactor bag consisting essentially of: an inside layer having a thickness between about 150 μιη to about 200 μιη; a barrier layer having a thickness between about 10 μιη to about 30 μιη; and an outside layer having a thickness between about 40 μιη to about 60 μιη.

[0091] Aspect 3. A multilayer bioreactor bag consisting of: an inside layer having a thickness between about 150 μιη to about 200 μιη; a barrier layer having a thickness between about 10 μιη to about 30 μιη; and an outside layer having a thickness between about 40 μιη to about 60 μιη.

[0092] Aspect 4. The multilayer bioreactor bag according to any of aspects 1-3, wherein the inside layer comprises a low density polyethylene, a linear low density polyethylene copolymer, or a combination thereof.

[0093] Aspect 5. The multilayer bioreactor bag according to aspect 4, wherein the linear low density polyethylene copolymer comprises linear low density ethylene-octene copolymer, linear low density ethylene-butene copolymer, or a combination thereof.

[0094] Aspect 6. The multilayer bioreactor bag according to any one of aspects 4-53, wherein the low density polyethylene comprises about 10 wt% to about 90 wt% of the inside layer.

[0095] Aspect 7. The multilayer bioreactor bag according to any one of aspects 2-4, wherein the linear low density polyethylene comprises about 10 wt% to about 90 wt% of the inside layer.

[0096] Aspect 8. The multilayer bioreactor bag according to aspect 4, wherein the low density polyethylene comprises about 85 wt% of the inside layer and the linear low density polyethylene comprises about 15 wt% of the inside layer.

[0097] Aspect 9. The multilayer bioreactor bag according to any one of aspects 1-8, wherein the inside layer has a thickness of about 180 μιη. [0098] Aspect 10. The multilayer bioreactor bag according to any one of aspects 1-9, wherein the inside layer is adhered directly to the barrier layer.

[0099] Aspect 11. The multilayer bioreactor bag according to any one of aspects 1-10, wherein the barrier layer is adhered directly to an internal surface of the inside layer, adhered directly to an internal surface of the outside layer, or both.

[00100] Aspect 12. The multilayer bioreactor bag according to any one of aspects 1-11, wherein the barrier layer has a thickness of about 20 μιη.

[00101] Aspect 13. The multilayer bioreactor bag according to any one of aspects 1-12, wherein the barrier layer comprises polyvinyl alcohol, acrylonitrile-butadiene copolymer, polyvinylidene chloride, polyalkylene carbonate, polyacrylonitrile, polypropylene, and polyethylene, or a combination thereof.

[00102] Aspect 14. The multilayer bioreactor bag according to any one of aspects 1-13, wherein the barrier layer comprises polyvinyl alcohol, poly( ethylene-co-vinyl alcohol), acrylonitrile-butadiene copolymer, polyvinylidene chloride, polyalkylene carbonate, polyacrylonitrile, polypropylene, and polyethylene, or a combination thereof.

[00103] Aspect 15. The multilayer bioreactor bag according to any one of aspects 1-14, wherein the barrier layer comprises poly( ethylene-co-vinyl alcohol).

[00104] Aspect 16. The multilayer bioreactor bag according to any one of aspects 1-15, wherein the outside layer comprises a low density polyethylene, a linear low density polyethylene copolymer, or a combination thereof.

[00105] Aspect 17. The multilayer bioreactor bag according to aspect 16, wherein the linear low density polyethylene copolymer comprises linear low density ethylene-octene copolymer, linear low density ethylene-butene copolymer, or a combination thereof.

[00106] Aspect 18. The multilayer bioreactor bag according to any one of aspects 16-17, wherein the low density polyethylene comprises about 70 wt% to about 100 wt% of the outside layer.

[00107] Aspect 19. The multilayer bioreactor bag according to aspects 16-17, wherein the linear low density polyethylene comprises about 1 wt% to about 30 wt% of the outside layer.

[00108] Aspect 20. The multilayer bioreactor bag according to aspect 16, wherein the low density polyethylene comprises about 85 wt% of the outside layer and the linear low density polyethylene comprises about 15 wt% of the outside layer.

[00109] Aspect 21. The multilayer bioreactor bag according to any one of aspects 1-20, wherein the outside layer has a thickness of about 55 μιη. [00110] Aspect 20. A multilayer bioreactor bag having an inside layer comprising a low density polyethylene/linear low density copolymer, a barrier layer comprising polyvinyl alcohol, and an outside layer comprising a low density polyethylene/linear low density copolymer.

[00111] Aspect 21. A multilayer bioreactor bag having an inside layer consisting essentially of: a low density polyethylene/linear low density copolymer, a barrier layer comprising polyvinyl alcohol, and an outside layer comprising a low density

polyethylene/linear low density copolymer.

[00112] Aspect 22. A multilayer bioreactor bag having an inside layer consisting of: a low density polyethylene/linear low density copolymer, a barrier layer comprising polyvinyl alcohol, and an outside layer comprising a low density polyethylene/linear low density copolymer.

[00113] Aspect 23. A multilayer bioreactor bag comprising: an first layer having a thickness between about 150 μιη to about 200 μιη; a second layer interposed between the first layer and the third layer and having a thickness between about 10 μιη to about 30 μιη; and an third layer having a thickness between about 40 μιη to about 60 μιη.

[00114] Aspect 24. A multilayer bioreactor bag consisting essentially of: an first layer having a thickness between about 150 μπι to about 200 μπι; a second layer interposed between the first layer and the third layer and having a thickness between about 10 μπι to about 30 μπι; and an third layer having a thickness between about 40 μπι to about 60 μπι.

[00115] Aspect 25. A multilayer bioreactor bag consisting of: an first layer having a thickness between about 150 μπι to about 200 μπι; a second layer interposed between the first layer and the third layer and having a thickness between about 10 μπι to about 30 μπι; and an third layer having a thickness between about 40 μπι to about 60 μπι.

[00116] Aspect 26. The multilayer bioreactor bag according to any one of aspects 1-25, further comprising a structural layer.

[00117] Aspect 27. The multilayer bioreactor bag according to aspect 26, wherein the structural layer comprising a structural layer comprises anhydride-modified polyethylene.

[00118] Aspect 28. The multilayer bioreactor bag according to any one of aspects 26-27, wherein the structural layer has a thickness of about 3 μπι.

[00119] Aspect 29. The multilayer bioreactor bag according to any one of aspects 1-28, further comprising vitamin E. [00120] Aspect 30. The multilayer bioreactor bag according to any of aspects 1- 29, further comprising a-tocopherol.

[00121] Aspect 31. The multilayer bioreactor bag according to any of aspects 1-29 , further comprising (synthetic) dl-a-tocopherol.

[00122] Aspect 32. A multilayer bioreactor bag consisting of: an inside layer having a thickness between about 150 μιη to about 200 μιη, wherein the inside layer comprises a linear low density polyethylene or a linear low density polyethylene copolymer or a combination thereof; a barrier layer having a thickness between about 10 μιη to about 30 μιη; and an outside layer having a thickness between about 40 μιη to about 60 μιη, wherein the multilayer bioreactor bag is free of bis(2,4-di-tert-butylphenyl)phosphate and/or tris(2,4-di-tert- butylphenyl)phosphite .

[00123] Aspect 33. A multilayer bioreactor bag consisting of: an inside layer having a thickness between about 150 μιη to about 200 μιη, wherein the inside layer comprises a linear low density polyethylene or a linear low density polyethylene copolymer or a combination thereof; a barrier layer having a thickness between about 10 μιη to about 30 μιη; and an outside layer having a thickness between about 40 μιη to about 60 μιη, wherein the multilayer bioreactor bag is free of tris(2,4-di-tert-butylphenyl)phosphite.

[00124] Aspect 34. A multilayer bioreactor bag consisting of: an inside layer having a thickness between about 150 μιη to about 200 μιη, wherein the inside layer comprises a linear low density polyethylene or a linear low density polyethylene copolymer or a combination thereof; a barrier layer having a thickness between about 10 μιη to about 30 μιη; and an outside layer having a thickness between about 40 μιη to about 60 μιη, wherein the multilayer bioreactor bag is free of bis(2,4-di-tert-butylphenyl)phosphate and tris(2,4-di-tert- butylphenyl)phosphite .

[00125] Aspect 35. The multilayer bioreactor bag of any of the foregoing aspects wherein the multilayer bioreactor bag is formed from a composition free of bis(2,4-di-tert- butylphenyl)phosphate and tris(2,4-di-tert-butylphenyl)phosphite.

[00126] Aspect 36. The multilayer bioreactor bag of any of the foregoing aspects wherein the multilayer bioreactor bag is free of bis(2,4-di-tert-butylphenyl)phosphate and tris(2,4-di- tert-butylphenyl)phosphite .

Definitions

[00127] It is to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. As used in the specification and in the claims, the term "comprising" may include the aspects "consisting of ' and "consisting essentially of." Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined herein.

[00128] As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polycarbonate" includes mixtures of two or more such poly carbonates. Furthermore, for example, reference to a filler includes mixtures of two or more such fillers.

[00129] Ranges can be expressed herein as from one value (first value) to another value (second value). When such a range is expressed, the range includes in some aspects one or both of the first value and the second value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[00130] As used herein, the terms "optional" or "optionally" mean that the subsequently described event, condition, component, or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[00131] As used herein, the term or phrase "effective," "effective amount," or "conditions effective to" refers to such amount or condition that is capable of performing the function or property for which an effective amount is expressed. As will be pointed out below, the exact amount or particular condition required will vary from one aspect to another, depending on recognized variables such as the materials employed and the processing conditions observed. Thus, it is not always possible to specify an exact "effective amount" or "condition effective to." However, it should be understood that an appropriate effective amount will be readily determined by one of ordinary skill in the art using only routine experimentation. [00132] As used herein, "free of or "substantially free of may refer to less than 0.5 wt. % or less than about 0.5 wt.% present in a given composition or component. In another aspect, substantially free of can be less than 0.1 wt. %, or less than about 0.1 wt.%. In another aspect, substantially free of can be less than 0.01 wt. %, or less than about 0.01 wt.%. In yet another aspect, substantially free of can be less than 100 parts per million (ppm), or less than about 100 ppm. In yet another aspect, substantially free can refer to an amount, if present at all, below a detectable level. "Free of or "substantially free of may indicate that the given component has not been explicitly added to the composition. For example, the disclosed single use reactor systems may be free of or substantially free of bis(2,4-di-tert- butylphenyl)phosphate. As a further example, the disclosed single use reactor systems may be free of or substantially free of tris(2,4-di-tert-butylphenyl)phosphite.

[00133] Disclosed are component materials to be used to prepare disclosed compositions of the disclosure as well as the compositions themselves to be used within methods disclosed herein. These and other materials are disclosed herein, and it is understood that when

[00134] combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds cannot be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the compounds are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the compositions of the disclosure. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific aspect or combination of aspects of the methods of the disclosure. [00135] References in the specification and concluding claims to parts by weight, of a particular element or component in a composition or article denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a composition containing 2 parts by weight of component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.

Claims

CLAIMS claimed:

A multilayer bioreactor bag comprising: a. an inside layer having a thickness between about 150 μιη to about 200 μιη; b. a barrier layer having a thickness between about 10 μιη to about 30

μιη; and an outside layer having a thickness between about 40 μιη to about 60 μιη.

The multilayer bioreactor bag according to claim 1, wherein the inside layer comprises a low density polyethylene, a linear low density polyethylene copolymer, or a combination thereof.

The multilayer bioreactor bag according to claim 2, wherein the linear low density polyethylene copolymer comprises linear low density ethylene-octene copolymer, linear low density ethylene-butene copolymer, or a combination thereof.

The multilayer bioreactor bag according to any one of claims 2-3, wherein the low density polyethylene comprises about 10 wt% to about 90 wt% of the inside layer.

The multilayer bioreactor bag according to any one of claims 2-4, wherein the linear low density polyethylene comprises about 10 wt% to about 90 wt% of the inside layer.

The multilayer bioreactor bag according to claim 2, wherein the low density polyethylene comprises about 85 wt% inside and the linear low density

polyethylene comprises about 15 wt% of the inside layer.

The multilayer bioreactor bag according to any one of claims 1-6, wherein the inside layer comprises about 180 μιη.

8. The multilayer bioreactor bag according to any one of claims 1-7, wherein the inside layer is adhered directly to the barrier layer.

9. The multilayer bioreactor bag according to any one of claims 1-8, wherein the

barrier layer is adhered directly to an internal surface of the inside layer, adhered directly to an internal surface of the outside layer, or both.

10. The multilayer bioreactor bag according to any one of claims 1-9, wherein the

barrier layer comprises about 20 μιη.

11. The multilayer bioreactor bag according to any one of claims 1-10, wherein the barrier layer comprises polyvinyl alcohol, aery lonitrile -butadiene copolymer, polyvinylidene chloride, polyalkylene carbonate, polyacrylonitrile, polypropylene, and polyethylene, or a combination thereof.

12. The multilayer bioreactor bag according to any one of claims 1-11, wherein the barrier layer comprises polyvinyl alcohol, poly( ethylene-co-vinyl alcohol), acrylonitrile- butadiene copolymer, polyvinylidene chloride, polyalkylene carbonate, polyacrylonitrile, polypropylene, and polyethylene, or a combination thereof.

13. The multilayer bioreactor bag according to any one of claims 1-12, wherein the barrier layer comprises poly( ethylene-co-vinyl alcohol).

14. The multilayer bioreactor bag according to any one of claims 1-13, wherein the outside layer comprises a low density polyethylene, a linear low density polyethylene copolymer, or a combination thereof.

15. The multilayer bioreactor bag according to claim 14, wherein the linear low density polyethylene copolymer comprises linear low density ethylene-octene copolymer, linear low density ethylene-butene copolymer, or a combination thereof.

16. The multilayer bioreactor bag according to any one of claims 14-15, wherein the low density polyethylene comprises about 70 wt% to about 100 wt% of the outside layer.

17. The multilayer bioreactor bag according to claims 14-16, wherein the linear low density polyethylene comprises about 1 wt% to about 30 wt% of the outside layer.

18. The multilayer bioreactor bag according to claim 14, wherein the low density

polyethylene comprises about 85 wt% of the outside and the linear low density polyethylene comprises about 15 wt% of the outside layer.

19. The multilayer bioreactor bag according to any one of claims 1-18, wherein the outside layer has a thickness of about 55 μιη.

20. A multilayer bioreactor bag having an inside layer comprising a low density

polyethylene/linear low density copolymer, a barrier layer comprising polyvinyl alcohol, and an outside layer comprising a low density polyethylene/linear low density copolymer.